WO2001092331A2 - Compositions et methodes de traitement de maladies d'origine immune - Google Patents
Compositions et methodes de traitement de maladies d'origine immune Download PDFInfo
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- WO2001092331A2 WO2001092331A2 PCT/US2001/017092 US0117092W WO0192331A2 WO 2001092331 A2 WO2001092331 A2 WO 2001092331A2 US 0117092 W US0117092 W US 0117092W WO 0192331 A2 WO0192331 A2 WO 0192331A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
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 in 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.
- T lymphocytes are an important component of a mammalian immune response. T cells recognize antigens which are associated with a self-molecule encoded by genes within the major histocompatibility complex (MHC). The antigen may be displayed together with MHC molecules on the surface of antigen presenting cells, virus infected cells, cancer cells, grafts, etc. The T cell system eliminates these altered cells which pose a health threat to the host mammal. T cells include helper T cells and cytotoxic T cells. Helper T cells proliferate extensively following recognition of an antigen -MHC complex on an antigen presenting cell. Helper T cells also secrete a variety of cytokines, i.e., lymphokines, which play a central role in the activation of B cells, cytotoxic T cells and a variety of other cells which participate in the immune response.
- MHC major histocompatibility complex
- helper T cell activation is initiated by the interaction of the T cell receptor (TCR) - CD3 complex with an antigen-MHC on the surface of an antigen presenting cell. This interaction mediates a cascade of biochemical events that induce the resting helper T cell to enter a cell cycle (the GO to Gl transition) and results in the expression of a high affinity receptor for IL-2 and sometimes IL-4.
- TCR T cell receptor
- the activated T cell progresses through the cycle proliferating and differentiating into memory cells or effector cells.
- activation of T cells involves additional costimulation induced by cytokines released by the antigen presenting cell or through interactions with membrane bound molecules on the antigen presenting cell and the T cell.
- the cytokines JX-1 and E -6 have been shown to provide a costimulatory signal.
- the interaction between the B7 molecule expressed on the surface of an antigen presenting cell and CD28 and CTLA-4 molecules expressed on the T cell surface effect T cell activation.
- Activated T cells express an increased number of cellular adhesion molecules, such as ICAM-1, integrins, VLA-4, LFA-1, CD56, etc.
- T-cell proliferation in a mixed lymphocyte culture or mixed lymphocyte reaction is an established indication of the ability of a compound to stimulate the immune system.
- MLR mixed lymphocyte reaction
- inflammatory cells infiltrate the site of injury or infection.
- the migrating cells may be neutrophilic, eosinophilic, monocytic or lymphocytic as can be determined by histologic examination of the affected tissues.
- Immune related diseases could be treated by suppressing the immune response. Using neutralizing antibodies that inhibit molecules having immune stimulatory activity would be beneficial in the treatment of immune-mediated and inflammatory 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.
- 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 either stimulate or inhibit 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 T-lymphocytes in a mammal in need thereof in response to an antigen, (d) stimulating the activity of T-lymphocytes 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 T-lymphocytes or (d) decreasing the proliferation of T-lymphocytes 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 polyneuropafhy, hepato
- 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;
- container containing said composition a 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: (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
- 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. In a preferred aspect, this method comprises the steps of:
- 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 for increasing the infiltration of inflammatory cells from the vasculature into a tissue of 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 infiltration of inflammatory cells from the vasculature in the mammal is increased.
- the invention provides a method for decreasing the infiltration of inflammatory cells from the vasculature into a tissue of 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 infiltration of inflammatory cells from the vasculature in the mammal is decreased.
- the invention provides a method of increasing the activity of T- lymphocytes 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 T-lymphocytes in the mammal is increased.
- the invention provides a method of decreasing the activity of T- lymphocytes 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 T-lymphocytes in the mammal is decreased.
- the invention provides a method of increasing the proliferation of T- lymphocytes 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 T- lymphocytes in the mammal is increased.
- the invention provides a method of decreasing the proliferation of T- lymphocytes 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 T- lymphocytes in the mammal is decreased.
- the invention provides a method of enhancing the infiltration of mononuclear cells, eosinophils or polymorphonuclear neutrophils (PMN) into a tissue of a mammal, said method comprising contacting said tissue with a PRO 1030 polypeptide or agonist thereof, wherein said infiltration is enhanced.
- the invention provides a method of inhibiting the inflammatory immune response, said method comprising contacting a T-cell line containing a reporter construct with a PR0221, PR0617, or PRO4302 polypeptide or agonist thereof, wherein said inflammatory immune response is inhibited and measured.
- 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 9
- 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 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.
- FIG. 1 shows a nucleotide sequence (SEQ ID NO:l) of a native sequence PR0221 cDNA, wherein SEQ ID NO:l is a clone designated herein as "DNA33089-1132".
- Figure 2 shows the amino acid sequence (SEQ ID NO:2) derived from the codihg sequence of SEQ ID NO:l shown in Figure 1.
- Figure 3 shows a nucleotide sequence (SEQ ID NO:3) of a native sequence PR0617 cDNA, wherein SEQ ID N0:3 is a clone designated herein as "DNA48309-1280".
- Figure 4 shows the amino acid sequence (SEQ ID N0:4) derived from the coding sequence of SEQ ID NO:3 shown in Figure 3.
- Figure 5 shows a nucleotide sequence (SEQ ID N0:5) of a native sequence PRO1030 cDNA, wherein SEQ ID NO:5 is a clone designated herein as "DNA59485- 1336" .
- Figure 6 shows the amino acid sequence (SEQ ID NO:6) derived from the coding sequence of SEQ ID NO:5 shown in Figure 5.
- Figure 7 shows a nucleotide sequence (SEQ ID NO:7) of a native sequence PRO4302 cDNA, wherein SEQ ID NO:7 is a clone designated herein as "DNA92218-2554".
- Figure 8 shows the amino acid sequence (SEQ ID NO:8) derived from the coding sequence of SEQ
- 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.
- 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 isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
- 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.
- PRO polypeptide also includes variants of the PRO/number polypeptides disclosed herein.
- a “native sequence PRO polypeptide” comprises a polypeptide having the same amino acid 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 variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide.
- 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 amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream 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.
- 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 acid encoding them, are contemplated by the present invention.
- 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.
- a PRO polypeptide variant will have 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 full-length
- 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, alternatively at least about 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 (%) amino acid sequence identity with respect to the PRO polypeptide sequences identified herein is defined as the percentage 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 necessary, 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 available 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.
- % 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 documentation 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.
- the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y
- Tables 2 and 3 demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "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.
- a % amino acid sequence identity 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.
- 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)).
- 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.
- the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y
- 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 identity 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.
- a PRO variant polynucleotide will have 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 9
- 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, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more.
- Percent (%) nucleic acid sequence identity 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 determining percent nucleic acid sequence identity can be achieved in various 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.
- % nucleic 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 documentation 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.
- the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows:
- 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.
- 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.
- 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)).
- 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.
- % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows:
- 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.
- 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 acid 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.
- 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.
- 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 include a promoter, optionally an operator sequence, and a ribosome binding site.
- Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
- Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
- 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.
- "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
- 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, antagonist, and neutralizing antibodies
- anti-PRO antibody compositions with polyepitopic specificity single chain anti-PRO antibodies
- fragments of anti-PRO antibodies see below.
- the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are 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 % poly vinylpyrrolidone/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 ⁇ g/ml), 0.1% SDS, and 10% dextran sul
- Modely stringent conditions may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory 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.
- washing solution and hybridization conditions e.g., temperature, ionic strength and %SDS
- 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.
- 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 other 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).
- immunoadhesin designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains.
- 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.
- 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.
- 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.
- agonist is used in the broadest sense and includes 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
- 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.
- 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 dexfrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
- buffers such as phosphate, citrate, and other organic acids
- antioxidants including ascorbic acid
- proteins such as serum albumin
- Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
- 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.
- 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 V H -V L 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 bind antigen, altliough 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 thiol 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.
- 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.
- immunoglobulins 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 V H and V domains of antibody, wherein these domains are present in a single polypeptide chain.
- the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
- a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
- 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.
- 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.
- 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.
- solid phase is meant a non-aqueous matrix to which the antibody of the present invention can adhere.
- solid phases encompassed herein include those formed partially or entirely of glass
- 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.
- 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.
- T cell mediated disease means a disease in which T cells directly or indirectly mediate or otherwise contribute to a morbidity in a mammal.
- the T cell mediated disease may be associated with cell mediated effects, lymphokine mediated effects, etc., and even effects associated with B cells if the B cells are stimulated, for example, by the lymphokines secreted by T cells.
- immune-related and inflammatory diseases examples include systemic lupus erythematosis, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sj ⁇ gren'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
- an “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.
- 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.
- cytotoxic agent 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., I 131 , I 125 , Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
- chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
- 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, Rh ⁇ ne-Poulenc Rorer, Antony, France), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carmino
- 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.
- the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase.
- 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.
- DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
- DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
- cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
- cytokines 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; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-oc and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor; transforming growth factors (TGFs) such as TGF- and TGF- ⁇ ; insulin-like growth factor-I and
- growth hormone
- immunoadhesin designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains.
- 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.
- immunoglobulin such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
- inflammatory cells designates cells that enhance the inflammatory response such as mononuclear cells, eosinophils, macrophages, and polymorphonuclear neutrophils (PMN).
- filel and file2 are two dna or two protein sequences.
- 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
- the program may create a tmp file in /tmp to hold info about traceback.
- dumpblockO dump a block of lines with numbers, stars: pr_align() * nums() — put out a number line: dumpblockO
- *po[i] *ps[i]; if (islower(*ps[i]))
- *ps[i] toupper(*ps[i]); po[i]++; ps[i]++;
- *py++ *px; else if (islower(*px))
- *py++ toupper(*px); if (index("ATGCU",*(py-l))) natgc++;
- 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.
- cDNAs encoding various PRO polypeptides have been identified and isolated, as disclosed in further detail in the Examples below. It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed.
- PRO/number 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.
- 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.
- 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.
- 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 determined 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- terminus 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 die desired termini of the DNA fragment are employed at the 5' and 3 'primers in die PCR. Preferably, PRO polypeptide fragments share at least one biological and/or immunological activity with the native PRO polypeptide disclosed herein.
- PCR polymerase chain reaction
- 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.
- 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;
- 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.
- preferred scanning amino acids are relatively small, neutral amino acids.
- 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, Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
- 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.
- crosslinking agents include, e.g., l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3 -dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
- 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.
- 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.
- 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. Enzymol.. 138:350 (1987).
- PRO polypeptide
- nonproteinaceous polymers e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes
- PEG polyethylene glycol
- polypropylene glycol polypropylene glycol
- 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.
- 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.
- tag polypeptides and their respective antibodies are well known in the art.
- poly-histidine poly-his
- poly-histidine-glycine poly-his-gly
- 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., J. Biol. Chem.,
- the chimeric molecule may comprise a fusion of the PRO with an immunoglobulin or a particular region of an immunoglobulin.
- an immunoglobulin also referred to as an "immunoadhesin”
- 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.
- the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGl molecule.
- PRO sequence or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques
- 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.
- 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
- Probes such as antibodies to the PRO or oligonucleotides of at least about 20-80 bases
- 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
- 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 is well known in the art, and include the use of radiolabels like 32 P-labeled ATP, biotinylation or enzyme labeling.
- Radiolabels like 32 P-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, principles, 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, CaCl 2 , CaP0 4 , liposome-mediated and electroporation.
- transformation is performed using standard techniques 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.
- 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.
- polycations e.g., polybrene, polyornithine.
- 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).
- 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. aeruginosa, 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.
- 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 kan r ;
- E. coli W3110 strain 37D6 which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan r ;
- 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.
- in vitro methods of cloning e.g., PCR or other nucleic acid polymerase reactions, are suitable.
- 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. Bacterio 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. drosophilarum (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.
- Candida Trichoderma 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. Biophys. Res. Commun..
- 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.
- invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.
- 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.
- mice sertoli cells TM4, Mather, Biol. Reprod.. 23:243-251 (1980)
- human lung cells W138, ATCC CCL 75
- human liver cells Hep G2, HB 8065
- mouse mammary tumor MMT 060562, ATCC CCL51. The selection of the appropriate host cell is deemed to be within the skill in the art.
- 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.
- a replicable vector for cloning (amplification of the DNA) or for expression.
- 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 recombinantly 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.
- 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.
- 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.
- the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces ⁇ - 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.
- 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 2 ⁇ 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.
- 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.
- the trpl 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 ⁇ -lactamase and lactose promoter systems [Chang et al., Nature. 275:615 (1978); Goeddel et al., Nature.
- trp tryptophan
- Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.
- suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al., J. Biol.
- glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 -phosphogly cerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
- 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, cytomegalovirus, 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.
- 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, cytomegalovirus, a retrovirus, he
- 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, oc-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 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.
- 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.
- 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 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.
- 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.
- a suitable detergent solution e.g. Triton-X 100
- 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.
- 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,
- 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.
- 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.
- 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 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.
- the activity of the PRO polypeptides can be further verified by antibody binding studies, in which the ability of anti-PRO antibodies to inhibit 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 hereinbelow.
- 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.
- 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.
- 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.
- 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).
- sandwich assay is an ELIS A assay, in which case the detectable moiety is an enzyme.
- the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
- 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.
- 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 T-cell 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.
- transgenic animals 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 al, Mol. Cell. Biol. 5: 642-648 [1985]).
- MLR mixed lymphocyte reaction
- T cells Since the majority of T cells respond to and produce IL-2 upon activation, differences in responsiveness in this assay in part reflect differences in IL-2 production by the responding cells.
- the MLR results can be verified by a standard lymphokine (IL-2) detection assay. Current Protocols in Immunology, above, 3.15, 6.3.
- IL-2 lymphokine
- a proliferative T cell response in an MLR assay may be due to direct mitogenic properties of an assayed molecule or to external antigen induced activation. Additional verification of the T cell stimulatory activity of the PRO polypeptides can be obtained by a costimulation assay.
- T cell activation requires an antigen specific signal mediated through the T-cell receptor (TCR) and a costimulatory signal mediated through a second ligand binding interaction, for example, the B7 (CD80, CD86)/CD28 binding interaction.
- CD28 crosslinking increases lymphokine secretion by activated T cells.
- T cell activation has both negative and positive controls through the binding of ligands which have a negative or positive effect.
- CD28 and CTLA-4 are related glycoproteins in the Ig superfamily which bind to B7. CD28 binding to B7 has a positive costimulation effect of T cell activation; conversely, CTLA-4 binding to B7 has a T cell deactivating effect. Chambers, C. A. and Allison, J. P., Curr. Opin.
- PRO polypeptides as well as other compounds of the invention, which are stimulators (costimulators) of T cell proliferation and agonists, e.g., agonist antibodies, thereto as determined by MLR and costimulation assays, for example, are useful in treating immune related diseases characterized by poor, suboptimal or inadequate immune function. These diseases are treated by stimulating the proliferation and activation of T cells (and T cell mediated immunity) and enhancing the immune response in a mammal through administration of a stimulatory compound, such as the stimulating PRO polypeptides.
- the stimulating polypeptide may, for example, be a PRO polypeptide or an agonist antibody thereof. Direct use of a stimulating compound as in the invention has been validated in experiments with 4-
- 1BB glycoprotein a member of the tumor necrosis factor receptor family, which binds to a ligand (4-1BBL) expressed on primed T cells and signals T cell activation and growth. Alderson, M. E. et al, J. Immunol. (1994) 24:2219.
- an agonist stimulating compound has also been validated experimentally. Activation of 4- IBB 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.
- An immune stimulating or enhancing effect can also be achieved by antagonizing or blocking the activity of a PRO which has been found to be inhibiting in the MLR assay. Negating the inhibitory activity of the compound produces a net stimulatory effect.
- Suitable antagonists/blocking compounds are antibodies or fragments thereof which recognize and bind to the inhibitory protein, thereby blocking the effective interaction of the protein with its receptor and inhibiting signaling through the receptor. This effect has been validated in experiments using anti-CTLA-4 antibodies which enhance T cell proliferation, presumably by removal of the inhibitory signal caused by CTLA-4 binding. Walunas, T. L. et al, Immunity (1994) 1:405.
- an immune stimulating or enhancing effect can also be achieved by administration of a PRO which has vascular permeability enhancing properties.
- Enhanced vacuolar permeability would be beneficial to disorders which can be attenuated by local infiltration of immune cells (e.g., monocytes, eosinophils, PMNs) and inflammation.
- immune cells e.g., monocytes, eosinophils, PMNs
- PRO polypeptides, as well as other compounds of the invention which are direct inhibitors of T cell 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.
- compounds 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 T cell mediated immune response by inhibiting T cell proliferation/activation and/or lymphokine secretion. Blocking the stimulating effect of the polypeptides suppresses the immune response of the mammal.
- This use has been validated in experiments using an anti-IL2 antibody. In these experiments, the antibody binds to IL2 and blocks binding of IL2 to its receptor thereby achieving a T cell inhibitory effect.
- the results of the cell based in vitro assays can be further verified using in vivo animal models and assays for T-cell 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 T cell reactivity against MHC antigens and minor transplant antigens. A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.3.
- An animal model for skin allograft rejection is a means of testing the ability of T cells to mediate in vivo tissue destruction and a measure of their role in transplant rejection.
- the most common and accepted models use murine tail-skin grafts.
- Repeated experiments have shown that skin allograft rejection is mediated by T cells, helper T cells and killer-effector T cells, and not antibodies.
- a suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.4.
- transplant rejection models which can be used to test the compounds of the invention are the allogeneic heart transplant models described by Tanabe, M. et al, Transplantation (1994) 58:23 and Tinubu, S. A. et al, J. Immunol. (1994) 4330-4338.
- Delayed type hypersensitivity reactions are a T cell mediated in vivo immune response characterized by inflammation which does not reach a peak until after a period of time has elapsed after challenge with an antigen. These reactions also occur in tissue specific autoimmune diseases such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE, a model for MS). A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.5.
- MS multiple sclerosis
- EAE experimental autoimmune encephalomyelitis
- EAE is a T cell mediated autoimmune disease characterized by T cell and mononuclear cell inflammation and subsequent demyelination of axons in the central nervous system.
- EAE is generally considered to be a relevant animal model for MS in humans. Bolton, C, Multiple Sclerosis (1995) 1:143. Both acute and relapsing-remitting models have been developed.
- the compounds of the invention can be tested for T cell stimulatory or inhibitory activity against immune mediated demyelinating disease using the protocol described in Current Protocols in Immunology, above, units 15.1 and 15.2. See also the models for myelin disease in which oligodendrocytes or Schwann cells are grafted into the central nervous system as described in Duncan, I. D. et al, Molec.
- 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.
- 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) .
- An animal model for arthritis is collagen-induced arthritis. This model shares clinical, histological and immunological characteristics of human autoimmune rheumatoid arthritis and is an acceptable model for human autoimmune arthritis.
- Mouse and rat models are characterized by synovitis, erosion of cartilage and subchondral bone.
- the compounds of the invention can be tested for activity against autoimmune arthritis using the protocols described in Current Protocols in Immunology, above, units 15.5. See also the model using a monoclonal antibody to CD18 and VLA-4 integrins described in Issekutz, A.C. et at, Immunology (1996) 88:569.
- a model of asthma has been described in which antigen-induced airway hyper-reactivity, pulmonary eosinophilia and inflammation are induced by sensitizing an animal with ovalbumin and then challenging the animal with the same protein delivered by aerosol.
- Several animal models (guinea pig, rat, non-human primate) show symptoms similar to atopic asthma in humans upon challenge with aerosol antigens.
- Murine models have many of the features of human asthma. Suitable procedures to test the compounds of the invention for activity and effectiveness in the treatment of asthma are described by Wolyniec, W. W. et al, Am. J. Respir. Cell Mol. Biol. (1998) 18:777 and the references cited therein.
- the compounds of the invention can be tested on animal models for psoriasis like diseases. Evidence suggests a T cell pathogenesis for psoriasis.
- the compounds of the invention can be tested in the scid/scid mouse model described by Schon, M. P. et al, Nat. Med. (1997) 3:183, in which the mice demonstrate histopathologic skin lesions resembling psoriasis.
- Another suitable model is the human skin/scid mouse chimera prepared as described by Nickoloff, B. J. et al, Am. J. Path. (1995) 146:580.
- 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.
- 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.
- 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 techniques 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 T cell 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.
- "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.
- 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.
- flanking DNA typically, several kilobases 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.
- the immuno stimulating compounds of the invention can be used in immunoadjuvant therapy for the treatment of tumors (cancer). It is now well established that T cells recognize human tumor specific antigens.
- T cells recognize human tumor specific antigens.
- DeSmet, C. et al are the immunoadjuvant therapy for the treatment of tumors.
- 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 T cell 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.
- 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.
- 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-immunoglobulin 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.
- the interaction is binding and the complex formed can be isolated or detected in the reaction mixture.
- the polypeptide encoded by die gene identified herein or the drug 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.
- 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.
- the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected.
- the detection of label immobilized on the surface indicates that complexing occurred.
- complexing can be detected, for example, by using a labelled antibody specifically binding the immobilized complex.
- 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.
- assays include traditional approaches, such as, cross-linking, co- immunoprecipitation, and co-purification through gradients or chromatographic columns.
- 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.
- 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-lacZ 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 ⁇ - galactosidase.
- a complete kit (MATCHMAKERTM) 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.
- 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.
- the reaction is run in the absence and in the presence of the test compound.
- 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.
- 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, T cell proliferation/activation, lymphokine release, or immune cell infiltration.
- antisense RNA and RNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
- antisense DNA 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 techniques. 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 deoxy nucleotides.
- 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.
- base pairing rules which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
- the present invention further provides anti-PRO antibodies.
- Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate 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, the 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.
- immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
- 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.
- 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
- a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
- the lymphocytes may be immunized in vitro.
- the immunizing agent will typically include the PRO polypeptide or a fusion protein thereof.
- PBLs peripheral blood lymphocytes
- 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.
- the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
- HGPRT hypoxanthine guanine phosphoribosyl transferase
- Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications. Marcel Dekker, Inc., New York, (1987) pp. 51-63].
- the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO.
- 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).
- RIA radioimmunoassay
- ELISA enzyme-linked immunoabsorbent assay
- 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).
- 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.
- 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 the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
- the monoclonal antibodies 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.
- 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.
- 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., supra] or by co valently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
- non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody 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.
- the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
- 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 non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
- CDR complementary determining region
- 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.
- 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)].
- Fc immunoglobulin constant region
- 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.
- 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 non-human species.
- 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)].
- 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.
- 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.
- Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
- one of the binding specificities is for the PRO, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
- bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two 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 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 die site necessary for light- chain binding present in at least one of the fusions.
- CHI first heavy-chain constant region
- the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
- the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
- one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
- Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
- Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared can be prepared using chemical linkage. Brennan et al, Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
- TAB thionitrobenzoate
- 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 humanized 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.
- 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.
- 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 (V ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain.
- V H and VL domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
- 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, trispecif ⁇ c antibodies can be prepared. Tutt et al, J. Immunol. 147:60 (1991).
- bispecific antibodies may bind to two different epitopes on a given PRO polypeptide herein.
- 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 (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular 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.
- 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).
- Heteroconjugate antibodies are also within the scope of the present invention.
- Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089].
- the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
- 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
- the antibody of the invention 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.
- 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, 1 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.
- 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).
- the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
- a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
- Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
- a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
- Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein- coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
- SPDP N-succinimidyl
- 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 W094/11026.
- 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).
- a "receptor” such streptavidin
- a ligand e.g., avidin
- cytotoxic agent e.g., a radionucleotide
- 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
- M. Pharmaceutical Compositions such as Doxorubicin
- the active PRO molecules of the invention can be administered 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; resorcinol; 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,
- 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.
- the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent.
- 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-(methylmefhacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
- colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
- macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
- the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
- sustained-release preparations 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.
- sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
- copolymers of L-glutamic acid and ⁇ -efhyl-L- glutamate non-degradable ethylene-vinyl acetate
- degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
- poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
- encapsulated antibodies 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 sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. N. Methods of Treatment
- polypeptides, antibodies and other active compounds of the present invention may be used to treat various immune related diseases and conditions, such as T cell mediated diseases, including those characterized by infiltration of inflammatory cells into a tissue, stimulation of T- cell proliferation, inhibition of T-cell proliferation, increased or decreased vascular permeability or the inhibition thereof.
- T cell mediated diseases including those characterized by infiltration of inflammatory cells into a tissue, stimulation of T- cell proliferation, inhibition of T-cell proliferation, increased or decreased vascular permeability or the inhibition 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), Sj ⁇ gren'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, tubul
- T lymphocytes have not been shown to be directly involved in tissue damage, T lymphocytes are required for the development of auto-reactive antibodies. The genesis of the disease is thus T lymphocyte dependent. Multiple organs and systems are affected clinically including kidney, lung, musculoskeletal system, mucocutaneous, eye, central nervous system, cardiovascular system, gastrointestinal tract, bone marrow and blood.
- Rheumatoid arthritis 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 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.
- 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 monropenia, thrombocytopenia and splenomegaly. This can be accompanied by vasculitis in multiple organs with formations of infarcts, skin ulcers and gangrene.
- RA rheumatoid nodules
- pericarditis pleuritis
- coronary arteritis intestitial pneumonitis with pulmonary fibrosis
- keratoconjunctivitis sicca and rhematoid nodules.
- 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.
- the cell most implicated as key to induction of the disease is the CD8+ T lymphocyte, a cell which targets antigen presented by class I MHC molecules.
- CD8+ T cells may react against the class I MHC allele HLA- B27 as if it were a foreign peptide expressed by MHC class I molecules.
- 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.
- 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.
- Sj ⁇ gren'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.
- Vasculitides 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.
- 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.
- MLNS mucocutaneous lymph node syndrome
- isolated CNS vasculitis Behet's disease
- thromboangiitis obliterans Buerger's disease
- 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
- 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 thyroiditis, 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).
- Type I diabetes mellitus or insulin-dependent diabetes is the autoimmune destruction of pancreatic islet ⁇ cells; this destruction is mediated by auto-antibodies and auto-reactive T cells.
- Antibodies to insulin or the insulin receptor can also produce the phenotype of insulin-non-responsiveness.
- Immune mediated renal diseases are the result of antibody or T lymphocyte mediated injury to renal tissue either directly as a result of the production of autoreactive antibodies or T cells against renal antigens or indirectly as a result of the deposition of antibodies and/or immune complexes in the kidney that are reactive against other, non-renal antigens.
- immune-mediated diseases that result in the formation of immune-complexes can also induce immune mediated renal disease as an indirect sequelae.
- Both direct and indirect immune mechanisms result in inflammatory response that produces/induces lesion development in renal tissues with resultant organ function impairment and in some cases progression to renal failure. Both humoral and cellular immune mechanisms can be involved in the pathogenesis of lesions.
- Demyelinating diseases of the central and peripheral nervous systems including Multiple Sclerosis; idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome; and Chronic Inflammatory Demyelinating Polyneuropathy, are believed to have an autoimmune basis and result in nerve demyelination as a result of damage caused to oligodendrocytes or to myelin directly.
- MS there is evidence to suggest that disease induction and progression is dependent on T lymphocytes.
- Multiple Sclerosis is a demyelinating disease that is T lymphocyte-dependent and has either a relapsing-remitting course or a chronic progressive course.
- T lymphocyte mediated, microglial cells and infiltrating macrophages CD4+ T lymphocytes are the predominant cell type at lesions.
- the mechanism of oligodendrocyte cell death and subsequent demyelination is not known but is likely T lymphocyte driven.
- 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.
- Autoimmune or Immune-mediated Skin Disease including Bullous Skin Diseases, Erythema Multiforme, and Contact Dermatitis are mediated by auto-antibodies, the genesis of which is T lymphocyte- dependent.
- Psoriasis is a T lymphocyte-mediated inflammatory disease. Lesions contain infiltrates of T lymphocytes, macrophages and antigen processing cells, and some neutrophils.
- Allergic diseases including asthma; allergic rhinitis; atopic dermatitis; food hypersensitivity; and urticaria are T lymphocyte dependent. These diseases are predominantly mediated by T lymphocyte induced inflammation, IgE mediated-inflammation or a combination of both.
- Transplantation associated diseases including Graft rejection and Graft-Versus-Host-Disease
- GVHD T lymphocyte-dependent; inhibition of T lymphocyte function is ameliorative.
- 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 MLR can be utilized therapeutically to enhance the immune response to infectious agents), diseases of immunodeficiency (molecules/derivatives/agonists) which stimulate the MLR 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.
- Molecules that inhibit the lymphocyte response in the MLR 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.
- 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 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, intracerobrospinal, subcutaneous, infra-articular, intrasynovial, intrathecal, oral, topical, or inhalation (intranasal, intrapulmonary) routes. Intravenous or inhaled adminisfration of polypeptides and antibodies is preferred.
- an anti-cancer agent may be combined with the administration of the proteins, antibodies or compounds of the instant invention.
- the patient to be treated with a the immunoadjuvant of the invention may also receive an anti-cancer agent (chemotherapeutic agent) or radiation therapy.
- chemotherapeutic agent chemotherapeutic agent
- Preparation and dosing schedules for such chemotiierapeutic 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.
- 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.
- the PRO polypeptides are coadministered with a growth inhibitory agent.
- the growth inhibitory agent may be administered first, followed by a PRO polypeptide.
- 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.
- 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.
- about 1 ⁇ g/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 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
- the treatment is sustained until a desired suppression of disease symptoms occurs.
- other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
- an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above.
- 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.
- 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.
- 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.
- Cell surface proteins such as proteins which are overexpressed in certain immune related diseases, are excellent targets for drug candidates or disease freatment.
- 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.
- 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.
- antibodies 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 other 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.
- 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.
- polypeptide-encoding nucleic acid sequences were identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc. (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e.g., GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, CA) databases.
- the signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration.
- the nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons.
- the second is not examined. If neither meets the requirement, the candidate sequence is not scored.
- the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals. Use of this algorithm resulted in the identification of numerous polypeptide-encoding nucleic acid sequences.
- EXAMPLE 2 Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNA Encoding Therefor
- the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases.
- the EST databases included public databases (e.g., Dayhoff, GenBank), and proprietary databases (e.g. LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA).
- the search was performed using the computer program BLAST or BLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons with a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap” (Phil Green, University of Washington, Seattle, WA).
- Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length.
- the probe sequences are typically 40-55 bp in length.
- additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 KB.
- DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
- the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA.
- the cDNA was primed with oligo dT containing a NotI site, linked with blunt to Sail hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al, Science. 253:1278-1280 (1991)) in the unique Xhol and NotI sites.
- a suitable cloning vector such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al, Science. 25
- EXAMPLE 3 Isolation of cDNA Clones Using Amylase Yeast Screening.
- RNA was used to generate an oligo dT primed cDNA library in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System). In this procedure, the double stranded cDNA was sized to greater than 1000 bp and the Sall/NotI tinkered cDNA was cloned into XhoI/NotI cleaved vector.
- pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sfil restriction enzyme site preceding the XhoI/NotI cDNA cloning sites.
- a secondary cDNA library was generated in order to preferentially represent the 5 'ends of the primary cDNA clones.
- Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY.O using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above). In this procedure the double stranded cDNA was sized to 500-1000 bp, linkered with blunt NotI adaptors, cleaved with Sfil, and cloned into Sfil/Notl cleaved vector.
- pSST-AMY.O is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDNA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites.
- cDNAs cloned into this vector that are fused in frame with the amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies.
- DNA from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was then elecfroporated as recommended by the manufacturer. Subsequently, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37°C for 30 minutes. The transformants were then plated onto 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37°C). Positive colonies were scraped off the plates and the DNA was isolated from the bacterial pellet using standard protocols, e.g. CsCl-gradient. The purified DNA was then carried on to the yeast protocols below.
- the yeast methods were divided into three categories: (1) Transformation of yeast with the plasmid/cDNA combined vector; (2) Detection and isolation of yeast clones secreting amylase; and (3) PCR amplification of the insert directly from the yeast colony and purification of the DNA for sequencing and further analysis.
- yeast strain used was HD56-5A (ATCC-90785). This strain has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-l 12, his3-l 1, his3-15, MAL + , SUC + , GAL + .
- yeast mutants can be employed that have deficient post-translational pathways. Such mutants may have translocation deficient alleles in seel I, secll, sec62, with truncated seel I being most preferred.
- antagonists including antisense nucleotides and/or ligands which interfere with the normal operation of these genes, other proteins implicated in this post translation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJlp or SSAlp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast.
- the cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li 2 OOCCH 3 ), and resuspended into LiAc/TE (2.5 ml).
- LiAc/TE 10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li 2 OOCCH 3
- Transformation took place by mixing the prepared cells (100 ⁇ l) with freshly denatured single stranded salmon testes DNA (Lof strand Labs, Gaithersburg, MD) and transforming DNA (1 ⁇ g, vol. ⁇ 10 ⁇ l) in microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 ⁇ l, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li 2 OOCCH 3 , pH 7.5) was added. This mixture was gently mixed and incubated at 30°C while agitating for 30 minutes.
- PEG/TE 600 ⁇ l, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li 2 OOCCH 3 , pH 7.5
- the cells were then heat shocked at 42°C for 15 minutes, and the reaction vessel cenfrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TE (500 ⁇ l, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followed by recentrifugation. The cells were then diluted into TE (1 ml) and aliquots (200 ⁇ l) were spread onto the selective media previously prepared in 150 mm growth plates (VWR).
- VWR 150 mm growth plates
- the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly.
- the selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al, Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 208-210 (1994). Transformants were grown at 30°C for 2-3 days.
- the detection of colonies secreting amylase was performed by including red starch in the selective growth media.
- Starch was coupled to the red dye (Reactive Red-120, Sigma) as per the procedure described by Biely et al, Anal. Biochem.. 172: 176-179 (1988).
- the coupled starch was incorporated into the SCD- Ura agar plates at a final concentration of 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0 (50-100 mM final concentration).
- the positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies.
- Well isolated single colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD-Ura agar. Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly.
- PCR was then performed as follows: a. Denature 92°C, 5 minutes b. 3 cycles of: Denature 92°C, 30 seconds
- the underlined regions of the oligonucleotides disclosed above annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY.O when no insert was present.
- the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers.
- the total product of the PCR reaction from an empty vector was 343 bp.
- signal sequence-fused cDNA resulted in considerably longer nucleotide sequences.
- a cDNA sequence of interest isolated in the above screen is identified. Probes were then generated from the sequence of the DNA molecule and used to screen a human library prepared as described in paragraph 1 above.
- the cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al, Science, 253:1278-1280 (1991)), and the cDNA size cut was less than 2800 bp.
- the oligonucleotides probes were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence.
- Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length.
- the probe sequences are typically 40-55 bp in length.
- DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, supra, with the PCR primer pair.
- a positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
- a full length clone was identified that contained a single open reading frame with an apparent translational initiation site and a stop signal.
- This assay shows that certain PRO polypeptides stimulate an immune response and induce inflammation by inducing mononuclear cell, eosinophil and PMN infiltration at the site of injection of the animal.
- This skin vascular permeability assay is conducted as follows. Hairless guinea pigs weighing 350 grams or more are anesthetized with ketamine (75-80 mg/Kg) and 5 mg/Kg Xylazine intramuscularly (IM). A sample of purified PRO polypeptide or a conditioned media test sample is injected intradermally onto the backs of the test animals with 100 ⁇ L per injection site. It is possible to have about 10-30, preferably about 16-24, injection sites per animal.
- Evans blue dye 1% in physiologic buffered saline
- Blemishes at the injection sites are then measured (mm diameter) at lhr, 6 hrs and 24 hrs post injection. Blemishes that are greater than 0 mm diameter or negative control injection are considered positive. Animals were sacrificed at the appropriate time after injection. Each skin injection site is biopsied and fixed in paraformaldehyde. The skins are then prepared for histopathalogic evaluation. At least a minimal perivascular infiltrate at the injection site is scored as positive, no infiltrate at the site of injection is scored as negative. Results are given in Table 8.
- PRO polypeptides are active as inhibitors of the inflammatory immune response.
- the PRO polypeptides are screened for inhibition of NF-kB which will identify novel suppressors of inflammatory immune responses.
- Jurkat cells (E6-1) [ATCC No. TIB-152] are transfected with a NF-kB- firefly luciferase reporter plasmid [Sfratagene] and co-ttansfected with a confrol renilla luciferase pRL-TK [Promega] plasmid. 15 hours post-transfection cells are seeded with PRO polypeptides [at 1% and 0.1% of the stock PRO polypeptide concentrations]. After a 1 hour incubation, TNF-alpha [0.25 ng/ml] is added.
- a molecule is considered a hit when the ratio of firefly luciferase to Renilla luciferase is 70% or less than that of the blank wells without SPDI molecules added, or the firefly luciferase is 70% or less of that of the blank wells without SPDI molecules added.
- a molecule is assigned a score of "1" when it is a hit and a score of "0" when it is not a hit. Results are shown in Table 9.
- EXAMPLE 7 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 O.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 techniques known in the art.
- EXAMPLE 8 Expression of PRO in E. coli
- This example illustrates preparation of an unglycosylated form of PRO by recombinant expression in 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 resfriction 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 tefracycline resistance.
- the vector is digested with resfriction 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 franscriptional 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.
- the cells After culturing the cells for several more hours, the cells can be harvested by centtifugation.
- 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 resfriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, 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(lacIq).
- 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 (NH 4 ) 2 S0 4 , 0.71 g sodium cittate*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 MgS0 4 ) and grown for approximately 20-30 hours at 30°C with shaking.
- CRAP media prepared by mixing 3.57 g (NH 4 ) 2 S0 4 , 0.71 g sodium cittate*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,
- 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 concenfration 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 concenfration 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 concenfration of 0.4% (pH of approximately 3).
- 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.
- 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.
- 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 nitrogen 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.
- 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.
- the PRO DNA is ligated into pRK5 with selected resfriction 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.
- 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 ⁇ g pRK5-PRO DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene [Thimmappaya et al, Cell. 31:543 (1982)] and dissolved in 500 ⁇ l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl 2 .
- the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml 35 S-cysteine and 200 ⁇ Ci ml 35 S- methionine.
- culture medium alone
- culture medium containing 200 ⁇ Ci/ml 35 S-cysteine and 200 ⁇ Ci ml 35 S- 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.
- 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 ⁇ g pRK5-PRO DNA is added. The cells are first concentrated from the spinner flask by cenfrifugation and washed with PBS. The DNA-dexttan 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 ⁇ g/ml bovine insulin and 0.1 ⁇ g/ml bovine fransferrin. After about four days, the conditioned media is cenfrifuged 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.
- PRO in another embodiment, can be expressed in CHO cells.
- the pRK5-PRO can be transfected into CHO cells using known reagents such as CaP0 or DEAE-dextran.
- the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35 S-methionine.
- the culture medium may be replaced with serum free medium.
- 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 Baculo virus 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.
- 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 Ni 2+ -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.
- 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 fransfection.
- Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available fransfection 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 ⁇ m filtered PS20 with 5% 0.2 ⁇ m 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 10 5 cells/mL.
- the cell media is exchanged with fresh media by centrifugation and resuspension in production medium.
- a 3L production spinner is seeded at 1.2 x 10 6 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 ⁇ m filter. The filtrate was either stored at 4°C or immediately loaded onto columns for purification.
- 10% antifoam e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion
- the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concenfration 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.
- Ni-NTA column Qiagen
- 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 ⁇ l 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.
- EXAMPLE 10 Expression of PRO in Yeast
- the following method describes recombinant expression of PRO in yeast.
- yeast expression vectors are constructed for infracellular 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 infracellular expression of PRO.
- 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
- yeast cells 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% frichloroacetic 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.
- the following method describes recombinant expression of PRO in Baculo virus-infected insect cells.
- the sequence coding for PRO is fused upstream of an epitope tag contained within a baculo virus expression vector.
- epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG).
- plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen).
- sequence encoding PRO or the desired portion of the coding sequence of PRO such as the sequence encoding the exfracellular 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 the 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-fransfecting the above plasmid and BaculoGoldTM virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin
- 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 Ni 2+ -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).
- Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl 2 ; 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 ⁇ m filter.
- a Ni 2+ -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 A 28 o 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 A 28 o baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer.
- a secondary wash buffer 50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0
- IgG tagged (or Fc tagged) PRO can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
- EXAMPLE 12 Preparation of Antibodies that Bind PRO This example illustrates preparation of monoclonal antibodies which can specifically bind PRO.
- 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 infraperitoneally in an amount from 1-100 micrograms.
- the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research,
- 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.
- 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 infraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO monoclonal antibodies.
- 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 13 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 SEPHAROSETM (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.
- a chromatographic resin such as CnBr-activated SEPHAROSETM (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).
- 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).
- 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 chaofrope such as urea or thiocyanate ion), and PRO polypeptide is collected.
- a low pH buffer such as approximately pH 2-3
- a high concentration of a chaofrope such as urea or thiocyanate ion
- This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a variety of drug 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 inttacellularly.
- One method of drug 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.
- 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.
- 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 drug 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 witi ⁇ 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 drug 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 drug 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 15 Rational Drug Design
- the goal of rational drug 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)).
- 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 sfructure 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 sfructure of homologous proteins. In both cases, relevant sttuctural 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 sfructure. 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.
- anti-ids anti-idiotypic antibodies
- 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.
- PRO polypeptide may be made available to perform such analytical studies as X-ray crystallography.
- 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.
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Abstract
Priority Applications (274)
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AU2001265019A AU2001265019A1 (en) | 2000-05-30 | 2001-05-25 | Compositions and methods for the treatment of immune related diseases |
AU6531101A AU6531101A (en) | 2000-06-02 | 2001-05-31 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
CA002591583A CA2591583A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
CA002591590A CA2591590A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
PCT/US2001/017800 WO2001093983A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
EP04005726A EP1489095A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptide, acides nucléiques le codant, et leur utilisation pour le diagnostic du cancer |
EP05024028A EP1686174A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides sécrétés et transmembranaires, ainsi que les acides nucléiques codant pour ceux-ci |
EP05024038A EP1702928A3 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secrétées ou transmembranaire, ainsi que les acides nucléiques qui les codent |
CA002591656A CA2591656A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
EP05024037A EP1700867A3 (fr) | 2000-06-02 | 2001-06-01 | Polypeptide secrétée et transmembranaire, ainsi que les acides nucléiques qui les codent |
EP05024031A EP1666492A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci |
EP05024035A EP1666594A3 (fr) | 2000-06-02 | 2001-06-01 | Polypeptide, acides nucléiques le codant, et leur utilisation pour le diagnostic du cancer |
CA002591929A CA2591929A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
CA002594659A CA2594659A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
EP05024025A EP1666597A3 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci |
EP01939834A EP1286749A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
CA002591930A CA2591930A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
EP05024033A EP1666493A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci |
JP2002501551A JP2004510409A (ja) | 2000-06-02 | 2001-06-01 | 分泌及び膜貫通ポリペプチドとそれをコードする核酸 |
AU2001265311A AU2001265311A1 (en) | 2000-06-02 | 2001-06-01 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
EP05024032A EP1659177A3 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci |
EP05024027A EP1666596A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci |
EP05024030A EP1666491A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci |
CA002410162A CA2410162A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptide pro 4332 marqueur de tumeurs du poumon, du colon, du rectum et du sein et acide nucleique codant pour ce polypeptide |
EP05024034A EP1666497A3 (fr) | 2000-06-02 | 2001-06-01 | Polypeptide, acides nucléiques le codant, et leur utilisation pour le diagnostic du cancer |
CA002591630A CA2591630A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
CA002585822A CA2585822A1 (fr) | 2000-06-02 | 2001-06-01 | Marqueur polypeptidique pro19628 pour tumeur du sein, du rectum, du colon ou du poumon et acide nucleique encodant |
EP05024023A EP1683864A3 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci |
CA002591841A CA2591841A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
CA002591814A CA2591814A1 (fr) | 2000-06-02 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
EP09004418A EP2075253A1 (fr) | 2000-06-23 | 2001-06-20 | Méthodes et composés pour la diagnose et le traitement de troubles associés à l'angiogenèse |
EP01957073A EP1309620A2 (fr) | 2000-06-23 | 2001-06-20 | Compositions et procedes de diagnose et traitement de maladies faisant intervenir l'angiogenese |
CA002648048A CA2648048A1 (fr) | 2000-06-23 | 2001-06-20 | Compositions et procedes de diagnostic et de traitement de troubles dont l'angiogenese |
AU2001278852A AU2001278852A1 (en) | 2000-06-23 | 2001-06-20 | Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis |
EP09004415A EP2168980A1 (fr) | 2000-06-23 | 2001-06-20 | Compositions et procédés pour le traitement et le diagnostic des troubles impliquant une angiogenèse |
CA002648051A CA2648051A1 (fr) | 2000-06-23 | 2001-06-20 | Compositions et procedes de diagnostic et de traitement de troubles dont l'angiogenese |
CA2709771A CA2709771A1 (fr) | 2000-06-23 | 2001-06-20 | Compositions et methode de diagnostic et de traitement des troubles mettant en cause l'angiogenese |
JP2002505812A JP2004506413A (ja) | 2000-06-23 | 2001-06-20 | 血管形成に関与する疾患の診断と治療のための組成物と方法 |
CA002648046A CA2648046A1 (fr) | 2000-06-23 | 2001-06-20 | Compositions et procedes de diagnostic et de traitement de troubles dont l'angiogenese |
PCT/US2001/019692 WO2002000690A2 (fr) | 2000-06-23 | 2001-06-20 | Compositions et procedes de diagnostic et de traitement de troubles dont l'angiogenese |
EP09004417A EP2077276A1 (fr) | 2000-06-23 | 2001-06-20 | Compositions et procédés pour le traitement et le diagnostic des troubles impliquant une angiogenèse |
CA002412211A CA2412211A1 (fr) | 2000-06-23 | 2001-06-20 | Compositions et procedes de diagnostic et de traitement de troubles dont l'angiogenese |
PCT/US2001/021066 WO2002008288A2 (fr) | 2000-07-20 | 2001-06-29 | Polypeptides secretes et transmembranaires et acides nucleiques codant ces polypeptides |
AU2001273150A AU2001273150A1 (en) | 2000-07-20 | 2001-06-29 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
CA002416456A CA2416456A1 (fr) | 2000-07-20 | 2001-06-29 | Polypeptides secretes et transmembranaires et acides nucleiques codant ces polypeptides |
JP2002514192A JP2004514420A (ja) | 2000-07-20 | 2001-06-29 | 分泌及び膜貫通ポリペプチドとそれをコードする核酸 |
EP01952391A EP1354040A2 (fr) | 2000-07-20 | 2001-06-29 | Polypeptides secretes et transmembranaires et acides nucleiques codant ces polypeptides |
AU7315001A AU7315001A (en) | 2000-07-20 | 2001-07-02 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
EP01951036A EP1309685A2 (fr) | 2000-07-20 | 2001-07-09 | COMPOSITIONs ET PROCEDEs DE DIAGNOSE ET TRAITEMENT DE MALADIES IMPLICANT ANGIOGENESIS |
AU2001271973A AU2001271973A1 (en) | 2000-07-20 | 2001-07-09 | Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis |
CA002416538A CA2416538A1 (fr) | 2000-07-20 | 2001-07-09 | Compositions et methodes diagnostiques et therapeutiques de troubles impliquant l'angiogenese |
PCT/US2001/021735 WO2002008284A2 (fr) | 2000-07-20 | 2001-07-09 | Compositions et methodes diagnostiques et therapeutiques de troubles impliquant l'angiogenese |
JP2002514188A JP2004516013A (ja) | 2000-07-20 | 2001-07-09 | 血管形成に関連する障害を診断及び治療するための組成物と方法 |
US09/918,585 US20030060406A1 (en) | 1997-10-17 | 2001-07-30 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
CA002632702A CA2632702A1 (fr) | 2000-09-01 | 2001-08-29 | Polypeptides secretes transmembranaires et acides nucleiques codant pour ceux-ci |
EP07022176A EP1944317A3 (fr) | 2000-09-01 | 2001-08-29 | Polypeptides sécrétés et transmembranaires, et acides nucléiques les codant |
PCT/US2001/027099 WO2002024888A2 (fr) | 2000-09-01 | 2001-08-29 | Polypeptides secretes transmembranaires et acides nucleiques codant pour ceux-ci |
AU2002216610A AU2002216610A1 (en) | 2000-09-01 | 2001-08-29 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
EP01985272A EP1341814A2 (fr) | 2000-09-01 | 2001-08-29 | Polypeptides secretes transmembranaires et acides nucleiques codant pour ceux-ci |
CA002421056A CA2421056A1 (fr) | 2000-09-01 | 2001-08-29 | Polypeptide servant de marqueur diagnostique de presence de tumeurs du colon |
JP2002529483A JP4451059B2 (ja) | 2000-09-01 | 2001-08-29 | 分泌及び膜貫通ポリペプチドとそれをコードする核酸 |
AU1661002A AU1661002A (en) | 2000-09-01 | 2001-09-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,193 US20030073624A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,299 US20030199435A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,194 US20030195333A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,189 US6972325B2 (en) | 1997-10-17 | 2001-10-15 | PRO273 polypeptides |
US09/978,192 US20020177553A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,298 US20030134785A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,191 US20030050239A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,295 US20020156006A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,188 US20030139328A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,585 US20030049633A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,681 US20030195148A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,403 US20030050240A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,665 US7294700B2 (en) | 1997-10-17 | 2001-10-16 | Anti-PRO846 antibodies |
US09/978,757 US20030083248A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,697 US20020169284A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,375 US7196165B2 (en) | 1997-10-17 | 2001-10-16 | PRO363 polypeptides |
US09/978,643 US20030104998A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/981,915 US20030054986A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,423 US20030069178A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,802 US20030199674A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,564 US7195760B2 (en) | 1997-10-17 | 2001-10-16 | Anti-pro363 antibodies |
US09/981,915 US7285623B2 (en) | 1997-10-17 | 2001-10-16 | PRO337 polypeptides |
US09/978,544 US20030199436A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,824 US20030055216A1 (en) | 1997-10-17 | 2001-10-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/210,028 US20030203446A1 (en) | 1998-10-07 | 2001-10-18 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/152,388 US20040223964A1 (en) | 1998-03-17 | 2001-10-18 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/164,749 US20040029218A1 (en) | 1998-10-07 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/165,067 US7279553B2 (en) | 1998-05-13 | 2001-10-19 | PRO1083 polypeptides |
US10/165,247 US7112657B2 (en) | 1998-10-07 | 2001-10-19 | PRO697 polypeptides |
US10/145,124 US20030190701A1 (en) | 1998-04-30 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/164,829 US20030194780A1 (en) | 1998-04-29 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/164,929 US20030194781A1 (en) | 1998-03-30 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/145,017 US20030186365A1 (en) | 1998-03-26 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/164,728 US20030186368A1 (en) | 1998-05-13 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/165,036 US20050227342A1 (en) | 1998-10-07 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/143,029 US7105640B2 (en) | 1997-10-17 | 2001-10-19 | Anti-pro792 antibodies |
US10/160,502 US7220835B2 (en) | 1998-07-30 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/145,089 US7208575B2 (en) | 1998-10-07 | 2001-10-19 | PRO531 polypeptides |
US10/162,521 US7067628B2 (en) | 1998-03-17 | 2001-10-19 | PRO788 polypeptides |
US10/166,709 US20030104536A1 (en) | 1998-10-07 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,922 US20030195345A1 (en) | 1997-10-17 | 2001-10-21 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,083 US20030148376A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/999,832 US7132283B2 (en) | 1997-10-17 | 2001-10-24 | PRO273 polypeptides |
US09/999,834 US20030064407A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,081 US20030049684A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/999,833 US6916648B2 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/002,967 US20030148373A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/999,830 US20030077700A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,084 US20030203402A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,085 US6974696B2 (en) | 1997-10-17 | 2001-10-24 | PRO853 nucleic acids |
US10/020,445 US20030198994A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,086 US7122375B2 (en) | 1997-10-17 | 2001-10-24 | PRO274 nucleic acids |
US09/999,832 US20020192706A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/999,829 US20030195344A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,926 US7074593B2 (en) | 1998-04-01 | 2001-10-25 | PRO 703 nucleic acids |
US10/013,927 US7189529B2 (en) | 1997-10-17 | 2001-10-25 | PRO792 nucleic acids |
US10/013,925 US7037710B2 (en) | 1997-10-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,917 US7029874B2 (en) | 1998-03-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/016,177 US20030073131A1 (en) | 1997-10-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,923 US7169912B2 (en) | 1997-10-17 | 2001-10-25 | PRO1017 nucleic acids |
US10/013,929 US7019124B2 (en) | 1997-10-17 | 2001-10-25 | PRO788 nucleic acids |
US10/013,920 US20040006219A1 (en) | 1997-10-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,921 US20030068648A1 (en) | 1997-10-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,928 US20030215905A1 (en) | 1998-10-07 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,918 US20030211091A1 (en) | 1997-10-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,456 US20020137890A1 (en) | 1997-03-31 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/001,054 US20020192209A1 (en) | 1997-09-17 | 2001-11-30 | Methods and compositions for inhibiting neoplastic cell growth |
US09/978,187 US20030096744A1 (en) | 1997-10-17 | 2002-01-28 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/081,056 US20040043927A1 (en) | 1997-09-19 | 2002-02-20 | Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis |
US10/119,480 US20040087769A1 (en) | 1998-09-10 | 2002-04-09 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,041 US20030077776A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,045 US20030073210A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,040 US20030082759A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,046 US20030194791A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,047 US20030077778A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,051 US20030092147A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,042 US20030096386A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,044 US20030190717A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,059 US20030190721A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,052 US20030199052A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,048 US20030199051A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,055 US20030190718A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,049 US20030022239A1 (en) | 1997-06-18 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,043 US7220831B2 (en) | 1997-03-31 | 2002-04-12 | PRO235 polypeptides |
US10/121,058 US20030190720A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,062 US20030077779A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,050 US20030054516A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,060 US20030190722A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,063 US20030199055A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,057 US20030190719A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,056 US20030082760A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,053 US20030199053A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,054 US20030199054A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,061 US20030082761A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,108 US7635478B2 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,262 US20030049816A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,291 US20030199058A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,154 US20030190724A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,235 US20030082762A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,212 US7276577B2 (en) | 1997-03-31 | 2002-04-15 | PRO1866 polypeptides |
US10/123,236 US20030068795A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,157 US20030190725A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,214 US7343721B2 (en) | 1997-03-31 | 2002-04-15 | PRO4406 polypeptide |
US10/123,261 US20030068796A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,155 US20030068794A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,292 US20030073211A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,213 US7193048B2 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,215 US7291329B2 (en) | 1997-03-31 | 2002-04-15 | Antibodies against PRO4406 |
US10/123,322 US20030199059A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,213 US20030199057A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,109 US20030190723A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,156 US20030194792A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,771 US20030199060A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,907 US7084258B2 (en) | 1997-03-31 | 2002-04-16 | Antibodies against the PRO862 polypeptides |
US10/123,910 US7329404B2 (en) | 1997-03-31 | 2002-04-16 | Antibodies against PRO1310 |
US10/123,913 US20030203462A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,905 US7285625B2 (en) | 1997-06-18 | 2002-04-16 | PRO536 polypeptides |
US10/123,902 US20030077781A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,906 US20030190726A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,903 US20030073212A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,911 US7408032B2 (en) | 1997-03-31 | 2002-04-16 | PRO1188 polypeptides |
US10/123,912 US20030100087A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,909 US7193049B2 (en) | 1997-03-31 | 2002-04-16 | PRO862 polypeptides |
US10/123,905 US20030087344A1 (en) | 1997-06-18 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,904 US20030022328A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,908 US7335728B2 (en) | 1997-03-31 | 2002-04-16 | PRO1310 polypeptides |
US10/124,816 US20030190728A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,813 US7312307B2 (en) | 1997-03-31 | 2002-04-17 | PRO1056 polypeptides |
US10/124,823 US20030199062A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,824 US20030077659A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/125,795 US7304131B2 (en) | 1997-03-31 | 2002-04-17 | PRO1483 polypeptides |
US10/125,704 US7357926B2 (en) | 1997-03-31 | 2002-04-17 | Antibodies against PRO1879 and the use thereof |
US10/124,818 US20030082763A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,822 US7109305B2 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,817 US20030077786A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/125,805 US20030194794A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,820 US20030190729A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,819 US7285626B2 (en) | 1997-03-31 | 2002-04-17 | PRO1076 polypeptides |
US10/124,814 US7105335B2 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,821 US20030199023A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/125,924 US7342097B2 (en) | 1997-03-31 | 2002-04-19 | PRO1309 polypeptides |
US10/125,927 US20030190731A1 (en) | 1997-03-31 | 2002-04-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/125,922 US7309762B2 (en) | 1997-03-31 | 2002-04-19 | PRO1360 polypeptides |
US10/125,932 US7317079B2 (en) | 1997-03-31 | 2002-04-19 | PRO812 polypeptides |
US10/125,931 US20030199063A1 (en) | 1997-03-31 | 2002-04-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/127,831 US20030082689A1 (en) | 1997-03-31 | 2002-04-22 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/128,689 US20030087365A1 (en) | 1997-03-31 | 2002-04-23 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/131,823 US7304132B2 (en) | 1997-03-31 | 2002-04-24 | PRO1693 polypeptides |
US10/131,817 US7291701B2 (en) | 1997-03-31 | 2002-04-24 | PRO1777 polypeptides |
US10/131,825 US7282566B2 (en) | 1997-03-31 | 2002-04-24 | PRO1779 polypeptide |
US10/137,868 US20030082764A1 (en) | 1997-03-31 | 2002-05-03 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/137,865 US20030032155A1 (en) | 1997-03-31 | 2002-05-03 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/137,867 US20030207349A1 (en) | 1997-03-31 | 2002-05-03 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/139,963 US7288625B2 (en) | 1997-03-31 | 2002-05-06 | PRO4395 polypeptides |
US10/139,980 US7247710B2 (en) | 1997-03-31 | 2002-05-06 | PRO4395 antibodies |
US10/140,023 US20030207416A1 (en) | 1997-03-31 | 2002-05-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,470 US20030022331A1 (en) | 1997-03-31 | 2002-05-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,020 US20030207415A1 (en) | 1997-03-31 | 2002-05-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,474 US20030032156A1 (en) | 1997-03-31 | 2002-05-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,024 US20040058424A1 (en) | 1997-03-31 | 2002-05-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,865 US20030207420A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,925 US20030073215A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,805 US20030207417A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,808 US7425621B2 (en) | 1997-03-31 | 2002-05-07 | Antibodies against the PRO4401 polypeptide |
US10/140,921 US7317080B2 (en) | 1997-03-31 | 2002-05-07 | PRO4303 polypeptides |
US10/140,928 US20030068798A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,809 US20030207418A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,864 US20030207419A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,860 US7307151B2 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/141,756 US7488586B2 (en) | 1997-03-31 | 2002-05-08 | PRO4409 polypeptides |
US10/141,754 US7361732B2 (en) | 1997-03-31 | 2002-05-08 | PRO4400 polypeptides |
US10/141,755 US7297764B2 (en) | 1997-03-31 | 2002-05-08 | PRO4318 polypeptides |
US10/141,760 US7342104B2 (en) | 1997-03-31 | 2002-05-08 | Antibodies against the PRO4320 polypeptide |
US10/141,701 US20030207421A1 (en) | 1997-03-31 | 2002-05-08 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/143,114 US20030036180A1 (en) | 1997-03-31 | 2002-05-09 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/142,417 US7304133B2 (en) | 1997-03-31 | 2002-05-09 | PRO4389 polypeptides |
US10/142,430 US7309766B2 (en) | 1997-03-31 | 2002-05-09 | PRO5774 polypeptides |
US10/143,113 US7329730B2 (en) | 1997-03-31 | 2002-05-09 | PRO4348 polypeptides |
US10/142,425 US20030207424A1 (en) | 1997-03-31 | 2002-05-09 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/143,032 US7408033B2 (en) | 1997-03-31 | 2002-05-10 | PRO5995 polypeptides |
US10/142,431 US7285629B2 (en) | 1997-03-31 | 2002-05-10 | Pro5005 polypeptides |
US10/142,423 US20030049817A1 (en) | 1997-03-31 | 2002-05-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/142,419 US7153941B2 (en) | 1997-03-31 | 2002-05-10 | Antibodies that bind PRO4994 polypeptides |
US10/146,730 US20030207427A1 (en) | 1997-03-31 | 2002-05-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/146,792 US20030207428A1 (en) | 1997-03-31 | 2002-05-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/147,528 US20030219885A1 (en) | 1997-03-31 | 2002-05-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/147,492 US20030082765A1 (en) | 1997-03-31 | 2002-05-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/147,536 US20040077064A1 (en) | 1997-03-31 | 2002-05-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/147,519 US20030077791A1 (en) | 1997-03-31 | 2002-05-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/152,395 US7189534B2 (en) | 1997-03-31 | 2002-05-21 | PRO4320 polynucleotide |
US10/153,934 US20030129695A1 (en) | 1997-03-31 | 2002-05-22 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/156,843 US20030207805A1 (en) | 1997-06-18 | 2002-05-28 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/157,782 US20030077792A1 (en) | 1997-03-31 | 2002-05-29 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/157,786 US20030208055A1 (en) | 1997-03-31 | 2002-05-29 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/158,782 US20030082766A1 (en) | 1997-03-31 | 2002-05-30 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/158,791 US20030207429A1 (en) | 1997-03-31 | 2002-05-30 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/160,498 US20030073216A1 (en) | 1997-03-31 | 2002-05-30 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/176,913 US20030022298A1 (en) | 1997-09-15 | 2002-06-20 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/197,942 US20030175882A1 (en) | 1998-09-10 | 2002-07-18 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/232,225 US20030096969A1 (en) | 2000-06-02 | 2002-08-29 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/242,505 US20030138898A1 (en) | 1998-12-01 | 2002-09-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/244,990 US20030113852A1 (en) | 2000-09-01 | 2002-09-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/805,667 US20050037458A1 (en) | 1999-03-10 | 2004-07-23 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US11/341,175 US7468427B2 (en) | 1997-03-31 | 2006-01-27 | Antibodies to PRO1275 polypeptide |
JP2006024604A JP2006223304A (ja) | 2000-09-01 | 2006-02-01 | 分泌及び膜貫通ポリペプチドとそれをコードする核酸 |
JP2006266069A JP2007049999A (ja) | 2000-06-02 | 2006-09-28 | 分泌及び膜貫通ポリペプチドとそれをコードする核酸 |
US11/786,466 US20080182275A1 (en) | 1998-12-22 | 2007-04-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
JP2008194764A JP2009039119A (ja) | 2000-07-20 | 2008-07-29 | 血管形成に関連する障害を診断及び治療するための組成物と方法 |
JP2008194721A JP2009039118A (ja) | 2000-07-20 | 2008-07-29 | 血管形成に関連する障害を診断及び治療するための組成物と方法 |
JP2008194678A JP2009039117A (ja) | 2000-07-20 | 2008-07-29 | 血管形成に関連する障害を診断及び治療するための組成物と方法 |
JP2011173336A JP6033531B2 (ja) | 2000-07-20 | 2011-08-08 | 血管形成に関連する障害を診断及び治療するための組成物と方法 |
JP2014187275A JP2015037408A (ja) | 2000-07-20 | 2014-09-16 | 血管形成に関連する障害を診断及び治療するための組成物と方法 |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
USPCT/US00/14941 | 2000-05-30 | ||
PCT/US2000/014941 WO2000073348A2 (fr) | 1999-06-02 | 2000-05-30 | Procedes et compositions visant a inhiber la proliferation des cellules cancereuses |
PCT/US2000/015264 WO2000073452A2 (fr) | 1999-06-02 | 2000-06-02 | Compositions et methodes de traitement de maladies liees a l'immunite |
USPCT/US00/15264 | 2000-06-02 | ||
PCT/US2000/032678 WO2001040466A2 (fr) | 1999-12-01 | 2000-12-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant ces polypeptides |
USPCT/US00/32678 | 2000-12-01 |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/854,208 Continuation-In-Part US7217412B2 (en) | 1997-03-31 | 2001-05-10 | IL-17C related mammalian cytokine polypeptides |
US09/854,280 Continuation-In-Part US7115398B2 (en) | 1997-03-31 | 2001-05-10 | IL-17 homologous polypeptides and therapeutic uses thereof |
Related Child Applications (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US87203501A Continuation | 1996-11-06 | 2001-06-01 | |
PCT/US2001/017800 Continuation WO2001093983A1 (fr) | 1996-11-06 | 2001-06-01 | Polypeptides secretes et transmembranaires et acides nucleiques codant lesdits polypeptides |
US10/081,056 Continuation US20040043927A1 (en) | 1997-09-19 | 2002-02-20 | Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis |
US10/119,480 Continuation US20040087769A1 (en) | 1998-09-10 | 2002-04-09 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/197,942 Continuation US20030175882A1 (en) | 1998-03-27 | 2002-07-18 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
Publications (2)
Publication Number | Publication Date |
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WO2001092331A2 true WO2001092331A2 (fr) | 2001-12-06 |
WO2001092331A8 WO2001092331A8 (fr) | 2003-02-13 |
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PCT/US2001/017092 WO2001092331A2 (fr) | 1997-03-31 | 2001-05-25 | Compositions et methodes de traitement de maladies d'origine immune |
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JP2011207903A (ja) * | 2011-06-03 | 2011-10-20 | Jichi Medical Univ | ミトコンドリア膜タンパク質群およびそれらをコードする遺伝子群 |
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JP2011207903A (ja) * | 2011-06-03 | 2011-10-20 | Jichi Medical Univ | ミトコンドリア膜タンパク質群およびそれらをコードする遺伝子群 |
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