WO2004041170A2 - Compositions and methods for the treatment of immune related diseases - Google Patents

Compositions and methods for the treatment of immune related diseases Download PDF

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Publication number
WO2004041170A2
WO2004041170A2 PCT/US2003/034312 US0334312W WO2004041170A2 WO 2004041170 A2 WO2004041170 A2 WO 2004041170A2 US 0334312 W US0334312 W US 0334312W WO 2004041170 A2 WO2004041170 A2 WO 2004041170A2
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WIPO (PCT)
Prior art keywords
polypeptide
jit
pro
acid sequence
antibody
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PCT/US2003/034312
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French (fr)
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WO2004041170A9 (en
Inventor
Hilary Clark
Jill Schoenfeld
Menno Van Lookeren
P. Mickey Williams
William I. Wood
Thomas D. Wu
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Genentech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to CA002503390A priority Critical patent/CA2503390A1/en
Priority to AU2003284357A priority patent/AU2003284357A1/en
Priority to EP03776595A priority patent/EP1578367A4/en
Priority to JP2004550183A priority patent/JP2006515747A/en
Priority to US10/533,519 priority patent/US20060263774A1/en
Publication of WO2004041170A2 publication Critical patent/WO2004041170A2/en
Priority to US12/153,928 priority patent/US20090098131A1/en
Publication of WO2004041170A9 publication Critical patent/WO2004041170A9/en
Priority to AU2010212279A priority patent/AU2010212279A1/en
Priority to US12/891,793 priority patent/US20120083420A1/en

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Definitions

  • the present invention relates to compositions and methods useful for the diagnosis and treatment of immune related diseases.
  • Immune related and inflammatory diseases are the manifestation or consequence of fairly complex, often multiple interconnected biological pathways which i normal physiology are critical to respond to insult or injury, initiate repair from insult or injury, and mount innate and acquired defense against foreign organisms. Disease or pathology occurs when these normal physiological pathways cause additional insult or injury either as directly related to the intensity of the response, as a consequence of abnormal regulation or excessive stimulation, as a reaction to self, or as a combination of these.
  • therapeutic intervention can occur by either antagonism of a detrimental process/pathway or stimulation of a beneficial process/pathway.
  • immune related diseases include immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasia, etc.
  • Immune related diseases could be treated by suppressing the immune response. Using neutralizing antibodies that inliibit molecules having immune stimulatory activity would be beneficial in the treatment of immune-mediated and hiflammatory diseases.
  • Molecules which inhibit the immune response can be utilized (proteins directly or via the use of antibody agonists) to inhibit the immune response and thus ameliorate immune related disease.
  • Macrophages represent an ubiquitously distributed population of fixed and circulating mononuclear phagocytes that express a variety of functions including cytokine production, killing of microbes and tumor cells and processing and presentation of antigens. Macrophages originate in the bone marrow from stem cells that give rise to a bipotent granulocyte/macrophage cell population. Distinct granulocyte and macrophage colony forming cell lineages arise from GM-CSF under the influence of specific cytokines. Upon division, monoblasts give rise to promonocytes and monocytes in the bone marrow. From there, monocytes enter the circulation. In response to particular stimuli (e.g. infection or foreign bodies) monocytes migrate into tissues and organs where they differentiate into macrophages.
  • stimuli e.g. infection or foreign bodies
  • Macrophages in various tissues vary in their morphology and function and have been assigned different names, e.g. Kupffer cells in the liver, pulmonary and alveolar macrophages in the lung and microglial cells in the central nervous system.
  • Kupffer cells in the liver e.g. pulmonary and alveolar macrophages in the lung and microglial cells in the central nervous system.
  • tissue macrophages e.g. monocytes were differentiated into macrophages by adherence to plastic in the presence of a combination of human and bovine serum.
  • monocytes-derived macrophages display features typical of differentiated tissue macrophages including their ability to phagocytose opsonized particles, secretion of TNF-alpha upon lipopolysaccharide (LPS) stimulation, formation of processes and the presence of macrophage cell surface markers.
  • LPS lipopolysaccharide
  • gene transcripts from non-differentiated monocytes harvested before adhering were compared with those at 1 day and 7 days in culture.
  • Genes selectively expressed in monocytes or macrophages could be used for the diagnosis and treatment of various chronic inflammatory or autoimmune diseases in the human.
  • surface expressed molecules or transmembrane receptors involved in monocyte/macrophage adhesion and endothelial cell transmigration could provide novel targets to treat chronic inflammation by interference with the homing of these cells to the site of inflammation.
  • transmembrane inhibitory receptors could be used to down-regulate monocyte/macrophage effector functions.
  • Therapeutic molecules can be antibodies, peptides, fusion proteins or small molecules.
  • the present invention concerns compositions and methods useful for the diagnosis and treatment of immune related disease in mammals, including humans.
  • the present invention is based on the identification of proteins (including agonist and antagonist antibodies) which are a result of stimulation of the immune response in mammals.
  • Immune related diseases can be treated by suppressing or enhancing the immune response. Molecules that enhance the immune response stimulate or potentiate the immune response to an antigen. Molecules which stimulate the immune response can be used therapeutically where enhancement of the immune response would be beneficial.
  • molecules that suppress the immune response attenuate or reduce the immune response to an antigen e.g., neutralizing antibodies
  • attenuation of the immune response would be beneficial e.g., inflammation
  • the PRO polypeptides, agonists and antagonists thereof are also useful to prepare medicines and medicaments for the treatment of immune-related and inflammatory diseases.
  • such medicines and medicaments comprise a therapeutically effective amount of a PRO polypeptide, agonist or antagonist thereof with a pharmaceutically acceptable carrier.
  • the admixture is sterile.
  • the invention concerns a method of identifying agonists or antagonists to a PRO polypeptide which comprises contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide.
  • the PRO polypeptide is a native sequence PRO polypeptide.
  • the PRO agonist or antagonist is an anti-PRO antibody.
  • the invention concerns a composition of matter comprising a PRO polypeptide or an agonist or antagonist antibody which binds the polypeptide in admixture with a carrier or excipient.
  • the composition comprises a therapeutically effective amount of the polypeptide or antibody.
  • the composition when the composition comprises an immune stimulating molecule, the composition is useful for: (a) increasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) stimulating or enhancing an immune response in a mammal in need thereof, (c) increasing the proliferation of monocytes/macrophages in a mammal in need thereof in response to an antigen, (d) stimulating the activity of monocytes/macrophages or (e) increasing the vascular permeability.
  • the composition when the composition comprises an immune inhibiting molecule, the composition is useful for: (a) decreasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) inhibiting or reducing an immune response in a mammal in need thereof, (c) decreasing the activity of monocytes/macrophages or (d) decreasing the proliferation of monocytes/macrophages in a mammal in need thereof in response to an antigen.
  • the composition comprises a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent.
  • the composition is sterile.
  • the invention concerns a method of treating an immune related disorder in a mammal in need thereof, comprising administering to the mammal an effective amount of a PRO polypeptide, an agonist thereof, or an antagonist thereto.
  • the immune related disorder is selected from the group consisting of: systemic lupus erythematosis, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis, idiopathic inflammatory myopathies, Sj ⁇ gren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renal disease, demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobili
  • the invention provides an antibody which specifically binds to any of the above or below described polypeptides.
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
  • the present invention concerns an isolated antibody which binds a PRO polypeptide.
  • the antibody mimics the activity of a PRO polypeptide (an agonist antibody) or conversely the antibody inhibits or neutralizes the activity of a PRO polypeptide (an antagonist antibody).
  • the antibody is a monoclonal antibody, which preferably has nonhuman complementarity determining region (CDR) residues and human framework region (FR) residues.
  • CDR complementarity determining region
  • FR human framework region
  • the antibody may be labeled and may be immobilized on a solid support.
  • the antibody is an antibody fragment, a monoclonal antibody, a single-chain antibody, or an anti-idiotypic antibody.
  • the present invention provides a composition comprising an anti-PRO antibody in admixture with a pharmaceutically acceptable carrier.
  • the composition comprises a therapeutically effective amount of the antibody.
  • the composition is sterile.
  • the composition may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability.
  • the antibody is a monoclonal antibody, an antibody fragment, a humanized antibody, or a single-chain antibody.
  • the invention concerns an article of manufacture, comprising:
  • composition of matter comprising a PRO polypeptide or agonist or antagonist thereof;
  • composition may comprise a therapeutically effective amount of the PRO polypeptide or the agonist or antagonist thereof.
  • the present invention concerns a method of diagnosing an immune related disease in a mammal, comprising detecting the level of expression of a gene encoding a PRO polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level in the test sample as compared to the control sample indicates the presence of immune related disease in the mammal from which the test tissue cells were obtained.
  • the present invention concerns a method of diagnosing an immune disease in a mammal, comprising (a) contacting an anti-PRO antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and a PRO polypeptide, in the test sample; wherein the formation of said complex is indicative of the presence or absence of said disease.
  • the detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type.
  • a larger quantity of complexes formed in the test sample indicates the presence or absence of an immune disease in the mammal from which the test tissue cells were obtained.
  • the antibody preferably carries a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art.
  • the test sample is usually obtained from an individual suspected of having a deficiency or abnormality of the immune system.
  • the invention provides a method for determining the presence of a PRO polypeptide in a sample comprising exposing a test sample of cells suspected of containing the PRO polypeptide to an anti-PRO antibody and determining the binding of said antibody to said cell sample.
  • the sample comprises a cell suspected of containing the PRO polypeptide and the antibody binds to the cell.
  • the antibody is preferably detectably labeled and/or bound to a solid support.
  • the present invention concerns an immune-related disease diagnostic kit, comprising an anti-PRO antibody and a carrier in suitable packaging.
  • the kit preferably contains instructions for using the antibody to detect the presence of the PRO polypeptide.
  • the carrier is pharmaceutically acceptable.
  • the present invention concerns a diagnostic kit, containing an anti-PRO antibody in suitable packaging.
  • the kit preferably contains instructions for using the antibody to detect the PRO polypeptide.
  • the invention provides a method of diagnosing an immune-related disease in a mammal which comprises detecting the presence or absence or a PRO polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of the PRO polypeptide in said test sample is indicative of the presence of an immune-related disease in said mammal.
  • the present invention concerns a method for identifying an agonist of a
  • PRO polypeptide comprising:
  • the invention concerns a method for identifying a compound capable of inhibiting the activity of a PRO polypeptide comprising contacting a candidate compound with a PRO polypeptide under conditions and for a time sufficient to allow these two components to interact and determining whether the activity of the PRO polypeptide is inhibited.
  • either the candidate compound or the PRO polypeptide is immobilized on a solid support.
  • the non- immobilized component carries a detectable label.
  • this method comprises the steps of: (a) contacting cells and a test compound to be screened in the presence of a PRO polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide; and (b) determining the induction of said cellular response to determine if the test compound is an effective antagonist.
  • the invention provides a method for identifying a compound that inhibits the expression of a PRO polypeptide in cells that normally express the polypeptide, wherein the method comprises contacting the cells with a test compound and determining whether the expression of the PRO polypeptide is inhibited. In a preferred aspect, this method comprises the steps of:
  • the present invention concerns a method for treating an immune-related disorder in a mammal that suffers therefrom comprising administering to the mammal a nucleic acid molecule that codes for either (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide or (c) an antagonist of a PRO polypeptide, wherein said agonist or antagonist may be an anti-PRO antibody.
  • the mammal is human.
  • the nucleic acid is administered via ex vivo gene therapy.
  • the nucleic acid is comprised within a vector, more preferably an adenoviral, adeno-associated viral, lentiviral or retroviral vector.
  • the invention provides a recombinant viral particle comprising a viral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptide of a PRO polypeptide, or (c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein the viral vector is in association with viral structural proteins.
  • the signal sequence is from a mammal, such as from a native PRO polypeptide.
  • the invention concerns an ex vivo producer cell comprising a nucleic acid construct that expresses retroviral structural proteins and also comprises a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptide of a PRO polypeptide or (c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein said producer cell packages the retroviral vector in association with the structural proteins to produce recombinant retroviral particles.
  • the invention provides a method of increasing the activity of monocytes/macrophages in a mammal comprising administering to said mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the activity of monocytes/macrophages in the mammal is increased.
  • the invention provides a method of decreasing the activity of monocytes/macrophages in a mammal comprising administering to said mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the activity of monocytes/macrophages in the mammal is decreased.
  • the invention provides a method of increasing the proliferation of monocytes/macrophages in a mammal comprising administering to said mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the proliferation of monocytes/macrophages in the mammal is increased.
  • the invention provides a method of decreasing the proliferation of monocytes/macrophages in a mammal comprising administering to said mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the proliferation of monocytes/macrophages in the mammal is decreased.
  • the invention provides vectors comprising DNA encoding any of the herein described polypeptides.
  • Host cell comprising any such vector are also provided.
  • the host cells may be CHO cells, E. coli, or yeast.
  • a process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
  • the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence.
  • Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin.
  • the invention provides an antibody which specifically binds to any of the above or below described polypeptides.
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
  • the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide.
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence
  • the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%) nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86%) nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about
  • Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains of the herein described PRO polypeptides are contemplated.
  • Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO antibody or as antisense oligonucleotide probes.
  • nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 190 nucle
  • novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such PRO polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PRO polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody.
  • the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences herein above identified.
  • the invention concerns an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99%
  • the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% ammo acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93%o amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about
  • the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as herein before described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture. Another aspect the invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated.
  • Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
  • the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein.
  • the agonist or antagonist is an anti-PRO antibody or a small molecule.
  • the invention concerns a method of identifying agonists or antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide.
  • the PRO polypeptide is a native PRO polypeptide.
  • the invention concerns a composition of matter comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier.
  • the carrier is a pharmaceutically acceptable carrier.
  • Another embodiment of the present invention is directed to the use of a PRO polypeptide, or an agonist or antagonist thereof as herein before described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide, an agonist or antagonist thereof or an anti-PRO antibody.
  • cDNA sequences which are differentially expressed in differentiated macrophages as compared to normal undifferentiated monocytes are individually identified with a specific alphanumerical designation. These cDNA sequences are differentially expressed in monocytes that are specifically treated as described in Example 1 below. If start and/or stop codons have been identified in a cDNA sequence shown in the attached figures, they are shown in bold and underlined font, and the encoded polypeptide is shown in the next consecutive figure.
  • FIGS. 1-2517 show the nucleic acids of the invention and their encoded PRO polypeptides. Also included, for convenience is a List of Figures attached hereto as Appendix A, which gives the figure number and the corresponding DNA or PRO number. List of Figures
  • Figure 3 DNA304680, HSPCB, 200064_at
  • Figure 57A-B DNA328361, BAA92570.1, 47773_at
  • Figure 7A-B DNA328348, MAP4, 243_g_at
  • Figure 61 DNA328363, DNA328363, 52651_at
  • Figure 11 DNA272223, NP.004444.1, 33494_at Figure 65A-B: DNA327528, BAB33338.1, 55081.at
  • Figure 13 DNA327522, NP_000396.1, 33646_g.at
  • Figure 67 DNA225650, NP-057246.1, 48825.at
  • Figure 15 DNA328349, NP_004556.1, 33760- ⁇ t Figure 69: DNA328365, NP-060541.1, 58780-S.at
  • Figure 17A-B DNA328350, NP_056155.1, 34764_at
  • Figure 71 DNA328366, NP_079233.1, 59375-at
  • Figure 19 DNA328351, NP_006143.1, 35974_at
  • Figure 73 DNA328367, NP-079108.2, 6047 l_at
  • Figure 21 DNA328352, NP.004183.1, 36553_at
  • Figure 75 DNA327876, NP-005081.1, 60528_at
  • Figure 25 DNA326969, NP.036455.1, 36711.at Figure 79: DNA328369, BC007634, 90610_at
  • Figure 26 PR083282
  • Figure 80A-B DNA328370, NP_001273.1,
  • Figure 27 DNA304703, NP_005923.1, 36830_at 200615-s.at
  • Figure 29 DNA328353, AAB72234.1, 37079_at
  • Figure 82 DNA323806, NP.075385.1, 200644_at
  • Figure 31 DNA103289, NP_006229.1, 37152_at
  • Figure 84 DNA327532, GLUL, 200648.s_at
  • Figure 33A-B DNA255096, NP_055449.1, 37384_at
  • Figure 86 DNA227055, NP.002625.1, 200658.s_at
  • Figure 37 DNA328354, PARVB, 37965_at
  • Figure 90 DNA83172, NP-003109.1, 200665_s_at
  • Figure 39 DNA53531, NP.001936.1, 38037_at
  • Figure 92 DNA328371, NP-004347.1, 200675_at
  • Figure 41 DNA254127, NP.008925.1, 38241_at
  • Figure 94A-B DNA328372, 105551.7, 200685_at
  • Figure 43 DNA328355, NP.006471.2, 38290.at
  • Figure 96 DNA324633, BC000478, 200691 J3_at
  • Figure 45 DNA328356, BC013566, 39248_at
  • Figure 98 DNA324633, NP-004125.2, 200692.s_at
  • Figure 47 DNA328357, 1452321.2, 39582 _at
  • Figure 100 DNA88350, NP-000168.1, 200696-S.at
  • Figure 49A-B DNA328358, STK10, 40420_at
  • Figure 102 DNA328373, AB034747, 200704_at
  • Figure 51A-B DNA328359, BAA21572.1, 41386i_at
  • Figure 104 DNA328374, NP-004853.1, 200706.s_at
  • Figure 53A-D DNA328360, NP.055061.1, 41660_at
  • Figure 106 DNA328375, NP-002071.1, 200708_at
  • Figure 54 PRO84220 Figure 107: PRO80880 Figure 108: DNA328376, NP_001210.1, 200755_s_at Figure 161: DNA225878, NP_004334.1, 200935 Jtt
  • Figure 110A-B DNA269826, NP_003195.1, Figure 163: DNA328382, 160963.2, 20094 l it
  • Figure 111 PR058228 Figure 165: DNA328383, NP.004956.3, 200944 ;_at
  • Figure 112 DNA325414, NP_001900.1, 200766jtt Figure 166: PR084238
  • Figure 113 PR0292 Figure 167 A-B: DNA287217, NP_001750.1,
  • Figure 114A-C DNA188738, NP_002284.2, 200771 Jtt 200953-S-at
  • Figure 120 DNA272928, NP_055579.1, 200794jc_at Figure 173: DNA326040, NP_005715.1, 200973-SJtt
  • Figure 122A-B DNA327536, BC017197, 200797_s_at Figure 175: DNA324110, NP_005908.1, 200978 Jit
  • Figure 124 DNA287211, NP_002147.1, 200806_s_at
  • Figure 177 DNA328386, NP.000602.1, 200983-x_at
  • Figure 126 DNA326655, NP_002803.1, 200820 Jit
  • Figure 179 DNA275408, NP_001596.1, 20100 ⁇ Jit
  • Figure 128A-B DNA328378, AB032261, 200832.S S ⁇ Figure 181: DNA328387, NP.001760.1, 201005 tt
  • Figure 130 DNA103558, NP_005736.1, 200837 Jit Figure 183: DNA103593, NP-000174.1, 201007 Jit
  • Figure 132 DNA196817, NP_001899.1, 200838 Jit Figure 185: DNA304713, NP_006463.2, 201008.S it
  • Figure 134A-B DNA327537, NP_004437.1, Figure 187: DNA328388, BC010273, 201013-SJit
  • Figure 135 PR083581
  • Figure 189 DNA328389, NP_006861.1, 201021.SJtt
  • Figure 136 DNA323982, NP_004896.1, 200844.s_at Figure 190: PR084241
  • Figure 137 PRO80709
  • Figure 191 DNA328390, NP_002291.1, 201030jcjit
  • Figure 139 PRO80619
  • Figure 193 DNA196628, NP_005318.1, 201036_s-at
  • Figure 140A-B DNA228029, NP-055577.1, 200862j ⁇ t
  • Figure 194 PR025105
  • Figure 141 PR038492
  • Figure 195 DNA287372, NP_002618.1, 201037 Jit
  • Figure 142 DNA328379, BC015869, 200878 _at Figure 196: PR069632
  • Figure 143 PR084234
  • Figure 197 DNA328391, NP.004408.1, 201041-SJrt
  • Figure 146A-B DNA274281, NPD36347.1
  • Figure 200 DNA227143, NP-036400.1, 201050jtt
  • Figure 147 PRO62204 Figure 202: DNA328392, 1500938.11, 201051 Jit
  • Figure 149 PR036491
  • Figure 204 DNA328261, AF130103, 201060_x_at
  • Figure 150 DNA326819, NP.000678.1, 200903j>_at Figure 205: DNA325001, NP_002794.1, 201068_sjtt
  • Figure 152 DNA328380, HSHLAEHCM, 200904 Jit Figure 207: DNA328393, NP_001651.1, 201096.sj ⁇ t
  • Figure 153 DNA328381, NP_005507.1, 200905_ ⁇ ⁇ t
  • Figure 208 PRO81010
  • Figure 155 DNA272695, NP.001722.1, 200920_s_at Figure 210A-B: DNA328395, NP_056198.1,
  • Figure 160 PRO38005 Figure 214: DNA328397, NPJJ02622.1, 201118 ⁇ t
  • Figure 217 PR034737
  • Figure 271 DNA150781, NP_001414.1, 201324j ⁇ t
  • Figure 218 DNA325398, NP_004083.2, 201135 ⁇ t Figure 272: PRO 12467
  • Figure 219 PRO81930
  • Figure 273 DNA328409, NP_002075.2, 201348 ⁇ t
  • Figure 221 PR02824
  • Figure 275 DNA324475, NPD04172.2, 201387 _sj ⁇ t
  • Figure 223 PR081141
  • Figure 277 DNA226353, NP.005769.1, 201395 jit
  • Figure 224 DNA151802, NP-003661.1, 201169_s _at Figure 278: PR036816
  • Figure 225 PRO 12890
  • Figure 279 DNA328410, NP.004519.1, 201403_sj ⁇ t
  • Figure 226 DNA226662, NP_057043.1, 201175 jit Figure 280: PRO60174
  • Figure 227 PR037125
  • Figure 281A-B DNA328411, 1400253.2, 201408 it
  • Figure 229 PR02638
  • Figure 283 DNA328412, NP_060428.1, 201411 j; ⁇ t
  • Figure 230 DNA273342, NP_005887.1, 201193 tt Figure 284: PR084257
  • Figure 231 PR061345
  • Figure 285 DNA273517, NP_000169.1, 201415 jit
  • Figure 232 DNA328399, NP_003000.1, 201194 tt Figure 286: PR061498
  • Figure 233 PR084248
  • Figure 287 DNA327550, NP_001959.1, 201435 _s.at
  • Figure 235 PRO4780
  • Figure 290 DNA325049, NP.005605.1, 201453.x Jit
  • Figure 237 PRO 1409 Figure 292: DNA274343, NP_000894.1, 201467_SJit
  • Figure 240 DNA103488, NP-002583.1, 201202_at Figure 295: PR084258
  • Figure 241 PR04815
  • Figure 296 DNA328414, NP.003891.1, 201471 _s_at
  • Figure 243A-B DNA328402, NP-073713.1, Figure 298: DNA103320, NP.002220.1, 201473j ⁇ t
  • Figure 244 PR084249
  • Figure 300 DNA88608, NP_002893.1, 201485 js it
  • Figure 246 PR081914
  • Figure 302 DNA304459, BC005020, 201489 Jit
  • Figure 248 PRO37078
  • Figure 304 DNA304459, NP_005720.1, 201490 _s_at
  • Figure 250 PRO84250 Figure 306: DNA253807, NP_065390.1, 201502 jsjit
  • Figure 253A-B DNA328404, NP_003321.1 , 201266 tt Figure 309: PRO60207
  • Figure 254 PR084251
  • Figure 310 DNA328416, NP.002613.2, 201507 it
  • Figure 256 PR03637
  • Figure 312 DNA271931, NP.005745.1, 201514jij ⁇ t
  • Figure 260 PR084252
  • Figure 316 DNA328417, ATP6V1F, 201527 jit
  • Figure 264 PR084254
  • Figure 320 DNA328419, NP.002779.1, 201532j ⁇ t
  • Figure 266 PR084255
  • Figure 322 DNA328420, BC002682, 20l537js.at
  • Figure 268 PRO38010 Figure 324: DNA88464, NP.005552.2, 201551 -s_at Figure 325: PRO2804 Figure 375: PR036359
  • Figure 326A-B DNA290226, NP-039234.1
  • Figure 376 DNA151017, NP_004835.1, 201810_s_at
  • Figure 327 PRO70317
  • Figure 378 DNA328429, NP_079106.2, 201818j ⁇ t
  • Figure 328 DNA227071, NP-000260.1, 201577.at Figure 379: PRO81201
  • Figure 329 PR037534
  • Figure 380 DNA328430, NP.005496.2, 201819_at
  • Figure 330A-B DNA227307, NP-009115.1, Figure 381: PR084267
  • Figure 332 DNA255406, NP-005533.1, 201625 J3_at
  • Figure 384 DNA150650, NP_057086.1, 201825 s_at
  • Figure 334A-B DNA328421, 475621.10, 201646 Jit Figure 386: DNA304710, NP.001531.1, 201841 _sj ⁇ t
  • Figure 336A-B DNA220748, PJ000201.1, 201656 ⁇ t
  • Figure 388 DNA88450, NP.000226.1, 201847 Jit
  • Figure 340A-B DNA328422, NP_004448.1, Figure 392: DNA272066, NP .002931.1, 201872_s_at
  • Figure 341 PR084263
  • Figure 394 DNA328431, NP_001817.1, 201897 js_at
  • Figure 343 PR02121
  • Figure 396 DNA103214, NP.006057.1, 201900 JSJit
  • Figure 345 PR061148
  • Figure 398 DNA227112, NP_006397.1, 201923j ⁇ t
  • Figure 347 PR059291
  • Figure 400 DNA83046, NP-000565.1, 201926_sj ⁇ t
  • Figure 348 DNA271223, NP-005070.1, 201689 js.at Figure 401 : PR02569
  • Figure 349 PR059538
  • Figure 402 DNA273014, NP.000117.1, 201931 Jit
  • Figure 350A-B DNA323965, NP_002848.1, Figure 403: PRO61085
  • Figure 352 DNA270883, NP_001061.1, 201714 ⁇ t Figure 406: DNA274167, NP.006422.1, 201946-S.at
  • Figure 354A-B DNA328425, NP.065207.2, Figure 408 A-B: DNA327562, HSMEMD, 201951 Jit
  • Figure 356 DNA328426, NP.000582.1, 201743 Jtt Figure 411: DNA227290, NP_055861.1, 201965 J3-at
  • Figure 358 DNA150429, NP-002813.1, 201745 Jit Figure 413A-B: DNA328432, NP-005768.1, 201967 Jit
  • Figure 360 DNA272465, NP_004543.1, 201757 Jit Figure 415A-B: DNA328433, ATP6V1A1,
  • Figure 362 DNA328427, NP_061109.1, 201760.S _at Figure 416: PR084268
  • Figure 367 PRO80670
  • Figure 421A-D DNA328434, NP-055816.2
  • Figure 368 DNA88619, NP.002924.1, 201785 tt 201996.s_at
  • Figure 370A-B DNA328428, NP_038479.1
  • Figure 423 DNA328435, NP-002481.1, 202001 jj_at
  • Figure 373 PRO38026 Figure 427: DNA327841, NP.068813.1, 202005 Jit
  • Figure 430 PRO84270 Figure 481 : DNA304716, NP 510867.1 , 202284 jjjtt
  • Figure 432 PR083593
  • Figure 483 DNA270142, NP.005947.2, 202309 _at
  • Figure 435A-B DNA270997, NPJ305047.1
  • Figure 486 PR062362
  • Figure 437A-B DNA327565, NP.056392.1, Figure 489: DNA106239, DNA106239, 202351 Jtt
  • Figure 439A-B DNA327566, NP_000373.1
  • Figure 492 DNA327074, FLJ21174, 202371 jit
  • Figure 440 PR083595
  • Figure 494 DNA 149091, DNA149091, 202377 jvt
  • Figure 441 DNA226116, NP_002990.1, 202071j ⁇ t
  • Figure 495A-B DNA151045, NP_005376.2
  • Figure 444 PR084272
  • Figure 497 A-B DNA200236, NP_003807.1, 202381 _at
  • Figure 448 PRO70409
  • Figure 501 DNA290234, NP_002914.1, 202388j ⁇ t
  • Figure 450 PR02683
  • Figure 503 DNA269766, NP_005646.1, 202393-S_at
  • Figure 451 DNA328440, NP_004517.1, 202107 _s_at Figure 504: PR058175
  • Figure 452 PR084274
  • Figure 505 DNA227612, NPD56230.1, 202427 _s_at
  • Figure 454 PRO60884 Figure 507: DNA324171, NP.065438.1, 202428 j jit
  • Figure 456 PROO Figure 509A-B: DNA327576, NP_000095.1,
  • Figure 459A-C DNA328443, NP.004371.1, 202160.at Figure 511A-D: DNA328450, NP_077719.1,
  • Figure 461A-C DNA271201, NP_005881.1, Figure 512: PRO84280
  • Figure 463 DNA328258, SLC16A1, 202236 _s _at Figure 515: DNA227921, NP-003789.1, 202468-SJit
  • Figure 468 PRO70754
  • Figure 520 PRO36029 A-B: DNA103449, NP.008862.1,
  • Figure 473 DNA256533, NP_006105.1, 202264j>Jit Figure 525A-B: DNA274893, NP_006282.1,
  • Figure 478 PR059821
  • Figure 529 DNA219229, NP-002189.1, 202531j ⁇ t
  • Figure 479 DNA328447, NP_000393.2, 202275 Jit Figure 530: PR034544
  • Figure 531A-B DNA274852, NP_004115.1, 202752-X_at
  • Figure 532 PRO62605
  • Figure 585A-C DNA328462, HSA303079,
  • Figure 533 DNA328453, NP_003752.2, 202546 ⁇ t 202759 J; Jit
  • Figure 535A-B DNA328454, NP_057525.1, Figure 587A-C: DNA328463, NP_009134.1,
  • Figure 537 DNA150817, NP_000840.1, 202554 _s_at Figure 589: DNA226080, NP..001601.1, 202767 Jit
  • Figure 539 DNA227994, NP_009107.1, 202562 jjjit Figure 591A-B: DNA150977, NP.006723.1, 202768 Jit
  • Figure 545 DNA328456, NP_000467.1, 202587.S Jit Figure 597A-B: DNA103521, NP_004163.1, 202800 Jit
  • Figure 547 DNA328457, NP_036422.1 , 202606 _s_at
  • Figure 599A-B DNA327583, ABCCl, 202805-S_at
  • Figure 548 PRO70421
  • Figure 600 PRO83604
  • Figure 549 DNA103245, NP_002341.1, 202626j;.at Figure 601: DNA328465, NP-005639.1, 202823 Jit
  • Figure 551 DNA83141, NP.000593.1, 202627 js_at Figure 603: DNA225865, NP.004986.1, 202827 -S.at
  • Figure 553 DNA254129, NP.006001.1, 202655 jit
  • Figure 605 DNA225926, NP_000138.1, 202838 Jtt
  • Figure 555 DNA270379, NP-002792.1, 202659 Jit Figure 607: DNA328466, NP.004554.1 , 202847 Jit
  • Figure 561 DNA273542, NP-002991.1, 202675 Jit Figure 613: DNA328467, SP100, 202864 _sj ⁇ t
  • Figure 568 PRO59409
  • Figure 620 PR084295
  • Figure 569A-B DNA150467, NP_055513.1
  • Figure 621A-B DNA255318, NP_036204.1
  • Figure 571 A-B DNA328459, NP-004332.2, 202715 ⁇ t Figure 623A-B: DNA328470, NP_055620.1, 202909 Jit
  • Figure 575 DNA328460, NP_004190.1, 202733jtt
  • Figure 627 DNA272425, NP.001489.1, 202923.sjit
  • Figure 577 DNA150713, NP.006570.1, 202735 Jit
  • Figure 629 DNA328471, ZMPSTE24, 202939 Jit
  • Figure 579A-B DNA328461, 350230.2, 202741 jtt
  • Figure 631 DNA269481, NP_001976.1, 202942 Jit
  • Figure 581 DNA271973, NP_002722.1, 202742 _s_at Figure 633: DNA328472, NP.000482.2, 202953 Jit
  • Figure 583A-B DNA150943, NP_036376.1, Figure 635A-B: DNA328473, NP_006473.1, 202968ji.at Figure 687: DNA328487, AF251295, 203299 JS_at
  • Figure 637A-C DNA328474, 1501914.1, 202969 Jit Figure 689: DNA328488, NP-003907.2, 203300_x jit
  • Figure 639 DNA325915, ZAP128, 202982j>_at Figure 691: DNA328489, NP.006511.1, 203303 Jit
  • Figure 641 DNA271272, NP_000366.1, 203031 S
  • a Figure 693A-B DNA328490, NP_000120.1, 203305 Jtt
  • Figure 645A-B DNA271865, NP_055566.1, Figure 697: DNA328491, ICAP-1A, 203336 3_at
  • Figure 646 PRO60145 Figure 699A-B: DNA328492, NP_056125.1,
  • Figure 651 DNA256830, NP-004815.1, 203100_s_at Figure 703: DNA328494, RPS6KA1, 203379 Jit
  • Figure 656 PRO38045 Figure 709A-B: DNA254616, NP_004473.1,
  • Figure 659A-B DNA328478, NP_055720.2,
  • Figure 711 DNA326892, NP-003711.1, 203405 Jit
  • Figure 660 PRO84304 Figure 713: DNA323927, NP-005563.1, 203411.sjit
  • Figure 664 PRO84305
  • Figure 717 DNA273410, NP-004036.1, 203454.sjit
  • Figure 665A-C DNA328480, NP_001990.1, 203184 jit Figure 718: PRO61409
  • Figure 666 PRO84306 Figure 719: DNA328495, NP_055578.1, 203465 _at
  • Figure 667A-B DNA271010, NP_055552.1, 203185_at Figure 720: PR058967
  • Figure 668 PR059339
  • Figure 721 DNA328496, NP_002428.1, 203466 jit
  • Figure 672 PRO84307
  • Figure 724 PRO50686
  • Figure 675 DNA328483, NP-061163.1, 203255j ⁇ t Figure 727
  • A-C DNA328498, AF285167, 203505 Jit
  • Figure 677 DNA227127, NP.003571.1, 203269 Jit Figure 729 A-B: DNA188400, NP_001057.1, 203508 Jtt
  • Figure 681 DNA302020, NP.005564.1, 203276 Jit Figure 733: DNA272911, NP-006545.1, 203517j ⁇ t
  • Figure 683A-B DNA328485, BHC80, 203278 -S.at Figure 735A-D: DNA328500, NP_000072.1,
  • Figure 686 PRO60119 Figure 737A-B: DNA103296, NP_006369.1, 203528j ⁇ t Figure 738: PR04626 Figure 791A-B: DNA272451, HSU86453, 203879 Jit
  • Figure 739 DNA323910, NPJ002956.1, 203535 _at Figure 792: PRO60700
  • Figure 740 PRO80648
  • Figure 793 DNA82429, NP_003011.1, 203889 Jit
  • Figure 742 PRO60653
  • Figure 796 PR037815
  • Figure 743 DNA328501, NP-076984.1, 203545j ⁇ t
  • Figure 797 DNA150974, NP_005684.1, 203920 Jit
  • Figure 749 DNA328503, NP-000272.1 , 203557 JS _at Figure 803: DNA227232, NP_001850.1, 203971 Jit
  • Figure 761A-B DNA328504, 1400155.1, 203608_at Figure 815: DNA226342, NP_000305.1, 204054 jit
  • Figure 769 DNA254642, NP_004100.1, 203646j ⁇ t Figure 823A-C: DNA328519, NP.075463.1,
  • Figure 772 PR04761
  • Figure 825 DNA328520, NP-079353.1, 204080 Jit
  • Figure 773A-B DNA272998, NP.055548.1, 203651 _at Figure 826: PR084334
  • Figure 774 PRO61070 Figure 827 A-B: DNA150739, NP_006484.1,
  • Figure 777 DNA255298, NP_004394.1, 203695 _sj ⁇ t
  • Figure 829 DNA227130, NP.002551.1, 204088 Jit
  • Figure 779 DNA227020, NP_001416.1, 203729 Jit Figure 831: DNA328521, NP-003069.1, 204099 _at
  • Figure 781 DNA328509, NP.006739.1, 203760 _s Jit Figure 833: DNA328522, NP.001769.2, 204118 Jit
  • Figure 785A-B DNA194602, NP-006370.1, Figure 837: DNA328524, NP_057097.1, 204125 Jit
  • Figure 786 PR023944
  • Figure 839 DNA328525, BC021224, 204131 _s.at
  • Figure 788 PR057838
  • Figure 841 DNA103532, NP.003263.1, 204137 Jit
  • Figure 846 PRO58901
  • Figure 900 PR011581
  • Figure 847 DNA328526, NP.000841.1, 204149_s_at
  • Figure 901 DNA328254, NP_000934.1, 204518_s_at
  • Figure 849A-B DNA150497, DNA150497, Figure 903A-B: DNA328535, NP_009147.1, 204544 Jit
  • Figure 850 PRO 12296
  • Figure 905 DNA225993, NP-000646.1, 204563 Jit
  • Figure 851 A-B DNA328527, NP_055751.1, Figure 906: PR036456
  • Figure 853 DNA328528, MLC1SA, 204173 Jit Figure 909: DNA151910, NP_004906.2, 204567 _sj ⁇ t
  • Figure 855 DNA328529, NP_001620.2, 204174_at Figure 911: DNA270564, NP_004499.1, 204615-XJit
  • Figure 857 DNA226380, NP-001765.1, 204192j ⁇ t
  • Figure 913 DNA328536, 1099945.20, 204619 _s Jit
  • Figure 859 DNA273070, NP-005189.2, 204193j ⁇ t Figure 915A-D: DNA328537, NP_004376.2,
  • Figure 862 PR037977
  • Figure 917 DNA151048, NP_006177.1, 204621 _s_at
  • Figure 866 PR071356
  • Figure 921A-B DNA88429, NP-000203.1,
  • Figure 867A-B DNA188734, NP_001261.1, 204258 jtt 204628-S.at
  • Figure 869 DNA226577, NP_071390.1, 204265-SJit Figure 923: DNA226079, NP_001602.1, 204638j ⁇ t
  • Figure 871 DNA273802, NP-066950.1, 204285_sj ⁇ t
  • Figure 925 DNA272078, NP.003019.1, 204657 Ji_at
  • Figure 875 DNA328531, NP-037542.1, 204348 _s_at Figure 929A-B: DNA328539, NP.000121.1,
  • Figure 878 PR084339
  • Figure 931 DNA328540, NP_006144.1, 204725 JSJit
  • Figure 880 PR036213 Figure 933A-B: DNA325192, NP.038203.1,
  • Figure 881 DNA328533, NP_003647.1, 204392j ⁇ t 204744 _s_at
  • Figure 883 DNA272469, NP.005299.1, 204396_SJit Figure 935: DNA328541, NP-004503.1, 204773j ⁇ t
  • Figure 887 DNA225756, NP_001636.1, 204416-x.at
  • Figure 939 DNA327050, NP_009199.1, 204787_at
  • Figure 891A-B DNA88476, NP_002429.1, 204438j ⁇ t
  • Figure 943 DNA272121, NP.005895.1, 204790j ⁇ t
  • Figure 892 PR02811 Figure 944: PRO60391 Figure 893 DNA 150972, NP.005252.1, 204472 _at Figure 945: DNA324799, NP_061823.1, 204806. ⁇ j ⁇ t Figure 894 PR012162 Figure 946: PR081414 Figure 895 DNA194652, NPJOOI 187.1, 204493 Jit Figure 947: DNA154704, DNA154704, 204807 Jit Figure 896 PR023974 Figure 948: DNA328544, NP_006673.1, 204834 Jit Figure 897 DNA328534, NP J56307.1, 204494.sj ⁇ t Figure 949: PR084347 Figure 898: PR084341 Figure 950: DNA225661, NP_001944.1, 204858 >Jit Figure 951: PR036124 Figure 1003: PR084354
  • Figure 952 DNA328545, NP-064525.1, 204859 _s_at Figure 1004: DNA328555, NP_001241.1, 205153j>Jit
  • Figure 954A-B DNA227629, NP-004527.1, Figure 1006: DNA80896, NP.001100.1, 205180-SJit
  • Figure 955 PRO38092 Figure 1008: DNA328556, NP-004568.1, 205194 Jit
  • Figure 962A-B DNA76503, NP.001549.1, 204912 Jit Figure 1015: PRO1910
  • Figure 965 PRO208 Figure 1018: DNA227081, NP_000390.2, 205249 Jit
  • Figure 967 PR038477
  • Figure 1020 DNA328557, NP_001098.1, 205260 _s -at
  • Figure 975 PRO84350 Figure 1028: DNA325783, NP.002558.1, 205353 JSJit
  • Figure 977 PRO70371
  • Figure 1030 DNA88215, NP_001919.1, 205382 3j ⁇ t
  • Figure 982A-B DNA273686, NP-055520.1, 205003 _at Figure 1036: DNA327638, NP_005516.1, 205404 jit
  • Figure 984 DNA272427, NP-004799.1, 205005 JS Jit Figure 1038: DNA328562, NP_000010.1, 205412j ⁇ t
  • Figure 986 DNA194830, NP.055437.1, 205011j ⁇ t
  • Figure 1040A-B DNA328563, NPU05329.2,
  • Figure 990A-B DNA328552, NP_055886.1, Figure 1043: PR084361
  • Figure 992 DNA328553, NP-061944.1, 205070 Jit Figure 1046: DNA328565, NP_057070.1, 205474j ⁇ t
  • Figure 994 DNA194627, NP-003051.1, 205074 ⁇ t
  • Figure 1048 DNA226153, NP.002649.1, 205479.s_at
  • Figure 996 DNA272181, NP-006688.1, 205076-SJit Figure 1050: DNA287224, NP_005092.1, 205483 _s_at
  • Figure 998 DNA254216, NPD02020.1, 205119-SJit
  • Figure 1052 DNA328566, NP_060446.1, 205510jsJit
  • Figure 1000 DNA299899, NP.002148.1, 205133 3_at Figure 1054: DNA328567, NP_006797.2, 205548 s ⁇ t
  • Figure 1002 DNA328554, NP-038202.1, 205147 _x Jit Figure 1056: DNA227535, NP_066190.1, 205568 Jit Figure 1057: PR037998 Figure 1 PR04944
  • Figure 1058A-B DNA327643, NP-055712.1, Figure 1 DNA328576, HSU20350, 205898 Jit
  • Figure 1060A-C DNA328568, NP-006720.1, Figure 1 PR059588
  • Figure 1062 DNA324324, NP_000679.1, 205633_sj ⁇ t
  • Figure 1 13 PRO25031
  • Figure 1065 PR084365 .
  • Figure 1 16A-B DNA270867, NP-006217.1,
  • Figure 1068 DNA287317, NP-003724.1, 205660 it Figure 1 18: DNA76516, NP_000556.1, 205945j ⁇ t
  • Figure 1070 DNA328570, NP-004040.1, 205681 it Figure 1 20: DNA196439, NP-003865.1, 205988 Jit
  • Figure 1072 DNA327644, NP_060395.2, 205684 _s Jit Figure 1 22: DNA36722, NP_000576.1, 205992 j>_at
  • Figure 1074 DNA150621, NP-036595.1, 205704 j;_at Figure 1 24: DNA328579, BC020082, 206020jat
  • Figure 1076 DNA328571, NP-001254.1, 205709.sjit Figure 1 26: DNA328580, HSU27699, 206058 Jit
  • Figure 1078 DNA88106, NP_004325.1, 205715 ⁇ t
  • Figure 1 28 DNA328581, NP-002122.1, 206074_sj ⁇ t
  • Figure 1080 DNA270401, NP-003140.1, 205743 jit Figure 1 30: DNA328582, NP-001865.1, 206100JU
  • Figure 1082 DNA275620, NP.000628.1, 205770 it Figure 1 32: DNA226105, NP_002925.1, 206111 jit
  • Figure 1084 DNA88187, NP-001757.1, 205789 it Figure 1 34: DNA225764, NP.000037.1, 206129 JSJit
  • Figure 1086 DNA76517, NP_002176.1, 205798 tt Figure 1 36: DNA328583, ASGR2, 206130 3 Jit Figure 1087: PR02541 Figure 1 37: PR084372
  • Figure 1088A-B DNA271915, NP_056191.1, Figure 1 38: DNA327656, NP_055294.1, 206134j ⁇ t
  • Figure 1089 PRO60192 Figure 1 40A-B: DNA271837, NP.055497.1,
  • Figure 1092 DNA328572, NPD04309.2, 205808 jit Figure 1 42: DNA328584, NP.001148.1, 206200.S Jit
  • Figure 1094 DNA328573, NP-006761.1, 205819j ⁇ t Figure 1 44: DNA226058, NP.005075.1, 206214 Jit
  • Figure 1096A-B DNA328574, NP.004963.1, Figure 1 46: DNA218691, NP_003832.1, 206222j ⁇ t
  • Figure 1097 PR084368 Figure 1 48A-C: DNA328585, AF286028,
  • Figure 1100 DNA328575, NP-071754.2, 205872 _ ⁇ ⁇ t Figure 1 50: PR084373
  • Figure 1101 PR084369
  • Figure 1 51 DNA328587, NP-002612.1, 206380_s ⁇ t
  • Figure 1102A-B DNA220746, NP_000876.1, Figure 1 52: PR02854
  • Figure 1104A-B DNA273962, NP-055605.1, Figure 1 55: DNA328588, NP_060823.1, 206500 -S_at
  • Figure 1105 PRO61910 Figure 1 57: DNA270444, NP-004824.1, 206513 it
  • Figure 1106 DNA93423. NP-000667.1, 205891 it Figure 1 58: PR058823 Figure 59: DNA196614. NPJ001158.1, 206536-s_at Figure 1212: PR084381
  • Figure 60 PRO25091
  • Figure 1213 DNA328598, NP-055146.1, 207528 _s_at
  • Figure 63 DNA327663, NP-006771.1, 206565_ ⁇ j ⁇ t Figure 1216: PR084382
  • Figure 64 PR083654 Figure 1217: DNA328600, NP_004839.1, 207571 jcjit
  • Figure 68 PR083141
  • Figure 1221 DNA328602, NP_002261.1, 207657 jt it
  • Figure 70 PR084375
  • Figure 1223 DNA226278, NP_005865.1, 207697 j
  • Figure 72 PR02691
  • Figure 1225 DNA227395, NP-005331.1, 207721_x_at
  • Figure 74 PR083657
  • Figure 1227 DNA325654, NP-054752.1, 207761_s_at
  • Figure 76 PR058498 Figure 1229: DNA226930, NP_004152.l, 207791_sj ⁇ t
  • Figure 78 PR051592
  • Figure 1231 DNA328603, NP_000304.1, 207808_s_at
  • Figure 80 PR084376 Figure 1233: DNA328604, NP.001174.2, 207809 j> jit
  • Figure 83 DNA188289, NP-001548.1, 207008 ⁇ t Figure 1237: DNA36708, NP-002081.1, 207850-at
  • Figure 84 PRO21820 Figure 1238: PR034256
  • Figure 85 DNA328592, AB015228, 207016.S Jit Figure 1239: DNA199788, NP,002981.1, 207861 Jit
  • Figure 89 DNA327673, NP_002188.1, 207071_SJit Figure 1243: DNA256523, NP_006854.1, 207872 ;.at
  • Figure 91A-B DNA328593, CIASl, 207075 it Figure 1245: DNA218651, NP_003798.1, 207907 jit
  • Figure 93A-B DNA328594, CSF1, 207082j ⁇ t
  • Figure 1247 DNA275286, NP.009205.1, 208002_s_at
  • Figure 201 DNA226996, NP.000239.1, 207233_SJit Figure 1255: DNA328608, NP.006264.2, 208075_s Jit
  • Figure 203 A-B DNA226536, NP-003225.1, Figure 1257: DNA255376, NP.l 10423.1, 20809 l_s Jit
  • Figure 204 PR036999 Figure 1259: DNA327686, NPJ0O5898.1, 208116-SJit Figure 205, DNA227668, NP-000158.1, 207387 js_at Figure 1260: PRO83670 Figure 206 PR038131 Figure 1261 A-B: DNA328609, NP.109592.1, Figure 207 DNA328596, DEGS, 207431 s_at 208121 i_at Figure 208 PR037741
  • Figure 1262 PR084391 Figure 209 DNA274829, NP-003653.1, 207469 S Jit Figure 1263: DNA328610, NP_U2601.1, 208146_s_at Figure 210 PR062588 Figure 1264: PR084392 Figure 211 DNA328597, NP-001680.1, 207507 J ⁇ Jit Figure 1265A-B: DNA226706, NP_003777.2, 208161j;_at Figure 1318: PR082662
  • Figure 1268 PR084393
  • Figure 1321 DNA326042, NP_031390.1, 208837 Jit
  • Figure 1269 DNA328612, NP-000166.2, 208308 jijit Figure 1322: PRO1078
  • Figure 1270 PR084394
  • Figure 1323A-B DNA328623, NP_056107.1,
  • Figure 1273 DNA227614, NP-004859.1, 208336 jj_at
  • Figure 1325 DNA227874, NP_003320.1, 208864-S-at
  • Figure 1275 DNA327690, NP-004022.1, 208436-s_at Figure 1327: DNA328624, BC003562, 208891 Jit
  • Figure 1277 DNA328613, NP-056953.2, 208510_s_at
  • Figure 1329 DNA328625, NP.073143.1, 208892_s_at
  • Figure 1279 A-C DNA328614, SRRM2, 208610-S-at Figure 1331 : DNA328626, NP_057078.1, 208898 Jit
  • Figure 1281A-C DNA328615, NP_003118.1, Figure 1333: DNA327700, BC015130, 208905jtt
  • Figure 1282 PR084397
  • Figure 1335 DNA325472, NP_116056.2, 208906Jit
  • Figure 1291 DNA328617, AF299343, 208653-SJit
  • Figure 1345 DNA290261, NP.001291.2, 208960-SJit
  • Figure 1293A-C DNA328618, NP-003307.2
  • Figure 1347 A-B DNA325478, NP-037534.2
  • Figure 1295 DNA304686, NP_002565.1, 208680j ⁇ t
  • Figure 1349 DNA328629, NP_006079.1, 208977-XJit
  • Figure 1297 DNA304499, NP_006588.1, 208687_x_at
  • Figure 1351 DNA328630, NP.036293.1, 209004-s_at
  • Figure 1299A-B DNA328619, BC001188, 208691 Jit
  • Figure 1353 DNA328631, AK027318, 209006_SJit
  • Figure 1301 DNA287189, NP_002038.1, 208693-S_at Figure 1355: DNA328632, DJ465N24.2.1Homo,
  • Figure 1305 DNA327696, AF228339, 208763_s_at Figure 1358A-B: DNA328634, NP.006594.1,
  • Figure 1308 PRO84402
  • Figure 1360 DNA328635, BC020946, 209026.x Jit
  • Figure 1310 PRO84403
  • Figure 1362 DNA274202. NP.006804.1, 209034 Jit
  • Figure 1311 DNA287169, CAA42052.1, 208805 _at Figure 1363: PR062131
  • Figure 1312 PR010404
  • Figure 1364 DNA328636, PAPSS1, 209043 Jit
  • Figure 1314 PR081185
  • Figure 1366A-C DNA328637, HSA7042, 209053 _s Jit
  • Figure 1316 PRO61502
  • Figure 1368 DNA326406, NP_005315.1, 209069 JSJit
  • Figure 1317 DNA328622, BC000835, 208827 Jit Figure 1369: PROl 1403
  • Figure 1370 DNA227289, NP.006532.1, 209080.x Jit Figure 1424: PRO50332
  • Figure 1372 DNA274180, NP_009005.1, 209083 ⁇ t 209281.s-at
  • Figure 1374 DNA327707, NPD00148.1, 209093_s-at Figure 1427: DNA328650, 200118.10, 209286 Jit
  • Figure 1376 DNA226564, NP-000099.1, 209095 Jit Figure 1429: DNA274883, NP.000058.1, 209301 _at
  • Figure 1378 DNA325163, NP_001113.1, 209122jit Figure 1431: DNA328651, AF087853, 209305-S-at
  • Figure 1380 DNA328638, BC000576, 209123_at
  • Figure 1433 DNA327718, CASP4, 209310-S.at
  • Figure 1382 DNA274723, AAB62222.1, 209129 _at
  • Figure 1435 DNA328652, NP.077298.1, 209321 _s_at
  • Figure 1384 DNA328639, HSM801840, 209132_s_at
  • Figure 1437 DNA328653, AF063020, 209337 -at
  • Figure 1388 DNA327713, BC010653, 209146j ⁇ t
  • Figure 1441 DNA328655, 346677.3, 209341.sjit
  • Figure 1390 DNA271937, NP.055419.1, 209154 Jit Figure 1443: DNA269630, NP.003281.1, 209344.at
  • Figure 1392 DNA328641, NP-001840.2, 209156-S_at Figure 1445A-B: DNA328656, HSA303098,
  • Figure 1396A-B DNA328642, AF073310, 209346_s_at
  • Figure 1397 PR084418 Figure 1449A-B: DNA328658, AF055376,
  • Figure 399 PR084419 Figure 1451: DNA327719, NP-003704.2, 209355-SJit Figure 400: DNA189700, NP_005243.1, 209189 Jit Figure 1452: PR083698 Figure 401: PR025619 Figure 1453: DNA328659, ECM1, 209365-S-at Figure 402: DNA327715, NP_115914.1, 209191 Jit Figure 1454: PR084433 Figure 403: PR083694 Figure 1455: DNA225952, NP.001267.1, 209395 Jit Figure 404: DNA103520, NP-002639.1, 209193 Jit Figure 1456: PR036415 Figure 405: PR04847 Figure 1457: DNA275366.
  • Figure 1476 PRO 1917 Figure 1529A-B: DNA328680, NP_062541.1,
  • Figure 1481 DNA328669, NP_005882.1, 209608_5_at
  • Figure 1533 DNA328681, NP_005089.1, 209928_s_at
  • Figure 1483A-B DNA328670, BC001618, Figure 1535: DNA272326, NP_006154.1, 209930j>_at
  • Figure 1484 PRO70011 Figure 1537: DNA328682, AF225981, 209935 Jit
  • Figure 1486 PR051256
  • Figure 1539 DNA327754, NP_150634.1, 209970 _x_at
  • Figure 1487A-B DNA272671, HSU26710, 209682j ⁇ t Figure 1540: PR04526
  • Figure 1488 PRO60796 Figure 1541: DNA328683, NP_000399.1, 210007 s_at
  • Figure 1490 PROl 1886
  • Figure 1543 DNA227660, NP_001327.1, 210042_sj ⁇ t
  • Figure 1492 PRO83705
  • Figure 1545 DNA327739, AF092535, 210058 Jtt
  • Figure 1500 PR084445
  • Figure 1553 DNA328686, NP_000566.1, 210118_s_at
  • Figure 1501A-B DNA304800, BC002538, 209723 Jit Figure 1554: PR064
  • Figure 1502 PR069458 Figure 1555: DNA227757, NP_000743.1, 210128 _s Jit
  • Figure 1503 A-B DNA328674, NP_056011.1, Figure 1556: PRO38220
  • Figure 1505 DNA324250, NP-536349.1, 209761 JSJit Figure 1559: DNA328687, AF004231, 210146-X_at
  • Figure 1506 PRO80934
  • Figure 1560 PR084456
  • Figure 1507 A-B DNA328675, ADAM19, 209765 _at Figure 1561 A-B: DNA328688, NP_006838.2,
  • Figure 1510 PRO83707
  • Figure 1563 DNA328689, NP_003259.2, 210166j ⁇ t
  • Figure 1512 PR084448
  • Figure 1565 DNA270196, HUMZFM1B, 210172jit
  • Figure 1514 PR024988
  • Figure 1567 DNA328690, NP_524145.1, 210240-S.at
  • Figure 1515 DNA328677, AF060511, 209836.x Jit Figure 1568: PRO59660
  • Figure 1516 PR084449
  • Figure 1569 DNA326963, HRIHFB2122, 210276.S Jit
  • Figure 1518 PR081419
  • Figure 1571 DNA328691, NP_065717.1, 210346-s.at
  • Figure 1520 PR061867
  • Figure 1573 DNA227652, NP.002549.1, 21040 ljit
  • Figure 1522 PRO70536
  • Figure 1575 DNA225514, NP_003864.1, 210510_s_at
  • Figure 1524 PR062586
  • Figure 1577 DNA216517, NP_005055.1, 210549.s_at
  • Figure 1526 PRO84450 Figure 1579: DNA327746, HUMGCBA, 210589 3Jit Figure 580: PRO83720 Figure 1633: PR084466 Figure 581: DNA328692, AF025529, 210660j ⁇ t Figure 1634: DNA226582, NP_003863.1, 211844_sj ⁇ t Figure 582: PR084459 Figure 1635: PRO37045 Figure 583: DNA272127, NP-003928.1, 210663-s_at Figure 1636: DNA151912, BAA06683.1, 211935j ⁇ t Figure 584: PRO60397 Figure 1637: PR012756 Figure 585: DNA326525, NP-006330.1, 210719j>_at Figure 1638: DNA325941, NP_005339.1, 211968_s_at Figure 586: PR082894 Figure 1639: PR082388 Figure 587: DNA226183, NP-001453.1, 210773_s_at Figure 1640: DNA
  • Figure 610 PR084463
  • Figure 1662 DNA88630, AAA52701.1, 212154 jit
  • Figure 611 DNA327752, HSDHACTYL, Figure 1663: PR02877
  • Figure 612A-B DNA328700, SCD, 211162_x_at Figure 1665: DNA328716, HSM800707, 212179 Jit Figure 613: PR084464 Figure 1666A-C: DNA255018, CAB61363.1, Figure 614: DNA328701, PSEN2, 211373 _s_at 212207 ⁇ t Figure 615: PRO80745 Figure 1667: PRO50107 Figure 616: DNA328702, NP_036519.1, 211413-S_at Figure 1668A-B: DNA328717, CAB70761.1, Figure 617: PR084465 212232j ⁇ t Figure 618: DNA256637, NP-008849.1, 211423_s_at Figure 1669: PR084473 Figure 619: PR051621 Figure 1670: DNA196116, DNA196116, 212246 Jit Figure 620: DNA328703, NP-003956.1, 211434_s .at Figure 1671 A-B: DNA254262,
  • Figure 1684 PR084477
  • Figure 1732A-B DNA328739, PTPRC, 212587_s_at
  • Figure 1687 DNA326808, BC019307, 212312j ⁇ t
  • Figure 1736 DNA151487, DNA151487, 212594 Jit
  • Figure 1689A-B DNA124122, NP-005602.2, Figure 1738A-B: DNA328740, BAA76781.1,
  • Figure 1691 DNA287190, CAB43217.1, 212333Jit Figure 1740: DNA81753, DNA81753, 212613j ⁇ t
  • Figure 1699A-B DNA327773, BAA25456.1
  • Figure 1748A-B DNA328742, 244522.6, 212628 Jit
  • Figure 1700 PR083739
  • Figure 1750 DNA270683, NP_006247.1, 212629 _s_at
  • Figure 1701A-C DNA328725, AB007923, 212390j ⁇ t
  • Figure 1751 PRO59047
  • Figure 1702A-B DNA150950, BAA07645.1
  • Figure 1752A-D DNA327777, HSIL1RECA
  • Figure 1703 PR012554
  • Figure 1753A-B DNA150762, BAA13197.1,
  • Figure 1704A-B DNA328726, BAA25466.2, 212658-at
  • Figure 1705 PRO84480 Figure 1755: DNA327838, NP.000568.1, 212659 j;j ⁇ t
  • Figure 1707 A-B DNA328728, 481567.2, 212458j ⁇ t
  • Figure 1757 DNA328743, 1234685.2, 212667 _at
  • Figure 1709 DNA151348, DNA151348, 212463 Jit Figure 1759: DNA328744, AF318364, 212680j j ⁇ t
  • FIG. 1710 PROl 1726 Figure 1760: PR084496
  • Figure 1711A- DNA328729, D80001, 212486-S-at Figure 1761: DNA328745, 482138.6, 212687 it
  • Figure 1713A-B DNA328730, BAA74899.2
  • Figure 1763 DNA324378, NP_000523.1, 212694 _s_at
  • Figure 1714 PR084483
  • Figure 1765 DNA328746, CAB43213.1, 212698-S.at
  • Figure 1715A-B DNA328731, 234169.5, 212500j ⁇ t Figure 1766: PR084498
  • Figure 1716 PR084484 Figure 1767A-B: DNA328747, BAA83030.1,
  • Figure 1717 DNA328732, NP_116193.1, 212502j ⁇ t 212765-at
  • Figure 1719 DNAO, AF038183, 212527 Jit Figure 1769A-B: DNA328748, HSJ001388, 212774 it
  • FIG. 1720 PRO Figure 1770: PRO59570
  • Figure 1721 DNA328734, AAH01171.1, 212539 Jit Figure 1771: DNA328749, HSM802266, 212779 Jit
  • Figure 1722 PR084487
  • Figure 1772 DNA328750, 7689361.1, 212812j ⁇ t
  • Figure 1723 DNA328735, PHIP, 212542_s _at Figure 1773: PRO84500
  • Figure 1724 PR084488 Figure 1774A-B: DNA328751, AF012086,
  • Figure 1726 PR084489
  • Figure 1775 DNA328752, CAA76270.1, 212864j ⁇ t
  • Figure 1727A-D DNA328737, 148650.1, 212560j ⁇ t
  • Figure 1776 PRO84501
  • Figure 1728 PRO84490 Figure 1777A-B: DNA328753, BAA13212.1,
  • Figure 1730A-B DNA328738, BAA31625.1, Figure 1778: PRO84502
  • Figure 1779 DNA271630, DNA271630, 212907 Jit Figure 832: DNA225974, NP.000864.1, 213620_s_at
  • Figure 1781 PRO84503
  • Figure 834 DNA328769, CAA69330.1, 213624j ⁇ t
  • Figure 1786 DNA154982, DNA154982, 213034j ⁇ t
  • Figure 840 PRO 12745
  • Figure 1790 PRO84506 Figure 844: DNA328772, AAC19149.1, 213761 jit
  • Figure 1792 DNA272600, NP-057259.1, 213112_s_3t
  • Figure 846 DNA328773, BC001528, 213766. ⁇ j ⁇ t
  • Figure 1794 DNA326217, NP_004474.1, 213129 ⁇ Jit Figure 848: DNA328774, NP.004263.1, 213793 j;_at
  • Figure 1796 DNA228053, DNA228053, 213158 Jit Figure 850A-B: DNA328775, NP.006540.2,
  • Figure 1797 A-G DNA103535, AF027153, 213164j ⁇ t 213812J3 at
  • Figure 1801 DNA328759, FJUMLPACI09, 213258 Jit Figure 854A-B: DNA328777, IDN3, 213918-SJit
  • Figure 1803 PRO84508 Figure 856: DNA196110, DNA196110, 214016-SJtt
  • Figure 1806 DNA260974, NP.006065.1, 213293.s_at
  • Figure 860 DNA328778, 234498.37, 214093 s.at
  • Figure 1808 DNA328762, AAL30845.1, 213338_3t
  • Figure 862A-B DNA272292, NP.055459.1,
  • Figure 1810 DNA327789, 1449824.5, 213348_at Figure 863 PRO60550
  • Figure 1811 PR083753
  • Figure 864 DNA82378, NP_002695.1, 214146_sj ⁇ t
  • Figure 1812 DNA328763, NP_001219.2, 213373 j>_at Figure 865 PRO 1725
  • Figure 1813 PR084511 Figure 866A-B: DNA328779, 332730.12,
  • Figure 1816 DNA328765, 411350.1, 213391 Jit Figure 868: DNA304659, NP_002023.1, 214211 jit
  • Figure 1818 DNA106195, DNA106195, 213454j ⁇ t
  • Figure 870 DNA256662, NP_009112.1, 214219_ ⁇ j ⁇ t
  • Figure 1820 DNA328766, NP_006077.1, 213476_ ⁇ j ⁇ t
  • Figure 872A-B DNA328780, 480940.15, 214285j ⁇ t
  • Figure 1824 DNA254264, HSM800224, 213546 Jit Figure 876: DNA273174, NP.001951.1, 214394 j _at
  • Figure 1826 DNA328768, 1194561.1, 213572_s_at Figure 878: DNA328782, 337794.1, 214405 Jit
  • Figure 1828 DNA327800, 1251176.10, 213593 -S_at Figure 880: DNA287630, NP_000160.1, 214430j ⁇ t
  • Figure 1830 DNA151422, DNA151422, 213605_s_at
  • Figure 882 DNA227376, NP_005393.1, 214435 j _at
  • Figure 1831 PRO 11792 Figure 883: PR037839 Figure 884: DNA273138, NP.005495.1, 214452 Jit Figure 1938: DNA328801, 407831.1, 215392j ⁇ t Figure 885: PR061182 Figure 1939: PR084543 Figure 886: DNA327812, NP_006408.2, 214453 -SJit Figure 1940A-B: DNA328802, C6orf5, 215411 _s_at Figure 887: PR083773 Figure 1941: PR084544 Figure DNA302598, NP_066361.1, 214487 _s_at Figure 1942: DNA275385, NP.002085.1, 215438-XJit Figure 889: PR06251 1 Figure 1943: PRO63048 Figure 890 DNA328783, NP.002021.2, 214560 Jit Figure 1944: DNA328803.
  • Figure 2011 A-B DNA324921, NP-073585.6
  • Figure 2066 DNA227498, NP_002125.3, 218120-SJit
  • Figure 2016 PRO36067 Figure 2072: DNA327857, NP.057386.1, 218142jj_at
  • Figure 2020 PRO50225 Figure 2076: DNA328846, NP.060522.2, 218169 Jit
  • Figure 2021A-B DNA328825, 1398762.11, 217886j ⁇ t Figure 2077: PR084578
  • Figure 2022 PR084563
  • Figure 2078 DNA228094, NP_079416.1, 218175j ⁇ t
  • Figure 2024 PRO25402
  • Figure 2080 DNA328847, NP_056338.1, 218194 it
  • Figure 2026 PR084564
  • Figure 2082 DNA150593, NP-054747.1, 218196j ⁇ t
  • Figure 2028 PR021784
  • Figure 2084 DNA256555, NP_060042.1, 218205 _s_at
  • Figure 2031 DNA328829, NP-057230.1, 217959 >_at Figure 2087: PRO84580
  • Figure 2032 PR084566 Figure 2088: DNA271622, NP_006020.3, 218224j ⁇ t
  • Figure 2034 PR084567
  • Figure 2090 DNA324353, NP_004538.2, 218226-S .at
  • Figure 2036 PR083367
  • Figure 2092 DNA328849, NP.057075.1, 218232j ⁇ t
  • Figure 2038 PR0233 Figure 2094: DNA328850, NP.057187.1, 218254_s_at
  • Figure 2040 PR084568
  • Figure 2096 DNA273230, NP_060914.1, 218273_S-at
  • Figure 2042 PR084569
  • Figure 2098 DNA328851, NP_068590.1, 218276_s_at
  • Figure 2043 DNA328834, AF220656, 217997 Jit Figure 2099: PR084582 Figure 2 00: DNA323953, NP.003507.1, 218280.x jit .152: DNA328869, NP_060892.1, 218613 Jit Figure 2 01: PRO80685 1153: PR084596 Figure 2 02: DNA254824, AF267865, 218294_sj ⁇ t 1154: DNA328870, NP.060639.1, 218614 Jit Figure 2 03: PRO49920 1155: PR084597 Figure 2 04A-B: DNA328852, NP_003609.2, 1156: DNA256870, NP_073600.1, 218618-SJit 21831 : ⁇ _at 1157: PRO51800 Figure 2 05: PR084583 1158: DNA254898, NP_060840.1, 218627 _at Figure 2 06A-B: DNA328853, NP_065702.2, 1159: PR04

Abstract

The present invention relates to compositions containing novel proteins and methods of using those compositions for the diagnosis and treatment of immune related diseases.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF IMMUNE RELATED DISEASES
Field of the Invention
The present invention relates to compositions and methods useful for the diagnosis and treatment of immune related diseases.
Background of the Invention Immune related and inflammatory diseases are the manifestation or consequence of fairly complex, often multiple interconnected biological pathways which i normal physiology are critical to respond to insult or injury, initiate repair from insult or injury, and mount innate and acquired defense against foreign organisms. Disease or pathology occurs when these normal physiological pathways cause additional insult or injury either as directly related to the intensity of the response, as a consequence of abnormal regulation or excessive stimulation, as a reaction to self, or as a combination of these.
Though the genesis of these diseases often involves multistep pathways and often multiple different biological systems/pathways, intervention at critical points in one or more of these pathways can have an ameliorative or therapeutic effect. Therapeutic intervention can occur by either antagonism of a detrimental process/pathway or stimulation of a beneficial process/pathway. Many immune related diseases are known and have been extensively studied. Such diseases include immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasia, etc.
Immune related diseases could be treated by suppressing the immune response. Using neutralizing antibodies that inliibit molecules having immune stimulatory activity would be beneficial in the treatment of immune-mediated and hiflammatory diseases. Molecules which inhibit the immune response can be utilized (proteins directly or via the use of antibody agonists) to inhibit the immune response and thus ameliorate immune related disease.
Macrophages represent an ubiquitously distributed population of fixed and circulating mononuclear phagocytes that express a variety of functions including cytokine production, killing of microbes and tumor cells and processing and presentation of antigens. Macrophages originate in the bone marrow from stem cells that give rise to a bipotent granulocyte/macrophage cell population. Distinct granulocyte and macrophage colony forming cell lineages arise from GM-CSF under the influence of specific cytokines. Upon division, monoblasts give rise to promonocytes and monocytes in the bone marrow. From there, monocytes enter the circulation. In response to particular stimuli (e.g. infection or foreign bodies) monocytes migrate into tissues and organs where they differentiate into macrophages.
Macrophages in various tissues vary in their morphology and function and have been assigned different names, e.g. Kupffer cells in the liver, pulmonary and alveolar macrophages in the lung and microglial cells in the central nervous system. However, the relationship between blood monocytes and tissue macrophages remains unclear. In the present study monocytes were differentiated into macrophages by adherence to plastic in the presence of a combination of human and bovine serum. After 7 days in culture, monocytes-derived macrophages display features typical of differentiated tissue macrophages including their ability to phagocytose opsonized particles, secretion of TNF-alpha upon lipopolysaccharide (LPS) stimulation, formation of processes and the presence of macrophage cell surface markers.
Using microarray technologies, gene transcripts from non-differentiated monocytes harvested before adhering were compared with those at 1 day and 7 days in culture. Genes selectively expressed in monocytes or macrophages could be used for the diagnosis and treatment of various chronic inflammatory or autoimmune diseases in the human. In particular, surface expressed molecules or transmembrane receptors involved in monocyte/macrophage adhesion and endothelial cell transmigration could provide novel targets to treat chronic inflammation by interference with the homing of these cells to the site of inflammation. In addition, transmembrane inhibitory receptors could be used to down-regulate monocyte/macrophage effector functions. Therapeutic molecules can be antibodies, peptides, fusion proteins or small molecules.
Despite the above research in monocyte/macrophages, there is a great need for additional diagnostic and therapeutic agents capable of detecting the presence of monocyte/macrophage mediated disorders in a mammal and for effectively reducing these disorders. Accordingly, it is an objective of the present invention to identify polypeptides that are differentially expressed in macrophages as compared to non-differentiated monocytes, and to use those polypeptides, and their encoding nucleic acids, to produce compositions of matter useful in the therapeutic treatment and diagnostic detection of monocyte/macrophage mediated disorders in mammals.
Summary of the Invention A. Embodiments
The present invention concerns compositions and methods useful for the diagnosis and treatment of immune related disease in mammals, including humans. The present invention is based on the identification of proteins (including agonist and antagonist antibodies) which are a result of stimulation of the immune response in mammals. Immune related diseases can be treated by suppressing or enhancing the immune response. Molecules that enhance the immune response stimulate or potentiate the immune response to an antigen. Molecules which stimulate the immune response can be used therapeutically where enhancement of the immune response would be beneficial. Alternatively, molecules that suppress the immune response attenuate or reduce the immune response to an antigen (e.g., neutralizing antibodies) can be used therapeutically where attenuation of the immune response would be beneficial (e.g., inflammation).
Accordingly, the PRO polypeptides, agonists and antagonists thereof are also useful to prepare medicines and medicaments for the treatment of immune-related and inflammatory diseases. In a specific aspect, such medicines and medicaments comprise a therapeutically effective amount of a PRO polypeptide, agonist or antagonist thereof with a pharmaceutically acceptable carrier. Preferably, the admixture is sterile.
In a further embodiment, the invention concerns a method of identifying agonists or antagonists to a PRO polypeptide which comprises contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide. Preferably, the PRO polypeptide is a native sequence PRO polypeptide. In a specific aspect, the PRO agonist or antagonist is an anti-PRO antibody.
In another embodiment, the invention concerns a composition of matter comprising a PRO polypeptide or an agonist or antagonist antibody which binds the polypeptide in admixture with a carrier or excipient. In one aspect, the composition comprises a therapeutically effective amount of the polypeptide or antibody. In another aspect, when the composition comprises an immune stimulating molecule, the composition is useful for: (a) increasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) stimulating or enhancing an immune response in a mammal in need thereof, (c) increasing the proliferation of monocytes/macrophages in a mammal in need thereof in response to an antigen, (d) stimulating the activity of monocytes/macrophages or (e) increasing the vascular permeability. In a further aspect, when the composition comprises an immune inhibiting molecule, the composition is useful for: (a) decreasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) inhibiting or reducing an immune response in a mammal in need thereof, (c) decreasing the activity of monocytes/macrophages or (d) decreasing the proliferation of monocytes/macrophages in a mammal in need thereof in response to an antigen. In another aspect, the composition comprises a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent. Preferably, the composition is sterile.
In another embodiment, the invention concerns a method of treating an immune related disorder in a mammal in need thereof, comprising administering to the mammal an effective amount of a PRO polypeptide, an agonist thereof, or an antagonist thereto. In a preferred aspect, the immune related disorder is selected from the group consisting of: systemic lupus erythematosis, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis, idiopathic inflammatory myopathies, Sjδgren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renal disease, demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious, autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease, gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, immunologic diseases of the lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft -versus-host-disease.
In another embodiment, the invention provides an antibody which specifically binds to any of the above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody. In one aspect, the present invention concerns an isolated antibody which binds a PRO polypeptide. In another aspect, the antibody mimics the activity of a PRO polypeptide (an agonist antibody) or conversely the antibody inhibits or neutralizes the activity of a PRO polypeptide (an antagonist antibody). In another aspect, the antibody is a monoclonal antibody, which preferably has nonhuman complementarity determining region (CDR) residues and human framework region (FR) residues. The antibody may be labeled and may be immobilized on a solid support. In a further aspect, the antibody is an antibody fragment, a monoclonal antibody, a single-chain antibody, or an anti-idiotypic antibody.
In yet another embodiment, the present invention provides a composition comprising an anti-PRO antibody in admixture with a pharmaceutically acceptable carrier. In one aspect, the composition comprises a therapeutically effective amount of the antibody. Preferably, the composition is sterile. The composition may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability. Alternatively, the antibody is a monoclonal antibody, an antibody fragment, a humanized antibody, or a single-chain antibody. In a further embodiment, the invention concerns an article of manufacture, comprising:
(a) a composition of matter comprising a PRO polypeptide or agonist or antagonist thereof;
(b) a container containing said composition; and
(c) a label affixed to said container, or a package insert included in said container referring to the use of said PRO polypeptide or agonist or antagonist thereof in the treatment of an immune related disease. The composition may comprise a therapeutically effective amount of the PRO polypeptide or the agonist or antagonist thereof.
In yet another embodiment, the present invention concerns a method of diagnosing an immune related disease in a mammal, comprising detecting the level of expression of a gene encoding a PRO polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level in the test sample as compared to the control sample indicates the presence of immune related disease in the mammal from which the test tissue cells were obtained.
In another embodiment, the present invention concerns a method of diagnosing an immune disease in a mammal, comprising (a) contacting an anti-PRO antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and a PRO polypeptide, in the test sample; wherein the formation of said complex is indicative of the presence or absence of said disease. The detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type. A larger quantity of complexes formed in the test sample indicates the presence or absence of an immune disease in the mammal from which the test tissue cells were obtained. The antibody preferably carries a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. The test sample is usually obtained from an individual suspected of having a deficiency or abnormality of the immune system.
In another embodiment, the invention provides a method for determining the presence of a PRO polypeptide in a sample comprising exposing a test sample of cells suspected of containing the PRO polypeptide to an anti-PRO antibody and determining the binding of said antibody to said cell sample. In a specific aspect, the sample comprises a cell suspected of containing the PRO polypeptide and the antibody binds to the cell. The antibody is preferably detectably labeled and/or bound to a solid support.
In another embodiment, the present invention concerns an immune-related disease diagnostic kit, comprising an anti-PRO antibody and a carrier in suitable packaging. The kit preferably contains instructions for using the antibody to detect the presence of the PRO polypeptide. Preferably the carrier is pharmaceutically acceptable.
In another embodiment, the present invention concerns a diagnostic kit, containing an anti-PRO antibody in suitable packaging. The kit preferably contains instructions for using the antibody to detect the PRO polypeptide.
In another embodiment, the invention provides a method of diagnosing an immune-related disease in a mammal which comprises detecting the presence or absence or a PRO polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of the PRO polypeptide in said test sample is indicative of the presence of an immune-related disease in said mammal. In another embodiment, the present invention concerns a method for identifying an agonist of a
PRO polypeptide comprising:
(a) contacting cells and a test compound to be screened under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide; and
(b) deterrnining the induction of said cellular response to determine if the test compound is an effective agonist, wherein the induction of said cellular response is indicative of said test compound being an effective agonist.
In another embodiment, the invention concerns a method for identifying a compound capable of inhibiting the activity of a PRO polypeptide comprising contacting a candidate compound with a PRO polypeptide under conditions and for a time sufficient to allow these two components to interact and determining whether the activity of the PRO polypeptide is inhibited. In a specific aspect, either the candidate compound or the PRO polypeptide is immobilized on a solid support. In another aspect, the non- immobilized component carries a detectable label. In a preferred aspect, this method comprises the steps of: (a) contacting cells and a test compound to be screened in the presence of a PRO polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide; and (b) determining the induction of said cellular response to determine if the test compound is an effective antagonist. In another embodiment, the invention provides a method for identifying a compound that inhibits the expression of a PRO polypeptide in cells that normally express the polypeptide, wherein the method comprises contacting the cells with a test compound and determining whether the expression of the PRO polypeptide is inhibited. In a preferred aspect, this method comprises the steps of:
(a) contacting cells and a test compound to be screened under conditions suitable for allowing expression of the PRO polypeptide; and
(b) determining the inhibition of expression of said polypeptide.
In yet another embodiment, the present invention concerns a method for treating an immune-related disorder in a mammal that suffers therefrom comprising administering to the mammal a nucleic acid molecule that codes for either (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide or (c) an antagonist of a PRO polypeptide, wherein said agonist or antagonist may be an anti-PRO antibody. In a preferred embodiment, the mammal is human. In another preferred embodiment, the nucleic acid is administered via ex vivo gene therapy. In a further preferred embodiment, the nucleic acid is comprised within a vector, more preferably an adenoviral, adeno-associated viral, lentiviral or retroviral vector. In yet another aspect, the invention provides a recombinant viral particle comprising a viral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptide of a PRO polypeptide, or (c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein the viral vector is in association with viral structural proteins. Preferably, the signal sequence is from a mammal, such as from a native PRO polypeptide.
In a still further embodiment, the invention concerns an ex vivo producer cell comprising a nucleic acid construct that expresses retroviral structural proteins and also comprises a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptide of a PRO polypeptide or (c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein said producer cell packages the retroviral vector in association with the structural proteins to produce recombinant retroviral particles.
In a still further embodiment, the invention provides a method of increasing the activity of monocytes/macrophages in a mammal comprising administering to said mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the activity of monocytes/macrophages in the mammal is increased.
In a still further embodiment, the invention provides a method of decreasing the activity of monocytes/macrophages in a mammal comprising administering to said mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the activity of monocytes/macrophages in the mammal is decreased. In a still further embodiment, the invention provides a method of increasing the proliferation of monocytes/macrophages in a mammal comprising administering to said mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the proliferation of monocytes/macrophages in the mammal is increased.
In a still further embodiment, the invention provides a method of decreasing the proliferation of monocytes/macrophages in a mammal comprising administering to said mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the proliferation of monocytes/macrophages in the mammal is decreased.
B. Additional Embodiments
In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli, or yeast. A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture. In other embodiments, the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence. Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin. In another embodiment, the invention provides an antibody which specifically binds to any of the above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences.
In other embodiments, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full- length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99%) nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PRO polypeptide cDNA as disclosed herein, the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
In a further aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%) nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86%) nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDNAs as disclosed herein, or (b) the complement of the DNA molecule of (a).
Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains of the herein described PRO polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO antibody or as antisense oligonucleotide probes. Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternatively at least about 200 nucleotides in length, alternatively at least about 250 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternatively at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such PRO polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PRO polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody.
In another embodiment, the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences herein above identified.
In a certain aspect, the invention concerns an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein. In a further aspect, the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% ammo acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93%o amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to an amino acid sequence encoded by any of the human protein cDNAs as disclosed herein.
In a specific aspect, the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as herein before described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture. Another aspect the invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture. In yet another embodiment, the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein. In a particular embodiment, the agonist or antagonist is an anti-PRO antibody or a small molecule.
In a further embodiment, the invention concerns a method of identifying agonists or antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide. Preferably, the PRO polypeptide is a native PRO polypeptide.
In a still further embodiment, the invention concerns a composition of matter comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier. Another embodiment of the present invention is directed to the use of a PRO polypeptide, or an agonist or antagonist thereof as herein before described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide, an agonist or antagonist thereof or an anti-PRO antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
In the list of figures for the present application, specific cDNA sequences which are differentially expressed in differentiated macrophages as compared to normal undifferentiated monocytes are individually identified with a specific alphanumerical designation. These cDNA sequences are differentially expressed in monocytes that are specifically treated as described in Example 1 below. If start and/or stop codons have been identified in a cDNA sequence shown in the attached figures, they are shown in bold and underlined font, and the encoded polypeptide is shown in the next consecutive figure.
The Figures 1-2517 show the nucleic acids of the invention and their encoded PRO polypeptides. Also included, for convenience is a List of Figures attached hereto as Appendix A, which gives the figure number and the corresponding DNA or PRO number. List of Figures
Figure 1: DNA227321, NP_001335.1, 200046_at Figure 55: DNA327526, BC001698, 45288_at
Figure 2: PR037784 Figure 56: PR083574
Figure 3: DNA304680, HSPCB, 200064_at Figure 57A-B: DNA328361, BAA92570.1, 47773_at
Figure 4: PR071106 Figure 58: PR084221
Figure 5: DNA328347, NP_002146.1, 117_at Figure 59: DNA328362, NP.060312.1, 48106 jt
Figure 6: PR058142 Figure 60: PR084222
Figure 7A-B: DNA328348, MAP4, 243_g_at Figure 61: DNA328363, DNA328363, 52651_at
Figure 8: PRO84209 Figure 62: PR084685
Figure 9: DNA83128, NP.002979.1, 32128-at Figure 63: DNA328364, NP-068577.1, 52940_at
Figure 10: PRO2601 Figure 64: PR084223
Figure 11: DNA272223, NP.004444.1, 33494_at Figure 65A-B: DNA327528, BAB33338.1, 55081.at
Figure 12: PRO60485 Figure 66: PR083576
Figure 13: DNA327522, NP_000396.1, 33646_g.at Figure 67: DNA225650, NP-057246.1, 48825.at
Figure 14: PR02874 Figure 68: PR036113
Figure 15: DNA328349, NP_004556.1, 33760-ϋt Figure 69: DNA328365, NP-060541.1, 58780-S.at
Figure 16: PRO84210 Figure 70: PR084224
Figure 17A-B: DNA328350, NP_056155.1, 34764_at Figure 71: DNA328366, NP_079233.1, 59375-at
Figure 18: PR084211 Figure 72: PR084225
Figure 19: DNA328351, NP_006143.1, 35974_at Figure 73: DNA328367, NP-079108.2, 6047 l_at
Figure 20: PR084212 Figure 74: PR084226
Figure 21: DNA328352, NP.004183.1, 36553_at Figure 75: DNA327876, NP-005081.1, 60528_at
Figure 22: PR084213 Figure 76: PR083815
Figure 23: DNA271996, NP.004928.1, 36566_at Figure 77A-B: DNA328368, 1503444.3, 87100_at
Figure 24: PRO60271 Figure 78: PR084227
Figure 25: DNA326969, NP.036455.1, 36711.at Figure 79: DNA328369, BC007634, 90610_at
Figure 26: PR083282 Figure 80A-B: DNA328370, NP_001273.1,
Figure 27: DNA304703, NP_005923.1, 36830_at 200615-s.at
Figure 28: PR071129 Figure 81: PR084228
Figure 29: DNA328353, AAB72234.1, 37079_at Figure 82: DNA323806, NP.075385.1, 200644_at
Figure 30: PR084214 Figure 83: PRO80555
Figure 31: DNA103289, NP_006229.1, 37152_at Figure 84: DNA327532, GLUL, 200648.s_at
Figure 32: PR04619 Figure 85: PR071134
Figure 33A-B: DNA255096, NP_055449.1, 37384_at Figure 86: DNA227055, NP.002625.1, 200658.s_at
Figure 34: PRO50180 Figure 87: PR037518
Figure 35: DNA256295, NP_002310.1, 37796_at Figure 88: DNA325702, NP-001771.1, 200663 Jit
Figure 36: PR051339 Figure 89: PR0283
Figure 37: DNA328354, PARVB, 37965_at Figure 90: DNA83172, NP-003109.1, 200665_s_at
Figure 38: PR084215 Figure 91: PRO2120
Figure 39: DNA53531, NP.001936.1, 38037_at Figure 92: DNA328371, NP-004347.1, 200675_at
Figure 40: PR0131 Figure 93 : PR04866
Figure 41: DNA254127, NP.008925.1, 38241_at Figure 94A-B: DNA328372, 105551.7, 200685_at
Figure 42: PR049242 Figure 95: PR084229
Figure 43: DNA328355, NP.006471.2, 38290.at Figure 96: DNA324633, BC000478, 200691 J3_at
Figure 44: PR084216 Figure 97: PR081277
Figure 45: DNA328356, BC013566, 39248_at Figure 98: DNA324633, NP-004125.2, 200692.s_at
Figure 46: PR038028 Figure 99: PR081277
Figure 47: DNA328357, 1452321.2, 39582 _at Figure 100: DNA88350, NP-000168.1, 200696-S.at
Figure 48: PR084217 Figure 101: PR02758
Figure 49A-B: DNA328358, STK10, 40420_at Figure 102: DNA328373, AB034747, 200704_at
Figure 50: PRO84218 Figure 103: PRO84230
Figure 51A-B: DNA328359, BAA21572.1, 41386i_at Figure 104: DNA328374, NP-004853.1, 200706.s_at
Figure 52: PR084219 Figure 105: PR084231
Figure 53A-D: DNA328360, NP.055061.1, 41660_at Figure 106: DNA328375, NP-002071.1, 200708_at
Figure 54: PRO84220 Figure 107: PRO80880 Figure 108: DNA328376, NP_001210.1, 200755_s_at Figure 161: DNA225878, NP_004334.1, 200935 Jtt
Figure 109: PRO1015 Figure 162: PR036341
Figure 110A-B: DNA269826, NP_003195.1, Figure 163: DNA328382, 160963.2, 20094 l it
200758 5_at Figure 164: PR084237
Figure 111: PR058228 Figure 165: DNA328383, NP.004956.3, 200944 ;_at
Figure 112: DNA325414, NP_001900.1, 200766jtt Figure 166: PR084238
Figure 113: PR0292 Figure 167 A-B: DNA287217, NP_001750.1,
Figure 114A-C: DNA188738, NP_002284.2, 200771 Jtt 200953-S-at
Figure 115: PRO25580 Figure 168: PR036766
Figure 116: DNA328377, NP_003759.1, 200787 _sJtt Figure 169: DNA328384, NP.036380.2, 200961 Jit
Figure 117: PR084232 Figure 170: PR084239
Figure 118: DNA270954, NP-001089.1, 200793_s_at Figure 171: DNA328385, AK001310, 200972jιt
Figure 119: PR059285 Figure 172: PRO730
Figure 120: DNA272928, NP_055579.1, 200794jc_at Figure 173: DNA326040, NP_005715.1, 200973-SJtt
Figure 121: PRO61012 Figure 174: PRO730
Figure 122A-B: DNA327536, BC017197, 200797_s_at Figure 175: DNA324110, NP_005908.1, 200978 Jit
Figure 123: PRO37003 Figure 176: PR04918
Figure 124: DNA287211, NP_002147.1, 200806_s_at Figure 177: DNA328386, NP.000602.1, 200983-x_at
Figure 125: PR069492 Figure 178: PR02697
Figure 126: DNA326655, NP_002803.1, 200820 Jit Figure 179: DNA275408, NP_001596.1, 20100θJit
Figure 127: PRO83005 Figure 180: PRO63068
Figure 128A-B: DNA328378, AB032261, 200832.S SΛ Figure 181: DNA328387, NP.001760.1, 201005 tt
Figure 129: PR084233 Figure 182: PR04769
Figure 130: DNA103558, NP_005736.1, 200837 Jit Figure 183: DNA103593, NP-000174.1, 201007 Jit
Figure 131: PR04885 Figure 184: PR04917
Figure 132: DNA196817, NP_001899.1, 200838 Jit Figure 185: DNA304713, NP_006463.2, 201008.S it
Figure 133: PR03344 Figure 186: PR071139
Figure 134A-B: DNA327537, NP_004437.1, Figure 187: DNA328388, BC010273, 201013-SJit
200842_s_at Figure 188: PRO84240
Figure 135: PR083581 Figure 189: DNA328389, NP_006861.1, 201021.SJtt
Figure 136: DNA323982, NP_004896.1, 200844.s_at Figure 190: PR084241
Figure 137: PRO80709 Figure 191: DNA328390, NP_002291.1, 201030jcjit
Figure 138: DNA323876, NP_006612.2, 200850-S_at Figure 192: PR082116
Figure 139: PRO80619 Figure 193: DNA196628, NP_005318.1, 201036_s-at
Figure 140A-B: DNA228029, NP-055577.1, 200862jιt Figure 194: PR025105
Figure 141: PR038492 Figure 195: DNA287372, NP_002618.1, 201037 Jit
Figure 142: DNA328379, BC015869, 200878 _at Figure 196: PR069632
Figure 143: PR084234 Figure 197: DNA328391, NP.004408.1, 201041-SJrt
Figure 144: DNA325584, NP_002005.1, 200895_s ιt Figure 198: PR084242
Figure 145: PR059262 Figure 199: DNA196484, DNA196484, 201042jιt
Figure 146A-B: DNA274281, NPD36347.1, Figure 200: DNA227143, NP-036400.1, 201050jtt
200899-s.at Figure 201: PRO37606
Figure 147: PRO62204 Figure 202: DNA328392, 1500938.11, 201051 Jit
Figure 148: DNA226028, NP.002346.1, 200900 s_at Figure 203: PR084243
Figure 149: PR036491 Figure 204: DNA328261, AF130103, 201060_x_at
Figure 150: DNA326819, NP.000678.1, 200903j>_at Figure 205: DNA325001, NP_002794.1, 201068_sjtt
Figure 151: PR083152 Figure 206: PR081592
Figure 152: DNA328380, HSHLAEHCM, 200904 Jit Figure 207: DNA328393, NP_001651.1, 201096.sjιt
Figure 153: DNA328381, NP_005507.1, 200905_χ ιt Figure 208: PRO81010
Figure 154: PR084236 Figure 209: DNA328394, AF131738, 201103 c ιt
Figure 155: DNA272695, NP.001722.1, 200920_s_at Figure 210A-B: DNA328395, NP_056198.1,
Figure 156: PRO60817 201104_x_at
Figure 157: DNA327255, NP.002385.2, 200924_s_at Figure 211: PR084245
Figure 158: PR057298 Figure 212: DNA328396, NP_002076.1, 201106jιt
Figure 159: DNA327540. NP-006818.1, 200929.at Figure 213: PR084246
Figure 160: PRO38005 Figure 214: DNA328397, NPJJ02622.1, 201118 ιt Figure 215: PR084247 • Figure 269: DNA255078, NP_006426.1, 201315-χ ιt
Figure 216: DNA328398, NP_002204.1, 201125.S Jit Figure 270: PRO50165
Figure 217: PR034737 Figure 271: DNA150781, NP_001414.1, 201324jιt
Figure 218: DNA325398, NP_004083.2, 201135 ιt Figure 272: PRO 12467
Figure 219: PRO81930 Figure 273: DNA328409, NP_002075.2, 201348 ιt
Figure 220: DNA88520, NP-002501.1, 201141 Jit Figure 274: PR081281
Figure 221: PR02824 Figure 275: DNA324475, NPD04172.2, 201387 _sjιt
Figure 222: DNA324480, NP_001544.1, 201163.S Jit Figure 276: PR081137
Figure 223: PR081141 Figure 277: DNA226353, NP.005769.1, 201395 jit
Figure 224: DNA151802, NP-003661.1, 201169_s _at Figure 278: PR036816
Figure 225: PRO 12890 Figure 279: DNA328410, NP.004519.1, 201403_sjιt
Figure 226: DNA226662, NP_057043.1, 201175 jit Figure 280: PRO60174
Figure 227: PR037125 Figure 281A-B: DNA328411, 1400253.2, 201408 it
Figure 228: DNA88066, NP.002328.1, 201186 Jit Figure 282: PR084256
Figure 229: PR02638 Figure 283: DNA328412, NP_060428.1, 201411 j; ιt
Figure 230: DNA273342, NP_005887.1, 201193 tt Figure 284: PR084257
Figure 231 : PR061345 Figure 285: DNA273517, NP_000169.1, 201415 jit
Figure 232: DNA328399, NP_003000.1, 201194 tt Figure 286: PR061498
Figure 233: PR084248 Figure 287: DNA327550, NP_001959.1, 201435 _s.at
Figure 234A-B: DNA103453, HUME16GEN, Figure 288: PR081164
201195.s_at Figure 289: DNA273396, DNA273396, 201449_at
Figure 235: PRO4780 Figure 290: DNA325049, NP.005605.1, 201453.x Jit
Figure 236: DNA328400, NP.003842.1, 201200_at Figure 291: PR037938
Figure 237: PRO 1409 Figure 292: DNA274343, NP_000894.1, 201467_SJit
Figure 238: DNA327542, NP.000091.1, 201201 Jit Figure 293: PR062259
Figure 239: PR083582 Figure 294: DNA328413, NP_004823.1, 201470 ιt
Figure 240: DNA103488, NP-002583.1, 201202_at Figure 295: PR084258
Figure 241: PR04815 Figure 296: DNA328414, NP.003891.1, 201471 _s_at
Figure 242: DNA328401, BC013678, 201212 ιt Figure 297: PR081346
Figure 243A-B: DNA328402, NP-073713.1, Figure 298: DNA103320, NP.002220.1, 201473jιt
201220 -x Jit Figure 299: PRO4650
Figure 244: PR084249 Figure 300: DNA88608, NP_002893.1, 201485 js it
Figure 245: DNA325380, NP-004995.1, 201226 Jit Figure 301: PR02864
Figure 246: PR081914 Figure 302: DNA304459, BC005020, 201489 Jit
Figure 247: DNA226615, NP.001668.1, 201242j?_at Figure 303: PRO37073
Figure 248: PRO37078 Figure 304: DNA304459, NP_005720.1, 201490 _s_at
Figure 249: DNA328403, NP.037462.1 , 201243 _s -at Figure 305: PRO37073
Figure 250: PRO84250 Figure 306: DNA253807, NP_065390.1, 201502 jsjit
Figure 251: DNA270950, NP-003182.1, 201263 Jit Figure 307: PRO49210
Figure 252: PR059281 Figure 308: DNA328415, BC006997, 201503 tt
Figure 253A-B: DNA328404, NP_003321.1 , 201266 tt Figure 309: PRO60207
Figure 254: PR084251 Figure 310: DNA328416, NP.002613.2, 201507 it
Figure 255: DNA97290, NP.002503.1, 201268 jit Figure 3 U: PR084259
Figure 256: PR03637 Figure 312: DNA271931, NP.005745.1, 201514jijιt
Figure 257: DNA325028, NP.001619.1, 201272 jit Figure 313: PRO60207
Figure 258: PR081617 Figure 314A-B: DNA150463, NP_055635.1, 201519 Jit
Figure 259: DNA328405, NP.l 12556.1, 201277.S Jit Figure 315: PR012269
Figure 260: PR084252 Figure 316: DNA328417, ATP6V1F, 201527 jit
Figure 261: DNA328406, NP.001334.1, 20I279-S_at Figure 317: PRO84260
Figure 262: PR084253 Figure 318: DNA328418, NPJ303398.1, 201531 Jit
Figure 263: DNA328407, WSB 1, 201296.S _at Figure 319: PR084261
Figure 264: PR084254 Figure 320: DNA328419, NP.002779.1, 201532jιt
Figure 265: DNA328408, NP_060713.1, 201308.S _at Figure 321: PR084262
Figure 266: PR084255 Figure 322: DNA328420, BC002682, 20l537js.at
Figure 267: DNA325595, NP.001966.1, 201313_at Figure 323: PR058245
Figure 268: PRO38010 Figure 324: DNA88464, NP.005552.2, 201551 -s_at Figure 325: PRO2804 Figure 375: PR036359
Figure 326A-B: DNA290226, NP-039234.1, Figure 376: DNA151017, NP_004835.1, 201810_s_at
201559_S-at Figure 377: PRO 12841
Figure 327: PRO70317 Figure 378: DNA328429, NP_079106.2, 201818jιt
Figure 328: DNA227071, NP-000260.1, 201577.at Figure 379: PRO81201
Figure 329: PR037534 Figure 380: DNA328430, NP.005496.2, 201819_at
Figure 330A-B: DNA227307, NP-009115.1, Figure 381: PR084267
201591-s-at Figure 382: DNA324015, NP_006326.1, 201821-S_at
Figure 331: PRO37770 Figure 383: PRO80735
Figure 332: DNA255406, NP-005533.1, 201625 J3_at Figure 384: DNA150650, NP_057086.1, 201825 s_at
Figure 333: PRO50473 Figure 385: PR012393
Figure 334A-B: DNA328421, 475621.10, 201646 Jit Figure 386: DNA304710, NP.001531.1, 201841 _sjιt
Figure 335: PR051048 Figure 387: PR071136
Figure 336A-B: DNA220748, PJ000201.1, 201656 ιt Figure 388: DNA88450, NP.000226.1, 201847 Jit
Figure 337: PR034726 Figure 389: PR02795
Figure 338: DNA269791, NP_001168.1, 201659 _s_at Figure 390: DNA150725, NP.001738.1, 201850jιt
Figure 339: PR058197 Figure 391: PRO 12792
Figure 340A-B: DNA328422, NP_004448.1, Figure 392: DNA272066, NP .002931.1, 201872_s_at
201661 s_at Figure 393: PRO60337
Figure 341: PR084263 Figure 394: DNA328431, NP_001817.1, 201897 js_at
Figure 342: DNA328423, NP-003245.1, 201666 it Figure 395: PRO45093
Figure 343: PR02121 Figure 396: DNA103214, NP.006057.1, 201900 JSJit
Figure 344: DNA273090, NP.002347.4, 201670 S Jit Figure 397: PR04544
Figure 345: PR061148 Figure 398: DNA227112, NP_006397.1, 201923jιt
Figure 346: DNA328424, NP-005142.1, 201672j_at Figure 399: PR037575
Figure 347: PR059291 Figure 400: DNA83046, NP-000565.1, 201926_sjιt
Figure 348: DNA271223, NP-005070.1, 201689 js.at Figure 401 : PR02569
Figure 349: PR059538 Figure 402: DNA273014, NP.000117.1, 201931 Jit
Figure 350A-B: DNA323965, NP_002848.1, Figure 403: PRO61085
201706_s_at Figure 404: DNA254147, NP-000512.1, 201944 Jit
Figure 351: PRO80695 Figure 405: PR049262
Figure 352: DNA270883, NP_001061.1, 201714 ιt Figure 406: DNA274167, NP.006422.1, 201946-S.at
Figure 353: PR059218 Figure 407: PRO62097
Figure 354A-B: DNA328425, NP.065207.2, Figure 408 A-B: DNA327562, HSMEMD, 201951 Jit
201722.s-at Figure 409A-B: DNA327563, NP.066945.1, 201963 Jit
Figure 355: PR084264 Figure 410: PR083592
Figure 356: DNA328426, NP.000582.1, 201743 Jtt Figure 411: DNA227290, NP_055861.1, 201965 J3-at
Figure 357: PR0384 Figure 412: PR037753
Figure 358: DNA150429, NP-002813.1, 201745 Jit Figure 413A-B: DNA328432, NP-005768.1, 201967 Jit
Figure 359: PR012769 Figure 414: PR061793
Figure 360: DNA272465, NP_004543.1, 201757 Jit Figure 415A-B: DNA328433, ATP6V1A1,
Figure 361: PRO60713 201971-s_at
Figure 362: DNA328427, NP_061109.1, 201760.S _at Figure 416: PR084268
Figure 363 : PR084265 Figure 417: DNA327073, NP_036418.1, 201994 Jit
Figure 364: DNA287167, NP-006627.1, 201761 Jit Figure 418: PR083365
Figure 365: PR059136 Figure 419: DNA226878, NP.000118.1, 201995jιt
Figure 366: DNA323937, NP-005689.2, 201771 Jtt Figure 420: PR037341
Figure 367: PRO80670 Figure 421A-D: DNA328434, NP-055816.2,
Figure 368: DNA88619, NP.002924.1, 201785 tt 201996.s_at
Figure 369: PR02871 Figure 422: PR084269
Figure 370A-B: DNA328428, NP_038479.1, Figure 423: DNA328435, NP-002481.1, 202001 jj_at
201798_s_at Figure 424: PRO60236
Figure 371: PR084266 Figure 425: DNA275246, NP.006102.1, 202003 _s.at
Figure 372: DNA227563, NP.004946.1, 201801-S.at Figure 426: PR062933
Figure 373: PRO38026 Figure 427: DNA327841, NP.068813.1, 202005 Jit
Figure 374: DNA225896, NP.000109.1, 201808 _sJit Figure 428: PR012377 Figure 429: DNA328436, 1171619.4, 202007 Jit Figure 480: PR084279
Figure 430: PRO84270 Figure 481 : DNA304716, NP 510867.1 , 202284 jjjtt
Figure 431: DNA327564, NP.000111.1, 202017jtt Figure 482: PR071142
Figure 432: PR083593 Figure 483: DNA270142, NP.005947.2, 202309 _at
Figure 433: DNA328437, AF083441, 202021 _χjιt Figure 484: PR058531
Figure 434: PR084271 Figure 485: DNA328448, NP.000777.1, 202314jιt
Figure 435A-B: DNA270997, NPJ305047.1, Figure 486: PR062362
202040.s.at Figure 487: DNA325115, NP_001435.1, 2023'45_s jit
Figure 436: PR059326 Figure 488: PR081689
Figure 437A-B: DNA327565, NP.056392.1, Figure 489: DNA106239, DNA106239, 202351 Jtt
202052_s_at Figure 490: DNA270502, NP.002807.1, 202352 jjjvt
Figure 438: PR083594 Figure 491: PRO58880
Figure 439A-B: DNA327566, NP_000373.1, Figure 492: DNA327074, FLJ21174, 202371 jit
202053_s_at Figure 493 : PR083366
Figure 440: PR083595 Figure 494: DNA 149091, DNA149091, 202377 jvt
Figure 441: DNA226116, NP_002990.1, 202071jιt Figure 495A-B: DNA151045, NP_005376.2,
Figure 442: PR036579 202379.s-at
Figure 443A-B: DNA328438, 100983.30, 202073 Jit Figure 496: PR012587
Figure 444: PR084272 Figure 497 A-B: DNA200236, NP_003807.1, 202381 _at
Figure 445: DNA328439, NP_068815.1, 202074_sjιt Figure 498: PR034137
Figure 446: PR084273 Figure 499: DNA328449, NP_005462.1, 202382_sjιt
Figure 447: DNA290272, NP_004898.1, 202081 Jtt Figure 500: PRO60304
Figure 448: PRO70409 Figure 501: DNA290234, NP_002914.1, 202388jιt
Figure 449: DNA327569, NPJJ01903.1, 202087.S Jit Figure 502: PRO70333
Figure 450: PR02683 Figure 503: DNA269766, NP_005646.1, 202393-S_at
Figure 451: DNA328440, NP_004517.1, 202107 _s_at Figure 504: PR058175
Figure 452: PR084274 Figure 505: DNA227612, NPD56230.1, 202427 _s_at
Figure 453: DNA272777, NP.000276.1, 202108 Jit Figure 506: PRO38075
Figure 454: PRO60884 Figure 507: DNA324171, NP.065438.1, 202428 j jit
Figure 455A-B: DNA328441, AL136139, 202149 Jit Figure 508: PRO60753
Figure 456: PROO Figure 509A-B: DNA327576, NP_000095.1,
Figure 457: DNA328442, NP-006078.2, 202154 JC _at 202434_s_at
Figure 458: PR084275 Figure 510: PRO83600
Figure 459A-C: DNA328443, NP.004371.1, 202160.at Figure 511A-D: DNA328450, NP_077719.1,
Figure 460: PR084276 202443jc_at
Figure 461A-C: DNA271201, NP_005881.1, Figure 512: PRO84280
20219 lJS-at Figure 513: DNA225809, NP.000387.1, 202450_s_at
Figure 462: PR059518 Figure 514: PR036272
Figure 463: DNA328258, SLC16A1, 202236 _s _at Figure 515: DNA227921, NP-003789.1, 202468-SJit
Figure 464: PR084151 Figure 516: PR038384
Figure 465: DNA328444, MGC14458, 202246 J>JH Figure 517: DNA150942, HSY18007, 202475jιt
Figure 466: PR084277 Figure 518: PR012549
Figure 467: DNA294794, NP.002861.1, 202252jtt Figure 519: DNA225566, NP_004744.1, 202481 Jit
Figure 468: PRO70754 Figure 520: PRO36029 A-B: DNA103449, NP.008862.1,
Figure imgf000016_0001
Figure 470: PR037639 Figure 522: PR04776
Figure 471: DNA325823, NP_055702.1, 202258 _s_at Figure 523: DNA328451, NP.000007.1, 202502jιt
Figure 472: PR082289 Figure 524: PR062139
Figure 473: DNA256533, NP_006105.1, 202264j>Jit Figure 525A-B: DNA274893, NP_006282.1,
Figure 474: PR051565 2025 IOJS Jit
Figure 475: DNA328445, NP-057698.1, 202266 _at Figure 526: PR062634
Figure 476: PR084278 Figure 527: DNA328452, NP_000394.1, 202528 it
Figure 477: DNA328446, NP_003896.1, 202268 J; Jtt Figure 528: PR063289
Figure 478: PR059821 Figure 529: DNA219229, NP-002189.1, 202531jιt
Figure 479: DNA328447, NP_000393.2, 202275 Jit Figure 530: PR034544 Figure 531A-B: DNA274852, NP_004115.1, 202752-X_at
202543-s.at Figure 584: PRO12550
Figure 532: PRO62605 Figure 585A-C: DNA328462, HSA303079,
Figure 533: DNA328453, NP_003752.2, 202546 ιt 202759 J; Jit
Figure 534: PR084281 Figure 586: PR084288
Figure 535A-B: DNA328454, NP_057525.1, Figure 587A-C: DNA328463, NP_009134.1,
20255 l_s_at 202760j;_at
Figure 536: PRO4330 Figure 588: PR084289
Figure 537: DNA150817, NP_000840.1, 202554 _s_at Figure 589: DNA226080, NP..001601.1, 202767 Jit
Figure 538: PRO12808 Figure 590: PR036543
Figure 539: DNA227994, NP_009107.1, 202562 jjjit Figure 591A-B: DNA150977, NP.006723.1, 202768 Jit
Figure 540: PR038457 Figure 592: PR012828
Figure 541: DNA328455, AY007134, 202573 « Figure 593A-B: DNA328464, 977954.20, 202769 Jit
Figure 542: PR084282 Figure 594: PRO84290
Figure 543: DNA323923, NP_001869.1, 202575 Jit Figure 595: DNA226578, NP.004345.1, 202770 SJit
Figure 544: PRO80657 Figure 596: PRO37041
Figure 545: DNA328456, NP_000467.1, 202587.S Jit Figure 597A-B: DNA103521, NP_004163.1, 202800 Jit
Figure 546: PR084283 Figure 598: PR04848
Figure 547: DNA328457, NP_036422.1 , 202606 _s_at Figure 599A-B: DNA327583, ABCCl, 202805-S_at
Figure 548: PRO70421 Figure 600: PRO83604
Figure 549: DNA103245, NP_002341.1, 202626j;.at Figure 601: DNA328465, NP-005639.1, 202823 Jit
Figure 550: PR04575 Figure 602: PR084291
Figure 551: DNA83141, NP.000593.1, 202627 js_at Figure 603: DNA225865, NP.004986.1, 202827 -S.at
Figure 552: PRO2604 Figure 604: PR036328
Figure 553: DNA254129, NP.006001.1, 202655 jit Figure 605: DNA225926, NP_000138.1, 202838 Jtt
Figure 554: PR049244 Figure 606: PR036389
Figure 555: DNA270379, NP-002792.1, 202659 Jit Figure 607: DNA328466, NP.004554.1 , 202847 Jit
Figure 556: PR058763 Figure 608: PR084292
Figure 557: DNA326896, NP_003672.1, 20267 l_s_at Figure 609: DNA103394, NP-004198.1, 202855 _s_at
Figure 558: PR069486 Figure 610: PR04722
Figure 559: DNA289526, NP_004015.2, 202672.S Jit Figure 611: DNA275144, NP-000128.1, 202862jιt
Figure 560: PRO70282 Figure 612: PR062852
Figure 561: DNA273542, NP-002991.1, 202675 Jit Figure 613: DNA328467, SP100, 202864 _sjιt
Figure 562: PR061522 Figure 614: PR084293
Figure 563: DNA328458, NP_037458.2, 202679 Jit Figure 615: DNA287289, NP-058132.1, 202869 Jit
Figure 564: PR084284 Figure 616: PR069559
Figure 565: DNA84130, NP_003801.1, 202687 3_at Figure 617: DNA328468, BC010960, 202872jιt
Figure 566: PRO1096 Figure 618: PR084294
Figure 567: DNA271085, NP_004751.1, 202693_sjιt Figure 619: DNA328469, NP-001686.1, 202874 3_at
Figure 568: PRO59409 Figure 620: PR084295
Figure 569A-B: DNA150467, NP_055513.1, Figure 621A-B: DNA255318, NP_036204.1,
202699 -s_at 202877 -s Jit
Figure 570: PR012272 Figure 622: PRO50388
Figure 571 A-B: DNA328459, NP-004332.2, 202715 ιt Figure 623A-B: DNA328470, NP_055620.1, 202909 Jit
Figure 572: PR084285 Figure 624: PR084296
Figure 573: DNA273290, NP_002047.1, 202722 _s_at Figure 625: DNA327584, NP-002955.2, 202917.s_at
Figure 574: PRO61300 Figure 626: PRO80649
Figure 575: DNA328460, NP_004190.1, 202733jtt Figure 627: DNA272425, NP.001489.1, 202923.sjit
Figure 576: PR084286 Figure 628: PRO60677
Figure 577: DNA150713, NP.006570.1, 202735 Jit Figure 629: DNA328471, ZMPSTE24, 202939 Jit
Figure 578: PRO 12082 Figure 630: PR084297
Figure 579A-B: DNA328461, 350230.2, 202741 jtt Figure 631: DNA269481, NP_001976.1, 202942 Jit
Figure 580: PR084287 Figure 632: PRO57901
Figure 581: DNA271973, NP_002722.1, 202742 _s_at Figure 633: DNA328472, NP.000482.2, 202953 Jit
Figure 582: PRO60248 Figure 634: PR084298
Figure 583A-B: DNA150943, NP_036376.1, Figure 635A-B: DNA328473, NP_006473.1, 202968ji.at Figure 687: DNA328487, AF251295, 203299 JS_at
Figure 636: PR084299 Figure 688: PR084312
Figure 637A-C: DNA328474, 1501914.1, 202969 Jit Figure 689: DNA328488, NP-003907.2, 203300_x jit
Figure 638: PRO84300 Figure 690: PR084313
Figure 639: DNA325915, ZAP128, 202982j>_at Figure 691: DNA328489, NP.006511.1, 203303 Jit
Figure 640: PR082369 Figure 692: PR084314
Figure 641: DNA271272, NP_000366.1, 203031 S A Figure 693A-B: DNA328490, NP_000120.1, 203305 Jtt
Figure 642: PR059583 Figure 694: PR084315
Figure 643: DNA324049, FH, 203032_s_at Figure 695: DNA327593, NP_006205.1, 203335 Jit
Figure 644: PRO62607 Figure 696: PR059733
Figure 645A-B: DNA271865, NP_055566.1, Figure 697: DNA328491, ICAP-1A, 203336 3_at
203037jj_at Figure 698: PR061323
Figure 646: PRO60145 Figure 699A-B: DNA328492, NP_056125.1,
Figure 647: DNA328475, LAMP2, 203042jιt 203354-S_at
Figure 648: PRO84301 Figure 700: PR084316
Figure 649 A-B: DNA328476, AF074331, 203058 JSJit Figure 701: DNA328493, NP_008957.1, 203367 Jit
Figure 650: PRO84302 Figure 702: PR084317
Figure 651: DNA256830, NP-004815.1, 203100_s_at Figure 703: DNA328494, RPS6KA1, 203379 Jit
Figure 652: PR051761 Figure 704: PR084318
Figure 653: DNA272867, NP.003960.1, 203109 Jit Figure 705: DNA274960, NP-008856.1, 203380-χ ιt
Figure 654: PRO60960 Figure 706: PR062694
Figure 655A-B: DNA227582, NP_000608.1, Figure 707: DNA88084, NP_000032.1, 203381 j;jιt
203124j;_at Figure 708: PR02644
Figure 656: PRO38045 Figure 709A-B: DNA254616, NP_004473.1,
Figure 657: DNA328477, NP_003767.1, 203152jιt 203397 jj-at
Figure 658: PRO84303 Figure 710: PR049718
Figure 659A-B: DNA328478, NP_055720.2, Figure 711: DNA326892, NP-003711.1, 203405 Jit
203158-s-at Figure 712: PR083213
Figure 660: PRO84304 Figure 713: DNA323927, NP-005563.1, 203411.sjit
Figure 661: DNA226136, NP.003246.1, 203167 Jit Figure 714: PRO80660
Figure 662: PR036599 Figure 715: DNA151037, NP_036461.1, 203414jιt
Figure 663: DNA328479, NPJ301473.1, 203178jιt Figure 716: PR012586
Figure 664: PRO84305 Figure 717: DNA273410, NP-004036.1, 203454.sjit
Figure 665A-C: DNA328480, NP_001990.1, 203184 jit Figure 718: PRO61409
Figure 666: PRO84306 Figure 719: DNA328495, NP_055578.1, 203465 _at
Figure 667A-B: DNA271010, NP_055552.1, 203185_at Figure 720: PR058967
Figure 668: PR059339 Figure 721: DNA328496, NP_002428.1, 203466 jit
Figure 669: DNA270448, NP_002487.1, 203189jsjιt Figure 722: PRO80786
Figure 670: PR058827 Figure 723A-B: DNA255622, NP_009187.1,
Figure 671A-B: DNA328481, MTMR2, 203211 _s_at 203472jsjιt
Figure 672: PRO84307 Figure 724: PRO50686
Figure 673A-C: DNA328482, NP_000426.1, Figure 725A-C: DNA328497, NP_005493.1,
203238_s_at 203504 _s_at
Figure 674: PRO84308 Figure 726: PR084319
Figure 675: DNA328483, NP-061163.1, 203255jιt Figure 727 A-C: DNA328498, AF285167, 203505 Jit
Figure 676: PRO84309 Figure 728: PRO84320
Figure 677: DNA227127, NP.003571.1, 203269 Jit Figure 729 A-B: DNA188400, NP_001057.1, 203508 Jtt
Figure 678: PRO37590 Figure 730: PR021928
Figure 679: DNA328484, UNC119, 203271.sjit Figure 731A-B: DNA328499, NP.003096.1, 203509 Jit
Figure 680: PR084310 Figure 732: PR084321
Figure 681: DNA302020, NP.005564.1, 203276 Jit Figure 733: DNA272911, NP-006545.1, 203517jιt
Figure 682: PRO70993 Figure 734: PRO60997
Figure 683A-B: DNA328485, BHC80, 203278 -S.at Figure 735A-D: DNA328500, NP_000072.1,
Figure 684: PR084311 203518 Jit
Figure 685: DNA328486, NP-000149.1, 203282 ιt Figure 736: PR084322
Figure 686: PRO60119 Figure 737A-B: DNA103296, NP_006369.1, 203528jιt Figure 738: PR04626 Figure 791A-B: DNA272451, HSU86453, 203879 Jit
Figure 739: DNA323910, NPJ002956.1, 203535 _at Figure 792: PRO60700
Figure 740: PRO80648 Figure 793: DNA82429, NP_003011.1, 203889 Jit
Figure 741A-B: DNA272399, NP_001197.1, Figure 794: PR02558
203543_s_at Figure 795: DNA328513, NP_057367.1, 203893 Jit
Figure 742: PRO60653 Figure 796: PR037815
Figure 743: DNA328501, NP-076984.1, 203545jιt Figure 797: DNA150974, NP_005684.1, 203920 Jit
Figure 744: PR084323 Figure 798: PR012224
Figure 745: DNA88453, NP_000228.1, 203548 JSJit Figure 799: DNA271676, NP_002052.1, 203925 Jit
Figure 746: PR02797 Figure 800: PR059961
Figure 747: DNA328502, NP_006566.2, 203553 JS Jit Figure 801: DNA88239, NP.004985.1, 203936.sjit
Figure 748: PR084324 Figure 802: PR02711
Figure 749: DNA328503, NP-000272.1 , 203557 JS _at Figure 803: DNA227232, NP_001850.1, 203971 Jit
Figure 750: PRO 10850 Figure 804: PR037695
Figure 751: DNA327594, NP_003869.1, 203560 Jit Figure 805: DNA328514, NP-005186.1, 203973 JSJU
Figure 752: PR083611 Figure 806: PR084329
Figure 753: DNA225916, NP-067674.1, 203561 Jit Figure 807: DNA328515, NP.000775.1, 203979 Jit
Figure 754: PR036379 Figure 808: PRO84330
Figure 755: DNA273676, NP.055488.1, 203584 Jit Figure 809: DNA327608, NP_001433.1, 203980 Jit
Figure 756: PR061644 Figure 810: PR083617
Figure 757: DNA83085, NP_000751.1, 20359 l s it Figure 811: DNA328516, NP_005833.1, 204011 Jit
Figure 758: PR02583 Figure 812: PR012323
Figure 759: DNA271003, NP_003720.1, 203594 jit Figure 813: DNA328517, NP_003558.1, 204032jιt
Figure 760: PR059332 Figure 814: PR084331
Figure 761A-B: DNA328504, 1400155.1, 203608_at Figure 815: DNA226342, NP_000305.1, 204054 jit
Figure 762: PR084325 Figure 816: PRO36805
Figure 763: DNA328505, NP_002484.1, 203613 sJtt Figure 817: DNA327609, 1448428.2, 204058_at
Figure 764: PR062117 Figure 818: PR083618
Figure 765: DNA328506, NP_001046.1, 203615.x Jtt Figure 819: DNA328518, MEl, 204059 S it
Figure 766: PR084326 Figure 820: PR084332
Figure 767: DNA225774, NP_005079.1, 203624 it Figure 821: DNA226737, NP_004576.1, 204070 jit
Figure 768: PR036237 Figure 822: PRO37200
Figure 769: DNA254642, NP_004100.1, 203646jιt Figure 823A-C: DNA328519, NP.075463.1,
Figure 770: PR049743 204072_s_at
Figure 771: DNA328507, NP.006395.1, 203650jtt Figure 824: PR084333
Figure 772: PR04761 Figure 825: DNA328520, NP-079353.1, 204080 Jit
Figure 773A-B: DNA272998, NP.055548.1, 203651 _at Figure 826: PR084334
Figure 774: PRO61070 Figure 827 A-B: DNA150739, NP_006484.1,
Figure 775: DNA328508, NP_003368.1, 203683_s_at 204084js_at
Figure 776: PR035975 Figure 828: PR012442
Figure 777: DNA255298, NP_004394.1, 203695 _sjιt Figure 829: DNA227130, NP.002551.1, 204088 Jit
Figure 778: PRO50371 Figure 830: PR037593
Figure 779: DNA227020, NP_001416.1, 203729 Jit Figure 831: DNA328521, NP-003069.1, 204099 _at
Figure 780: PR037483 Figure 832: PR062553
Figure 781: DNA328509, NP.006739.1, 203760 _s Jit Figure 833: DNA328522, NP.001769.2, 204118 Jit
Figure 782: PR057996 Figure 834: PR02696
Figure 783: DNA328510, NP-055066.1, 203775 Jit Figure 835: DNA328523, NP-006712.1, 204119_s ιt
Figure 784: PR084327 Figure 836: PR084335
Figure 785A-B: DNA194602, NP-006370.1, Figure 837: DNA328524, NP_057097.1, 204125 Jit
203789 -s-at Figure 838: PR084336
Figure 786: PR023944 Figure 839: DNA328525, BC021224, 204131 _s.at
Figure 787: DNA328511, NP.031397.1, 203825 Jit Figure 840: PR084337
Figure 788: PR057838 Figure 841: DNA103532, NP.003263.1, 204137 Jit
Figure 789A-B: DNA328512, NP_005772.2, Figure 842: PR04859
203839-s.at Figure 843: DNA324816, NP_001060.1, 204141 Jit
Figure 790: PR084328 Figure 844: PR081429 Figure 845: DNA270524, NP.059982.1, 204142 Jit Figure 899: DNA328254, BC002678, 204517jιt
Figure 846: PRO58901 Figure 900: PR011581
Figure 847: DNA328526, NP.000841.1, 204149_s_at Figure 901: DNA328254, NP_000934.1, 204518_s_at
Figure 848: PR037856 Figure 902: PROl 1581
Figure 849A-B: DNA150497, DNA150497, Figure 903A-B: DNA328535, NP_009147.1, 204544 Jit
204155_s_at Figure 904: PRO60044
Figure 850: PRO 12296 Figure 905: DNA225993, NP-000646.1, 204563 Jit
Figure 851 A-B: DNA328527, NP_055751.1, Figure 906: PR036456
204160 -s Jit Figure 907: DNA287284, NP_060943.1, 204565jιt
Figure 852: PR04351 Figure 908: PR059915
Figure 853: DNA328528, MLC1SA, 204173 Jit Figure 909: DNA151910, NP_004906.2, 204567 _sjιt
Figure 854: PRO60636 Figure 910: PR012754
Figure 855: DNA328529, NP_001620.2, 204174_at Figure 911: DNA270564, NP_004499.1, 204615-XJit
Figure 856: PR049814 Figure 912: PR058939
Figure 857: DNA226380, NP-001765.1, 204192jιt Figure 913: DNA328536, 1099945.20, 204619 _s Jit
Figure 858: PR04695 Figure 914: PR084342
Figure 859: DNA273070, NP-005189.2, 204193jιt Figure 915A-D: DNA328537, NP_004376.2,
Figure 860: PRO70107 204620_s_at
Figure 861: DNA227514, NP.000152.1, 204224ji_at Figure 916: PR084343
Figure 862: PR037977 Figure 917: DNA151048, NP_006177.1, 204621 _s_at
Figure 863: DNA270434, NP.006434.1, 204238 jj_at Figure 918: PRO12850
Figure 864: PR058814 Figure 919A-B: DNA328538, 351122.2, 204627 _s Jit
Figure 865: DNA307936, NP.004926.1, 204247_s_at Figure 920: PR084344
Figure 866: PR071356 Figure 921A-B: DNA88429, NP-000203.1,
Figure 867A-B: DNA188734, NP_001261.1, 204258 jtt 204628-S.at
Figure 868: PR022296 Figure 922: PR02344
Figure 869: DNA226577, NP_071390.1, 204265-SJit Figure 923: DNA226079, NP_001602.1, 204638jιt
Figure 870: PRO37040 Figure 924: PR036542
Figure 871: DNA273802, NP-066950.1, 204285_sjιt Figure 925: DNA272078, NP.003019.1, 204657 Ji_at
Figure 872: PR061763 Figure 926: PRO60348
Figure 873: DNA328530, NP.009198.2, 204328 Jit Figure 927: DNA227425, NP_001038.1, 204675jιt
Figure 874: PR024118 Figure 928: PR037888
Figure 875: DNA328531, NP-037542.1, 204348 _s_at Figure 929A-B: DNA328539, NP.000121.1,
Figure 876: PR084338 204713.s-at
Figure 877: DNA328532, LHVIKl, 204357-S _at Figure 930: PR084345
Figure 878: PR084339 Figure 931: DNA328540, NP_006144.1, 204725 JSJit
Figure 879: DNA225750, NP.000254.1, 204360ji jit Figure 932: PR012168
Figure 880: PR036213 Figure 933A-B: DNA325192, NP.038203.1,
Figure 881: DNA328533, NP_003647.1, 204392jιt 204744 _s_at
Figure 882: PRO84340 Figure 934: PR081753
Figure 883: DNA272469, NP.005299.1, 204396_SJit Figure 935: DNA328541, NP-004503.1, 204773jιt
Figure 884: PRO60717 Figure 936: PR04843
Figure 885: DNA226462, NP.002241.1, 204401 jit Figure 937: DNA328542, NP.055025.1, 204774jιt
Figure 886: PR036925 Figure 938: PR02577
Figure 887: DNA225756, NP_001636.1, 204416-x.at Figure 939: DNA327050, NP_009199.1, 204787_at
Figure 888: PR036219 Figure 940: PRO34043
Figure 889: DNA226286, NP_001657.1, 204425 Jit Figure 941 : DNA328543, NP_005883.1, 204789_at
Figure 890: PR036749 Figure 942: PR084346
Figure 891A-B: DNA88476, NP_002429.1, 204438jιt Figure 943: DNA272121, NP.005895.1, 204790jιt
Figure 892: PR02811 Figure 944: PRO60391 Figure 893 DNA 150972, NP.005252.1, 204472 _at Figure 945: DNA324799, NP_061823.1, 204806.χjιt Figure 894 PR012162 Figure 946: PR081414 Figure 895 DNA194652, NPJOOI 187.1, 204493 Jit Figure 947: DNA154704, DNA154704, 204807 Jit Figure 896 PR023974 Figure 948: DNA328544, NP_006673.1, 204834 Jit Figure 897 DNA328534, NP J56307.1, 204494.sjιt Figure 949: PR084347 Figure 898: PR084341 Figure 950: DNA225661, NP_001944.1, 204858 >Jit Figure 951: PR036124 Figure 1003: PR084354
Figure 952: DNA328545, NP-064525.1, 204859 _s_at Figure 1004: DNA328555, NP_001241.1, 205153j>Jit
Figure 953: PR084348 Figure 1005: PR034457
Figure 954A-B: DNA227629, NP-004527.1, Figure 1006: DNA80896, NP.001100.1, 205180-SJit
204860 j;-at Figure 1007: PR01686
Figure 955: PRO38092 Figure 1008: DNA328556, NP-004568.1, 205194 Jit
Figure 956: DNA328546, NP-005249.1, 204867 Jit Figure 1009: PR084355
Figure 957: PR084349 Figure 1010: DNA273535, NP.004217.1, 205214jιt
Figure 958: DNA255993, NP.008936.1, 204872 it Figure 1011: PR061515
Figure 959: PR051044 Figure 1012: DNA93504, NPΛ06009.1, 205220jιt
Figure 960: DNA273666, NP.003349.1, 204881-S Jit Figure 1013: PR04923
Figure 961: PR061634 Figure 1014: DNA325255, NP_001994.2, 205237 Jit
Figure 962A-B: DNA76503, NP.001549.1, 204912 Jit Figure 1015: PRO1910
Figure 963 : PR02536 Figure 1016: DNA327634. NP-005129.1, 205241 Jit
Figure 964: DNA328547, TLR2, 204924 Jit Figure 1017: PR083636
Figure 965: PRO208 Figure 1018: DNA227081, NP_000390.2, 205249 Jit
Figure 966: DNA228014, NP_002153.1, 204949 Jit Figure 1019: PR037544
Figure 967: PR038477 Figure 1020: DNA328557, NP_001098.1, 205260 _s -at
Figure 968: DNA328548, NP-006298.1, 204955 _at Figure 1021: PR084356
Figure 969: PR02618 Figure 1022: DNA328558, BC016618, 205269 it
Figure 970: DNA103283, NP_002423.1, 204959 Jit Figure 1023: PR084357
Figure 971: PR04613 Figure 1024: DNA328559, NP„005556.1, 205270-SJit
Figure 972: DNA227091, NP-000256.1, 204961_s_at Figure 1025: PR084358
Figure 973: PR037554 Figure 1026A-B: DNA227505, NP_003670.1,
Figure 974A-B: DNA328549, NP_002897.1, 205306-X_at
204969-SJit Figure 1027: PR037968
Figure 975: PRO84350 Figure 1028: DNA325783, NP.002558.1, 205353 JSJit
Figure 976: DNA328301, NP-005204.1, 204971 tt Figure 1029: PRO59001
Figure 977: PRO70371 Figure 1030: DNA88215, NP_001919.1, 205382 3jιt
Figure 978A-B: DNA328550, NP.001439.2, Figure 1031: PRO2703
204983j;_at Figure 1032: DNA328560. NP.003650.1, 20540 ljit
Figure 979: PR0937 Figure 1033: PR084359
Figure 980: DNA269665, NP-002454.1, 204994 Jit Figure 1034: DNA328561, NP_004624.1, 205403 Jit
Figure 981: PRO58076 Figure 1035: PRO2019
Figure 982A-B: DNA273686, NP-055520.1, 205003 _at Figure 1036: DNA327638, NP_005516.1, 205404 jit
Figure 983: PR061653 Figure 1037: PR083639
Figure 984: DNA272427, NP-004799.1, 205005 JS Jit Figure 1038: DNA328562, NP_000010.1, 205412jιt
Figure 985: PRO60679 Figure 1039: PRO84360
Figure 986: DNA194830, NP.055437.1, 205011jιt Figure 1040A-B: DNA328563, NPU05329.2,
Figure 987: PRO24094 205425-at
Figure 988: DNA328551, NP-.003823.1, 205048_s_at Figure 1041: PR081554
Figure 989: PR084351 Figure 1042: DNA328564, HPCALl, 205462js_at
Figure 990A-B: DNA328552, NP_055886.1, Figure 1043: PR084361
205068_s_at Figure 1044: DNA196825, NP_005105.1, 205466_s_at
Figure 991: PR084352 Figure 1045: PR025266
Figure 992: DNA328553, NP-061944.1, 205070 Jit Figure 1046: DNA328565, NP_057070.1, 205474jιt
Figure 993: PR084353 Figure 1047: PR084362
Figure 994: DNA194627, NP-003051.1, 205074 ιt Figure 1048: DNA226153, NP.002649.1, 205479.s_at
Figure 995: PR023962 Figure 1049: PR036616
Figure 996: DNA272181, NP-006688.1, 205076-SJit Figure 1050: DNA287224, NP_005092.1, 205483 _s_at
Figure 997: PRO60446 Figure 1051: PRO69503
Figure 998: DNA254216, NPD02020.1, 205119-SJit Figure 1052: DNA328566, NP_060446.1, 205510jsJit
Figure 999: PR049328 Figure 1053: PR084363
Figure 1000: DNA299899, NP.002148.1, 205133 3_at Figure 1054: DNA328567, NP_006797.2, 205548 s ιt
Figure 1001: PRO62760 Figure 1055: PR084364
Figure 1002: DNA328554, NP-038202.1, 205147 _x Jit Figure 1056: DNA227535, NP_066190.1, 205568 Jit Figure 1057: PR037998 Figure 1 PR04944
Figure 1058A-B: DNA327643, NP-055712.1, Figure 1 DNA328576, HSU20350, 205898 Jit
205594 tt Figure 1 PRO4940
Figure 1059: PR083644 Figure 1 DNA328577, NP.003905.1, 205899 Jit
Figure 1060A-C: DNA328568, NP-006720.1, Figure 1 PR059588
205603_sjιt Figure 1 12A-B: DNA 196549, NP.003034.1,
Figure 1061: PR059731 205920-at t
Figure 1062: DNA324324, NP_000679.1, 205633_sjιt Figure 1 13: PRO25031
Figure 1063: PR081000 Figure 1 14: DNA328578, NP-004656.2, 205922 jit
Figure 1064: DNA328569, NP_077274.1, 205634 _χjιt Figure 1 15: PR07426
Figure 1065: PR084365 . Figure 1 16A-B: DNA270867, NP-006217.1,
Figure 1066: DNA88076, NP-.001628.1, 205639 jvt 205934jιt
Figure 1067: PRO2640 Figure 1 17: PRO59203
Figure 1068: DNA287317, NP-003724.1, 205660 it Figure 1 18: DNA76516, NP_000556.1, 205945jιt
Figure 1069: PR069582 Figure 1 19: PRO2022
Figure 1070: DNA328570, NP-004040.1, 205681 it Figure 1 20: DNA196439, NP-003865.1, 205988 Jit
Figure 1071: PR037843 Figure 1 21: PR024934
Figure 1072: DNA327644, NP_060395.2, 205684 _s Jit Figure 1 22: DNA36722, NP_000576.1, 205992 j>_at
Figure 1073: PR083645 Figure 1 23: PR077
Figure 1074: DNA150621, NP-036595.1, 205704 j;_at Figure 1 24: DNA328579, BC020082, 206020jat
Figure 1075: PR012374 Figure 1 25: PRO84370
Figure 1076: DNA328571, NP-001254.1, 205709.sjit Figure 1 26: DNA328580, HSU27699, 206058 Jit
Figure 1077: PR084366 Figure 1 27: PR04627
Figure 1078: DNA88106, NP_004325.1, 205715 ιt Figure 1 28: DNA328581, NP-002122.1, 206074_sjιt
Figure 1079: PR02655 Figure 1 29: PR034536
Figure 1080: DNA270401, NP-003140.1, 205743 jit Figure 1 30: DNA328582, NP-001865.1, 206100JU
Figure 1081 : PR058784 Figure 1 31: PR084371
Figure 1082: DNA275620, NP.000628.1, 205770 it Figure 1 32: DNA226105, NP_002925.1, 206111 jit
Figure 1083: PR063244 Figure 1 33: PR036568
Figure 1084: DNA88187, NP-001757.1, 205789 it Figure 1 34: DNA225764, NP.000037.1, 206129 JSJit
Figure 1085: PR02689 Figure 1 35: PR036227
Figure 1086: DNA76517, NP_002176.1, 205798 tt Figure 1 36: DNA328583, ASGR2, 206130 3 JitFigure 1087: PR02541 Figure 1 37: PR084372
Figure 1088A-B: DNA271915, NP_056191.1, Figure 1 38: DNA327656, NP_055294.1, 206134jιt
20580 l_sjιt Figure 1 39: PR036117
Figure 1089: PRO60192 Figure 1 40A-B: DNA271837, NP.055497.1,
Figure 1090: DNA194766, NP-079504.1, 205804.s it 206135jttt
Figure 1091: PRO24046 Figure 1 4L PRO60117
Figure 1092: DNA328572, NPD04309.2, 205808 jit Figure 1 42: DNA328584, NP.001148.1, 206200.S Jit
Figure 1093: PR084367 Figure 1 43: PR04833
Figure 1094: DNA328573, NP-006761.1, 205819jιt Figure 1 44: DNA226058, NP.005075.1, 206214 Jit
Figure 1095: PRO 1559 Figure 1 45: PR036521
Figure 1096A-B: DNA328574, NP.004963.1, Figure 1 46: DNA218691, NP_003832.1, 206222jιt
205842 s.at Figure 1 47: PR034469
Figure 1097: PR084368 Figure 1 48A-C: DNA328585, AF286028,
Figure 1098: DNA327651, NP_005612.1, 205863 Jit 206239.S .at
Figure 1099: PR083649 Figure 1 49: DNA328586, NP-002369.2, 206267 _s Jit
Figure 1100: DNA328575, NP-071754.2, 205872 _χ ιt Figure 1 50: PR084373
Figure 1101 : PR084369 Figure 1 51: DNA328587, NP-002612.1, 206380_s ιt
Figure 1102A-B: DNA220746, NP_000876.1, Figure 1 52: PR02854
205884 tt Figure 1 53: DNA255814, NP_005840.1, 206420 ιt
Figure 1103: PR034724 Figure 1 54: PRO50869
Figure 1104A-B: DNA273962, NP-055605.1, Figure 1 55: DNA328588, NP_060823.1, 206500 -S_at
205888-S ιt Figure 1 56: PR084374
Figure 1105: PRO61910 Figure 1 57: DNA270444, NP-004824.1, 206513 it
Figure 1106: DNA93423. NP-000667.1, 205891 it Figure 1 58: PR058823 Figure 59: DNA196614. NPJ001158.1, 206536-s_at Figure 1212: PR084381
Figure 60: PRO25091 Figure 1213: DNA328598, NP-055146.1, 207528 _s_at
Figure 61: DNA270019, NP-036351.1, 206538_at Figure 1214: PR023276
Figure 62:tPR058414 Figure 1215: DNA328599, NFKB2, 207535 j>jιt
Figure 63: DNA327663, NP-006771.1, 206565_χjιt Figure 1216: PR084382
Figure 64: PR083654 Figure 1217: DNA328600, NP_004839.1, 207571 jcjit
Figure 65: DNA327665, NP.002099.1, 206643 jit Figure 1218: PR084383
Figure 66: PR083655 Figure 1219: DNA328601, NP-056490.1, 207574_sjιt
Figure 67: DNA328589, BCL2L1, 206665 5_at Figure 1220: PR084384
Figure 68: PR083141 Figure 1221: DNA328602, NP_002261.1, 207657 jt it
Figure 69: DNA328590, C6orf32, 206707 jcat Figure 1222: PR084385
Figure 70: PR084375 Figure 1223: DNA226278, NP_005865.1, 207697 j „at
Figure 71A-B: DNA88191, NP_001234.1, 206729 Jit Figure 1224: PR036741
Figure 72: PR02691 Figure 1225: DNA227395, NP-005331.1, 207721_x_at
Figure 73: DNA327669, NP.000914.1, 206792jcjιt Figure 1226: PR037858
Figure 74: PR083657 Figure 1227: DNA325654, NP-054752.1, 207761_s_at
Figure 75: DNA270107, NP-006856.1, 206881 _s it Figure 1228: PR04348
Figure 76: PR058498 Figure 1229: DNA226930, NP_004152.l, 207791_sjιt
Figure 77: DNA256561, NP-062550.1, 206914jιt Figure 1230: PR037393
Figure 78: PR051592 Figure 1231: DNA328603, NP_000304.1, 207808_s_at
Figure 79: DNA328591, NP-006635.1, 206976_SJit Figure 1232: PR084386
Figure 80: PR084376 Figure 1233: DNA328604, NP.001174.2, 207809 j> jit
Figure 81 A-B: DNA227659, NP-000570.1, Figure 1234: PR084387
206991 it Figure 1235: DNA327682, NP_001905.1, 207843 jc_at
Figure 82: PR038122 Figure 1236: PR083666
Figure 83: DNA188289, NP-001548.1, 207008 ιt Figure 1237: DNA36708, NP-002081.1, 207850-at
Figure 84: PRO21820 Figure 1238: PR034256
Figure 85: DNA328592, AB015228, 207016.S Jit Figure 1239: DNA199788, NP,002981.1, 207861 Jit
Figure 86: PR084377 Figure 1240: PRO34107
Figure 87: DNA227531, NP.004722.1, 207057 Jit Figure 1241: DNA328605, ST7, 207871_sjιt
Figure 88: PR037994 Figure 1242: PR084388
Figure 89: DNA327673, NP_002188.1, 207071_SJit Figure 1243: DNA256523, NP_006854.1, 207872 ;.at
Figure 90: PRO83660 Figure 1244: PR051557
Figure 91A-B: DNA328593, CIASl, 207075 it Figure 1245: DNA218651, NP_003798.1, 207907 jit
Figure 92: PR084378 Figure 1246: PR034447
Figure 93A-B: DNA328594, CSF1, 207082jιt Figure 1247: DNA275286, NP.009205.1, 208002_s_at
Figure 94: PR084379 Figure 1248: PR062967
Figure 95: DNA88291, NP_001965.1, 207111 Jit Figure 1249 A-B: DNA328606, CBFA2T3, 208056 _s Jit
Figure 96: PR02729 Figure 1250: PR084389
Figure 97 A-B: DNA327674, NP-002739.1, Figure 1251A-B: DNA328607, NP.003639.1,
207121 .at 208072-s.at
Figure 98: PR083661 Figure 1252: PRO84390
Figure 99: DNA328595. NP-001045.1, 207122 jιt Figure 1253: DNA327685, NP-067586.1, 208074_s_at
Figure 200: PRO84380 Figure 1254: PR083669
Figure 201: DNA226996, NP.000239.1, 207233_SJit Figure 1255: DNA328608, NP.006264.2, 208075_s Jit
Figure 202: PR037459 Figure 1256: PR09932
Figure 203 A-B: DNA226536, NP-003225.1, Figure 1257: DNA255376, NP.l 10423.1, 20809 l_s Jit
207332-S-at Figure 1258: PRO50444
Figure 204 PR036999 Figure 1259: DNA327686, NPJ0O5898.1, 208116-SJit Figure 205, DNA227668, NP-000158.1, 207387 js_at Figure 1260: PRO83670 Figure 206 PR038131 Figure 1261 A-B: DNA328609, NP.109592.1, Figure 207 DNA328596, DEGS, 207431 s_at 208121 i_at Figure 208 PR037741 Figure 1262: PR084391 Figure 209 DNA274829, NP-003653.1, 207469 S Jit Figure 1263: DNA328610, NP_U2601.1, 208146_s_at Figure 210 PR062588 Figure 1264: PR084392 Figure 211 DNA328597, NP-001680.1, 207507 JϊJit Figure 1265A-B: DNA226706, NP_003777.2, 208161j;_at Figure 1318: PR082662
Figure 1266: PR037169 Figure 1319: DNA227556, NP.001670.1, 208836_at
Figure 1267: DNA328611. RASGRP2, 208206-S_at Figure 1320: PRO38019
Figure 1268: PR084393 Figure 1321: DNA326042, NP_031390.1, 208837 Jit
Figure 1269: DNA328612, NP-000166.2, 208308 jijit Figure 1322: PRO1078
Figure 1270: PR084394 Figure 1323A-B: DNA328623, NP_056107.1,
Figure 1271: DNA270558, NP.006734.1, 208319j;jιt 208858_s_at
Figure 1272: PR058933 Figure 1324: PR061321
Figure 1273: DNA227614, NP-004859.1, 208336 jj_at Figure 1325: DNA227874, NP_003320.1, 208864-S-at
Figure 1274: PRO38077 Figure 1326: PR038337
Figure 1275: DNA327690, NP-004022.1, 208436-s_at Figure 1327: DNA328624, BC003562, 208891 Jit
Figure 1276: PR083673 Figure 1328: PRO59076
Figure 1277: DNA328613, NP-056953.2, 208510_s_at Figure 1329: DNA328625, NP.073143.1, 208892_s_at
Figure 1278: PR084395 Figure 1330: PRO84404
Figure 1279 A-C: DNA328614, SRRM2, 208610-S-at Figure 1331 : DNA328626, NP_057078.1, 208898 Jit
Figure 1280: PR084396 Figure 1332: PR061768
Figure 1281A-C: DNA328615, NP_003118.1, Figure 1333: DNA327700, BC015130, 208905jtt
208611-s.at Figure 1334: PR083683
Figure 1282: PR084397 Figure 1335: DNA325472, NP_116056.2, 208906Jit
Figure 1283A-C: DNA328616, NP_001448.1, Figure 1336: PR081995
208613js_at Figure 1337A-B: DNA328627, FLI13052, 208918 js_at
Figure 1284: PR084398 Figure 1338: PRO84405
Figure 1285: DNA326362, VATI, 208626 _s_at Figure 1339: DNA325473, NP-006353.2, 208922-S_at
Figure 1286: PR082758 Figure 1340: PROS 1996
Figure 1287: DNA325912, NP.001093.1, 208637 jcjit Figure 1341: DNA287238, NP.000425.1, 208926 Jit
Figure 1288: PR082367 Figure 1342: PR069515
Figure 1289: DNA271268, NP-009057.1, 208649 SJit Figure 1343: DNA328628, NP_060542.2, 208933_sjιt
Figure 1290: PR059579 Figure 1344: PRO84406
Figure 1291: DNA328617, AF299343, 208653-SJit Figure 1345: DNA290261, NP.001291.2, 208960-SJit
Figure 1292: PR084399 Figure 1346: PRO70387
Figure 1293A-C: DNA328618, NP-003307.2, Figure 1347 A-B: DNA325478, NP-037534.2,
208664 _s_at 208962jj_at
Figure 1294: PRO84400 Figure 1348: PR081999
Figure 1295: DNA304686, NP_002565.1, 208680jιt Figure 1349: DNA328629, NP_006079.1, 208977-XJit
Figure 1296: PR071112 Figure 1350: PRO84407
Figure 1297: DNA304499, NP_006588.1, 208687_x_at Figure 1351: DNA328630, NP.036293.1, 209004-s_at
Figure 1298: PRO71063 Figure 1352: PRO84408
Figure 1299A-B: DNA328619, BC001188, 208691 Jit Figure 1353: DNA328631, AK027318, 209006_SJit
Figure 1300: PRO84401 Figure 1354: PRO84409
Figure 1301: DNA287189, NP_002038.1, 208693-S_at Figure 1355: DNA328632, DJ465N24.2.1Homo,
Figure 1302: PR069475 209007-s_at
Figure 1303: DNA324217, ATIC, 208758 Jit Figure 1356: DNA328633, NP.004784.2, 209017-S Jit
Figure 1304: PRO80908 Figure 1357: PR084411
Figure 1305: DNA327696, AF228339, 208763_s_at Figure 1358A-B: DNA328634, NP.006594.1,
Figure 1306: PR083679 209023 _s_at
Figure 1307: DNA328620, AK000295, 208772 _at Figure 1359: PR084412
Figure 1308: PRO84402 Figure 1360: DNA328635, BC020946, 209026.x Jit
Figure 1309: DNA328621, NP.002788.1, 208799 Jit Figure 1361: PR084413
Figure 1310: PRO84403 Figure 1362: DNA274202. NP.006804.1, 209034 Jit
Figure 1311: DNA287169, CAA42052.1, 208805 _at Figure 1363: PR062131
Figure 1312: PR010404 Figure 1364: DNA328636, PAPSS1, 209043 Jit
Figure 1313: DNA324531, NP_002120.1, 208808_s_at Figure 1365: PR084414
Figure 1314: PR081185 Figure 1366A-C: DNA328637, HSA7042, 209053 _s Jit
Figure 1315: DNA273521, NP_002070.1, 208813 Jit Figure 1367: PR081109
Figure 1316: PRO61502 Figure 1368: DNA326406, NP_005315.1, 209069 JSJit
Figure 1317: DNA328622, BC000835, 208827 Jit Figure 1369: PROl 1403 Figure 1370: DNA227289, NP.006532.1, 209080.x Jit Figure 1424: PRO50332
Figure 1371: PR037752 Figure 1425A-B: DNA226827, NP_001673.1,
Figure 1372: DNA274180, NP_009005.1, 209083 ιt 209281.s-at
Figure 1373: PR062110 Figure 1426: PRO37290
Figure 1374: DNA327707, NPD00148.1, 209093_s-at Figure 1427: DNA328650, 200118.10, 209286 Jit
Figure 1375: PR083689 Figure 1428: PR084425
Figure 1376: DNA226564, NP-000099.1, 209095 Jit Figure 1429: DNA274883, NP.000058.1, 209301 _at
Figure 1377: PRO37027 Figure 1430: PR062628
Figure 1378: DNA325163, NP_001113.1, 209122jit Figure 1431: DNA328651, AF087853, 209305-S-at
Figure 1379: PRO81730 Figure 1432: PR082889
Figure 1380: DNA328638, BC000576, 209123_at Figure 1433: DNA327718, CASP4, 209310-S.at
Figure 1381: PR081129 Figure 1434: PR083697
Figure 1382: DNA274723, AAB62222.1, 209129 _at Figure 1435: DNA328652, NP.077298.1, 209321 _s_at
Figure 1383: PRO62502 Figure 1436: PR084426
Figure 1384: DNA328639, HSM801840, 209132_s_at Figure 1437: DNA328653, AF063020, 209337 -at
Figure 1385: PR084415 Figure 1438: PR084427
Figure 1386: DNA328640, ASPH, 209135_at Figure 1439: DNA328654, UAP1, 209340jιt
Figure 1387: PR084416 Figure 1440: PR084428
Figure 1388: DNA327713, BC010653, 209146jιt Figure 1441: DNA328655, 346677.3, 209341.sjit
Figure 1389: PR037975 Figure 1442: PR084429
Figure 1390: DNA271937, NP.055419.1, 209154 Jit Figure 1443: DNA269630, NP.003281.1, 209344.at
Figure 1391: PRO60213 Figure 1444: PRO58042
Figure 1392: DNA328641, NP-001840.2, 209156-S_at Figure 1445A-B: DNA328656, HSA303098,
Figure 1393: PR084417 209345-s_at
Figure 1394: DNA325285, AKR1C3, 209160_at Figure 1446: PRO84430
Figure 1395: PR081832 Figure 1447A-B: DNA328657, NP_060895.1,
Figure 1396A-B: DNA328642, AF073310, 209346_s_at
209184_s_at Figure 1448: PR084431
Figure 1397: PR084418 Figure 1449A-B: DNA328658, AF055376,
Figure 1398A-B: DNA328643, HUMHK1A, 209348-s_at
209186jιt Figure 1450: PR084432
Figure 399: PR084419 Figure 1451: DNA327719, NP-003704.2, 209355-SJit Figure 400: DNA189700, NP_005243.1, 209189 Jit Figure 1452: PR083698 Figure 401: PR025619 Figure 1453: DNA328659, ECM1, 209365-S-at Figure 402: DNA327715, NP_115914.1, 209191 Jit Figure 1454: PR084433 Figure 403: PR083694 Figure 1455: DNA225952, NP.001267.1, 209395 Jit Figure 404: DNA103520, NP-002639.1, 209193 Jit Figure 1456: PR036415 Figure 405: PR04847 Figure 1457: DNA275366. BC001851, 209444 jtt Figure 406A-B: DNA269816, MEF2C, 209199 _s_at Figure 1458: PRO63036 Figure 407: PR058219 Figure 1459: DNA328660, NP-003675.2, 209467.sjit Figure 408: DNA328644, 349746.9, 209200jtt Figure 1460: PR084434 Figure 409: PRO84420 Figure 1461A-B: DNA328661, NP.006304.1, Figure 410: DNA326891, NP.001748.1, 209213jιt 209475_at Figure 411: PR083212 Figure 1462: PR084435 Figure 412: DNA328645, NP_009006.1, 209216jιt Figure 1463: DNA328662, 0SBPL1A, 209485_s.at Figure 413: PR084421 Figure 1464: PR084436 Figure 414: DNA227483, NP_003120.1, 209218 Jit Figure 1465: DNA324899, NP-002938.1, 209507 Jit Figure 415: PR037946 Figure 1466: PR081503 Figure 416: DNA328646, NP.036517.1, 209230_s ιt Figure 1467: DNA274027, HSU38654, 209515j;_at Figure 417: PR084422 Figure 1468: PR061971 Figure 418A-C: DNA328647, AB017133, 209234_at Figure 1469: DNA328663, NP_057157.1, 209524_at Figure 419: PR084423 Figure 1470: PR036183 Figure 420A-B: DNA328648, D87075, 209236 _at Figure 1471 A-C: DNA328664, NP-009131.1, Figure 421: DNA328649, NP.l 16093.1, 20925 l_x.at 209534_x-at Figure 422: PR084424 Figure 1472: PR084437 Figure 423: DNA255255, NPJ371437.1, 209267 -S-at Figure 1473A-B: DNA328665, RGL, 209568_s-at Figure 1474: PR084438 Figure 1527: DNA328258, HSM802616, 209900.S _at
Figure 1475: DNA328666, AF084943, 209585 Ji-at Figure 1528: PR084J51
Figure 1476: PRO 1917 Figure 1529A-B: DNA328680, NP_062541.1,
Figure 1477: DNA328667, S69189, 209600_SJit 209907_s_at
Figure 1478: PR084439 Figure 1530: PR084451
Figure 1479: DNA328668, NP.003157.1, 209607.xJit Figure 1531: DNA299884, AB040875, 209921 Jit
Figure 1480: PRO84440 Figure 1532: PRO70858
Figure 1481: DNA328669, NP_005882.1, 209608_5_at Figure 1533: DNA328681, NP_005089.1, 209928_s_at
Figure 1482: PR084441 Figure 1534: PR084452
Figure 1483A-B: DNA328670, BC001618, Figure 1535: DNA272326, NP_006154.1, 209930j>_at
209610_s_at Figure 1536: PRO60583
Figure 1484: PRO70011 Figure 1537: DNA328682, AF225981, 209935 Jit
Figure 1485: DNA256209, NP-002259.1, 209653jιt Figure 1538: PR084453
Figure 1486: PR051256 Figure 1539: DNA327754, NP_150634.1, 209970 _x_at
Figure 1487A-B: DNA272671, HSU26710, 209682jιt Figure 1540: PR04526
Figure 1488: PRO60796 Figure 1541: DNA328683, NP_000399.1, 210007 s_at
Figure 1489: DNA151564, DNA151564, 209683jιt Figure 1542: PR084454
Figure 1490: PROl 1886 Figure 1543: DNA227660, NP_001327.1, 210042_sjιt
Figure 1491: DNA327727, NP_000308.1, 209694 _at Figure 1544: PR038123
Figure 1492: PRO83705 Figure 1545: DNA327739, AF092535, 210058 Jtt
Figure 1493: DNA328671, NP_000498.2, 209696jιt Figure 1546: PR083714
Figure 1494: PR084442 Figure 1547: DNA327740, NP_003944.1, 210087_s_at
Figure 1495: DNA327728, BC004492, 209703_x.at Figure 1548: PR01787
Figure 1496: PR04348 Figure 1549: DNA328684, BC001234, 210102_at
Figure 1497: DNA328672, CAA68871.1, 209707 Jit Figure 1550: PR084455
Figure 1498: PR084444 Figure 1551 A-B: DNA328685, NP_127497.1,
Figure 1499A-B: DNA328673, HUMCSDFl, 210113_sjιt
209716_at Figure 1552: PR034751
Figure 1500: PR084445 Figure 1553: DNA328686, NP_000566.1, 210118_s_at
Figure 1501A-B: DNA304800, BC002538, 209723 Jit Figure 1554: PR064
Figure 1502: PR069458 Figure 1555: DNA227757, NP_000743.1, 210128 _s Jit
Figure 1503 A-B: DNA328674, NP_056011.1, Figure 1556: PRO38220
209760-at Figure 1557: DNA227501, NP_000295.1, 210139 s_at
Figure 1504: PR084446 Figure 1558: PR037964
Figure 1505: DNA324250, NP-536349.1, 209761 JSJit Figure 1559: DNA328687, AF004231, 210146-X_at
Figure 1506: PRO80934 Figure 1560: PR084456
Figure 1507 A-B: DNA328675, ADAM19, 209765 _at Figure 1561 A-B: DNA328688, NP_006838.2,
Figure 1508: PR084447 210152-at
Figure 1509: DNA327731, NP.003302.1, 209803-S it Figure 1562: PR084457
Figure 1510: PRO83707 Figure 1563: DNA328689, NP_003259.2, 210166jιt
Figure 1511: DNA328676, IL16, 209827 _s_at Figure 1564: PR07521
Figure 1512: PR084448 Figure 1565: DNA270196, HUMZFM1B, 210172jit
Figure 1513A-B: DNA196499, AB002384, 209829 Jit Figure 1566: PR058584
Figure 1514: PR024988 Figure 1567: DNA328690, NP_524145.1, 210240-S.at
Figure 1515: DNA328677, AF060511, 209836.x Jit Figure 1568: PRO59660
Figure 1516: PR084449 Figure 1569: DNA326963, HRIHFB2122, 210276.S Jit
Figure 1517: DNA324805, NP.008978.1, 209846 _s_at Figure 1570: PR083276
Figure 1518: PR081419 Figure 1571: DNA328691, NP_065717.1, 210346-s.at
Figure 1519: DNA273915, NP_036215.1, 209864 jit Figure 1572: PR084458
Figure 1520: PR061867 Figure 1573: DNA227652, NP.002549.1, 21040 ljit
Figure 1521: DNA290585, NP.000573.1, 209875_s_at Figure 1574: PR038115
Figure 1522: PRO70536 Figure 1575: DNA225514, NP_003864.1, 210510_s_at
Figure 1523: DNA328678, NP_008843.1, 209882 jit Figure 1576: PR035977
Figure 1524: PR062586 Figure 1577: DNA216517, NP_005055.1, 210549.s_at
Figure 1525: DNA328679, 347423.1, 209892jtt Figure 1578: PR034269
Figure 1526: PRO84450 Figure 1579: DNA327746, HUMGCBA, 210589 3Jit Figure 580: PRO83720 Figure 1633: PR084466 Figure 581: DNA328692, AF025529, 210660jιt Figure 1634: DNA226582, NP_003863.1, 211844_sjιt Figure 582: PR084459 Figure 1635: PRO37045 Figure 583: DNA272127, NP-003928.1, 210663-s_at Figure 1636: DNA151912, BAA06683.1, 211935jιt Figure 584: PRO60397 Figure 1637: PR012756 Figure 585: DNA326525, NP-006330.1, 210719j>_at Figure 1638: DNA325941, NP_005339.1, 211968_s_at Figure 586: PR082894 Figure 1639: PR082388 Figure 587: DNA226183, NP-001453.1, 210773_s_at Figure 1640: DNA287433, NP_006810.1, 212009 j;_at Figure 588: PR036646 Figure 1641: PRO69690 Figure 589: DNA226078, NP-000296.1, 210830_s_at Figure 1642: DNA328708, NP_002678.1, 212036_s ιt Figure 590: PR036541 Figure 1643: PR084467 Figure 591: DNA226152, NP-002650.1, 210845j>_at Figure 1644: DNA103380, NP_003365.1, 212038 _s tt Figure 592: PR036615 Figure 1645: PRO4710 Figure 593: DNA328693, HSU03891, 210873-XJit Figure 1646: DNA328709. BC004151, 212048-SJit Figure 594: PRO84460 Figure 1647: PR037676 Figure 595: DNA328694, BC007810, 210944 _s.at Figure 1648A-B: DNA254751, AB018353, 212074_at Figure 596: PR084461 Figure 1649: DNA328710, HUMLAMA, 212086 CJit Figure 597: DNA213676, NP.004604.1, 211003-XJit Figure 1650A-B: DNA298616, NP.001839.1, Figure 598: PR035142 212091j;_at Figure 599: DNA328695, NP-002145.1, 211015-S.at Figure 1651: PRO71027 Figure 600: PRO61480 Figure 1652: DNA154139, DNA154139, 212099 Jit Figure 601: DNA328696, NP.009214.1, 211026js_at Figure 1653: DNA328711, AK023154, 212115 Jit Figure 602: PRO62720 Figure 1654: PR084468 Figure 603: DNA328697, NP_116112.1, 211038_s it Figure 1655: DNA328712, NP_006501.1, 212118jιt Figure 604: PR084462 Figure 1656: PR084469 Figure 605: DNA328698, BC006403, 211063.sJit Figure 1657: DNA328713, AF100737, 212130.x Jit Figure 606: PR012168 Figure 1658: PRO84470 Figure 607: DNA326712, NP.001285.1, 211136JS _at Figure 1659: DNA328714, HSM801966, 212146jιt Figure 608: PRO83054 Figure 1660A-B: DNA151915, BAA09764.1, Figure 609 A-B: DNA328699, AF189723, 212149_at
211137_s_at Figure 1661: PR012758
Figure 610: PR084463 Figure 1662: DNA88630, AAA52701.1, 212154 jit Figure 611: DNA327752, HSDHACTYL, Figure 1663: PR02877
211150_s.at Figure 1664: DNA328715, BC000950, 212160jιt
Figure 612A-B: DNA328700, SCD, 211162_x_at Figure 1665: DNA328716, HSM800707, 212179 Jit Figure 613: PR084464 Figure 1666A-C: DNA255018, CAB61363.1, Figure 614: DNA328701, PSEN2, 211373 _s_at 212207 ιt Figure 615: PRO80745 Figure 1667: PRO50107 Figure 616: DNA328702, NP_036519.1, 211413-S_at Figure 1668A-B: DNA328717, CAB70761.1, Figure 617: PR084465 212232jιt Figure 618: DNA256637, NP-008849.1, 211423_s_at Figure 1669: PR084473 Figure 619: PR051621 Figure 1670: DNA196116, DNA196116, 212246 Jit Figure 620: DNA328703, NP-003956.1, 211434_s .at Figure 1671 A-B: DNA254262, NP.055197.1, Figure 621: PR01873 212255_s_at Figure 622: DNA327755, NP-115957.1, 211458-S_at Figure 1672: PR049373 Figure 623: PR083725 Figure 1673: DNA327771, NP.l 09591.1, 212268_at Figure 624A-B: DNA328704, FGFR1, 211535_sJit Figure 1674: PR083737 Figure 625 PR034231 Figure 1675A-B: DNA328718, AAC39776.1, Figure 626 DNA324626, RIL, 211564-SJit 212285-SJit Figure 627 PR081272 Figure 1676: PR084474 Figure 628 DNA328705, NPJJ01345.1, 211653 _χjιt Figure 1677: DNA328719, BC012895, 212295 js_at Figure 629 PR062617 Figure 1678: PR084475 Figure 630 DNA328706, BC021909, 211714_χjιt Figure 1679: DNA271103, NP_005796.1, 212296 jit Figure 631 PRO 10347 Figure 1680: PR059425 Figure 632A-B: DNA328707, AF172264, Figure 1681A-B: DNA328720, HSA306929,
211828_s_at 212297 Jit Figure 1682: PR084476 212569-at
Figure 1683A-B: DNA328721, 1450005.12, 212298jιt Figure 1731: PR084491
Figure 1684: PR084477 Figure 1732A-B: DNA328739, PTPRC, 212587_s_at
Figure 1685A-B: DNA150464, BAA34466.1, Figure 1733: PR084492
212311JH Figure 1734: DNA327776, 1379302.1, 212593 s_at
Figure 1686: PRO 12270 Figure 1735: PR083742
Figure 1687: DNA326808, BC019307, 212312jιt Figure 1736: DNA151487, DNA151487, 212594 Jit
Figure 1688: PR083141 Figure 1737: PROl 1833
Figure 1689A-B: DNA124122, NP-005602.2, Figure 1738A-B: DNA328740, BAA76781.1,
212332 Jit 212611 Jit
Figure 1690: PR06323 Figure 1739: PR084493
Figure 1691: DNA287190, CAB43217.1, 212333Jit Figure 1740: DNA81753, DNA81753, 212613jιt
Figure 1692: PR069476 Figure 1741: PR09216 1742A-B: DNA253817, BAA20767.1,
Figure imgf000028_0001
Figure 1694: DNA328722, BC012469, 212341 Jit Figure 1743: PRO49220
Figure 1695: PR084478 Figure 1744A-B: DNA328741, 474863.12, 212622jιt
Figure 1696: DNA328723, S47833, 212360 Jit Figure 1745: PR084494
Figure 1697: PR036682 Figure 1746: DNA194679, BAA05062.1, 212623 Jit
Figure 1698A-B: DNA328724, AB007856, 212367 Jit Figure 1747: PR023989
Figure 1699A-B: DNA327773, BAA25456.1, Figure 1748A-B: DNA328742, 244522.6, 212628 Jit
212368jtt Figure 1749: PRO59047
Figure 1700: PR083739 Figure 1750: DNA270683, NP_006247.1, 212629 _s_at
Figure 1701A-C: DNA328725, AB007923, 212390jιt Figure 1751: PRO59047
Figure 1702A-B: DNA150950, BAA07645.1, Figure 1752A-D: DNA327777, HSIL1RECA,
212396-s_at 212657jiJit
Figure 1703: PR012554 Figure 1753A-B: DNA150762, BAA13197.1,
Figure 1704A-B: DNA328726, BAA25466.2, 212658-at
212443jιt Figure 1754: PR012455
Figure 1705: PRO84480 Figure 1755: DNA327838, NP.000568.1, 212659 j;jιt
Figure 1706: DNA328727, AB033105, 212453 Jit Figure 1756: PR083789
Figure 1707 A-B: DNA328728, 481567.2, 212458jιt Figure 1757: DNA328743, 1234685.2, 212667 _at
Figure 1708: PR084482 Figure 1758: PR084495
Figure 1709: DNA151348, DNA151348, 212463 Jit Figure 1759: DNA328744, AF318364, 212680j jιt
Figure 1710: PROl 1726 Figure 1760: PR084496
Figure 1711A-: DNA328729, D80001, 212486-S-at Figure 1761: DNA328745, 482138.6, 212687 it
Figure 1712: PR038526 Figure 1762: PR084497
Figure 1713A-B: DNA328730, BAA74899.2, Figure 1763: DNA324378, NP_000523.1, 212694 _s_at
212492_s_at Figure 1764: PRO81047
Figure 1714: PR084483 Figure 1765: DNA328746, CAB43213.1, 212698-S.at
Figure 1715A-B: DNA328731, 234169.5, 212500jιt Figure 1766: PR084498
Figure 1716: PR084484 Figure 1767A-B: DNA328747, BAA83030.1,
Figure 1717: DNA328732, NP_116193.1, 212502jιt 212765-at
Figure 1718: PR084485 Figure 1768: PR084499
Figure 1719: DNAO, AF038183, 212527 Jit Figure 1769A-B: DNA328748, HSJ001388, 212774 it
Figure 1720: PRO Figure 1770: PRO59570
Figure 1721: DNA328734, AAH01171.1, 212539 Jit Figure 1771: DNA328749, HSM802266, 212779 Jit
Figure 1722: PR084487 Figure 1772: DNA328750, 7689361.1, 212812jιt
Figure 1723: DNA328735, PHIP, 212542_s _at Figure 1773: PRO84500
Figure 1724: PR084488 Figure 1774A-B: DNA328751, AF012086,
Figure 1725: DNA328736, BC009846, 212552_at 212842 jc it
Figure 1726: PR084489 Figure 1775: DNA328752, CAA76270.1, 212864jιt
Figure 1727A-D: DNA328737, 148650.1, 212560jιt Figure 1776: PRO84501
Figure 1728: PRO84490 Figure 1777A-B: DNA328753, BAA13212.1,
Figure 1729: DNA270260, HSPDCE2, 212568_5_at 212873jιt
Figure 1730A-B: DNA328738, BAA31625.1, Figure 1778: PRO84502 Figure 1779: DNA271630, DNA271630, 212907 Jit Figure 832: DNA225974, NP.000864.1, 213620_s_at
Figure 1780: DNA328754, 1397726.9, 212912jιt Figure 833: PR036437
Figure 1781 : PRO84503 Figure 834: DNA328769, CAA69330.1, 213624jιt
Figure 1782A-B: DNA328755, BAA25490.1, Figure 835: PR084517
212946jιt Figure 836: DNA260173, DNA260173, 213638jιt
Figure 1783: PRO84504 Figure 837: PRO54102
838A-C: DNA273792, DNA273792,
Figure imgf000029_0001
Figure 1785: PRO84505 Figure 839: DNA151886, CAB43234.1, 213682jιt
Figure 1786: DNA154982, DNA154982, 213034jιt Figure 840: PRO 12745
Figure 1787: DNA327785, BC017336, 213061-SJit Figure 841: DNA227788, NP_002995.1, 213716jsjιt
Figure 1788: PR083749 Figure 842: PR038251
Figure 1789A-C: DNA328757, 475076.9, 213069 Jit Figure 843: DNA328771, HSMYOSIE, 213733 J t
Figure 1790: PRO84506 Figure 844: DNA328772, AAC19149.1, 213761 jit
Figure 1791 A-B: DNA328758, AB011123, 213109_at Figure 845: PR084519
Figure 1792: DNA272600, NP-057259.1, 213112_s_3t Figure 846: DNA328773, BC001528, 213766.χjιt
Figure 1793: PRO60737 Figure 847: PRO84520
Figure 1794: DNA326217, NP_004474.1, 213129 ϊJit Figure 848: DNA328774, NP.004263.1, 213793 j;_at
Figure 1795: PRO82630 Figure 849: PRO60536
Figure 1796: DNA228053, DNA228053, 213158 Jit Figure 850A-B: DNA328775, NP.006540.2,
Figure 1797 A-G: DNA103535, AF027153, 213164jιt 213812J3 at
Figure 1798: PR04862 Figure 851: PR084521
Figure 1799: DNA150875, CAB45717.1, 213246 Jit Figure 852: DNA328776, 407661.4, 213817jιt
Figure 1800: PROl 1589 Figure 853: PR084522
Figure 1801 : DNA328759, FJUMLPACI09, 213258 Jit Figure 854A-B: DNA328777, IDN3, 213918-SJit
Figure 1802: DNA328760, 1376674.1, 213274j3.at Figure 855: PR084523
Figure 1803: PRO84508 Figure 856: DNA196110, DNA196110, 214016-SJtt
Figure 1804A-B: DNA328761, BAA82991.1, Figure 857: PR024635
213280 Jit Figure 858: DNA150990, NP_003632.1, 214022j;jit
Figure 1805: PRO84509 Figure 859: PRO12570
Figure 1806: DNA260974, NP.006065.1, 213293.s_at Figure 860: DNA328778, 234498.37, 214093 s.at
Figure 1807: PRO54720 Figure 86L PR084524
Figure 1808: DNA328762, AAL30845.1, 213338_3t Figure 862A-B: DNA272292, NP.055459.1,
Figure 1809: PR084510 214130.S at
Figure 1810: DNA327789, 1449824.5, 213348_at Figure 863 PRO60550
Figure 1811: PR083753 Figure 864: DNA82378, NP_002695.1, 214146_sjιt
Figure 1812: DNA328763, NP_001219.2, 213373 j>_at Figure 865 PRO 1725
Figure 1813: PR084511 Figure 866A-B: DNA328779, 332730.12,
Figure 1814: DNA328764, NP_438169.1, 213375_sjιt 214155.S at
Figure 1815: PR084512 Figure 867: PR084525
Figure 1816: DNA328765, 411350.1, 213391 Jit Figure 868: DNA304659, NP_002023.1, 214211 jit
Figure 1817: PR084513 Figure 869: PRO71086
Figure 1818: DNA106195, DNA106195, 213454jιt Figure 870: DNA256662, NP_009112.1, 214219_χjιt
Figure 1819: DNA327795, BC014226, 213457 Jit Figure 871: PR051628
Figure 1820: DNA328766, NP_006077.1, 213476_χjιt Figure 872A-B: DNA328780, 480940.15, 214285jιt
Figure 1821: PR084514 Figure 873: PR084526
Figure 1822: DNA328767, BC008767, 213501 Jit Figure 874: DNA328781, 1453703.13, 214349 Jit
Figure 1823: PR084515 Figure 875: PR084527
Figure 1824: DNA254264, HSM800224, 213546 Jit Figure 876: DNA273174, NP.001951.1, 214394 j _at
Figure 1825: PR049375 Figure 877: PR061211
Figure 1826: DNA328768, 1194561.1, 213572_s_at Figure 878: DNA328782, 337794.1, 214405 Jit
Figure 1827: PR084516 Figure 879: PR084528
Figure 1828: DNA327800, 1251176.10, 213593 -S_at Figure 880: DNA287630, NP_000160.1, 214430jιt
Figure 1829: PR083763 Figure 881: PR02154
Figure 1830: DNA151422, DNA151422, 213605_s_at Figure 882: DNA227376, NP_005393.1, 214435 j _at
Figure 1831: PRO 11792 Figure 883: PR037839 Figure 884: DNA273138, NP.005495.1, 214452 Jit Figure 1938: DNA328801, 407831.1, 215392jιt Figure 885: PR061182 Figure 1939: PR084543 Figure 886: DNA327812, NP_006408.2, 214453 -SJit Figure 1940A-B: DNA328802, C6orf5, 215411 _s_at Figure 887: PR083773 Figure 1941: PR084544 Figure DNA302598, NP_066361.1, 214487 _s_at Figure 1942: DNA275385, NP.002085.1, 215438-XJit Figure 889: PR06251 1 Figure 1943: PRO63048 Figure 890 DNA328783, NP.002021.2, 214560 Jit Figure 1944: DNA328803. BAA91443.1, 215440js.at Figure 891 : PR084529 Figure 1945: PR084545 Figure 892: DNA324728, BC017730, 214581 _x_at Figure 1946: DNA328804, 403621.1, 215767 Jit Figure 893 PR0868 Figure 1947: PR084546 Figure 894A-B: DNA328784, 331045.1, 214582jιt Figure 1948A-B: DNA328805, BAA86482.1, Figure 895: PRO84530 215785 sjιt Figure 896: DNA328785, NP.004062.1, 214683 _s_at Figure 1949: PR084547 Figure 897: PR084531 Figure 1950: DNA328806, 208045.1, 216109 Jit Figure 898: DNA328786, BC017407, 214686 ιt Figure 1951: PR084548 Figure 899: PR084532 Figure 1952: DNA269532, NP_004802.1, 216250_s.at Figure 900: DNA271990, DNA271990, 214722_at Figure 1953: PR057948 Figure 901A-B: DNA274485, AB007863, 214735 Jit Figure 1954: DNA328807, AAH10129.1 , 216483-S.at Figure 902: DNA328787, 238292.8, 214746_s_at Figure 1955: PR084549 Figure 903: PR084533 Figure 1956: DNA188349, NP.002973.1, 216598 j;_at Figure 904: DNA328788, AK023937, 214763 Jit Figure 1957: PR021884 Figure 905: PR029183 Figure 1958: DNA328808, 1099517.2, 216607 JSJit Figure 906A-B: DNA328789, 344240.3, 214770jιt Figure 1959: PRO84550 Figure 907: PR084534 Figure 1960: DNA328809, PTPN12, 216915_sjιt Figure 908A-B: DNA328790, 481415.9, 214786jιt Figure 1961: PRO4803 Figure 909: PR084535 Figure 1962: DNA328810, NP.001770.1, 216942j>_at Figure 910: DNA328791, 1383762.1, 214790jιt Figure 1963: PR02557 Figure 911: PR084536 Figure 1964A-C: DNA328811, NP_002213.1, Figure 912: DNA328792, 7692351.10, 214830 Jit 216944-s-at Figure 913: PR084537 Figure 1965: PR084551 Figure 914: DNA328314, BC022780, 214841jιt Figure 1966: DNA328812. BAA86575.1, 216997.χjιt Figure 915: PR084182 Figure 1967: PR084552 Figure 916: DNA83102, DNA83102, 214866 Jit Figure 1968A-B: DNA328813, BAA76774.1, Figure 917: PR02591 217118_s Jit Figure 918: DNA161326, DNA161326, 214934jιt Figure 1969: PR084553 Figure 919: DNA328794, 1099353.2, 214974_χjιt Figure 1970A-B: DNA328814, HUMMHHLAJC, Figure 920: PR084539 217436jc_at Figure 921: DNA328795, AF057354, 214975-S.at Figure 1971A-B: DNA328815, 331104.2, 217521 Jit Figure 922: DNA328796, HSM800535, 215078 _at Figure 1972: PR084554 Figure 923: DNA328797, 000092.6, 215087 Jit Figure 1973: DNA328816, 1446567.1, 217526jιt Figure 924: PRO84540 Figure 1974: PR084555 Figure 925: DNA328798, NP_002088.1, 215091 _s it Figure 1975A-B: DNA255619, AF054589, Figure 926: PR084541 217599 _s_at Figure 927: DNA328799, BC008376, 215101-s.at Figure 1976: PRO50682 Figure 928: PR01721 Figure 1977: DNA327848, NP.005998.1, 217649 Jit Figure 929: DNA270522, NP.006013.1, 21511 l_s_at Figure 1978: PR083793 Figure 930: PR058899 Figure 1979: DNA328817, 1498470.1, 217678 Jit Figure 931 : DNA328800, 194537.1, 215224 jtt Figure 1980: PR084556 Figure 932: PR084542 Figure 1981: DNA328818, NP.071435.1 , 217730jtt Figure 933A-B: DNA327827, HSM800826, Figure 1982: PR038175
215235 Jit Figure 1983: DNA327935, NP_079422.1 , 217745-sJit
Figure 1934A-B: DNA226905, NPJJ55672.1, Figure 1984: PR083866
215342_s_at Figure 1985A-B: DNA88040, NP_000005.1, 217757 Jit
Figure 1935: PR037368 Figure 1986: PR02632
Figure 1936: DNA327831 , NP.076956.1, 215380_sjιt Figure 1987A-B: DNA88226, NP_000055.1, 217767 Jit
Figure 1937: PR083783 Figure 1988: PR02237 Figure 1989: DNA325821, NP_057016.1, 217769 _s_at Figure 2044: DNA326005, NP_057004.1, 218007 jj_at
Figure 1990: PR082287 Figure 2045: PR082446
Figure 1991: DNA227358, NP.057479.1, 217777_sjιt Figure 2046: DNA328835, NP.068760.1, 218019-SJit
Figure 1992: PR037821 Figure 2047: PR084571
Figure 1993: DNA328819, NP_057145.1, 217783-SJit Figure 2048: DNA328836, NP.054894.1, 218027 Jit
Figure 1994: PR084557 Figure 2049: PR084572
Figure 1995: DNA327850, NP.006546.1, 217785_s_at Figure 2050: DNA328837, NP_057149.1, 218046-SJit
Figure 1996: PRO60803 Figure 2051: PR081876
Figure 1997: DNA328303, NP_056525.1, 217807_s_at Figure 2052: DNA328838, NP.054797.2, 218049 _s.at
Figure 1998: PR084173 Figure 2053: PRO70319
Figure 1999: DNA328820, NP.077022.1, 217808_s_at Figure 2054: DNA328839, NP.057180.1, 218059 jit
Figure 2000: PR084558 Figure 2055: PR084573
Figure 2001: DNA328821, NP_006708.1, 217813_s it Figure 2056: DNA328840, NP.060481.1, 218067-S_at
Figure 2002: PR084559 Figure 2057: PR084574
Figure 2003: DNA328822, AK001511, 217830-s.at Figure 2058: DNA328841, NP.060557.2, 218073_SJit
Figure 2004: PRO84560 Figure 2059: PR084575
Figure 2005: DNA328823, NP_057421.1, 217838_sjrt Figure 2060A-C: DNA328842, 235943.8, 218098 Jit
Figure 2006: PR084561 Figure 2061: PR084576
Figure 2007: DNA226759, NP_054775.1, 217845-χjιt Figure 2062: DNA328843, NPJJ60939.1, 218099 Jit
Figure 2008: PR037222 Figure 2063: PR084577
Figure 2009: DNA327939, NP_060654.1, 217852_s _at Figure 2064: DNA328844, NP_061156.1, 218111_s_at
Figure 2010: PR083869 Figure 2065: PR082111
Figure 2011 A-B: DNA324921, NP-073585.6, Figure 2066: DNA227498, NP_002125.3, 218120-SJit
217853jit Figure 2067: PR037961
Figure 2012: PR081523 Figure 2068: DNA328845, NP_060615.1, 218126jιt
Figure 2013: DNA328824, DREV1, 217868_s Jit Figure 2069: PRO 10274
Figure 2014: PR084562 Figure 2070: DNA227264, LOC51312, 218136-sjιt
Figure 2015: DNA225604, NP-057226.1, 217869 _at Figure 2071: PR037727
Figure 2016: PRO36067 Figure 2072: DNA327857, NP.057386.1, 218142jj_at
Figure 2017: DNA326937, NP.002406.1, 217871_s_at Figure 2073 : PR083799
Figure 2018: PR083255 Figure 2074: DNA325852, NP_078813.1, 218153_at
Figure 2019: DNA255145, NP_060917.1, 217882jιt Figure 2075: PR082314
Figure 2020: PRO50225 Figure 2076: DNA328846, NP.060522.2, 218169 Jit
Figure 2021A-B: DNA328825, 1398762.11, 217886jιt Figure 2077: PR084578
Figure 2022: PR084563 Figure 2078: DNA228094, NP_079416.1, 218175jιt
Figure 2023: DNA189504, NP_064539.1, 217898jιt Figure 2079: PR038557
Figure 2024: PRO25402 Figure 2080: DNA328847, NP_056338.1, 218194 it
Figure 2025: DNA328826, NP-004272.2, 217911 _s Jit Figure 2081: PR084579
Figure 2026: PR084564 Figure 2082: DNA150593, NP-054747.1, 218196jιt
Figure 2027: DNA328827, NPJ376869.1, 217949 JSJit Figure 2083: PR012353
Figure 2028: PR021784 Figure 2084: DNA256555, NP_060042.1, 218205 _s_at
Figure 2029: DNA328828, NP_067027.1, 217956_sjιt Figure 2085: PR051586
Figure 2030: PR084565 Figure 2086: DNA328848, NP.004522.1, 218212_s_at
Figure 2031: DNA328829, NP-057230.1, 217959 >_at Figure 2087: PRO84580
Figure 2032: PR084566 Figure 2088: DNA271622, NP_006020.3, 218224jιt
Figure 2033: DNA328830, NP-061118.1, 217962 Jit Figure 2089: PRO59909
Figure 2034: PR084567 Figure 2090: DNA324353, NP_004538.2, 218226-S .at
Figure 2035: DNA327855, NP_057387.1, 217975 jit Figure 2091: PR081026
Figure 2036: PR083367 Figure 2092: DNA328849, NP.057075.1, 218232jιt
Figure 2037: DNA328831, NP_057329.1, 217989 Jit Figure 2093: PR04382
Figure 2038: PR0233 Figure 2094: DNA328850, NP.057187.1, 218254_s_at
Figure 2039: DNA328832, NP_067022.1, 217995jιt Figure 2095: PR084581
Figure 2040: PR084568 Figure 2096: DNA273230, NP_060914.1, 218273_S-at
Figure 2041: DNA328833, BC018929, 217996jιt Figure 2097: PR061257
Figure 2042: PR084569 Figure 2098: DNA328851, NP_068590.1, 218276_s_at
Figure 2043: DNA328834, AF220656, 217997 Jit Figure 2099: PR084582 Figure 2 00: DNA323953, NP.003507.1, 218280.x jit .152: DNA328869, NP_060892.1, 218613 Jit Figure 2 01: PRO80685 1153: PR084596 Figure 2 02: DNA254824, AF267865, 218294_sjιt 1154: DNA328870, NP.060639.1, 218614 Jit Figure 2 03: PRO49920 1155: PR084597 Figure 2 04A-B: DNA328852, NP_003609.2, 1156: DNA256870, NP_073600.1, 218618-SJit 21831 : ι_at 1157: PRO51800 Figure 2 05: PR084583 1158: DNA254898, NP_060840.1, 218627 _at Figure 2 06A-B: DNA328853, NP_065702.2, 1159: PR049988 218319-att 1160: DNA328871, NP_068378.1, 218631 Jit Figure 2 07: PR084584 1161: PR084598 Figure 2 08: DNA328854, NP.056979.1, 218350-SJit 1162: DNA328872, NP.036528.1, 218634 ιt Figure 2 09: PR084585 1163: PR084599 Figure 2 10: DNA328855, NPD76952.1, 218375_at 1164: DNA328873, NP.057041.1, 218698 jit Figure 2 1 L PR09771 1165: PRO84600 Figure 2 12: DNA328856, NPJJ68376.1, 218380jιt 1166: DNA324621, NP_054754.1, 218705 _s_at Figure 2 13: PR084586 1167: PR01285 Figure 2 14: DNA328857, NP_037481.1, 218407 _χjιt 1168: DNA328874, NP_054778.1, 218723 jjjit Figure 2 15: PR084587 1169: PRO84601 Figure 2 16: DNA324953, NP_057412.1, 218412_s_at 1170: DNA328875, NP_064554.2, 218729 Jit Figure 2 17: PRO81550 1171: PRO84602 Figure 2 18A-B: DNA255062, NP.060704.1, 1172: DNA328876, NP_060582.1, 218772_x_at 218424 j! Jit 1173: PRO84603 Figure 2 19: PRO50149 1174: DNA328877, BC020507, 218821 it Figure 2 20: DNA150661, NP-057162.1, 218446-S_at 1175: PRO84604 Figure 2 21: PR012398 1176: DNA328878, NP_060104.1, 218823-SJit Figure 2 22: DNA326218, NP-064573.1, 218447 _at 1177: PRO84605 Figure 2 23: PR082631 1178: DNA328879, NP_064570.1, 218845jιt Figure 2 24: DNA328858, HEBP1, 218450jιt 1179: PRO84606 Figure 2 25: PR084588 1180: DNA227367, NP_062456.1, 218853_s_at Figure 2 26: DNA327942, NP-060596.1, 218465Jit 1181: PRO37830 Figure 2 27: PRO83870 1182: DNA327872, NP-057713.1, 218856jit Figure 2 28: DNA328859, AF154054, 218468 JSJit 1183: PR083812 Figure 2 29: PRO1608 1184: DNA328880, NP_060369.1, 218872jιt Figure 2 30A-B: DNA328860, NP-037504.1, 1185: PRO84607 218469 Jit t 1186: DNA328881, NP_057706.1, 218890j jιt Figure 2 3 PRO1608 1187: PR049469 Figure 2 32: DNA328861, NP.057030.2, 218472j>_at 1188: DNA287166, NP-055129.1, 218943-s_at Figure 2 33: PR084589 1189: PR069459 Figure 2 34: DNA328862, NP_057626.2, 218499 Jit 1190: DNA328882, NP_109589.1, 218967 js_at Figure 2 35: PRO84590 1191: PR061822 Figure 2 36: DNA328863, NP_060264.1, 218503 Jit 1192: DNA327211, NP-075053.1, 218989-s.at Figure 2 37: PR084591 1193: PRO71052 Figure 2 38: DNA328864, NP 360726.2, 218512jιt 1194: DNA255929, NP.060935.1, 218992jtt Figure 2 39: PR084592 1195: PRO50982 Figure 2 40: DNA255432, NP-060283.1, 218516.s_at 1196: DNA328883, NP.037474.1, 218996jιt Figure 2 4L PRO50499 1197: PRO84608 Figure 2 42: DNA194326, NP_065713.1, 218538-SJit 1198: DNA227194, FLJ11000, 218999 Jit Figure 2 43: PRO23708 1199: PR037657 Figure 2 44: DNA328865, NP_064587.1, 218557 Jit Figure 2200: DNA328884, NP.054884.1, 219006 Jit Figure 2 45: PR084593 Figure 2201: PRO84609 Figure 2 46: DNA328866, NP.005691.1, 218567 ._at Figure 2202: DNA227187, NP_057703.1, 219014jιt Figure 2 47: PR069644 Figure 2203: PRO37650 Figure 2 48: DNA328867, NP_085053.1, 218600 Jit Figure 2204: DNA328885, NP_061108.2, 219017-at Figure 2 49: PR084594 Figure 2205: PRO50294 Figure 2 50: DNA328868, NP_057629.1, 218611 -at Figure 2206A-B: DNA255239, NP.004832.1, Figure 2 51: PR084595 219026 JS Jit Figure 2207: PRO50316 Figure 2259: PR083824
Figure 2208: DNA328886, NP_078811.1, 219040_at Figure 2260: DNA328901, FLJ20533, 219449 -S.at
Figure 2209: PRO84610 ' Figure 2261: PR084622
Figure 2210: DNA328887, NP_061907.1, 219045 ιt Figure 2262: DNA328902, NP_071750.1, 219452jit
Figure 2211: PR084611 Figure 2263: PR084623
Figure 2212: DNA328888, NP_060436.1, 219053_s.at Figure 2264: DNA328903, NP.002805.1, 219485 3_3t
Figure 2213: PR084612 Figure 2265: PR084624
Figure 2214: DNA328889, NP_006005.1, 219061.s it Figure 2266: DNA328904, NP_076941.1, 219491 Jit
Figure 2215: PR084613 Figure 2267: PR084625
Figure 2216: DNA328890, NP_060403.1, 219093jιt Figure 2268A-B: DNA328905, NP-075392.1,
Figure 2217: PR084614 219496_3t
Figure 2218: DNA327877, NP_065108.1, 219099 Jit Figure 2269: PR084626
Figure 2219: PR083816 Figure 2270: DNA328906, NP_078855.1, 219506 Jit
Figure 2220: DNA328891, NP-060263.1, 219143 j_at Figure 2271: PR084627
Figure 2221: PR084615 Figure 2272: DNA328907, NP_000067.1, 219534 _x_at
Figure 2222: DNA210216, NP_006860.1, 219150_s_at Figure 2273: PR084628
Figure 2223: PR033752 Figure 2274: DNA328908, 7691567.2, 219540 ιt
Figure 2224: DNA328892, NP D67643.2, 219165jιt Figure 2275: PR084629
Figure 2225: PR084616 Figure 2276: DNA225636, NP_065696.1, 219557 _s.at
Figure 2226A-B: DNA328893, NP_065699.1, Figure 2277: PRO36099
219201-S it Figure 2278A-B: DNA328909, NP_078800.2,
Figure 2227: PR09914 219558JU
Figure 2228: DNA287235, NP_060598.1, 219204 ji_at Figure 2279: PRO84630
Figure 2229: PR069514 Figure 2280: DNA328910, NP.057666.1, 219593 Jit
Figure 2230: DNA225594, NP_037404.1, 219229 Jit Figure 2281: PR038848
Figure 2231: PRO36057 Figure 2282: DNA328911, MS4A4A, 219607 JSJit
Figure 2232: DNA328894, NP.060796.1, 219243jιt Figure 2283 : PR084631
Figure 2233: PR084617 Figure 2284: DNA328912, NP-060287.1, 219622jιt
Figure 2234: DNA328895, NP.071762.2, 219259jιt Figure 2285: PR084632
Figure 2235: PR01317 Figure 2286: DNA328913, NPJ379213.1, 219631 Jit
Figure 2236: DNA328896, NP-079037.1, 219265 Jit Figure 2287: PR084633
Figure 2237: PR084618 Figure 2288: DNA328914, NP-060883.1, 219634jιt
Figure 2238: DNA328897, TRPV2, 219282JS Jit Figure 2289: PR036664 (
Figure 2239: PR012382 Figure 2290: DNA327892, NP-060470.1, 219648 Jit
Figure 2240: DNA273489, NP_055210.1, 219290-XJit Figure 2291: PR083828
Figure 2241 : PR061472 Figure 2292: DNA328915, NP_055056.2, 219654 Jit
Figure 2242A-B: DNA328898, NP.060261.1, Figure 2293: PR084634
219316j>_3t Figure 2294: DNA228002, NP_071744.1, 219666jit
Figure 2243: PR084619 Figure 2295: PR038465
Figure 2244: DNA328899, NP-061024.1, 219326 j_at Figure 2296: DNA328916, NP-071932.1, 219678_x_at
Figure 2245: PRO84620 Figure 2297: PR084635
Figure 2246A-B: DNA255889, NP_061764.1, Figure 2298: DNA287206, NP_060124.1, 219691 Jit
219340 j;_at Figure 2299: PR069488
Figure 2247: PRO50942 Figure 2300: DNA328917, NP.061838.1, 219725 Jit
Figure 2248: DNA328900, NP.060814.1, 219344 jtt Figure 2301: PRO7306
Figure 2249: PR084621 Figure 2302: DNA328918, NP_078935.1, 219770jιt
Figure 2250: DNA254518, NP-057354.1, 219371 _s.at Figure 2303: PR084636
Figure 2251: PR049625 Figure 2304: DNA328919, NP_078987.1, 219777 Jit
Figure 2252: DNA188342, NP_064510.1, 219385_at Figure 2305: PR084637
Figure 2253: PR021718 Figure 2306: DNA227152, NP_038467.1, 219788_at
Figure 2254: DNA256417, NP_077271.1, 219402 3Jit Figure 2307: PR037615
Figure 2255: PR051457 Figure 2308: DNA328920, NP.061129.1, 219837.S _at
Figure 2256A-B: DNA327887, NP.006656.1, Figure 2309: PR04425
219403j;_at Figure 2310: DNA256033, NP_060164.1, 219858 _s_at
Figure 2257: PR083823 Figure 231 PR051081
Figure 2258: DNA327888, NP-071732.1, 219412jιt Figure 2312: DNA254838, NP_078904.1, 219874jιt Figure 2313: PR049933 Figure 2366: DNA328935, NP-009002.1 , 220387 _s_at Figure 2314: DNA328921 , NP. .057079.1, 219878J5 Jit Figure 2367: PRO84650 Figure 2315: PR084638 Figure 2368: DNA254861, MC0LN3, 220484 _at Figure 2316: DNA256325, NP..005470.1, 219889 Jit Figure 2369: PR049953 Figure 2317: PR051367 Figure 2370: DNA328936, NP.066998.1, 220491 Jit Figure 2318: DNA328922, NP..037384.1 , 219890 Jit Figure 2371: PRO 1003 Figure 2319: PR084639 Figure 2372: DNA328937, PHEMX, 220558 j _3t Figure 2320: DNA328923, NP..075379.1, 219892jιt Figure 2373: PRO12380 Figure 2321: PRO84640 Figure 2374: DNA328938, NP-060617.1, 220643js.at Figure 2322: DNA256608, NP..060408.1, 219895jvt Figure 2375: PR084651 Figure 2323: PR051611 Figure 2376: DNA323756, NP-057267.2, 220688 _s Jit Figure 2324: DNA328924, NP..057150.2, 219933 Jit Figure 2377: PRO80512 Figure 2325: PR084641 Figure 2378: DNA328939, NP-008834.1, 220741 s_at Figure 2326: DNA255456, NP..057268.1, 219947 Jit Figure 2379: PR084652 Figure 2327: PRO50523 Figure 2380: DNA288247, NP-478059.1, 220892_s_at Figure 2328: DNA227804, NP..065394.1, 219952_sjιt Figure 2381: PRO70011 Figure 2329: PR038267 Figure 2382: DNA328940, NP.078893.1 , 220933.sort Figure 2330: DNA328925, NP..076403.1, 220005 Jit Figure 2383: PR084653 Figure 2331: PR084642 Figure 2384: DNA328941, NP-055218.2, 220937 _s_at Figure 2332: DNA256467, NP..079054.1, 220009 Jtt Figure 2385: PR084654 Figure 2333: PR051504 Figure 2386: DNA327953, NP_055182.2, 220942_x_at Figure 2334A-B: DNA292946, NP.061160.1, Figure 2387: PR083878 220023 Jit Figure 2388A-B: DNA323882, NP-000692.2,
Figure 2335: PRO70613 220948js_at Figure 2336: DNA171414, NP. .009130.1, 220034 Jit Figure 2389: PRO80625 Figure 2337: PRO20142 Figure 2390: DNA327917, NP_112240.1, 220966-XJ t Figure 2338: DNA328926, NP..064703.1, 220046-s Jit Figure 2391: PR083852 Figure 2339: PR084643 Figure 2392: DNA328942, NP_112216.2, 220985 _s_at Figure 2340A-B: DNA221079, NP_071445.1, Figure 2393: PR084655 220066 Jit Figure 2394: DNA328943, NP.036566.1, 221041 SJit
Figure 2341 : PR034753 Figure 2395: PR051680 Figure 2342: DNA256091, NP. .071385.1, 220094 >_at Figure 2396: DNA328944, NP_060554.1, 221078_sjιt Figure 2343: PR051141 Figure 2397: PR084656 Figure 2344: DNA328927, NP..078993.2, 220122jιt Figure 2398: DNA328945, NP-079177.2, 221081 _s_at Figure 2345: PR084644 Figure 2399: PR084657 Figure 2346: DNA328928, NP..068377.1, 220178 Jit Figure 2400: DNA328946, NP-055164.1, 221087 _s_3t Figure 2347: PR084645 Figure 2401: PR012343 Figure 2348: DNA324716, NP..463459.1, 220189-SJit Figure 2402: DNA328947, NPD55245.1, 221188.s_at Figure 2349: PR081347 Figure 2403: PR084658 Figure 2350: DNA228059, NP..073742.1, 220199 js.at Figure 2404: DNA257293, NP-110396.1, 221210jsjιt Figure 2351: PR038522 Figure 2405: PR051888 Figure 2352: DNA328929, NP..060375.1, 220240 _s Jit Figure 2406: DNA327920, NP_110431.1, 221245 _s_at Figure 2353: PR084646 Figure 2407: PR083855 Figure 2354A-B: DNA328930, NP_038465.1, Figure 2408A-C: DNA328287, NP.072174.2, 220253_s_at 221246.x Jit
Figure 2355: PR023525 Figure 2409: PR084163 Figure 2356: DNA328931, NP. .004226.1, 220266.s_at Figure 2410: DNA328948, NP_110437.1, 221253 sjit Figure 2357: PR084647 Figure 2411 : PR084659 Figure 2358: DNA328932, NP..079057.1, 220301 Jit Figure 2412: DNA256432, NP.110415.1, 221266_s_at Figure 2359: PR084648 Figure 2413: PR051471 Figure 2360: DNA328933, NP..057466.1, 220307 Jit Figure 2414: DNA328027, NP_112570.2, 221437 _s_at Figure 2361 : PR09891 Figure 2415: PR083944 Figure 2362: DNA256735, NP..060175.1, 220333 Jit Figure 2416A-B: DNA272014, AF084555, Figure 2363: PR051669 221482-s_at Figure 2364A-B: DNA328934, EML4, 220386 _s Jit Figure 2417: PRO60289 Figure 2365: PR084649 Figure 2418: DNA328949, AF157510, 221487-s.at Figure 2419: PRO84660 Figure 2466: PRO84670
Figure 2420: DNA328950, NP_057025.1, 221504-SJit Figure 2467: DNA328967, BC017905, 221815 ιt
Figure 2421: PR084661 Figure 2468: PR084671
Figure 2422A-B: DNA328951, HSM802232, Figure 2469: DNA274058, NP_057203.1, 221816_s_3t
221523 3_at Figure 2470: PR061999
Figure 2423: PR084662 Figure 2471A-B: DNA328968, 1322249.6, 221830 it
Figure 2424: DNA328952, NP_067067.1, 221524 _s_at Figure 2472: PR062511
Figure 2425: PR084663 Figure 2473: DNA272419, AF105036, 221841_sjιt
Figure 2426A-B: DNA273901, NP_110389.1, Figure 2474: PRO60672
221530.s-at Figure 2475: DNA299882, DNA299882, 221872jιt
Figure 2427: PR061855 Figure 2476: PRO70856
Figure 2428: DNA274676, DKFZp564A176Homo, Figure 2477: DNA328969, 334394.2, 221878jιt
221538_sjιt Figure 2478: PR084672
Figure 2429: DNA328953, NP_004086.1, 221539 Jit Figure 2479: DNA327933, 1452741.11, 221899 Jit
Figure 2430: PRO70296 Figure 2480: PR083865
Figure 2431 A-B: DNA328954, NP-113664.1, Figure 2481: DNA328970, NP.057696.1, 221920 jsjit
221541_3t Figure 2482: PR084673
Figure 2432: PR09851 Figure 2483: DNA328971, AK000472, 221923 s.at
Figure 2433 A-B : DNA269992, HUMACYLCOA, Figure 2484: PR084674
221561_at Figure 2485: DNA254787, AK023140, 221935 sjιt
Figure 2434: PR058388 Figure 2486: PR049885
Figure 2435: DNA328955, NP_054887.1, 221570.s_3t Figure 2487: DNA327114, NP-006004.1, 221989 Jit
Figure 2436: PR084664 Figure 2488: PR062466
Figure 2437A-B: DNA328956, AF110908, 221571 Jtt Figure 2489: DNA328972, BC009950, 222001 JtJit
Figure 2438: DNA188321, NP-004855.1, 221577 _x.at Figure 2490: DNA328973, NP_115538.1, 222024_s_3t
Figure 2439: PR021896 Figure 2491: PR082497
Figure 2440: DNA328957, WBSCR5, 221581 _s _at Figure 2492: DNA1 19482, DNA119482, 222108jιt
Figure 2441 : PR023859 Figure 2493: PRO9850
Figure 2442: DNA328958, BC001663, 221593 _s_at Figure 2494: DNA328974, NP.061893.1, 222116jj_at
Figure 2443: PR084665 Figure 2495: PR084676
Figure 2444: DNA328959, NP_077027.1, 221620 _s Jit Figure 2496: DNA287209, NP-056350.1, 222154 JSJit
Figure 2445: PRO4302 Figure 2497: PRO69490
Figure 2446: DNA254777, NP_055140.1, 221676_sj t Figure 2498: DNA328975, NP_078807.1, 222155 s_at
Figure 2447: PR049875 Figure 2499: PR047688
Figure 2448: DNA327526, NP-065727.2, 221679-S_at Figure 2500: DNA328976, BC019091, 222206.S Jit
Figure 2449: PR083574 Figure 2501: PR084677
Figure 2450: DNA328960, NP-076426.1 , 221692JS Jit Figure 2502: DNA256784, NP.075069.1, 222209 _s.at
Figure 2451 : PR084666 Figure 2503: PR051716
Figure 2452: DNA327929, AK001785, 221748.sjit Figure 2504: DNA328977, NP.071344.1, 222216_s_at
Figure 2453 : PR083861 Figure 2505: PR084678
Figure 2454: DNA328961, NP_443112.1, 221756jιt Figure 2506: DNA328978, NP_060373.1, 222244 _s_at
Figure 2455: PR084667 Figure 2507: PR084679
Figure 2456: DNA328962, BC021574, 221759 Jit Figure 2508A-B: DNA328979, 006242.19, 222266jιt
Figure 2457: PR082746 Figure 2509 PRO84680
Figure 2458A-B: DNA328963, 328765.9, 221760_at Figure 2510: DNA328980, 7692031.1, 222273 ιt
Figure 2459: PR084668 Figure 2511 PR084681
Figure 2460A-B: DNA327930, 1455324.9, 221765 Jit Figure 2512: DNA328981, AF443871, 222294 _s_at
Figure 2461: PR083862 Figure 2513 PR024633
Figure 2462: DNA328964, AK056028, 221770 ιt Figure 2514: DNA328982, 154391.1, 222313-at
Figure 2463: PR084669 Figure 2515 PR084682
Figure 2464A-C: DNA328965, AB051505, 221778 Jit Figure 2516: DNA328983, 206335.1, 222366 Jit
Figure 2465A-B: DNA328966, BAB14908.1, Figure 2517 PR084683 221790_s.at DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions
The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (i.e., PRO/number) refers to specific polypeptide sequences as described herein. The terms "PRO/number polypeptide" and "PRO/number" wherein the term "number" is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein). The PRO polypeptides described herein may be isokted from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. The term "PRO polypeptide" refers to each individual PRO/number polypeptide disclosed herein. All disclosures in this specification which refer to the "PRO polypeptide" refer to each of the polypeptides individually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the invention individually. The term "PRO polypeptide" also mcludes variants of the PRO/number polypeptides disclosed herein.
A "native sequence PRO polypeptide" comprises a polypeptide having the same smino scid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence PRO polypeptide" specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e.g., an extracellular domain sequence), naturally-occurring varisnt forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In various embodiments of the invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop codons are shown in bold font and underlined in the figures.
However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as arnino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstresm from the amino acid position 1 in the figures may be employed as the starting amino acid residue for the PRO polypeptides. The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have less than 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein. Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic scid encoding them, are contemplated by the present invention. The approximate location of the "signal peptides" of the various PRO polypeptides disclosed herein are shown in the present specification and/or the accompanying figures. It is noted, however, that the C- terminsl boundsry of a signal peptide may vary, but most likely by no more thsn about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
"PRO polypeptide variant" means an active PRO polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, alternatively 3t least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively 3t least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino scid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% 3mino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellulsr domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, PRO variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alteπrativeiy 3t lesst sbout 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or more.
"Percent (%) 3mino acid sequence identity" with respect to the PRO polypeptide sequences identified herein is defined as the percent3ge of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific PRO polypeptide sequence, after aligning the sequences and introducing gaps, if necess3ry, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly avaikble computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentstion in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of 3mino acid residues in B. It will be apprecisted thst where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations using this method, Tables 2 and 3 demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designsted "Comparison Protein" to the amino acid sequence designated "PRO", wherein "PRO" represents the amino acid sequence of a hypothetical PRO polypeptide of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "PRO" polypeptide of interest is being compared, and "X, " Y" and "Z" each represent different hypothetical amino acid residues.
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immedistely preceding paragraph using the ALIGN-2 computer program. However, % amino acid sequence identity values msy 3lso be obtained as described below by using the WU- BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span = 1 , overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequence identify value is determined by dividing (a) the number of matching identical amino acid residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (i.e., the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest. For example, in the statement "a polypeptide comprising an the amino acid sequence A which has or having at least 80% amino acid sequence identify to the amino acid sequence B", the amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.
Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, MD. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62. In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identify of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identify to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino scid sequence A is not equal to the length of amino acid sequence B, tire % amino acid sequence identity of A to B will not equal the % amino acid sequence identify of B to A.
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identify with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identify, alternatively at least about 81% nucleic acid sequence identity, altern3tively at least about 82% nucleic acid sequence identify, alternatively at least about 83%> nucleic acid sequence identify, alternatively at least about 84%) nucleic scid sequence identify, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86%> nucleic acid sequence identify, alternatively at least about 87%) nucleic acid sequence identify, alternatively at least about 88%) nucleic acid sequence identify, alternatively at least about 89% nucleic acid sequence identify, slternatively at least about 90% nucleic acid sequence identify, alternatively at least about 91% nucleic acid sequence identify, alternatively at least about 92% nucleic acid sequence identify, alternatively at least about 93% nucleic acid sequence identify, alternatively at least about 94% nucleic acid sequence identify, alternatively at least about 95% nucleic scid sequence identify, alternatively at least about 96% nucleic acid sequence identify, alternatively at least about 97% nucleic acid sequence identify, alternatively at least about 98% nucleic acid sequence identify and alternatively at least about 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full- length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Variants do not encompass the native nucleotide sequence. Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternstively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more.
"Percent (%) nucleic acid sequence identify" with respect to PRO-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the PRO nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of deteπnining percent nucleic acid sequence identify can be achieved in v3rious ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes herein, however, % nucleic acid sequence identify values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Tsble 1 below hss been filed with user document3tion in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identify of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identify to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identify of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO-DNA", wherein "PRO-DNA" represents a hypothetical PRO-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid molecule of interest is being compared, and "N", "L" and "V" each represent different hypothetical nucleotides.
Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. However, % nucleic acid sequence identity values may also be obtained as described below by using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzvmology 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap sρan= 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is employed, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (i.e., the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide-encoding nucleic acid molecule of interest. For example, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest.
Percent nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, MD. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters 3re set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62. In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the %> nucleic acid sequence identity of D to C.
In other embodiments, PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length PRO polypeptide as disclosed herein. PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide.
"Isolated," when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
An "isolated" PRO polypeptide-encoding nucleic scid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally sn operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenyktion signals, and enhancers. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. The term "antibody" is used in the broadest sense and specifically covers, for example, single anti-
PRO monoclonal antibodies (including agonist, amagonist, snd neutralizing antibodies), anti-PRO antibody compositions withpolyepitopic specificity, single chain anti-PRO sntibodies, and fragments of anti-PRO antibodies (see below). The term "monoclonal antibody" as used herein refers to an antibody obtained from a popuktion of substantially homogeneous antibodies, i.e., the individual antibodies comprising the popuktion sre identical except for possible naturally-occurring mutations that may be present in minor amounts.
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1%) sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by 3 high-stringency W3sh consisting of 0.1 x SSC contenting EDTA at 55°C.
"Moderately stringent conditions" may be identified as described by Sambrook et al., Molecular Cloning: A Lsboratorv Manual. New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising a PRO polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with otlier epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
"Active" or "activity" for the purposes herein refers to form(s) of a PRO polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring PRO, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally- occurring PRO other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO and an "immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally- occurring PRO.
The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO polypeptide disclosed herein. In a similar manner, the term "agonist" is used in the broadest sense and mcludes any molecule that mimics a biological activity of a native PRO polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native PRO polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying agonists or antagonists of a PRO polypeptide may comprise contacting a PRO polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PRO polypeptide.
"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
"Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human. Administration "in combination with" one or more further therapeutic agents includes simultaneous
(concurrent) and consecutive administration in any order.
"Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chekting agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.
"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al.. Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and - binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non- covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-V dimer. Collectively, the six CDRs confer antigen- binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bmd antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free tbiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) comiected to a light-chain variable domain (V ) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA. 90:6444-6448 (1993).
An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
An antibody that "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149.
A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a PRO polypeptide or antibody thereto) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. A "small molecule" is defined herein to have a molecular weight below about 500 Daltons.
The term "immune related disease" means a disease in which a component of the immune system of a mammal causes, mediates or otherwise contributes to a morbidity in the mammal. Also included are diseases in which stimulation or intervention of the immune response has an ameliorative effect on progression of the disease. Included within this term are immune-mediated inflammatory diseases, non- immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasia, etc.
The term "monocyte/macrophage mediated disease" means a disease in which monocytes/macrophages directly or indirectly mediate or otherwise contribute to a morbidity in a mammal. The monocyte/macrophage mediated disease may be associated with cell mediated effects, lymphokine mediated effects, etc., and even effects associated with other immune cells if the cells are stimulated, for example, by the lymphokines secreted by monocytes/macrophages.
Examples of immune-related and inflammatory diseases, some of which are immune mediated, which can be treated according to the invention include systemic lupus erythematosis, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjδgren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guilkin-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing chokngitis, inflammatory bowel disease (ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, immunologic diseases of the lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft -versus-host-disease. Infectious diseases including viral diseases such as AIDS (HIV infection), hepatitis A, B, C, D, and E, herpes, etc., bacterial infections, fungal infections, protozoal infections and parasitic infections. The term "effective amount" is a concentration or amount of a PRO polypeptide and/or agonist/antagonist which results in achieving a particular stated purpose. An "effective amount" of a PRO polypeptide or agonist or antagonist thereof may be determined empirically. Furthermore, a "therapeutically effective amount" is a concentration or amount of a PRO polypeptide and/or agonist/antagonist which is effective for achieving a stated therapeutic effect. This amount may also be determined empirically.
The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., I13 , I125, Y90 and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhόne-Poulenc Rorer, Antony, France), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (see U.S. Pat. No. 4,675,187), melphalan and other related nitrogen mustards. Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone.
A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressing any of the genes identified herein, either in vitro or in vivo. Thus, the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkykting agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.
The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorekxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; pro ctin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF- and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-l , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificify of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificify which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
Table 1
/*
* C-C increased from 12 to 15
* Z is average of EQ *B is average of ND
* match with stop is _M; stop-stop = 0; J (joker) match = 0 */
#define M -8 /* value of a match with a stop */ it day[26][26] = {
/* A' B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/
/*A*/ 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0},
/*B*/ 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2,_M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3, 1},
/*C*/ -2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5},
/*D*/ 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2},
/*E*/ 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 3},
/* p */ 4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5},
/*G*/ 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M,-l,-l,-3, 1, 0, 0,-1,-7, 0,-5, 0}, /*H*/ -1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,_M, 0, 3, 2,-1,-1, 0,-2,-3, 0, 0, 2}, /*τ*/ -1,-2,-2,-2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2,_M,-2,-2,-2,-l, 0, 0, 4,-5, 0,-1,-2},
/*J*/ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/*K*/ -1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1, 3, 0, 0, 0,-2,-3, 0,-4, 0},
/*L*/ -2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3,-3,-l, 0, 2,-2, 0,-1,-2},
/*M*/ ■1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2,_M,-2,-l, 0,-2,-1, 0, 2,-4, 0,-2,-1},
/*N*/ 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-2, 1},
/*0*/ _M,_M,_M,_M,_M,_M,_M,_M, M, M,_M,_M,_M,_M,
0,_M,_M M,_M,_M,_M,_M,_M,_M,_M,_M},
/* p */ 1,-1,-3 -1, -1 -5 -1 0 -2 0 -1 ,-3 -2 -1, _M, 6, 0, 0, 1, 0, 0,-1,-6, 0,-5, 0}, /*Q*/ 0, 1,-5 2, 2, -5, -i, 3, -2, 0, 1, -2, -1, 1, M, 0, 4, 1,-1,-1, 0,-2,-5, 0,-4, 3}, /*R*/ -2, 0,-4. -1, -1 -4 -3 2 -2 0 3, ,-3 0 0, ~M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-4, 0}, /*S*/ 1,0,0 0, 0, -3, 1, -1, -1, 0, 0, -3, -2, 1, M, 1,-1,0,2, 1,0,-1,-2,0,-3,0}, /* Ύ */ 1, 0,-2 0, 0, -3, 0, -1, 0, 0, 0, -1, -1, 0, M, 0,-1,-1, 1, 3, 0, 0,-5, 0,-3, 0},
/*u*/ 0,0,0 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/* v */ 0,-2,-2, -2, -2 -1 -1 -2 4 0 -2 ,2 2, -2, _M,-l,-2,-2,-l, 0, 0, 4,-6, 0,-2,-2},
/*w*/ -6,-5,-8 -7 -7 0 -7 -3 -5 0 -3 ,-2 -4 ,-4, _M,-6,-5, 2,-2,-5, 0,-6,17, 0, 0,-6}, /*x*/ 0,0,0 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/* Y */ -3,-3, 0 -4, -4 7, -5, 0, -1, 0, -4, ,-1 -2 -2, ,_M,-5,-4,-4,-3,-3, 0,-2, 0, 0,10,-4},
/*z*/ 0, 1,-5 2, 3, -5, 0, 2, -2, 0, 0, -2, -1, 1, M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4} };
Table 1 (conf)
/* */
^include < stdio.h> #include <ctype.h>
#define MAXJMP 16 /* max jumps in a diag */
Mefine MAXGAP 24 /* don't continue to penalize gaps larger than this */
#define JMPS 1024 /* max jmps in an path */
#define MX 4 /* save if there's at least MX-1 bases since last jmp */
#define DMAT 3 /* value of matching bases */
#defιne DMIS 0 /* penalty for mismatched bases */
#defϊne DINSO 8 /* penalty for a gap */
#define DINS1 1 /* penalty per base */
#define PINSO 8 /* penalty for a gap */
#define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /* size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. of jmp in seq x */
}; /* limits seq to 2" 16 -1 */ struct diag { int score; /* score at last jmp */ long offset; /* offset of prev block */ short ijmp; /* current jmp index */ struct jmp jp; /* list of jmps */
}; struct path { int spc; /* number of leading spaces */ short n[JMPS];/* size of jmp (gap) */ int x[JMPS];/* loc of jmp (last elem before gap) */
}; char *ofile; /* output file name */ char *namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for err msgs */ char *seqx[2]; /* seqs: getseqs() */ int dmax; /* best diag: nw() */ int dmaxO; /* final diag */ int dna; /* set if dna: main() */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int lenO, lenl; /* seq lens */ int ngapx, ngapy; /* total size of gaps */ int smax; /* max score: nw() */ int *xbm; /* bitmap for matching */ long offset; /* current offset in jmp file */ struct diag *dx; /* holds diagonals */ struct path PP[2]; /* holds path for seqs */ char *calloc(), *malloc(), *index(), *strcpy(); char *getseq(), *g_calloc(); Table 1 (conf)
/* Needleman-Wunsch alignment program *
* usage: progs filel file2
* where filel and file2 are two dna or two protein sequences. The sequences can be in upper- or lower-case an may contain ambiguity Any lines beginning with ';', ' > ' or ' < ' are ignored Max file length is 65535 (limited by unsigned short x in the jmp struct) A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA * Output is in the file "align. out"
*
* The program may create a tmp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650 */ #include "nw.h" #include "day.h" static _dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
static _pbval[26] = {
1, 2|(1 < <('D'-'A'))| (1 < <('N'-'A')), 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1 < < 10, 1 < < 11, 1 < < 12, 1 < < 13, 1 < < 14,
1 < < 15, 1 < < 16, 1 < < 17, 1 < < 18, 1 < < 19, 1 < <20, 1 < <21, 1 < <22,
1 < <23, 1 < <24, 1 < <25 | ( 1 < <('E,-'A')) |(1 < <('Q'-'A'))
}; main(ac, av) main int ac; char *av[ ]; prog = av[0]; if (ac != 3) { fprintf(stderr, "usage: %s filel file2\n", prog); fprintf(stderr, "where filel and file2 are two dna or two protein sequences. \n"); fprintf(stderr,"The sequences can be in upper- or lower-case\n"); fprintf(stderr,"Any lines beginning with ';' or ' < ' are ignored\n"); fprintf(stderr, "Output is in the file \"align.out\"\n"); exit(l);
} namex[0] = av[l]; namex[l] = av[2]; seqx[0] = getseq(namex[0], &len0); seqxfl] = getseq(namex[l], &lenl); xbm = (dna)? _dbval : bval; endgaps = 0; /* 1 to penalize endgaps */ ofile = "align.out"; /* output file */ nw(); /* fill in the matrix, get the possible jmps */ readjmpsO; /* get the actual jmps */ print(); /* print stats, alignment */ cleanup(O); /* unlink any tmp files */ Table 1 (conf)
/* do the alignment, return best score: main()
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y. nw
{ char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /* keep track of dely */ int ndelx, delx; /* keep track of delx */ int *tmp; /* for swapping rowO, rowl */ int mis; /* score for each type */ int insO, insl; /* insertion penalties */ register id; /* diagonal index */ register ij; /* jmp index */ register *col0, *coll; /* score for curr, last row */ register xx, yy; /* index into seqs */ dx = (struct diag *)g_calloc("to get diags", lenO+lenl + l, sizeof(struct diag)); ndely = (int *)g_calloc("to get ndely", lenl-t- 1, sizeof(int)); dely = (int *)g_calloc("to get dely", lenl-t- 1, sizeof(int)); colO = (int *)g_calloc("to get colO", lenl + 1, sizeof(int)); coll = (int *)g_calloc("to get coll", lenl + 1, sizeof(int)); insO = (dna)? DINS0 : PINS0; insl = (dna)? DINS1 : PINS1; smax = -10000; if (endgaps) { for (col0[0] = dely[0] = -insO, yy = 1; yy < = lenl; yy+ +) { col0[yy] = delyfyy] = col0[yy-l] - insl; ndelyfyy] = yy;
} col0[0] = 0; /* Waterman Bull Math Biol 84 */
} else for (yy = 1; yy < = lenl; yy+ +) dely[yy] = -insO;
/* fill in match matrix */ for (px = seqx[0], xx = 1; xx < = lenO; px+ +, xx+ +) { /* initialize first entry in col */ if (endgaps) { if (xx = = l) coll[0] = delx = -(insO+insl); else coll[0] = delx = col0[0] - insl; ndelx = xx;
} else { coll[0] = 0; delx = -insO; ndelx = 0; Table 1 (conf)
...nw seqxfl], yy = 1; yy < = lenl; py+ +, yy+ +) { mis = col0[yy-l]; if (dna) mis + = (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS; else mis + = _day[*px-'A'][*py-'A']; /* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps */ if (endgaps 1 1 ndely[yy] < MAXGAP) { if (col0[yy] - insO > = delyfyy]) { dely[yy] = col0[yy] - (insO+insl); ndelyfyy] = 1; } else { delyfyy] -= insl; ndelyfyy] + + ;
} } else { if (colOfyy] - (insO+insl) > = delyfyy]) { delyfyy] = colOfyy] - (insO+insl); ndelyfyy] = 1;
} else ndelyfyy] + + ; } /* update penalty for del in y seq;
* favor new del over ongong del */ if (endgaps | | ndelx < MAXGAP) { if (coll[yy-l] - insO > = delx) { delx = collfyy-1] - (insO+insl); ndelx = 1; } else { delx -= insl; ndelx + +; }
} else { if (coll[yy-l] - (insO+insl) > = delx) { delx = coll[yy-l] - (insO+insl); ndelx = 1; } else ndelx + +;
}
/* pick the maximum score; we're favoring * mis over any del and delx over dely
*/ Table 1 (conf)
...nw id = xx - yy + lenl - 1; if (mis > = delx && mis > = delyfyy]) collfyy] = mis; else if (delx > = delyfyy]) { collfyy] = delx; ij = dx[id].ijmp; if (dx[id].jp.n[0] && (Idna 1 1 (ndelx > = MAXJMP && xx > dx[id].jp.x[ij]+MX) 1 1 mis > dxfid]. score +DINS0)) { dx[id].ijmp+ + ; if (+ +ij > = MAXJMP) { writejmps(id); ij = dxfid]. ijmp = 0; dxfid]. off set = offset; offset + = sizeof(structjmp) + sizeof(offset);
}
} dxfid] .jp.nfij] = ndelx; dxfid] jp.xfij] = xx; dxfid]. score = delx;
} else { collfyy] = delyfyy]; ij = dxfid]. ijmp; if (dxfid] .jp.nfO] && (Idna 1 1 (ndelyfyy] > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) 1 1 mis > dxfid]. score +DINS0)) { dxfid]. ijmp + +; if (+ +ij > = MAXJMP) { writejmps(id); ij = dxfid]. ijmp = 0; dxfid]. offset = offset; offset + = sizeof(struct jmp) + sizeof(offset);
} } dx[id].jp.n[ij] = -ndelyfyy]; dx[id].jp.x[ij] = xx; dxfid]. score = delyfyy];
> if (xx = = lenO && yy < lenl) {
/* last col */ if (endgaps) collfyy] -= ins0+insl*(lenl-yy); if (collfyy] > smax) { smax = collfyy]; dmax = id; } } } if (endgaps && xx < lenO) coll[yy-l] -= ins0+insl*(len0-xx); if (collfyy- 1] > smax) { smax = coll[yy-l]; dmax = id;
} tmp = colO; colO = coll; coll = tmp;
}
(void) free((char *)ndely); (void) free((char *)dely);
(void) free((char *)col0) (void) free((char *)coll) Table 1 (conf)
/* *
* print() — only routine visible outside this module *
* static:
* getmatO — trace back best path, count matches: print()
* pr_align() — print alignment of described in array pf ]: print()
* dumpblockO — dump a block of lines with numbers, stars: pr_align() * nums() — put out a number line: dumpblockO
* putlineO — put out a line (name, [num], seq, [num]): dumpblockO
* stars() - - ut a line of stars: dumpblockO
* stripnameO — strip any path and prefix from a seqname */
#inchιde "nw.h"
#define SPC 3
#define P_LINE 256 /* maximum output line */ #define P_SPC 3 /* space between name or num and seq */ extern _day[26][26]; int olen; /* set output line length */
FILE *fx; /* output file */ printO print
{ int lx, ly, firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, "w")) = = 0) { fρrintf(stderr,"%s: can't write %s\n", prog, ofile); cleanup(l);
} fprintf(fx, " < first sequence: %s (length = %d)\n", namexfO], lenO); fprintf(fx, " <second sequence: %s (length = %d)\n", namexfl], lenl); olen = 60; lx = lenO; ly = lenl; firstgap = lastgap = 0; if (dmax < lenl - 1) { /* leading gap in x */ pp[0].sρc = firstgap = lenl - dmax - 1; ly -= pp[0].spc;
} else if (dmax > lenl - 1) { /* leading gap in y */ pp[l].sρc = firstgap = dmax - (lenl - 1); Ix -= pp[l].spc;
} if (dmaxO < lenO - 1) { /* trailing gap in x */ lastgap = lenO - dmaxO -1; lx -= lastgap;
} else if (dmaxO > lenO - 1) { /* trailing gap in y */ lastgap = dmaxO - (lenO - 1); ly -= lastgap;
} getmat(lx, ly, firstgap, lastgap); pr_align(); } Table 1 (conf)
/*
* trace back the best path, count matches */ static getmat( lx, ly, firstgap, lastg sap) getmat int lx, ly; /* "core" (minus endgaps) */ int firstgap, , lastgap; /* leading ; trailing overlap */ { int nm, iO, il, sizO, sizl; char outx[32]; double pet; register nO, nl; register char *pO, *pl;
/* g ;et total matches, score
*/ i0 = = il = sizO = = sizl = 0:
P0 : = seqxfO] + pp[l].spc; pι = = seqxfl] + pp[0].spc; nO ■■ = PPfl]-spc + 1; nl ■■ = pp[0].spc + l; nm = 0; while ( *p0 && *pl ) { if (sizO) { pl + + ; nl + + ; sizO— ;
> else if (sizl) { p0+ + ; n0+ +; sizl—;
} else { if (xbm[*p0-'A']&xbm[*pl-'A']) nm+ + ; if (nO + + = = pp[0] .xfiO]) sizO = ρp[0].n[iO+ +]; if (nl + + == pp[l].x[il]) sizl = pp[l].n[il + +]; p0+ + ; pl + +;
}
/* pet homology:
* if penalizing endgaps, base is the shorter seq * else, knock off overhangs and take shorter core
*/ if (endgaps) lx = (lenO < lenl)? lenO : lenl; else lx = (lx < ly)? lx : ly; pet = 100.*(double)nm/(double)lx; fprintf(fx, "\n"); fprintf(fx, " < %d match%s in an overlap of %d: %.2f percent similarity\n", nm, (nm = = 1)? "" : "es", lx, pet); Table 1 (conf) rintf(fx, " <gaps in first sequence: %d", gapx);
...getmat if (gapx) {
(void) sprintf(outx, " (%d %s%s)", ngapx, (dna)? "base": "residue", (ngapx = = 1)? "": "s"); fprintf(fx," %s", outx); fprintf(fx, ", gaps in second sequence: %d", gapy); if (gapy) {
(void) sprintf(outx, " (%d %s%s)", ngapy, (dna)? "base": "residue", (ngapy = = 1)? "": "s"); fprintf(fx," %s", outx);
} if (dna) fprintf(fx,
"\n< score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n", smax, DMAT, DMIS, DINS0, DESfSl); else fprintβfx,
"\n<score: %d (Dayhoff PAM 250 matrix, gap penalty = %d 4- d per residue)\n", smax, PINS0, PINS1); if (endgaps) fprintf(fx,
" <endgaps penalized, left endgap: %d %s s, right endgap: %d %s%s\n", firstgap, (dna)? "base" : "residue", (firstgap = = 1)? "" : "s", lastgap, (dna)? "base" : "residue", (lastgap = = 1)? "" : "s"); else fprintf(fx, " < endgaps not penalizedYn"); static nm; /* matches in core — for checking */ static lmax; /* lengths of stripped file names */ static ij[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static Dip]; /* current elem number — for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* set by stars() */
/*
* print alignment of described in struct path ppf ]
*/ static pr_align() pr align
{ int nn; /* char count */ int more; register i; for (i = 0, lmax = 0; i < 2; i+ +) { nn = stripname(namex[i]); if (nn > lmax) lmax = nn; ncfi] = 1; nifi] = 1; sizfi] = ijfi] = 0; psfi] = seqxfi]; pofi] = outfi]; } Table 1 (conf) for (nn = nm = 0, more = 1 ; more; ) {
...pralign for (i = more = 0; i < 2; i+ +) { /*
* do we have more of this sequence? */ if(!*ps[i]) continue; more+ + ; if (ppfij.spc) { /* leading space */ *po[i] + + = ' '; ppfij.spc--;
} else if (sizfi]) { /* in a gap */ *po[i] + + = '-'; sizfi]—; } else { /* we're putting a seq element
*/ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] = toupper(*ps[i]); po[i]++; ps[i] + + ;
/* * are we at next gap for this seq?
*/ if(ni[i] ==pp[i].x[ij[i]]){ /*
* we need to merge all gaps * at this location
*/ sizfi] =pp[i].n[ij[i] + +]; while (nifi] == ppfij.xfijfi]]) sizfi] += pp[i].n[ij[i] + +]; } ni[i] + + ; } } if (+ +nn = = olen 11 !more && nn) { dumpblockO; for(i = 0;i < 2;i++) po[i] = out[i]; nn = 0;
> }
/*
* dump a block of lines, including numbers, stars: pr_align() */ static dumpblockO dumpblock
{ register i; for(i = 0;i < 2; i++) *po[i]~ = '\0'; Table 1 (conf )
...dumpblock
(void) putc('\n', fx); for(i = 0; i < 2;i++){ if (*out[i] && (*out[i] != ' * 11 *(po[i]) !='')){ if(i==0) nums(i); if(i==0&&*out[l]) stars(); pudine(i); if(i ==0&&*out[l]) φrintf(fx, star); ϊf ===== i) nums(i);
/*
* put out a number line: dumpblockO */ static nums(ix) nums int ix; /* index in outf ] holding seq line */ char nlme[P_LINEJ; register i, j; register char *pn, *px, *py; for (pn = nline, i = 0; i < lmax+P_SPC; i+ + , pn++)
*pn = ' '; for (i = ncfix], py = outfix]; *py; py+ + , pn++) { if(*py==" II *py =='-') else { if (i 10 == 011 (i == 1 && ncfix] ! = 1»{ j = (i < 0)? -i : i; for (px = pn; j; j /= 10, px~)
*px =j%10 + '0'; if (i < 0)
*px = '-'
} else
*pn = ' '• i+ + ;
}
}
*pn = '\0'; nc[ix] = i; for (pn = nline; *pn; pn+ +)
(void) putc(*pn, fx);
(void) putc('\n', fx);
}
/*
* put out a line (name, [num], seq, [num]): dumpblockO */ static putline(ix) putline int ix; { Table 1 (conf)
...putline int i; register char *px; for (px = namexfix], i = 0; *px && *px != ':'; px+ +, i+ +)
(void) putc(*px, fx); for (; i < lmax+P_SPC; i+ +) (void) putc(' ', fx);
/* these count from 1:
* nif ] is current element (from 1)
* ncf ] is number at start of current line */ for (px = outfix]; *px; px+ +)
(void) putc(*px&0x7F, fx); (void) putc('\n', fx);
}
* put a line of stars (seqs always in outfO], outflj): dumpblockO
*/ static stars() stars
{ int i; register char *p0, *pl, ex, *px; if (!*out[0J 1 1 (*out[0J = = • ' && *(ρo[0]) = = ") !*out[l] 1 1 (*out[l] = = ' • && *(po[l]) = = ")) return; px = star; for (i = lmax+P_SPC; i; i~)
*px+ + = ' '; for (ρ0 = outfO], pi = outflj; *ρ0 && *pl; p0+ +, pl + +) { if (isalpha(*p0) && isalpha(*pl)) { if (xbm[*p0-'A']&xbm[*pl-'A']) { ex = '*'; nm+ + ;
} else if (!dna && day[*p0-'A'][*pl-'A'J > 0) cx = '.'; else ex = ' ';
} else ex = ' ';
*px+ + = ex;
}
*px+ + = '\n'; *px = '\0'; Table 1 (conf)
/*
* strip path or prefix from pn, return len: pr_align()
*/ static stripname(pn) stripname
/* file name (may be path) */ register char py = 0; for (px = pn; *px; px+ +) if (*px = = V) py = px + 1; if (py)
(void) strcpy(pn, py); return(strlen(pn)) ; }
Table 1 (conf)
/*
* cleanupO ~ cleanup any tmp file
* getseqO — read in seq, set dna, len, maxlen
* g_calloc() — calloc() with error checkin
* readjmpsO ~ get die good jmps, from tmp file if necessary
* writejmpsO — write a filled array of jmps to a tmp file: nw() */
#include "nw.h" #include <sys/file.h> char *jname = "/tmp/homgXXXXXX"; /* tmp file for jmps */ FILE *fj; int cleanupO; /* cleanup tmp file */ long lseek();
/* * remove any tmp file if we blow
*/ cleanup(i) cleanup int i;
{ if (fj)
(void) unlink(jname); exit(i);
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with ';', ' < ', or ' > '
* seq in upper or lower case */ char * getsc :q(file, len) getseq char *file; /* file name */ int *len; /* seq len */
{ char line[1024], *ρseq: register char *px, *py; int natgc, tlen;
FILE *fp; if ((fp = fopen(file,"r")) = = 0) { fρrintf(stderr,"%s: can't read %s\n", prog, file); exit(l);
} tlen = natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ';' | | *line == ' < ' 1 1 *line = = ' > ') continue; for (px = line; *px ! = '\n'; px+ +) if (isupper(*px) 1 1 islower(*px)) tlen+ + ;
} if ((pseq = malloc((unsigned)(tlen+6))) = = 0) { fprintf(stderr,"%s: malloc() failed to get %d bytes for s\n", prog, tlen+6, file); exit(l);
} pseq[0] = pseqfl] = pseq[2] = pseq[3] = '\0'; Table 1 (conf)
...getseq py = pseq + 4; *len = tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line = = ';' | | *line = = ' < ' | | *line = = ' > ') continue; for (px = line; *px != '\n'; px++) { if (isupper(*px))
*py+ + = *px; else if (islower(*px))
*py+ + = toupper(*px); if (index("ATGCU",*(py-l))) natgc + + ; } } *py+ + = '\0';
*py = '\0';
(void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4);
char * g_calloc(msg, nx, sz) gjcalloc char *msg; /* program, calling routine */ int nx, sz; /* number and size of elements */ char *px, *calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) { fprintf(stderr, "%s: g_calloc() failed %s (n= %d, sz= d)\n", prog, msg, nx, sz); exit(l); } } return(px); }
/*
* get final jmps from dx[ ] or tmp file, set pp[ J, reset dmax: main() */ readjmpsO readjmps
{ int fd = -1; int siz, iO, il; register i, j, xx; if (fj) {
(void) fclose(fj); if ((fd = open(jname, OJ DONLY, 0)) < 0) { fprintf(stderr, "%s: can't open() %s\n", prog, jname); cleanup(l); } } for (i = iO = il = 0, dmaxO = dmax, xx = lenO; ; i+ +) { while (1) { for (j = dxfdmaxj.ijmp; j > = 0 && dxfdmaxj.jp. xfj] > = xx; j-) Table 1 (conf)
...readjmps if (j < 0 && dxfdmaxj. offset && fj) {
(void) lseek(fd, dxfdmaxj. offset, 0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmaxj. offset, sizeof(dx[dmax]. offset)); dxfdmaxj.ijmp = MAXJMP-1;
} else break;
} if (i > = IMPS) { fprintf(stderr, " %s: too many gaps in alignmenΛn", prog); cleanup(l); } if (j > = 0) { siz = dxfdmaxj.jp.nfj]; xx = dxfdmaxj.jp. xfjj; dmax + = siz; if (siz < 0) { /* gap in second seq */ ppflj.nfil] = -siz; xx + = siz;
/* id = xx - yy + lenl - 1 */ ppflj.xfil] = xx - dmax + lenl - 1; gapy+ + ; ngapy -= siz; /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP 1 1 endgaps)? -siz : MAXGAP; il + + ;
} else if (siz > 0) { /* gap in first seq */ ppfOJ.nfiO] = siz; ppfOJ.xfiO] = xx; gapx+ + ; ngapx + = siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP 1 1 endgaps)? siz : MAXGAP; i0+ + ; }
} else break;
}
/* reverse the order of jmps
*/ for (j = 0, iO-; j < iO; j + + , i0~) { i = ppfOJ.nfj]; pp[0].n[j] = ppfOJ.nfiO]; ppfOJ.nfiO] = i; i = pp[0].x[j]; ppfOJ.xfj] = pp[0].x[i0]; pp[0].x[i0] = i;
} for (j = 0, il— ; j < il; j + + , il-) { i = ppflj.nfj]; ppflj.nfj] = ppflj.nfil]; pp[l].n[il] = i; i = ppflj.xfjj; ppflj.xfj] = ppflj.xfilj; ppflj.xfil] = i; } if (fd > = 0)
(void) close(fd); if (fj) {
(void) unlink(jname); fj = 0; offset = 0; } } Table 1 (conf)
/*
* write a filled jmp struct offset of the prev one (if any): nw() */ writejmps(ix) ritejmps int ix;
{ char *mktemp(); if (!fj) { if (mktemp(jname) < 0) { fprintf(stderr, "%s: can't mktempO %s\n", prog, jname); cleanup(l);
} if ((fj = fopen(jname, "w")) == 0) { fprintf(stderr, "%s: can't write %s\n", prog, jname); exit(l); }
}
(void) fwrite((char *)&dx[ixj.jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dxfixj. offset, sizeof(dx[ix]. offset), 1, fj);
}
Table 2
PRO xxxxxxxxxxxxxxx (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) = 5 divided by 15 = 33.3%
Table 3
PRO XXXXXXXXXX (Length = 10 arnino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) 5 divided by 10 = 50%
Table 4
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matchmg nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) = 6 divided by 14 = 42.9%
Table 5
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) = 4 divided by 12 = 33.3%
II. Compositions and Methods of the Invention
A. Full-Length PRO Polypeptides
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides. In particular, cDNAs encoding various PRO polypeptides have been identified and isolated, as disclosed in further detail in the Examples below. However, for sake of simplicity, in the present specification the protein encoded by the full length native nucleic acid molecules disclosed herein as well as all further native homologues and variants included in the foregoing definition of PRO, will be referred to as "PRO/number", regardless of their origin or mode of preparation.
As disclosed in the Examples below, various cDNA clones have been disclosed. The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill. For the PRO polypeptides and encoding nucleic acids described herein, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time. B. PRO Polypeptide Variants
In addition to the full-length native sequence PRO polypeptides described herein, it is contemplated that PRO variants can be prepared. PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA, and/or by synthesis of the desired PRO polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PRO or in various domains of the PRO described herein, can be made, for example, using any of the techniques and guidelines for conservative and non- conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO that results in a change in the amino acid sequence of the PRO as compared with the native sequence PRO. Optionally, the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determmed by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
PRO polypeptide fragments are provided herein. Such fragments may be truncated at the N- termmus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PRO polypeptide.
PRO fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PRO fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, PRO polypeptide fragments share at least one biological and/or immunological activity with the native PRO polypeptide disclosed herein.
In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened.
Table 6
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gin; asn lys
Asn (N) gin; his; lys; arg gin
Asp (D) glu glu
Cys (C) ser ser
Gln (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala ala
His (H) asn; gin; lys; arg arg lie (I) leu; val; met; ala; phe; norleucine leu
Leu (L) norleucine; ile; val; met; ala; phe ile
Lys (K) arg; gin; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala
Ser (S) tlir thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) tip; phe; thr; ser phe
Val (V) ile; leu; met; phe; ala; norleucine leu
Substantial modifications in function or immunological identity of the PRO polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res.. 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO variant DNA.
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used. C. Modifications of PRO Covalent modifications of PRO are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a PRO polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO. Derivatization with bifunctional agents is useful, for instance, for crosslinking PRO to a water- insoluble support matrix or surface for use in the method for purifying anti-PRO antibodies, and vice- versa. Commonly used crosslinking agents include, e.g., l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysucchifrnide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- ditMobis(succinimidylpropionate), bifunctional malefrnides such as bis-N-maleimido-l,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutamfnyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties. W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the PRO polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO (for O-linked glycosylation sites). The PRO amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids. Another means of increasing the number of carbohydrate moieties on the PRO polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophvs.. 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzvmol.. 138:350 (1987).
Another type of covalent modification of PRO comprises linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The PRO of the present invention may also be modified in a way to form a chimeric molecule comprising PRO fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PRO with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO. The presence of such epitope-tagged forms of the PRO can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology. 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering. 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology. 6: 1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an alpha-tubulin epitope peptide [Skinner et al., Biol. Chem.. 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA. 87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO polypeptide in place of at least one variable region within an Ig molecule.
In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGl molecule. For the production of immunoglobulin fusions see also US Patent No. 5,428,130 issued June 27, 1995.
D. Preparation of PRO
The description below relates primarily to production of PRO by culturing cells transformed or transfected with a vector containing PRO nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PRO. For instance, the PRO sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques
[see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969);
Merrifield, J. Am. Chem. Soc, 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions. Various portions of the PRO may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO.
1. Isolation of DNA Encoding PRO
DNA encoding PRO may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level. Accordingly, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the
Examples. The PRO-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).
Libraries can be screened with probes (such as antibodies to the PRO or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding PRO is to use PCR methodology [Sambrook et al., supra: Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein.
Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
2. Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors described herein for PRO production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, prmciples, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl2, CaP04, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techmques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Patent No. 4,399,216.
Transformations into yeast are typically carried out according to the method of Van Solingen et al., JL_ Bac . 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature. 336:348-352 (1988).
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aemginosa, and Streptomyces . These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1A2, which has the complete genotype tonA ; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kanr; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF- lac)169 degP ompT rbs7 ilvG kanr; E. coli W3110 strain 40B4, which is strain 37D6 with a non- kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al., Bio/Technology. 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2): 737-742 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophila m (ATCC 36,906; Van den Berg et al., Bio/Technology. 8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]); Candida; Tnchoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophvs. Res. Commun.. 112:284-289 [1983]; Tilburn et al., Gene, 26:205- 221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA. 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J.. 4:475-479 [1985]). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs. 269 (1982).
Suitable host cells for the expression of glycosylated PRO are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA. 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.. 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriate host cell is deemed to be within the skill in the art.
3. Selection and Use of a Replicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
The PRO may be produced recombinanfly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the PRO-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders. Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA. 77:4216 (1980). A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene. 10:157 (1980)]. The trp\ gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to the PRO-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem.. 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg.. 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
PRO transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalo virus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
Transcription of a DNA encoding the PRO by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the PRO coding sequence, but is preferably located at a site 5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO.
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO in recombinant vertebrate cell culture are described in Gefliing et al., Nature, 293:620-625 (1981); Mantei et al., Nature. 281:40-46 (1979); EP 117,060; and EP 117,058.
4. Detecting Gene Amplification/Expression
Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO DNA and encoding a specific antibody epitope.
5. Purification of Polypeptide
Forms of PRO may be recovered from culture medium or from host cell lysates. If membrane- bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of PRO can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents. It may be desired to purify PRO from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contammants such as IgG; and metal chelating columns to bind epitope-tagged forms of the PRO. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice. Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular PRO produced.
E. Tissue Distribution
The location of tissues expressing the PRO can be identified by determining mRNA expression in various human tissues. The location of such genes provides information about which tissues are most likely to be affected by the stimulating and inhibiting activities of the PRO polypeptides. The location of a gene in a specific tissue also provides sample tissue for the activity blocking assays discussed below.
As noted before, gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 [1980]), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
Gene expression in various tissues, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence of a PRO polypeptide or against a synthetic peptide based on the DNA sequences encoding the PRO polypeptide or against an exogenous sequence fused to a DNA encoding a PRO polypeptide and encoding a specific antibody epitope. General techniques for generating antibodies, and special protocols for Northern blotting and in situ hybridization are provided below.
F. Antibody Binding Studies
The activity of the PRO polypeptides can be further verified by antibody binding studies, in which the ability of anti-PRO antibodies to inliibit the effect of the PRO polypeptides, respectively, on tissue cells is tested. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereiribelow.
Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987). Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody. The amount of target protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies preferably are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.
Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g., US Pat No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. For immunohistochemistry, the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
G. Cell-Based Assays
Cell-based assays and animal models for immune related diseases can be used to further understand the relationship between the genes and polypeptides identified herein and the development and pathogenesis of immune related disease.
In a different approach, cells of a cell type known to be involved in a particular immune related disease are transfected with the cDNAs described herein, and the ability of these cDNAs to stimulate or inhibit immune function is analyzed. Suitable cells can be transfected with the desired gene, and monitored for immune function activity. Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit or stimulate immune function, for example to modulate monocyte/macrophage proliferation or inflammatory cell infiltration. Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of immune related diseases.
In addition, primary cultures derived from transgenic animals (as described below) can be used in the cell-based assays herein, although stable cell lines are preferred. Techniques to derive continuous cell lines from transgenic animals are well known in the art (see, e.g., Small et ah, Mol. Cell. Biol. 5: 642- 648 [1985]).
The use of an agonist stimulating compound has also been validated experimentally. Activation of 4-1BB by treatment with an agonist anti-4-lBB antibody enhances eradication of tumors. Hellstrom, I. and Hellstrom, K. E., Crit. Rev. Immunol. (1998) 18: 1. Immunoadjuvant therapy for treatment of tumors, described in more detail below, is another example of the use of the stimulating compounds of the invention. Alternatively, an immune stimulating or enhancing effect can also be achieved by administration of a PRO which has vascular permeability enhancing properties. Enhanced vascular permeability would be beneficial to disorders which can be attenuated by local infiltration of immune cells (e.g., monocytes/macrophages, eosinophils, PMNs) and inflammation. On the other hand, PRO polypeptides, as well as other compounds of the invention, which are direct inhibitors of monocyte/macrophage proliferation/activation, lymphokine secretion, and/or vascular permeability can be directly used to suppress the immune response. These compounds are useful to reduce the degree of the immune response and to treat immune related diseases characterized by a hyperactive, superoptimal, or autoimmune response. The use of compound which suppress vascular permeability would be expected to reduce inflammation. Such uses would be beneficial in treating conditions associated with excessive inflammation.
Alternatively, compounds, e.g., antibodies, which bind to stimulating PRO polypeptides and block the stimulating effect of these molecules produce a net inhibitory effect and can be used to suppress the monocyte/macrophage mediated immune response by inhibiting monocyte/macrophage proliferation/activation and/or lymphokine secretion. Blocking the stimulating effect of the polypeptides suppresses the immune response of the mammal.
H. Animal Models
The results of the cell based in vitro assays can be further verified using in vivo animal models and assays for monocyte/macrophage function. A variety of well known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of immune related disease, and to test the efficacy of candidate therapeutic agents, including antibodies, and other antagonists of the native polypeptides, including small molecule antagonists. The in vivo nature of such models makes them predictive of responses in human patients. Animal models of immune related diseases include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g., murine models. Such models can be generated by introducing cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, etc.
Graft-versus-host disease occurs when immunocompetent cells are transplanted into immunosuppressed or tolerant patients. The donor cells recognize and respond to host antigens. The response can vary from life threatening severe inflammation to mild cases of diarrhea and weight loss. Graft-versus-host disease models provide a means of assessing monocyte/macrophage reactivity against MHC antigens and minor transplant antigens. A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.3.
Animal models for delayed type hypersensitivity provides an assay of cell mediated immune function as well. In chronic Delayed type hypersensitivity (DTH) reactions, monocytes that have differentiated into macrophages lead to the destruction of host tissue which is replaced by fibrous tissue (fibrosis). Contact hypersensitivity is a simple delayed type hypersensitivity in vivo assay of cell mediated immune function. In this procedure, cutaneous exposure to exogenous haptens which gives rise to a delayed type hypersensitivity reaction which is measured and quantitated. Contact sensitivity involves an initial sensitizing phase followed by an elicitation phase. The elicitation phase occurs when the T lymphocytes encounter an antigen to which they have had previous contact. Swelling and inflammation occur, making this an excellent model of human allergic contact dermatitis. At this point, monocytes leave the blood and differentiate in to macrophages. A suitable procedure is described in detail in Current Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc., 1994, unit 4.2. See also Grabbe, S. and Schwarz, T, Immun. Today 19 (1): 37-44 (1998)
Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals. Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees and monkeys. Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U.S. Patent No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et al., Proc. Natl. Acad. Sci. USA 82, 6148-615 [1985]); gene targeting in embryonic stem cells (Thompson et al., Cell 56, 313-321 [1989]); electroporation of embryos (Lo, Mol. Cel. Biol. 3, 1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al, Cell 57, 717-73 [1989]). For review, see, for example, U.S. Patent No. 4,736,866.
For the purpose of the present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals"). The transgene can be integrated either as a single transgene, or in concatamers, e.g., head-to-head or head-to-tail tandems. Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al., Proc. Natl. Acad. Sci. USA 89, 6232-636 (1992).
The expression of the transgene in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene. The level of mRNA expression can then be analyzed using techmques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry. The animals may be further examined for signs of immune disease pathology, for example by histological examination to determine infiltration of immune cells into specific tissues. Blocking experiments can also be performed in which the transgenic animals are treated with the compounds of the invention to determine the extent of the monocytes/macrophage proliferation stimulation or inhibition of the compounds. In these experiments, blocking antibodies which bind to the PRO polypeptide, prepared as described above, are administered to the animal and the effect on immune function is determined.
Alternatively, "knock out" animals can be constructed which have a defective or altered gene encoding a polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal. For example, cDNA encoding a particular polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques. A portion of the genomic DNA encoding a particular polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several lobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas and Capecchi, Cell, 51_:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the polypeptide.
I. ImmunoAdjuvant Therapy
In one embodiment, the immunostimulating compounds of the invention can be used in immunoadjuvant therapy for the treatment of tumors (cancer). It is now well established that monocytes/macrophages recognize human tumor specific antigens. One group of tumor antigens, encoded by the MAGE, BAGE and GAGE families of genes, are silent in all adult normal tissues , but are expressed in significant amounts in tumors, such as melanomas, lung tumors, head and neck tumors, and bladder carcinomas. DeS et, C. et al, (1996) Proc. Natl. Acad. Sci. USA, 93:7149. It has been shown that stimulation of immune cells induces tumor regression and an antitumor response both in vitro and in vivo. Melero, I. et al, Nature Medicine (1997) 3:682; Kwon, E. D. et al., Proc. Natl. Acad. Sci. USA (1997) 94: 8099; Lynch, D. H. et al, Nature Medicine (1997) 3:625; Finn, O. J. and Lotze, M. T., J. Immunol. (1998) 21:114. The stimulatory compounds of the invention can be administered as adjuvants, alone or together with a growth regulating agent, cytotoxic agent or chemotherapeutic agent, to stimulate monocyte/macrophage proliferation/activation and an antitumor response to tumor antigens. The growth regulating, cytotoxic, or chemotherapeutic agent may be administered in conventional amounts using known administration regimes. Immunostimulating activity by the compounds of the invention allows reduced amounts of the growth regulating, cytotoxic, or chemotherapeutic agents thereby potentially lowering the toxicity to the patient. J. Screening Assays for Drug Candidates
Screening assays for drug candidates are designed to identify compounds that bind to or complex with the polypeptides encoded by the genes identified herein or a biologically active fragment thereof, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, (poly)peptide-rmmunoglobulin fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art. All assays are common in that they call for contacting the drug candidate with a polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the polypeptide encoded by the gene identified herein or the drag candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labelled antibody specifically binding the immobilized complex. If the candidate compound interacts with but does not bind to a particular protein encoded by a gene identified herein, its interaction with that protein can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, cross-linking, co- immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature (London) 340, 245-246 (1989); Chien et al, Proc. Natl. Acad. Sci. USA 88, 9578-9582 (1991)] as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl-ZαcZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein- protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
In order to find compounds that interfere with the interaction of a gene identified herein and other intra- or extracellular components can be tested, a reaction mixture is usually prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a test compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described above. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.
K. Compositions and Methods for the Treatment of Immune Related Diseases
The compositions useful in the treatment of immune related diseases include, without limitation, proteins, antibodies, small organic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc. that inhibit or stimulate immune function, for example, monocyte proliferation activation, lymphokine release, or immune cell infiltration.
For example, antisense RNA and RNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techmques. For further details see, e.g., Rossi, Current Biology 4, 469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997). Nucleic acid molecules in triple helix formation used to inhibit transcription should be single- stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551, supra. These molecules can be identified by any or any combination of the screening assays discussed above and/or by any other screening techniques well known for those skilled in the art. L. Anti-PRO Antibodies
The present invention further provides anti-PRO antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.
1. Polyclonal Antibodies
The anti-PRO antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, die immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the PRO polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation. 2. Monoclonal Antibodies
The anti-PRO antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include the PRO polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol.. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. , 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or purified from die culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Patent No. 4,816,567; Morrison et al., supraj or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
3. Human and Humanized Antibodies
The anti-PRO antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a nonhuman species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature. 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.. 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non- human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature. 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.. 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. , 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al, Bio/Technology 10. 779-783 (1992); Lonberg et al, Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
The antibodies may also be affinity matured using known selection and/or mutagenesis methods as described above. Preferred affinity matured antibodies have an affinity which is five times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the matured antibody is prepared.
4. Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy- chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature. 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy- chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzvmology, 121:210 (1986).
According to another approach described in WO 96/27011 , the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generatmg bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared can be prepared using chemical linkage. Brennan et al, Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent mtermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Fab' fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al , J. Exp. Med. 175:217-225 (1992) describe the production of a fully humamzed bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various technique for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al, J. Immunol. 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al, J. Immunol. 152:5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. , J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies may bind to two different epitopes on a given PRO polypeptide herein. Alternatively, an anti-PRO polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular PRO polypeptide. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PRO polypeptide. These antibodies possess a PRO-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the PRO polypeptide and further binds tissue factor (TF).
5. Heteroconjugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
6. Effector Function Engineering
It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g. , the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al. , J. Exp Med.. 176: 1191-1195 (1992) and Shopes, J. Immunol.. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design. 3: 219-230 (1989).
7. Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radiocoηjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein- coupling agents such as N-succinrmidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. , Science, 238: 1098 (1987). Carbon-14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
In another embodiment, the antibody may be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide). 8. Immunolipo somes
The antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA. 82: 3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA. 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al .. J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al. , J. National Cancer Inst., 81.(19): 1484 (1989).
M. Pharmaceutical Compositions
The active PRO molecules of the invention (e.g. , PRO polypeptides, anti-PRO antibodies, and/or variants of each) as well as other molecules identified by the screening assays disclosed above, can be admimstered for the treatment of immune related diseases, in the form of pharmaceutical compositions. Therapeutic formulations of the active PRO molecule, preferably a polypeptide or antibody of the invention, are prepared for storage by mixing the active molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcrnol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
Compounds identified by the screening assays disclosed herein can be formulated in an analogous manner, using standard techniques well known in the art.
Lipofections or liposomes can also be used to deliver the PRO molecule into cells. Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable region sequences of an antibody, peptide molecules can be designed which retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA 90, 7889-7893 [1993]). The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active PRO molecules may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techmques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. Sustained-release preparations or the PRO molecules may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyefhyl- methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfliydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. N. Methods of Treatment
It is contemplated that the polypeptides, antibodies and other active compounds of the present invention may be used to treat various immune related diseases and conditions, such as monocyte/macrophage diseases, including those characterized by infiltration of inflammatory cells into a tissue, stimulation of monocyte/macrophages, inliibition of monocytes/macrophages, increased or decreased vascular permeability or the inliibition thereof.
Exemplary conditions or disorders to be treated with the polypeptides, antibodies and other compounds of the invention, include, but are not limited to systemic lupus erythematosis, rheumatoid arthritis, juvenile chronic arthritis, osteoarthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjδgren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease (ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune- mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhiriitis, atopic dermatitis, food hypersensitivity and urticaria, immunologic diseases of the lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft - versus-host-disease .
Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease that mainly involves the synovial membrane of multiple joints with resultant injury to the articular cartilage. The pathogenesis is T lymphocyte dependent and is associated with the production of rheumatoid factors, auto- antibodies directed against self IgG, with the resultant formation of immune complexes that attain high levels in joint fluid and blood. These complexes in the joint may induce the marked infiltrate of lymphocytes and monocytes/macrophages into the synovium and subsequent marked synovial changes; the joint space/fluid if infiltrated by similar cells with the addition of numerous neutrophils. Tissues affected are primarily the joints, often in symmetrical pattern. However, extra-articular disease also occurs in two major forms. One form is the development of extra-articular lesions with ongoing progressive joint disease and typical lesions of pulmonary fibrosis, vasculitis, and cutaneous ulcers. The second form of extra-articular disease is the so called Felty's syndrome which occurs late in the RA disease course, sometimes after joint disease has become quiescent, and involves the presence of neutropenia, thrombocytopenia and splenomegaly. This can be accompanied by vasculitis in multiple organs with formations of infarcts, skin ulcers and gangrene. Patients often also develop rheumatoid nodules in the subcutis tissue overlying affected joints; the nodules late stage have necrotic centers surrounded by a mixed inflammatory cell infiltrate. Other manifestations which can occur in RA include: pericarditis, pleuritis, coronary arteritis, intestitial pneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, and rheumatoid nodules. The number and activation state of macrophages in the inflamed synovius correlates with the significance of RA (Kinne et al., 2000 Arthritis Res. 2: 189-202). As described above, macrophages are not believed to be involved in the early events of RA, but monocytes/macrophages have tissue destructive and tissue remodeling properties which may contribute to both acute and chronic RA. Juvenile chronic arthritis is a chronic idiopathic inflammatory disease which begins often at less than 16 years of age. Its phenotype has some similarities to RA; some patients which are rhematoid factor positive are classified as juvenile rheumatoid arthritis. The disease is sub-classified into three major categories: pauciarticular, polyarticular, and systemic. The arthritis can be severe and is typically destructive and leads to joint ankylosis and retarded growth. Other manifestations can include chronic anterior uveitis and systemic amyloidosis.
Spondyloarthropathies are a group of disorders with some common clinical features and the common association with the expression of HLA-B27 gene product. The disorders include: ankylosing sponylitis, Reiter's syndrome (reactive arthritis), arthritis associated with inflammatory bowel disease, spondylitis associated with psoriasis, juvenile onset spondyloarthropathy and undifferentiated spondyloarthropathy. Distinguishing features include sacroileitis with or without spondylitis; inflammatory asymmetric arthritis; association with HLA-B27 (a serologically defined allele of the HLA-B locus of class I MHC); ocular inflammation, and absence of autoantibodies associated with other rheumatoid disease. It was shown that CD 163+ macrophages were increased in the synovial lining and colonic mucosa in Spondyloarthropathy and correlates with the expression of HLA-DR and the production of TNF-alpha (Baeten et al., 2002 J Pathol 196(3):343-350).
Systemic sclerosis (scleroderma) has an unknown etiology. A hallmark of the disease is induration of the skin; likely this is induced by an active inflammatory process. Scleroderma can be localized or systemic; vascular lesions are common and endothelial cell injury in the microvasculature is an early and important event in the development of systemic sclerosis; the vascular injury may be immune mediated. An immunologic basis is implied by the presence of mononuclear cell infiltrates in the cutaneous lesions and the presence of anti-nuclear antibodies in many patients. ICAM-1 is often upregulated on the cell surface of fibroblasts in skin lesions suggesting that T cell interaction with these cells may have a role in the pathogenesis of the disease. As well as T cells, monocytes/macrophages are proposed to play a role in the progression of scleroderma by secreting fibrogenic cytokines (Yamamoto et al., 2001 J Dermatol Sci 26(2): 133-139). Other organs involved include: the gastrointestinal tract: smooth muscle atrophy and fibrosis resulting in abnormal peristalsis/motility; kidney: concentric subendothelial intimal proliferation affecting small arcuate and interlobular arteries with resultant reduced renal cortical blood flow, results in proteinuria, azotemia and hypertension; skeletal muscle: atrophy, interstitial fibrosis; inflammation; lung: interstitial pneumonitis and interstitial fibrosis; and heart: contraction band necrosis, scarring/fibrosis.
Idiopathic inflammatory myopathies including dermatomyositis, polymyositis and others are disorders of chronic muscle inflammation of unknown etiology resulting in muscle weakness. Muscle injury/inflammation is often symmetric and progressive. Autoantibodies are associated with most forms. These myositis-specific autoantibodies are directed against and inhibit the function of components, proteins and RNA's, involved in protein synthesis.
Sjδgren's syndrome is due to immune-mediated inflammation and subsequent functional destruction of the tear glands and salivary glands. The disease can be associated with or accompanied by inflammatory connective tissue diseases. The disease is associated with autoantibody production against Ro and La antigens, both of which are small RNA-protein complexes. Lesions result in keratoconjunctivitis sicca, xerostomia, with other manifestations or associations including bilary cirrhosis, peripheral or sensory neuropathy, and palpable purpura.
Systemic vasculitis are diseases in which the primary lesion is inflammation and subsequent damage to blood vessels which results in ischemia/necrosis/degeneration to tissues supplied by the affected vessels and eventual end-organ dysfunction in some cases. Vasculitis can also occur as a secondary lesion or sequelae to other immune-inflammatory mediated diseases such as rheumatoid arthritis, systemic sclerosis, etc. , particularly in diseases also associated with the formation of immune complexes. Diseases in the primary systemic vasculitis group include: systemic necrotizing vasculitis: polyarteritis nodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener's granulomatosis; lymphomatoid granulomatosis; and giant cell arteritis. Miscellaneous vasculitides include: mucocutaneous lymph node syndrome (MLNS or Kawasaki's disease), isolated CNS vasculitis, Behet's disease, thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizing venulitis. The pathogenic mechanism of most of the types of vasculitis listed is believed to be primarily due to the deposition of immunoglobulin complexes in the vessel wall and subsequent induction of an inflammatory response either via ADCC, complement activation, or both.
Sarcoidosis is a condition of unknown etiology which is characterized by the presence of epithelioid granulomas in nearly any tissue in the body; involvement of the lung is most common. The pathogenesis involves the persistence of activated macrophages and lymphoid cells at sites of the disease with subsequent chronic sequelae resultant from the release of locally and systemically active products released by these cell types.
Autoimmune hemolytic anemia including autoimmune hemolytic anemia, immune pancytopenia, and paroxysmal noctural hemoglobinuria is a result of production of antibodies that react with antigens expressed on the surface of red blood cells (and in some cases other blood cells including platelets as well) and is a reflection of the removal of those antibody coated cells via complement mediated lysis and/or ADCC/Fc-receptor-mediated mechanisms .
Thyroiditis including Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, and atrophic uiyroiditis, are the result of an autoimmune response against thyroid antigens with production of antibodies that react with proteins present in and often specific for the thyroid gland. Experimental models exist including spontaneous models: rats (BUF and BB rats) and chickens (obese chicken strain); inducible models: immunization of animals with either thyroglobulin, thyroid microsomal antigen (thyroid peroxidase). Inflammatory and Fibrotic Lung Disease, including Eosinophilic Pneumonias; Idiopathic
Pulmonary Fibrosis, and Hypersensitivity Pneumonitis may involve a disregulated immune-inflammatory response. Inhibition of that response would be of therapeutic benefit.
Psoriasis is a T lymphocyte-mediated inflammatory disease. Lesions contain infiltrates of T lymphocytes, macrophages and antigen processing cells, and some neutrophils. Other diseases in which intervention of the immune and/or inflammatory response have benefit are infectious disease including but not limited to viral infection (including but not limited to AIDS, hepatitis A, B, C, D, E and herpes) bacterial infection, fungal infections, and protozoal and parasitic infections. Molecules (or derivatives/agonists) which stimulate the immune reaction can be utilized therapeutically to enhance the immune response to infectious agents), diseases of immunodeficiency (molecules/derivatives/agonists) which stimulate the immune reaction can be utilized therapeutically to enhance the immune response for conditions of inherited, acquired, infectious induced (as in HIV infection), or iatrogenic (i.e., as from chemotherapy) immunodeficiency, and neoplasia.
It has been demonstrated that some human cancer patients develop an antibody and/or monocyte/macrophage response to antigens on neoplastic cells. It has also been shown in animal models of neoplasia that enhancement of the immune response can result in rejection or regression of that particular neoplasm. Molecules that enhance the monocyte/macrophage response have utility in vivo in enhancing the immune response against neoplasia. Molecules which enhance the monocyte/macrophage proliferative response (or small molecule agonists or antibodies that affected the same receptor in an agonistic fashion) can be used therapeutically to treat cancer. Molecules that inhibit the monocyte/macrophage response also function in vivo during neoplasia to suppress the immune response to a neoplasm; such molecules can either be expressed by the neoplastic cells themselves or their expression can be induced by the neoplasm in other cells. Antagonism of such inhibitory molecules (either with antibody, small molecule antagonists or other means) enhances immune-mediated tumor rejection.
Additionally, inhibition of molecules with proinflammatory properties may have therapeutic benefit in reperfusion injury; stroke; myocardial infarction; atherosclerosis; acute lung injury; hemorrhagic shock; burn; sepsis/septic shock; acute tubular necrosis; endometriosis; degenerative joint disease and pancreatis. The compounds of the present invention, e.g., polypeptides or antibodies, are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrosprnal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation (intranasal, intrapulmonary) routes. Intravenous or inhaled administration of polypeptides and antibodies is preferred.
In immunoadjuvant therapy, other therapeutic regimens, such administration of an anti-cancer agent, may be combined with the administration of the proteins, antibodies or compounds of the instant invention. For example, the patient to be treated with a the immunoadjuvant of the invention may also receive an anti-cancer agent (chemotherapeutic agent) or radiation therapy. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may precede, or follow administration of the immunoadjuvant or may be given simultaneously therewith. Additionally, an anti-estrogen compound such as tamoxifen or an anti-progesterone such as onapristone (see, EP 616812) may be given in dosages known for such molecules.
It may be desirable to also administer antibodies against other immune disease associated or tumor associated antigens, such as antibodies which bind to CD20, CDlla, CD18, ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be coadministered to the patient.
Sometimes, it may be beneficial to also administer one or more cytokines to the patient. In one embodiment, the PRO polypeptides are coadministered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by a PRO polypeptide. However, simultaneous administration or administration first is also contemplated. Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the PRO polypeptide.
For the treatment or reduction in the severity of immune related disease, the appropriate dosage of an a compound of the invention will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound, and the discretion of the attending physician. The compound is suitably administered to the patient at one time or over a series of treatments. For example, depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g.,
0.1-20 mg/kg) of polypeptide or antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above.
For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techmques and assays.
O. Articles of Manufacture
In another embodiment of the invention, an article of manufacture containing materials (e.g., comprising a PRO molecule) useful for the diagnosis or treatment of the disorders described above is provided. The article of manufacture comprises a container and an instruction. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is usually a polypeptide or an antibody of the invention. An instruction or label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
P. Diagnosis and Prognosis of Immune Related Disease
Cell surface proteins, such as proteins which are overexpressed in certain immune related diseases, are excellent targets for drug candidates or disease treatment. The same proteins along with secreted proteins encoded by the genes amplified in immune related disease states find additional use in the diagnosis and prognosis of these diseases. For example, antibodies directed against the protein products of genes amplified in multiple sclerosis, rheumatoid arthritis, or another immune related disease, can be used as diagnostics or prognostics.
For example, antibodies, including antibody fragments, can be used to qualitatively or quantitatively detect the expression of proteins encoded by amplified or overexpressed genes ("marker gene products"). The antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or otiier techniques known in the art. These techniques are particularly suitable, if the overexpressed gene encodes a cell surface protein
Such binding assays are performed essentially as described above. In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this purpose, a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in situ detection.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
EXAMPLES Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, VA.
EXAMPLE 1: Microarray analysis of monocyte/macrophages.
Nucleic acid microarrays, often containing thousands of gene sequences, are useful for identifying differentially expressed genes in diseased tissues as compared to their normal counterparts. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The cDNA probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes known to be expressed in certain disease states may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. If the hybridization signal of a probe from a test (in this instance, differentiated macrophages) sample is greater than hybridization signal of a probe from a control (in this instance, non-differentiated monocytes) sample, the gene or genes expressed in the test tissue are identified. The implication of this result is that an overexpressed protein in a test tissue is useful not only as a diagnostic marker for the presence of the disease condition, but also as a therapeutic target for treatment of the disease condition.
The methodology of hybridization of nucleic acids and microarray technology is well known in the art. In one example, the specific preparation of nucleic acids for hybridization and probes, slides, and hybridization conditions are all detailed in PCT Patent Application Serial No. PCT/US01/10482, filed on March 30, 2001 and which is herein incorporated by reference. In this experiment, CD14+ monocytes are selected by positive selection according to Miltenyi
MACS™ protocol. Lymphocytes in 100 ml heparinized blood are separated using Ficoll Paque™. Cells are washed twice in PBS/0.5% BSA/2 mM EDTA. In final wash, all gradients are pooled and volume is brought to approximately 10 ml. The cells are centrifuged, the supernatant is removed and the cell pellet is resuspended in buffer in a total volume of 10e7 cells per 80 μl buffer. Add 20 μl CD 14 microbeads per 10e7 total cells, mix and incubate 15 minutes at 6-12 C. Wash the cells by adding 20x labeling volume of buffer, spin pellet and resuspend in 500 ul buffer per 10e8 cells. Separate cells with MACS™ depletion column type D and check purity of cells by labeling with anti-CD45 and anti-CD14 antibodies (cell purity at this point is > 95 %). Lyse cells in RNA lysis buffer to obtain a timepoint of Day 0 monocytes, then plate remaining cells in 6 well plates in macrophage differentiation medium: DMEM 4.5 ug/ml glucose, Pen-Strep, L-glutamine, 20% FBS and 10% Human AB serum (Gemini, Cat # 100-512). Seed cells at 1.5 x 10e6 per well (6 well Costar cell culture plates) and grow at 37 C, 7% C02. After 24 hours in culture, the cells were harvested and lysed in RNA lysis buffer to obtain mRNA for the Day 1 timepoint. The remaining cells were kept in culture and until Day 7. After 7 days in culture, the cells were lysed in RNA lysis buffer to obtain Day 7 timepoint at which time the cells displayed gross macrophage morphology.
The mRNA was isolated by Qiagen miniprep and analysis run on Affimax™ (Affymetrix Inc. Santa Clara, CA) microarray chips and proprietary Genentech microarrays. The cells harvested at Day 0 timepoint, the Day 1 timepoint, and the Day 7 timepoint were subjected to the same analysis. Genes were compared whose expression was upregulated at Day 7 as compared to Day 0 and Day 1.
Below are the results of these experiments, demonstrating that various PRO polypeptides of the present invention are differentially expressed in differentiated macrophages at Day 7 as compared to non- differentiated monocytes at Day 0 and at Day 1. As described above, these data demonstrate that the PRO polypeptides of the present invention are useful not only as diagnostic markers for the presence of one or more immune disorders, but also serve as therapeutic targets for the treatment of those immune disorders.
Specifically, the cDNAs shown Figures 592, Figure 708, Figure 724, Figure 888, Figure 1095, Figure
1109, Figure 1456 and Figure 2331 are significantly overexpressed in differentiated macrophages as compared to non-differentiated monocytes at Day 0 and Day 1. The Figures 1-2517 show the nucleic acids of the invention and their encoded PRO polypeptides that are differentially expressed in differentiated macrophages at Day 7 as compared to non-differentiated monocytes at Day 0 and at Day 1.
EXAMPLE 2: Use of PRO as a hybridization probe The following method describes use of a nucleotide sequence encoding PRO as a hybridization probe.
DNA comprising the coding sequence of full-length or mature PRO as disclosed herein is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO) in human tissue cDNA libraries or human tissue genomic libraries. Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled PRO-derived probe to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20 hours.
Washing of the filters is performed in an aqueous solution of 0. lx SSC and 0.1 % SDS at 42°C. DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO can then be identified using standard techmques known in the art.
EXAMPLE 3: Expression of PRO in E. coli This example illustrates preparation of an unglycosylated form of PRO by recombinant expression E. coli.
The DNA sequence encoding PRO is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO coding region, lambda transcriptional terminator, and an argU gene.
The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing. Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.
After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PRO protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
PRO may be expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding PRO is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation imtiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(ladq). Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30°C with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH4)2S04, 0.71 g sodium citrate»2H20, 1.07 g KC1, 5 .36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgS04) and grown for approximately 20-30 hours at 30°C with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1M and 0.02 M, respectively, and the solution is stirred overnight at 4°C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4°C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence. The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml. The refolding solution is stirred gently at 4°C for 12-36 hours. The refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final concentration. The refolded protein is chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0.1 % TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from die desired form, the reversed phase step also removes endotoxin from the samples.
Fractions containing the desired folded PRO polypeptide are pooled and the acetonitrile removed using a gentle stream of mtrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.
Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
EXAMPLE 4: Expression of PRO in mammalian cells
This example illustrates preparation of a potentially glycosylated form of PRO by recombinant expression in mammalian cells. The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector. Optionally, the PRO DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO DNA using ligation methods such as described in Sambrook et al. , supra. The resulting vector is called pRK5-PRO. In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL
1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 μg pRK5-PRO DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25°C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days. Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml 35S-cysteine and 200 μCi/ml 35S- methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PRO polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
In an alternative technique, PRO may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci. , 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PRO can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
In another embodiment, PRO can be expressed in CHO cells. The pRK5-PRO can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35S-methionine. After determining the presence of PRO polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed PRO can then be concentrated and purified by any selected method. Epitope-tagged PRO may also be expressed in host CHO cells. The PRO may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged PRO insert can then be subcloned into a SV40 promoter/enhancer containing vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 promoter/enhancer containing vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged PRO can then be concentrated and purified by any selected method, such as by Ni2+-chelate affinity chromatography.
PRO may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure.
Stable expression in CHO cells is performed using the following procedure. The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins are fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form. Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used expression in CHO cells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.
Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Quiagen), Dosper® or Fugene (Boehringer Mannheim). The cells are grown as described in Lucas et al.. supra. Approximately 3 x IO"7 cells are frozen in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing 10 mL of media and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37°C. After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3 x 105 cells/mL. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Patent No. 5,122,469, issued June 16, 1992 may actually be used. A 3L production spinner is seeded at 1.2 x 106 cells/mL. On day 0, pH is determined. On day 1, the spinner is sampled and sparging with filtered air is commenced. On day 2, the spinner is sampled, the temperature shifted to 33°C, and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture is harvested by centrifugation and filtering through a 0.22 μm filter. The filtrate was either stored at 4°C or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μl of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly- His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
EXAMPLE 5: Expression of PRO in Yeast The following method describes recombinant expression of PRO in yeast.
First, yeast expression vectors are constructed for intracellular production or secretion of PRO from the ADH2/GAPDH promoter. DNA encoding PRO and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO. For secretion,
DNA encoding PRO can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PRO.
Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain. Recombinant PRO can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing PRO may further be purified using selected column chromatography resins. Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
EXAMPLE 6: Expression of PRO in Baculovirus-Infected Insect Cells
The following method describes recombinant expression of PRO in Baculovirus-infected insect cells.
The sequence coding for PRO is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PRO or the desired portion of the coding sequence of PRO such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to die 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold™ virus DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
Expressed poly-his tagged PRO can then be purified, for example, by Ni2+-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al.', Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1 % NP-40; 0.4 M KC1), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni2+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline A280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching A280 baseline again, die column is developed wiui a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni2+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His10-tagged PRO are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PRO can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography. Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
EXAMPLE 7: Preparation of Antibodies that Bind PRO
This example illustrates preparation of monoclonal antibodies which can specifically bind PRO. Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified PRO, fusion proteins containing PRO, and cells expressing recombinant PRO on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research,
Hamilton, MT) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti- PRO antibodies.
After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35 % polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.l, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against PRO. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PRO is within the skill in the art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed. EXAMPLE 8: Purification of PRO Polypeptides Using Specific Antibodies
Native or recombinant PRO polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PRO polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin.
Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
Such an immunoaffinity column is utilized in the purification of PRO polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble PRO polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown. A soluble PRO polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PRO polypeptide (e.g. , high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PRO polypeptide binding (e.g. , a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and PRO polypeptide is collected.
EXAMPLE 9: Drag Screening
This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a variety of drag screening techniques. The PRO polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. One method of drag screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between PRO polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PRO polypeptide and its target cell or target receptors caused by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any other agents which can affect a PRO polypeptide-associated disease or disorder. These methods comprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying (I) for the presence of a complex between the agent and the PRO polypeptide or fragment, or (ii) for the presence of a complex between the PRO polypeptide or fragment and the cell, by methods well known in the art. In such competitive binding assays, the PRO polypeptide or fragment is typically labeled. After suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell complex.
Another technique for drag screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PRO polypeptide, the peptide test compounds are reacted with PRO polypeptide and washed. Bound PRO polypeptide is detected by methods well known in the art. Purified PRO polypeptide can also be coated directly onto plates for use in the aforementioned drag screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drag screening assays in which neutralizing antibodies capable of binding PRO polypeptide specifically compete with a test compound for binding to PRO polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PRO polypeptide.
EXAMPLE 10: Rational Drug Design
The goal of rational drag design is to produce structural analogs of biologically active polypeptide of interest (i.e. , a PRO polypeptide) or of small molecules with which they interact, e.g. , agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the PRO polypeptide or which enhance or interfere with the function of the PRO polypeptide in vivo (c.f., Hodgson, Bio/Technology, 9: 19-21 (1991)).
In one approach, the three-dimensional structure of the PRO polypeptide, or of a PRO polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the PRO polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous PRO polypeptide-like molecules or to identify efficient inhibitors. Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al , J. Biochem., 113:742-746 (1993).
It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the PRO polypeptide may be made available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the PRO polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography. The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to diose skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

What is claimed:
1. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence identity to:
(a) die nucleotide sequence shown in any one of the Figures 1-2517 (SEQ ID NOS: 1- 2517); or
(b) the nucleotide sequence encoding the polypeptide shown in any one of the Figures 1-2517 (SEQ ID NOS: 1-2517).
2. A vector comprising the nucleic acid of Claim 1.
3. The vector of Claim 2 operably linked to control sequences recognized by a host cell transformed with the vector.
4. A host cell comprising the vector of Claim 2.
5. The host cell of Claim 4, wherein said cell is a CHO cell, an E.coli cell or a yeast cell.
6. A process for producing a PRO polypeptide comprising culturing the host cell of Claim 5 under conditions suitable for expression of said PRO polypeptide and recovering said PRO polypeptide from the cell culture.
7. An isolated polypeptide having at least 80% amino acid sequence identity to: (a) a polypeptide shown in any one of Figures 1-2517 (SEQ ID NOS: 1-2517); or
(b) a polypeptide encoded by the hill length coding region of the nucleotide sequence shown in any one of Figures 1-2517 (SEQ ID NOS: 1-2517).
8. A chimeric molecule comprising a polypeptide according to Claim 7 fused to a heterologous amino acid sequence.
9. The chimeric molecule of Claim 8, wherem said heterologous amino acid sequence is an epitope tag sequence or an Fc region of an immunoglobulin.
10. An antibody which specifically binds to a polypeptide according to Claim 7.
11. The antibody of Claim 10, wherein said antibody is a monoclonal antibody, a humamzed antibody or a single-chain antibody.
I l l
12. A composition of matter comprising (a) a polypeptide of Claim 7, (b) an agonist of said polypeptide, (c) an antagonist of said polypeptide, or (d) an antibody that binds to said polypeptide, in combination with a carrier.
13. The composition of matter of Claim 12, wherein said carrier is a pharmaceutically acceptable carrier.
14. The composition of matter of Claim 13 comprising a therapeutically effective amount of (a), (b), (c) or (d).
15. An article of manufacture, comprising: a container; a label on said container; and a composition of matter comprising (a) a polypeptide of Claim 7, (b) an agonist of said polypeptide, (c) an antagonist of said polypeptide, or (d) an antibody that binds to said polypeptide, contained within said container, wherein label on said container indicates that said composition of matter can be used for treating an immune related disease.
16. A method of treating an immune related disorder in a mammal in need thereof comprising administering to said mammal a therapeutically effective amount of (a) a polypeptide of Claim 7, (b) an agonist of said polypeptide, (c) an antagonist of said polypeptide, or (d) an antibody that binds to said polypeptide.
17. The method of Claim 16, wherem the immune related disorder is systemic lupus erythematosis, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, a spondyloarthropathy, systemic sclerosis, an idiopathic inflammatory myopathy, Sjδgren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renal disease, a demyelinating disease of the central or peripheral nervous system, idiopathic demyelinating polyneuropathy, Guillain-Barre syndrome, a chronic inflammatory demyelinating polyneuropathy, a hepatobiliary disease, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, inflammatory bowel disease, gluten-sensitive enteropathy, Whipple's disease, an autoimmune or immune-mediated skin disease, a bullous skin disease, erythema multiforme, contact dermatitis, psoriasis, an allergic disease, asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, urticaria, an immunologic disease of the lung, eosinophilic pneumonias, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, a transplantation associated disease, graft rejection or graft-versus-host-disease.
18. A method for determining the presence of a PRO polypeptide of the invention as described in Figures 1-2517 (SEQ ID NOS: 1-2517), in a sample suspected of containing said polypeptide, said method comprising exposing said sample to an anti-PRO antibody, where the and determining binding of said antibody to a component of said sample.
19. A method of diagnosing an immune related disease in a mammal, said method comprising detecting the level of expression of a gene encoding a PRO polypeptide of the invention as described in Figures 1-2517 (SEQ ID NOS: 1-2517), (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower level of expression of said gene in the test sample as compared to the control sample is indicative of the presence of an immune related disease in the mammal from which the test tissue cells were obtained.
20. A method of diagnosing an immune related disease in a mammal, said method comprising (a) contacting a PRO polypeptide of the invention as described in Figures 1-2517 (SEQ ID
NOS: 1-2517), anti-PRO antibody with a test sample of tissue cells obtained from said mammal and (b) detecting the formation of a complex between the antibody and the polypeptide in the test sample, wherein formation of said complex is indicative of the presence of an immune related disease in the mammal from which the test tissue cells were obtained.
21. A method of identifying a compound that inhibits the activity of a PRO polypeptide of the invention as described in Figures 1-2517 (SEQ ID NOS: 1-2517), said method comprising contacting cells which normally respond to said polypeptide with (a) said polypeptide and (b) a candidate compound, and determining the lack responsiveness by said cell to (a).
22. A method of identifying a compound that inhibits the expression of a gene encoding a PRO polypeptide of the invention as described in Figures 1-2517 (SEQ ID NOS: 1-2517), said mediod comprising contacting cells which normally express said polypeptide with a candidate compound, and determining the lack of expression said gene.
23. The method of Claim 22, wherein said candidate compound is an antisense nucleic acid.
24. A method of identifying a compound that mimics the activity of a PRO polypeptide of the invention as described in any one of Figures 1-2517 (SEQ ID NOS: 1-2517), said method comprising contacting cells which normally respond to said polypeptide with a candidate compound, and determining the responsiveness by said cell to said candidate compound.
25. A method of stimulating the immune response in a mammal, said method comprising administering to said mammal an effective amount of a PRO polypeptide of the invention as described in any one of Figures 1-2517 (SEQ ID NOS: 1-2517), antagonist, wherein said immune response is stimulated.
26. A method of diagnosing an inflammatory immune response in a mammal, said method comprising detecting the level of expression of a gene encoding a PRO polypeptide of the invention as described in any one of Figures 1-2517 (SEQ ID NOS: 1-2517), (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower level of expression of said gene in the test sample as compared to the control sample is indicative of the presence of an inflammatory immune response in the mammal from which the test tissue cells were obtained.
27. A method of differentiating monocytes comprising; (a) isolating a population of monocytes;
(b) contacting the monocytes with an effective amount of a PRO polypeptide of the invention as described in any of of Figures 1-2517 (SEQ ID NOS: 1-2517); and
(c) determining the differentiation of said monocytes to said PRO polypeptide.
PCT/US2003/034312 2002-11-01 2003-10-30 Compositions and methods for the treatment of immune related diseases WO2004041170A2 (en)

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