Definitions

• the present invention relates to compositions and methods 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-mediated inflammatory 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 recognise antigens which are associated with a self-molecule encoded by genes within the major histocompatibility complex (MHC).
• MHC major histocompatibility complex
• 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.
• helper T cell activation is initiate 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 G 1 transition) and results in the expression of a high affinity receptor for IL-2 and sometimes I -4 The activated T cell progresses through the cycle proliferating and differentiating into memory cells or effector cells
• T cells In addition to the signals mediated through the TCR, 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 IL- 1 and IL-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, mtegnns, 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
• inflammatory cells infiltrate the site of injury or infection
• the migrating cells may be neutrophihc, eosinophilic, monocytic or lymphocytic Histologic examination of the affected tissues provides evidence of an immune stimulating or inhibiting responseCurrent Protocols in Immunology, ed John E Coligan, 1994, John Wiley & Sons, Ine
• Immune related diseases can 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 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
• Such stimulatory- molecules can also be inhibited where suppression of the immune response would be of value
• Neutralizing antibodies are examples of molecules that inhibit molecules having immune stimulatory activity and which would be beneficial in the treatment of immune related and inflammatory diseases Molecules which inhibit the immune response can also be utilized (proteins directly or via the use of antibody agonists) to inhibit the immune response and thus ameliorate immune related disease
• the proteins of the invention encoded by the genes of the invention are useful for the diagnosis and/or treatment (including prevention) of immune related diseases.
• Antibodies which bind to stimulatory proteins are useful to suppress the immune system and the immune response.
• Antibodies which bind to inhibitory proteins are useful to stimulate the immune system and the immune response.
• the proteins and antibodies of the invention are also useful to prepare medicines and medicaments for the treatment of immune related and inflammatory diseases.
• the present invention concerns an isolated antibody which binds a PR0245, PR0217, PRO301, PR0266, PR0335. PR0331 or PR0326 polypeptide.
• the antibody mimics the activity of a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide (an agonist antibody) or conversely the antibody inhibits or neutralizes the activity of a PR0245, PR0217. PRO301, PR0266, PR0335, PR0331 or PR0326 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. The antibody may be labeled and may be immobilized on a solid support.
• the antibody is an antibody fragment, a single-chain antibody, or an anti-idiotypic antibody.
• the invention concerns a composition containing a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide or an agonist or antagonist antibody which binds the polypeptide in admixture with a carrier or excipient.
• the composition contains a therapeutically effective amount of the peptide or antibody.
• the composition when the composition contains 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, or (c) increasing the proliferation of T-lymphocytes in a mammal in need thereof in response to an antigen.
• the composition when the composition contains 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, or (c) decreasing the proliferation of T- lymphocytes in a mammal in need thereof in response to an antigen.
• the composition contains 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 the use of the polypeptides and antibodies of the invention to prepare a composition or medicament which has the uses described above.
• the invention concerns nucleic acid encoding an anti-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibody, and vectors and recombinant host cells comprising such nucleic acid.
• the invention concerns a method for producing such an antibody by culturing a host cell transformed with nucleic acid encoding the antibody under conditions such that the antibody is expressed, and recovering the
• the invention further concerns antagonists and agonists of a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide that inhibit one or more of the functions or activities of the PR0245, PR0217, PRO301. PR0266, PR0335, PR0331 or PR0326 polypeptide.
• the invention concerns isolated nucleic acid molecules that hybridize to the complement of the nucleic acid molecules encoding the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptides.
• the nucleic acid preferably is DNA, and hybridization preferably occurs under stringent conditions.
• Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein, which, in turn, can find use in the modulation of the respective amplified genes, or as antisense primers in amplification reactions.
• sequences can be used as part of ribozyme and/or triple helix sequence which, in turn, may be used in regulation of the amplified genes.
• the invention concerns a method for determining the presence of a PR0245, PR0217, PRO301, PR0266. PR0335, PR0331 or PR0326 polypeptide comprising exposing a cell suspected of containing the polypeptide to an anti-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibody and determining binding of the antibody to the cell.
• 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 PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 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 expression level in the test 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-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibody with a test sample of tissue cells obtained from the mammal , and (b) detecting the formation of a complex between the antibody and the PR0245, PR0217, PRO301, PR0266. PR0335, PR0331 or PR0326 polypeptide in the test sample.
• 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 of tumor 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 present invention concerns a diagnostic kit, containing an anti-
• PR0331 or PR0326 antibody and a carrier e.g. a buffer
• the kit preferably contains instructions for using the antibody to detect the PR0245, PR0217, PRO301, PR0266. PR0335. PR0331 or PR0326 polypeptide.
• the invention concerns an article of manufacture, comprising: a container; a label on the container; and a composition comprising an active agent contained within the container; wherein the composition is effective for stimulating or inhibiting an immune response in a mammal, the label on the container indicates that the composition can be used to treat an immune related disease, and the active agent in the composition is an agent stimulating or inhibiting the expression and/or activity of the PR0245, PR0217, PRO301, PR0266, PR0335. PR0331 or PR0326 polypeptide.
• the active agent is a PR0245, PR0217. PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide or an anti-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibody.
• a further embodiment is a method for identifying a compound capable of inhibiting the expression and/or activity of a PR0245. PR0217. PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide by contacting a candidate compound with a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide under conditions and for a time sufficient to allow these two components to interact.
• either the candidate compound or the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide is immobilized on a solid support.
• the non-immobilized component carries a detectable label.
• Figures 1 A-D and Table 4 show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity ( Figures 1 A-B) and % nucleic acid sequence identity ( Figures 1C-D) using the ALIGN-2 sequence comparison computer program, wherein "PRO” represents the amino acid sequence of a hypothetical polypeptide of the invention of interest, “Comparison Protein” represents the amino acid sequence of a polypeptide against which the "PRO” polypeptide of interest is being compared, “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, "X”, “Y” and “Z” each represent different hypothetical amino acid residues and "N", “L” and “V” each represent different hypothetical nucleotides.
• Figures 2A-P and Table 5 provide the complete source code for the ALIGN-2 sequence comparison computer program.
• This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program.
• Figure 3 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0245(UNQ219), wherein the nucleotide sequence (SEQ ID NO: 1) is a clone designated herein as "DNA35638" Also presented in bold font and underlined are the positions of the respective start and stop codons
• Figure 4 and Table 6 show the ammo acid sequence (SEQ ID NO 2) of a native sequence PR0245 polypeptide as derived from the coding sequence of Figure 3 Also shown are the approximate locations of vanous other important polypeptide domains if known
• Figure 5 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0217(UNQ 191 ), wherein the nucleotide sequence (SEQ ID NO 3) is a clone designated herein as "DNA33094" Also presented in bold font and underlined are the positions of the respective start and stop codons
• Figure 6 and Table 7 show the amino acid sequence (SEQ ID NO 4) of a native sequence PR0217 polypeptide as de ⁇ ved from the coding sequence of Figure 5 Also shown are the approximate locations of vanous other important polypeptide domains if known
• Figure 7 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PRO301(UNQ264), wherein the nucleotide sequence (SEQ ID NO 5) is a clone designated herein as "DNA40628" Also presented in bold font and underlined are the positions of the respective start and stop codons
• Figure 8 and Table 8 show the amino acid sequence (SEQ ID NO 6) of a native sequence PRO301 polypeptide as de ⁇ ved from the coding sequence of Figure 7 Also shown are the approximate locations of vanous other important polypeptide domams if known
• Figure 9 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0266 (UNQ233), wherein the nucleotide sequence (SEQ ID NO 7) is a clone designated herein as "DNA37150" Also presented in bold font and underlined are the positions of the respective start and stop codons
• Figure 10 and Table 9 show the amino acid sequence (SEQ ID NO 8) of a native sequence PR0266 polypeptide as de ⁇ ved from the coding sequence of Figure 9 Also shown are the approximate locations of vanous other important polypeptide domains if known
• Figure 11 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0335 (UNQ287V), wherein the nucleotide sequence (SEQ ID NO 9) is a clone designated herein as "DNA41388" Also presented in bold font and underlined are the positions of the respective start and stop codons
• Figure 12 and Table 10 show the ammo acid sequence (SEQ ID NO 10) of a native sequence PR0335 polypeptide as de ⁇ ved from the coding sequence of Figure 1 1 Also shown are the approximate locations of vanous other important polypeptide domains if known
• Figure 13 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0331 (UNQ292), wherein the nucleotide sequence (SEQ ID NO 1 1) is a clone designated herein as "DNA40981 " Also presented in bold font and underlined are the positions of the respective start and stop codons
• Figure 14 and Table 1 1 show the ammo acid sequence (SEQ ID NO 12) of a native sequence PR0331 polypeptide as de ⁇ ved from the coding sequence of Figure 13 Also shown are the approximate locations of vanous other important polypeptide domains if known
• Figure 15 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0326 (UNQ287), wherein the nucleotide sequence (SEQ ID NO 13) is a clone designated herein as "DNA37140" Also presented in bold font and underlined are the positions of the respective start and stop codons
• Figure 16 and Table 12 show the ammo acid sequence (SEQ ID NO 14) of a native sequence PR0331 polypeptide as de ⁇ ved from the coding sequence of Figure 15 Also shown are the approximate locations of vanous other important polypeptide domains if known
• immune related disease means a disease in which a component of the immune system of a mammal causes, mediates or otherwise contnbutes to a morbidity in the mammal
• 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 lymphokmes secreted by T cells
• immune-related and inflammatory diseases examples include systemic lupus erythematosis, rheumatoid arthntis, juvenile chronic arth ⁇ tis, spondyloarthropathies, systemic sclerosis (scleroderma).
• idiopathic inflammatory myopathies skin to muscle, muscle, muscle, muscle, muscle, muscle, muscle, muscle, muscle, muscle, muscle vascuhtis, 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.
• autoimmune hemolytic anemia immuno pancytopenia
• paroxysmal nocturnal hemoglobinuria paroxysmal nocturnal hemoglobinuria
• autoimmune thrombocytopenia idiopathic thrombocytopenic purpura. immune-mediated thrombocytopenia
• thyroiditis Gramve's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroid
• immune-mediated renal disease glomerulonephntis, tubulointerstitial neph ⁇ tis
• demvehnating diseases of the central and pe ⁇ pheral nervous systems such as multiple sclerosis, idiopathic demyehnatmg polyneuropathy or Guillam-Barre syndrome, and chronic inflammatory demyehnatmg polyneuropathy
• hepatobi ary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, pnmary biliary cirrhosis, granulomatous hepatitis, and sclerosmg cholangitis
• inflammatory and fibrotic lung diseases such as inflammatory bowel disease (ulcerative colitis Crohn's disease), gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact
• Treatment is an intervention performed with the intention of preventing the development or alte ⁇ ng the pathology of a disorder
• treatment refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those m which the disorder is to be prevented
• a therapeutic agent may directly decrease or increase the magnitude of response of a component of the immune response, or render the disease more susceptible to treatment by other therapeutic agents, e g antibiotics, antifungals, anti-mflammatory agents, chemotherapeutics, etc
• the "pathology" of an immune related disease includes all phenomena that compromise the well-being of the patient This includes, without limitation, abnormal or uncontrollable cell growth (neutrophihc, eosmophilic, monocytic, lymphocytic cells), antibody production, auto-antibody production, complement production, interference with the normal functioning of neighbonng cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of any inflammatory or immunological response, infiltration of inflammatory cells (neutrophihc. eosmophilic, monocytic, lymphocytic) into cellular spaces, etc
• 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, horses, cats, cows, etc Preferably, the mammal is human
• Administration "in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration m any order
• Carriers 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 earner is an aqueous pH buffered solution.
• physiologically acceptable earners 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 polyvinylpyrrohdone; amino acids such as glycme, glutamme, asparagine, arginme or lysine; monosaccha ⁇ des, disacchandes, and other carbohydrates including glucose, mannose, or dext ⁇ ns; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol, salt-forming countenons such as sodium; and/or nomonic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM
• 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 , 1 125 , Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacte ⁇ al, fungal, plant or animal ongin, or fragments thereof.
• chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
• chemotherapeutic agents include ad ⁇ amycm, doxorubicm, epirubicin, 5-fluorourac ⁇ l, cytosine arabmoside ("Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxm, taxoids, e.g.
• paclitaxel (Taxol, B ⁇ stol-Myers Squibb Oncology, P ⁇ nceton, NJ), and doxetaxel (Taxotere, Rhone- Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastme, bleomycm, etoposide, lfosfamide, mitomycin C. mitoxantrone, vmc ⁇ stine, vinorelbme, carboplatin, temposide, daunomycm, carmmomycm, ammoptenn.
• dactinomycin dactinomycin, mitomycins, esperamicins (see U.S. Pat. No. 4,675, 187), melphalan and other related nitrogen mustards.
• hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onap ⁇ stone.
• a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressmg 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 overexpressmg 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 (vincnstine and vinblastme), taxol, and topo II inhibitors such as doxorubicm, epirubicin, daunorubicm, etoposide, and bleomycm.
• DNA alkylating agents such as tamoxifen. pred sone, dacarbazine, mechlorethamme, cisplatin, methotrexate, 5-fluorouracil, and ara-C Further information can be found in The Molecular Basis of
• cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
• cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; 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-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; plate
• a "PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide” refers to a native sequence PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 having the same amino acid sequence as a PR0245, PR0217. PRO301, PR0266, PR0335, PR0331 or PR0326 derived from nature.
• Such native sequence PR0245, PR0217, PRO301, PR0266. PR0335, PR0331 or PR0326 can be isolated from nature or can be produced by recombinant and/or synthetic means.
• the term specifically encompasses naturally-occurring truncated or secreted forms (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326.
• the native sequence PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 is a mature or full- length native sequence PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 comprising the amino acid sequences shown in Figures 4, 6, 8, 10, 12, 14 and 16.
• polypeptide of the invention refers to each individual PR0245, PR0217, PRO301. PR0266, PR0335, PR0331 or PR0326 polypeptide. All disclosures in this specification which refer to the "polypeptide of the invention” or to "the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide" refer to each of the polypeptides individually as well as jointly. For example, descriptions of the preparation of, purification of.
• compound of the invention includes the polypeptide of the invention, as well as agonist antibodies for and antagonist antibodies to these polypeptide, peptides or small molecules having agonist or antagonist activity developed from the polypeptide, etc
• polypeptide of the invention also includes variants of the PR0245 PR0217. PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptides
• a "va ⁇ ant" polypeptide means an active polypeptide as defined below having at least about 80% ammo acid sequence identity with the amino acid sequence of the PR0245, PR0217. PRO301, PR0266.
• va ⁇ ant polypeptides include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and/or C-terminus, as well as within one or more internal domains
• a va ⁇ ant polypeptide will have at least about 80% ammo acid sequence identity, more preferably at least about 81 % amino acid sequence identity, more preferably at least about 82% ammo acid sequence identity, more preferably at least about 83% ammo acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% ammo acid sequence identity, more preferably at least about 88% ammo acid sequence identity, more preferably at least about 89% ammo acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least at least about
• va ⁇ ant polypeptides of the invention are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 ammo acids in length, more often at least about 40 amino acids in length, more often at least about 50 ammo acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids m length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids m length, more often at least about 300 ammo acids in length, or more
• Percent (%) amino acid sequence identity with respect to the polypeptide sequences identified herein is defined as the percentage of ammo acid residues in a candidate sequence that are identical with the ammo acid residues in a sequence of the PR0245, PR0217, PRO301.
• PR0266, PR0335, PR0331 or PR0326 polypeptides after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not conside ⁇ ng any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in vanous ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled in the art can determine approp ⁇ ate parameters for measu ⁇ ng alignment, including any algorithms needed to achieve maximal alignment over the full- length of the sequences being compared For purposes herein, however, % ammo acid sequence identity values are obtained as desc ⁇ bed below by using the sequence compa ⁇ son computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Figures 2A-P The ALIGN-2 sequence compa ⁇ son computer program was authored by Genentech, Ine and the source code
• the ALIGN-2 program is publicly available through Genentech, Ine , South San Francisco, California or may be compiled from the source code provided in Figures 2A-P
• the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D All sequence compa ⁇ son parameters are set by the ALIGN-2 program and do not vary
• % ammo acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
• % amino acid sequence identity values used herein are obtained as desc ⁇ bed above using the ALIGN-2 sequence companson computer program
• % ammo acid sequence identity may also be determined using the sequence companson program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res 25 3389-3402 (1997))
• % ammo acid sequence identity of a given ammo acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given ammo acid sequence A that has or comp ⁇ ses a certain % amino acid sequence identity to, with, or against a given ammo acid sequence B) is calculated as follows
• polypeptides of the invention are polypeptides which in the context of the amino acid sequence identity compansons performed as desc ⁇ bed above, include ammo acid residues in the sequences compared that are not only identical, but also those that have similar properties These polypeptides are termed "positives" Amino acid residues that score a positive value to an ammo acid residue of interest are those that are either identical to the ammo acid residue of interest or are a preferred substitution (as defined in Table 1 below) of the ammo acid residue of interest.
• the % value of positives of a given amino acid sequence A to, with, or against a given ammo acid sequence B (which can alternatively be phrased as a given ammo acid sequence A that has or comp ⁇ ses a certain % positives to, with, or against a given ammo acid sequence B) is calculated as follows
• invention vanant polynucleotide or " invention vanant nucleic acid sequence” means a nucleic acid molecule which encodes an active polypeptide of the invention as defined herein and which has at least about 80% nucleic acid sequence identity with the nucleotide acid sequence of DNA35638. DNA33094.
• an invention va ⁇ ant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity
• DNA40628, DNA37150, DNA41388, DNA40981. or DNA37140 will encode a polypeptide having an amino acid sequence which is identical to the ammo acid sequence of a PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 or the amino acid sequence encoded by the clones deposited with the ATCC desc ⁇ bed below
• invention va ⁇ ant polynucleotides are at least about 30 nucleotides m length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides m length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides m length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides m length, more often at least about 300 nucleotides m length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides m length, more often at least about 900 nucleotides in length, or more "Percent (%) nucleic acid sequence identity" with respect to the invention polypeptide- encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a
• 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, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full- length of the sequences being compared. For purposes herein, however, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Figures 2A-P. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc.
• the ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Figures 2A-P.
• 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.
• % 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:
• W is the number of nucleotides scored as identical matches by the sequence alignment program
• nucleic acid sequence identity is not equal to the length of nucleic acid sequence D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations.
• Figures 1C-D 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”.
• % 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 compa ⁇ son program may be downloaded from http //www ncbr nlm nih gov
• % 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
• invention va ⁇ ant polynucleotides are nucleic acid molecules that encode an active polypeptide of the invention and which are capable of hyb ⁇ dizmg, preferably under stnngent hyb ⁇ dization and wash conditions, to nucleotide sequences encoding the full-length invention polypeptide
• invention va ⁇ ant polypeptides include those that are encoded by an invention va ⁇ ant polynucleotide
• an "isolated" nucleic acid molecule encoding a polypeptide of the invention is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinanly associated in the natural source of the polypeptide-encodmg 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 nucleic acid molecules therefore are distinguished from the polypeptide- encodmg nucleic acid molecule as it exists in natural cells
• an isolated nucleic acid molecule encoding a polypeptide of the invention includes polypeptide-encodmg nucleic acid molecules contained m cells that ordinarily express a polypeptide of the invention 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 ot an operably linked coding sequence m a particular host organism
• the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence and a ⁇ bosome 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 preprotem that participates m the secretion of the polypeptide
• a promoter or enhancer is operably linked to a coding sequence if it affects the transc ⁇ ption of the sequence
• a ⁇ bosome 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. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient rest ⁇ ction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice
• “Stnngency" of hyb ⁇ dization reactions is readily determmable 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. Hybndization generally depends on the abrhty of denatured DNA to reanneal when complementary strands are present rn an environment below their melting temperature. The higher the degree of desired homology between the probe and hybndizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relatrve temperatures would tend to make the reaction conditions more stnngent, while lower temperatures less so. For additional details and explanation of stnngency of hyb ⁇ dization reactions, see Ausubel et al , Cmrent Protocols in Molecular Biology, Wiley Interscience Publishers, (1995)
• “Stnngent conditions” or “high stnngency 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 chlo ⁇ de/0.0015 M sodium c ⁇ trate/0 1% sodium dodecyl sulfate at 50C, (2) employ du ⁇ ng hybridization a denatu ⁇ ng agent, such as formamide, for example, 50% (v/v) formamide with 0 1% bovine serum album ⁇ n 0.1% F ⁇ coll/0 1% polyv ⁇ nylpyrrol ⁇ done/50mM sodium phosphate buffer at pH 6.5 wrth 750 mM sodium chlonde, 75 mM sodium citrate at 42C; 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, s
• Modely stnngent conditions may be identified as desc ⁇ bed by Sambrook et al , Molecular Cloning. A Laboratory Manual, New York Cold Spnng Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength and %SDS) less stnngent that those described above
• An example of moderately stringent conditions is overnight incubation at 37C m a solution compnsmg 20% formamide, 5 x SSC (150 mM NaCl, 15 mM tnsodium 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 37-50C
• the skilled artrsan wrll 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 chime ⁇ c polypeptide compnsmg a polypeptide of the invention 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 ammo acid residues (preferably, between about 10 and 20 ammo acid residues)
• activation or activity m the context of va ⁇ ants of the nolypeptide of the invention refers to form(s) of proteins of the invention which retain the biologic and or mimunologic activities of a native or naturally-occur ⁇ ng polypeptide of the invention
• Bio activity in the context of an antibody or another molecule that can be identified by the screening assays disclosed herein (e g an organic or inorganic small molecule, peptide, etc ) is used to refer to the abrlrty of such molecules to prise or rnhrbrt infiltration of inflammatory cells into a tissue, to stimulate or inhibit T-cell proliferation and to stimulate or inhibit lymphokine release by cells Another preferred activity is increased vascular permeability or the inhibition thereof
• antagonist is used in the broadest sense, and includes any molecule that partrally or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide of the invention disclosed herein
• agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or ammo acid sequence va ⁇ ants of native polypeptides of the invention , peptides, small organic molecules, etc
• a "small molecule” is defined herein to have a molecular weight below about 600 daltons
• Antibodies (Abs) and “immunoglobulins” (Igs) are glycoprotems having the same structural characte ⁇ stics While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules whrch lack antigen specificity
• polypeptides of the latter kind are. for example, produced at low levels by the lymph system and at increased levels by myelomas
• antibody is used m the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e g bispecific antibodres) formed from at least two tact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
• An anti- PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 antibody is an antibody which immunologically binds to a PR0245, PR0217, PRO301 , PR0266, PR0335. PR0331 or PR0326 polypeptide.
• the antibody may bind to any domain of the polypeptide of the invention which may be contacted by the antibody.
• the antibody may bind to any extracellular domain of the polypeptide and when the entire polypeptide is secreted, to any domain on the polypepetide which is available to the antibody for binding.
• “Native antibodies” and “native immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
• V H variable domain
• Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light- chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
• variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).
• CDRs complementarity-determining regions
• FR framework
• the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
• the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al, NIH Publ. No.91 -3242, Vol. I, pages 647-669 (1991)).
• the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
• variable region or “complementarity-determining regions” as used herein define a subregion within the variable region of extreme sequence variability of the antibody, which form the antigen-binding site and are the main determinants of antigen specificity. According to one definition, they can be, for example, residues (Kabat nomenclature) 24-34 (LI), 50-56 (L2) and
• the hypervariable region can be the "hypervariable loop", comprising, for example, residues (Chothia nomenclature) 26-32 (LI), 50-53 (L2), 91-96 (L3) in the light chain variable region and residue (Chothia nomenclature) 26- 32 (HI), 53-55 (L2) and 96-101 (L3); Chothia and Lesk, J. Mol. Biol. 196: 901-917 [1987].
• "Framework" or "FR" residues are those variable domain residues of relatively low sequence variability which lie in between the CDR regions.
• 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 multispecifc 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, whose PR0245, PR0217, PRO301, Pro266, pro335, pro331 or pro326 reflects its ability to crystallize readily.
• Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
• Fv is the minimum antibody fragment which contains a complete antigen-recognition and - binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VJJ-VL dimer.
• CDRs confer antigen-binding specificity to the antibody.
• 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, although at a lower affinity than the entire binding site.
• the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
• Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
• Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free 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 (K) 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.
• the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called called , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
• the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
• 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. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
• the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 [1975], or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
• the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature. 352:624-628 [1991] and Marks et al, J.
• the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA. 81:6851- 6855 [1984]).
• chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences
• 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 are human immunoglobulins (recipient antibody) in which residues from a complementanty-determinmg region (CDR) of the recipient are replaced by residues from a CDR of a non-human specres (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
• Fv framework regron (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues
• humamzed antibodies may compnse residues which are found neither m the recipient antibody nor in the imported CDR or framework sequences. These modrfications are made to further refine and maxrmize antibody performance.
• the humanized antibody will compnse substantially all of at least one, and typically two, vanable 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 sequence.
• the humanized antibody optimally also will compnse at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• the term "humanized antibody” includes a "pnmatized” antibody where the antigen-bmding region of the antibody is de ⁇ ved from an antibody produced by immunizing macaque monkeys with the antigen of interest. Antibodies containing residues from Old World monkeys are also possible within the invention. See, for example, U.S. Patent Nos. 5,658,570; 5,693,780; 5,681,722; 5,750,105; and 5,756,096.
• Antibodies and fragments thereof in this mvention also include "affinity matured" antibodies in which an antibody is altered to change the ammo acid sequence of one or more of the CDR regions and/or the framework regions to alter the affinity of the antibody or fragment thereof for the antigen to which it binds.
• Affinity maturation may result in an increase or in a decrease in the affinity of the matured antibody for the antrgen relative to the starting antibody.
• the starting antibody will be a humamzed, human, chimenc or murine antibody and the affinity matured antibody will have a higher affinity than the starting antibody Dunng the maturation process, one or more of the amino acid residues in the CDRs or in the framework regrons are changed to a different resrdue using any standard method.
• Suitable methods include point mutations using well known cassette mutagenesis methods (Wells et al., 1985, Gene, 34 315) or oligonucleotide mediated mutagenesis methods (Zoller et al., 1987, Nucleic Acids Res., 10 6487-6504).
• Affinity maturation may also be performed using known selection methods in which many mutations are produced and mutants having the desired affinity are selected from a pool or library of mutants based on improved affinity for the antigen or ligand
• Known phage display techniques can be conveniently used m this approach See, for example, U.S. 5,750,373, U.S. 5,223.409, etc.
• Human antibodies are also with in the scope of the antibodies of the invention. Human antibodies can be produced using ⁇ anous techniques known in the art. including phage display hbranes [Hoogenboom and Wrnter, 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); Boerner et al., J Immunol . 147(0.86-95 (1991), U. S.
• human antibodies can be made by introducing of human immunoglobulin loci mto transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, whrch closely resembles that seen rn humans rn all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is desc ⁇ bed, for example, in U.S. Patent Nos. 5,545.807; 5,545,806; 5,569,825; 5,625,126, 5,633,425; 5,661,016, and in the followmg screntrfrc publications.
• Single-chain Fv or “sFv” antibody fragments compnse the V H and V L domarns of antrbody, wherein these domams are present in a single polypeptide chain.
• the Fv polypeptrde further comp ⁇ ses a polypeptide linker between the VJ and VL domarns which enables the sFv to form the desrred structure for antrgen binding.
• diabodies refers to small antrbody fragments with two antigen-bmding srtes, whrch fragments compnse a heavy-chain vanable domain (Vjj) connected to a light-chain vanable domain (VjJ in the same polypeptide chain (Vjj - VjJ.
• Vjj heavy-chain vanable domain
• VjJ light-chain vanable domain
• linker that is too short to allow pai ⁇ ng between the two domains on the same chain, the domains are forced to pair with the complementary domarns of another chain and create two antigen-bmding sites.
• Diabodies are desc ⁇ bed more fully in, for example, EP 404,097; WO 93/11161; and Holhnger 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 matenals which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonprotemaceous solutes.
• the compound of the invention will be pu ⁇ fied (1) to greater than 95% by weight of the compound 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 nonreducrng condrtions using Coomassie blue or, preferably, silver stain.
• Isolated compound e.g. antibody or polypeptide. includes the compound in situ within recombinant cells since at least one component of the compound's natural environment will not be present Ordinarily, however, isolated compound will be prepared by at least one purification step
• label when used herein refers to a detectable compound or composition which is conjugated drrectly or indirectly to the compound, e g antibody or polypeptide, so as to generate a "labelled" compound
• 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 matnx to which the compound of the present invention can adhere
• solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysaccha ⁇ des (e g , agarose), polyacrylamrdes, polystyrene, polyvinyl alcohol and sihcones
• the solid phase can comprise the well of an assay plate, in others it is a punfication column (e g , an affinity chromatography column)
• Thrs term also m cludes a drscontmuous solrd phase of discrete partrcles, such as those descnbed m U S Patent No 4,275,149
• a "hposome” is a small vesicle composed of vanous types of hpids, phosphohpids and/or surfactant which is useful for delivery of a drug (such as the ant ⁇ -ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal
• a drug such as the ant ⁇ -ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent
• the components of the hposome are commonly arranged rn a brlayer formatron, similar to the lipid arrangement of biological membranes
• the term "lmmunoadhesm” designates antibody-like molecules which combine the binding specificity of a heterologous protem (an “adhesin”) with the effector functrons of immunoglobulin constant domains Structurally, the immunoadhesins compnse a fusron of an ammo acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (1 e , is "heterologous"), and an immunoglobulin constant domain sequence
• the adhesm part of an rmmunoadhesm molecule typically is a contiguous ammo acid sequence comprising at least the binding site of a receptor or a ligand
• the immunoglobulin constant domain sequence in the lmmunoadhesm may be obtained from any rmmunoglobuhn, such as IgG- 1 , IgG-2, IgG-3, or IgG-4 subtypes, IgA (including Ig
• the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides refe ⁇ ed to rn the present applrcation as PR0245, PR0217.
• PRO301, PR0266 PR0335, PR0331 or PR0326 (UNQ219, UNQ191, UNQ264, UNQ233, UNQ287V, UNQ292 or UNQ287 respectively)
• PR03 1 oi PR0326 polypeptide has been identified and isolated, as disclosed m 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 However, for sake of simplicity, in the present specification the protein encoded by DNA35638, DNA33094. DNA40628, DNA37150, DNA41388, DNA40981 and DN A37140 as well as all further native homologues and variants included in the foregoing definition of PR0245. PR0217. PRO301. PR0266, PR0335, PR0331 or PR0326, will be referred to as PR0245, PR0217, PRO301 , PR0266. PR0335, PR0331 or PR0326 or simply as "the polypeptide of the invention", regardless of their ongm or mode of preparation
• the desc ⁇ ption below relates pnma ⁇ ly to production of the polypeptide of the invention by cultunng cells transformed or transfected with a vector containing nucleic acid which encodes of the polypeptide of the invention
• a vector containing nucleic acid which encodes of the polypeptide of the invention may be employed to prepare of the polypeptrde of the mvention
• the polypeptide sequence, or portions thereof may be produced by drrect peptrde synthesrs usmg solid- phase technrques [see, e g , Stewart et al , Solrd-Phase Peptide Synthesis.
• variants can be prepared by introducing appropriate nucleotide changes into the DNA, and/or by synthesis of the desired polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the polypeptide of the mvention, such as changing the number or position of glycosylation sites or alternativeng the membrane anchoring characte ⁇ stics
• Vanations in the native full-length sequence or in vanous domains of the polypeptide of the invention descnbed herem can be made, for example, using any of the techniques and guidelines for conservatrve and non-conservatrve mutatrons set forth, for mstance, rn U S Patent No 5,364,934
• Va ⁇ atrons may be a substitution, deletion or insertion of one or more codons encoding the polypeptide that results in a change in the ammo acid sequence of the polypeptide as compared with the native sequence polypeptide sequence
• 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 polypeptide of the invention
• Guidance in determining which ammo acid residue may be inserted, substituted or deleted without adversely affecting the desired activrty may be found by compa ⁇ ng the sequence of the polypeptide of the invention with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology Amino
• Polypeptide fragments of the polypeptides of the invention are also withm the scope of the mventron Such fragments may be truncated at the N-termmus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein Certain fragments lack ammo acid residues that are not essential for a desrred brologrcal actrvrty of the invention polypeptide
• Polypeptide fragments may be prepared by any of a number of convent onal technrques Desrred peptide fragments may be chemrcally synthesized
• An alternative approach involves generating fragments by enzymatrc digestion, e g , by treating the protein with an enzyme known to cleave proteins at sites defined by particular ammo acid residues, or by digesting the DNA with suitable restnction enzymes and isolating the desired fragment
• Yet another surtable technrque involves isolating and amplifying a DNA fragment encodmg a desrred polypeptrde fragment, by polymerase chain reaction (PCR) Oligonucleotides that define the desired termrnr of the DNA fragment are employed at the 5' and 3' p ⁇ mers rn the PCR
• polypeptrde fragments share at least one biological and/or immunological activity with the native invention polypeptide
• Substantial modifications in function or immunological identity of the invention 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:
• hydrophobic norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic cys, ser, thr;
• Non-conservatrve substitutions will entail exchanging a member of one of these classes for another class Such substituted residues also may be introduced mto the conservatrve substitution sites or, more preferably, mto the remarnmg (non-conserved) srtes.
• the va ⁇ atrons can be made using methods known in the art such as olrgonucleotrde-medrated (srte-drrected) mutagenesis, alanine scanning, and PCR mutagenesis.
• Site-directed mutagenesis [Carter et al., Nucl Acids Res.. 13.4331 (1986), Zoller et al , Nucl. Acids Res . JO'6487 (1987)]
• cassette mutagenesis [Wells et al., Gene. 34:315 (1985)]
• restnction 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 va ⁇ ant DNA.
• Scannmg amino acid analysis can also be employed to identify one or more ammo acrds along a contrguous sequence.
• preferred scanning amino acids are relatively small, neutral amino acids
• Such amino acids include alanme, glycine, se ⁇ ne, 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 conformatron of the va ⁇ ant [Cunnmgham and Wells, Science. 244- 1081-1085 (1989)].
• Alamne is also typically preferred because rt rs the most common ammo acid Further, it is frequently found m both bu ⁇ ed and exposed positions [Creighton, The Proteins. (W H Freeman & Co , N.Y.); Chothia, J Mol Biol . 150: 1 (1976)]. If alanine substitution does not yield adequate amounts of va ⁇ ant, an isote ⁇ c amino acid can be used
• Covalent modifications of polypeptides of the invention are included withm the scope of this invention.
• One type of covalent modification includes reacting targeted amino acid residues of an invention polypeptide with an organic de ⁇ vatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the polypeptide.
• Denvatization with bifunctional agents is useful, for mstance, for crosslmking the invention polypeptide to a water-insoluble support matnx or surface for use rn the method for punfymg anti-polypeptide antibodies, and vice-versa
• Commonly used crosslmking agents include, e.g., 1 , 1 -b ⁇ s(d ⁇ azoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccmimide esters, for example, esters with 4-az ⁇ dosal ⁇ cyl ⁇ c acid, homobifunctional rmidoesters, including disuccimmidyl esters such as 3,3'-d ⁇ th ⁇ ob ⁇ s(succ ⁇ n ⁇ m ⁇ dylprop ⁇ onate), bifunctronal maleimides such as b ⁇ s-N-male ⁇ m ⁇ do-1.8-octane and agents such as methyl-3-[(p- az ⁇ dophenyl)d
• Another type of covalent modification of the invention 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 polypeptide (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.
• 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 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 polypeptide (for O-linked glycosylation sites).
• the amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the 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 polypeptide of the invention is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem.. pp. 259-306 (1981).
• Removal of carbohydrate moieties present on the polypeptide of the invention may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation.
• Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., U_8:131 (1981).
• Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. EnzvmoL 138:350 (1987).
• Another type of covalent modification comprises linking the invention polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
• the polypeptide of the present invention may also be modified in a way to form a chimenc molecule comprising the invention polypeptide fused to another, heterologous polypeptide or amino acid sequence.
• such a chimenc molecule compnses a fusion of the invention polypeptide 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 ammo- or carboxyl- terminus of the polypeptide of the invention. The presence of such epitope-tagged forms of the polypeptide of the invention can be detected using an antibody against the tag polypeptide.
• provision of the epitope tag enables the polypeptide of the invention to be readily punfied by affinity punfication using an anti-tag antibody or another type of affinity matnx that binds to the epitope tag. Vanous tag polypeptides and their respective antibodies are well known in the art.
• tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology. 6: 1204-1210 (1988)]; the KT3 eprtope peptrde [Martin et al, Scrence. 255: 192-194 (1992)]; an - tubulrn epitope peptide [Skmner et al., J. Biol. Chem.. 266: 15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al, Proc Natl. Acad. Ser. USA. 87:6393-6397 (1990)].
• the chimenc molecule may compnse a fusron of the polypeptide of the invention with an immunoglobulin or a particular region of an immunoglobulin.
• a fusron could be to the Fc regron of an IgG molecule.
• the Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of an invention polypeptide in place of at least one vanable region withm an Ig molecule.
• the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGl molecule.
• immunoglobulin fusrons see also US Patent No 5,428,130 issued June 27, 1995.
• DNA encoding the polypeptide of the invention may be obtained from a cDNA library prepared from trssue belreved to possess the polypeptide mRNA and to express it at a detectable level
• human DNA can be conveniently obtained from a cDNA hbrary prepared from human tissue, such as desc ⁇ bed in the Examples.
• the gene encoding the polypeptrde of the invention may also be obtained from a genomic library or by oligonucleotide synthesis
• Libra ⁇ es can be screened with probes (such as antibodies to the polypeptide of the invention or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding the polypeptide of the invention is to use PCR methodology [Sambrook et al., supra: Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
• the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.
• the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 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 through sequence alignment using computer software programs such as ALIGN, DNAstar, and INHERIT which employ various algorithms to measure homology.
• 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.
• Host cells are transfected or transformed with expression or cloning vectors described herein for production of the polypeptides of the invention 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 transfection are known to the ordinarily skilled artisan, for example, CaP ⁇ 4 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 or other cells that contain substantial cell-wall barriers.
• 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.
• the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed.
• General aspects of mammalian cell host system transformations have been described in U.S. Patent No.
• Transformations into yeast are typically carried out according to the method of Van Solingen et al, J. Bact.. 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA). 76:3829 (1979).
• other methods for introducing DNA into cells such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used.
• 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).
• eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding the polypeptides of the invention.
• Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.
• Suitable host cells for the expression of glycosylated polypeptides of the invention 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.
• the nucleic acid e.g., cDNA or genomic DNA
• a replicable vector for cloning (amplification of the DNA) or for expression.
• Various vectors are publicly available.
• the vector may, for example, be in the form of a plasmid, cosmid, viral particle, phagemid 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 polypeptide of the invention 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 DNA encoding the polypeptide of the invention 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 alpha-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 2u plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
• Selection genes 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 nucleic acid encoding the polypeptide of the invention, 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 trj ⁇ l gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature. 282:39 (1979); Kingsman et al, Gene.
• the trp I 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 nucleic acid sequence encoding the polypeptide of the invention 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 beta-lactamase and lactose promoter systems [Chang et al., Nature. 275:615 (1978); Goeddel et al., Nature. 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res..
• Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide of the invention.
• S.D. Shine-Dalgarno
• Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem.. 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg.. 7: 149 (1968); Holland, Biochemistry.
• J7:4900 (1978)] such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
• enolase such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
• 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.
• Transcription of the polypeptide of the invention 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 2,21 1,504 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 2,21 1,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus,
• 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 mammalran genes (globm, elastase, albumin, alpha- fetoprotem, and insulin).
• an enhancer from a eukaryotic cell virus examples include the SV40 enhancer on the late side of the replication ongm (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication ongm, and adenovirus enhancers
• the enhancer may be spliced mto the vector at a position 5' or 3' to the coding sequence of the polypeptide of the invention, but is preferably located at a site 5' from the promoter
• Expression vectors used m eukaryotic host cells will also contain sequences necessary for the termination of transcnptron and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasronally 3', untranslated regrons of eukaryotic or viral DNAs or cDNAs These regions contain nucleotide segments transcnbed as polyadenylated fragments rn the untranslated portion of the mRNA encoding the polypeptide of the invention
• vectors, and host cells suitable for adaptatron to the synthesis of the polypeptide of the invention in recombinant vertebrate cell culture are desc ⁇ bed rn Gethmg et al., Nature. 293:620-625 (1981); Mantel et al., Nature. 281 :40-46 (1979); EP 1 17,060; and EP 1 17,058.
• Gene expression may be measured in a sample directly, for example, by conventronal Southern blotting, Northern blotting to quantitate the transcnption of mRNA [Thomas, Proc Natl Acad Sci USA, 77"5201-5205 (1980)], dot blotting (DNA analysis), or in situ hyb ⁇ dization, using an approp ⁇ ately labeled probe, based on the sequences provided herein.
• antrbodies may be employed that can recognize specific duplexes, including DNA duplexes.
• RNA duplexes and DNA-RNA hybnd duplexes or DNA-protein duplexes
• the antibodies in turn may be labeled and the assay may be earned out where the duplex is bound to a surface, so that upon the formatron of duplex on the surface, the presence of antibody bound to the duplex can be detected
• Gene expressron 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 drrectly the expression of gene product
• immunological methods such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate drrectly the expression of gene product
• Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal.
• the antibodies may be prepared against a native sequence of the inventive polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to DNA encoding the polypeptide of the invention and encoding a specific antibody epitope lv Punfication of Polypeptide
• Forms of the polypeptide of the invention 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 the polypeptide of the invention can be disrupted by various physical or chemical means, such as freeze- thaw cycling, sonication, mechanical disruption, or cell lysing agents.
• polypeptide of the invention may be desired to purify from recombinant cell proteins or polypeptides.
• the following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS- PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the polypeptide of the invention .
• tissue expressing the polypeptides of the invention 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 polypeptides of the invention. 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 polypeptide of the invention or against a synthetic peptide based on the DNA sequences encoding the polypeptide of the invention or against an exogenous sequence fused to a DNA encoding a polypeptide of the invention 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 polypeptides of the invention can be further verified by antibody binding studies, in which the ability of anti-PR0245. PR0217, PRO301, PR0266. PR0335, PR0331 or PR0326 antibodies to inhibit the effect of the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptides 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).
• ком ⁇ онентs 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 ELISA 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. 4.
• 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 rmmune functron, for example to modulate T-cell prohferatron or rnflammatory 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.
• p ⁇ mary cultures denved from transgenic animals can be used in the cell-based assays herein, although stable cell lines are preferred Technrques to de ⁇ ve continuous cell lines from transgenrc anrmals are well known in the art (see, e.g. Small et al. Mol. Cell. Biol 5. 642-648 [1985]).
• MLR mixed lymphocyte reaction
• MLR mixed lymphocyte reaction
• the ability of a test compound to stimulate the proliferation of activated T cells is assayed.
• a suspension of responder T cells is cultured with allogeneic stimulator cells and the prohferatron of T cells is measured by uptake of t ⁇ tiated thymidme.
• This assay is a general measure of T cell reactivity.
• a prohferatrve T cell response rn an MLR assay may be due to drrect mitogemc propertres of an assayed molecule or to external antigen induced activation. Additional venficatron of the T cell stimulatory activity of the polypeptides of the invention 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 crosslmking 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 glycoprote s in the Ig superfamrly 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 negative T cell deactivating effect.
• the polypeptides of the invention are assayed for T cell costimulatory or rnhibitory activity
• Polypeptrdes of the mventron, as well as other compounds of the mvention, which are stimulators (costimulators) of T cell prohferatron 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 stimulatmg 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 polypeptides of the invention
• the stimulatmg polypeptide may, for example, be a PR0245 PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 polypeptide or an agonist antibody therefor
• an agonist stimulating compound has also been validated experimentally Activation of 4- IBB by treatment with an agomst ant ⁇ -4-lBB antibody enhances eradication of tumors Hellstrom, I and Hellstrom, K E , Cnt Rev Immunol (1998) 18 1 Immunoadjuvant therapy for treatment of tumors, descnbed n 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 achreved by antagomzrng or blocking the activity of a protein which has been found to be nhrbrtmg in the MLR assay Negatmg the inhibitory actrvrty of the compound produces a net strmulatory effect
• Suitable antagonists/blocking compounds are antibodies or fragments thereof whrch recognize and bind to the inhibitory protein, thereby blocking the effectrve mteractron of the protem with its receptor and inhibiting signaling through the receptor This effect has been validated in expenments usmg ant ⁇ -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 , Immunrty ( 1994) 1 405
• polypeptides of the invention as well as other compounds of the invention, which are direct inhibitors of T cell proliferation/activation and/or lymphokine secretion, 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 charactenzed by a hyperactive, superoptrmal, or autormmune response Thrs use of the compounds of the mventron has been valrdated by the expenments descnbed above in which CTLA-4 binding to receptor B7 deactivates T cells
• the direct inhibitory compounds of the invention functron in an analogous manner
• compounds, e g antibodies, which bind to stimulating polypeptides of the invention and block the stimulating effect of these molecules produce a net rnhrbrtory effect and can be used to suppress the T cell medrated rmmune response by rnhrbrtmg T cell prolrferatron/actrvation and or lymphokme secretion Blocking the stimulating effect of the polypeptides suppresses the immune response of the mammal This use has been validated in expenments usmg an antr-IL2 antrbody In these expenments, the antrbody bmds to IL2 and blocks brnding of IL2 to its receptor thereby achieving a T cell lnhibitorv effect 5.
• 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.
• An animal model for arth ⁇ tis is collagen-induced arth ⁇ tis Thrs model shares clinical, histological and immunological charactenstrcs of human autoimmune rheumatoid arthritis and is an acceptable model for human autormmune arthntrs
• Mouse and rat models are charactenzed by synovrtis, erosron of cartilage and subchondral bone
• the compounds of the invention can be tested for actrvity against autoimmune arthritis using the protocols desc ⁇ bed in Current Protocols m Immunology, above, units 15 5 See also the model using a monoclonal antibody to CD 18 and VLA- 4 integnns descnbed rn Issekutz. A C et al . Immunology (1996) 88 569
• a model of asthma has been descnbed in which antigen-induced airway hyper-reactivity, pulmonary eosmophiha and inflammation are induced bv sensitizing an animal with ovalbumm and then challenging the animal with the same protein delivered by aerosol
• Several animal models (guinea pig, rat, non-human pnmate) show symptoms similar to atopic asthma in humans upon challenge with aerosol antigens Munne models have many of the features of human asthma Suitable procedures to test the compounds of the mvention for actrvity and effectrveness n the treatment of asthma are descnbed by Wolynrec, W W et al , Am J Resprr Cell Mol Brol (1998) 18 777 and the references cited therein
• the compounds of the invention can be tested on animal models for psonasis like diseases Evidence suggests a T cell pathogenesis for psoriasis
• the compounds of the invention can be tested n the scrd/scrd mouse model descnbed Schon M P et al , Nat Med (1997) 3 183, in which the mice demonstrate histopathologic skm lesions resembling psonasis
• Another suitable model is the human skrn/scid mouse chrmera prepared as descnbed 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 identrf ⁇ ed herein mto 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 pnmates, e g baboons chimpanzees and monkeys
• Techniques known m the art to introduce a transgene mto such animals include pronucleic mrcromjectron (Hoppe and Wanger, U S Patent No 4,873,191), retrovrrus-medrated gene transfer mto germ lrnes (e g , Van der Putten et al , Proc Natl Acad Sci USA 82, 6148-615 [1985]), gene targeting in embryonic stem cells (Thompson et al , Cell 56, 313- 321
• transgenic animals include those that carry the transgene only rn part of therr cells ("mosaic anrmals")
• the transgene can be mtegrated either as a srngle transgene, or n concatamers, e g , head-to-head or head-to-tarl tandems
• Selectrve mtroduction of a transgene mto a particular cell type is also possible by followmg, for example, the technrque of Lasko et al , Proc Natl Acad Sci USA 89, 6232-636 (1992)
• transgene rn transgenic animals can be monitored by standard techniques For example, Southern blot analysis or PCR amplification can be used to venfy the mtegratron of the transgene The level of mRNA expressron can then be analyzed using techniques such as in situ hybndization, Northern blot analysrs, PCR, or rmmunocytochemrstry
• the animals may be further examined for srgns of rmmune drsease pathology, tor example by histological exammation to determine infiltration of immune cells mto specific tissues
• Blocking expenments can also be performed in which the transgenic anrmals are treated with the compounds of the invention to determine the extent of the T cell proliferation strmulation or inhibition of the compounds
• blocking antibodies which bind to the polypeptide of the mventron, prepared as desc ⁇ bed above, are admmrstered to the animal and the effect on immune functron is determmed
• "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 partrcular polypeptide can be deleted or replaced wrth another gene, such as a gene encoding a selectable marker which can be used to monitor integration
• several kilobases of unaltered flankrng DNA are included in the vector [see e.g., Thomas and Capecchi, Cell, 5J_:503 (1987) for a descnption of homologous recombination vectors]
• the vector is introduced into an embryo
• 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, m Teratocarcinomas and Embryonic Stem Cells A Practical Approach, E. J.
• a chrmenc embryo can then be rmplanted mto a suitable pseudopregnant female foster ammal and the embryo brought to term to create a "knock out" ammal
• Progeny harbo ⁇ ng the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed ammals in which all cells of the ammal contarn the homologously recombmed DNA.
• Knockout animals can be characterized for mstance, for their ability to defend agamst certain pathological conditions and for therr development of pathologrcal condrtions due to absence of the polypeptide.
• the rmmunostrmulatmg compounds of the mventron can be used rn rmmunoadjuvant therapy for the treatment of tumors (cancer).
• tumors cancer
• T cells recognize human tumor specific antigens.
• One group of tumor antigens, encoded by the MAGE, BAGE and GAGE families of genes, are silent in all adult normal tissues , but are expressed in significant amounts m tumors, such as melanomas, lung tumors, head and neck tumors, and bladder carcinomas. DeSmet, C. et al, (1996) Proc. Natl. Acad. Sci.
• 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 prohferatron/actrvatron and an antrtumor response to tumor antrgens.
• the growth regulatmg, cytotoxrc, or chemotherapeutrc 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 lowenng the toxicity to the patient
• Screening assays for drug candrdates are desrgned to rdentify compounds that bind or complex with the polypeptides encoded by the genes identified herein or a biologically active fragment thereof, or otherwrse interfere with the mteractron of the encoded polypeptrdes wrth other cellular proteins.
• Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candrdates
• Small molecules contemplated mclude synthetrc organic or inorganic compounds, including peptides, preferably soluble peptides, (poly)pept ⁇ de- ⁇ mmunoglobulm fusions, and.
• antrbodres including, without limitation, poly- and monoclonal antibodies and antibody fragments, smgle-cham antibodies, anti-idiotypic antibodies, and chimenc or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
• the assays can be performed rn a varrety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characte ⁇ zed in the art.
• the interaction is binding and the complex formed can be rsolated or detected rn the reaction mrxture.
• Non-covalent attachment generally is accomplished by coating the solid surface wrth a solution of the polypeptide and drymg.
• an rmmobihzed antibody e.g. a monoclonal antibody, specific for the polypeptrde to be rmmobrhzed can be used to anchor it to a solid surface.
• the assay rs performed by addmg the non-immobilized component, whrch may be labeled by a detectable label, to the rmmobrhzed 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.
• complexmg can be detected, for example, by usmg a labelled antrbody specifically binding the immobilized complex
• the candidate compound interacts with but does not bind to a particular protem encoded by a gene rdentrfred herein, its interaction with that protein can be assayed by methods well known for detecting protem-protem interactions.
• assays include traditional approaches, such as, cross- lrnkrng, co-rmmunoprecrprtatron, and co-punfication through gradients or chromatographic columns
• protem-protein interactions can be monitored by using a yeast-based genetic system desc ⁇ bed 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 .
• transc ⁇ ptronal activators such as yeast GAL4, consrst of two physically discrete modular domains, one acting as the DNA-bmdmg domain, while the other one functioning as the transcnption activation domain
• yeast expression system descnbed in the foregoing publications (generally referred to as the "two-hybnd system") takes advantage of this property, and employs two hybnd proteins, one m which the target protein is fused to the DNA- bindmg domain of GAL4, and another, in which candidate activating proteins are fused to the actrvation domain
• the expression of a GAL 1 -lacZ reporter gene under control of a G AL4-act ⁇ vated promoter depends on reconstitution of GAL4 activity via protem-protem rnteractron Colonres containing interacting polypeptides are detected with a chromogemc substrate for beta-galactosidase
• 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 rnteractron and binding of the two products
• a placebo may be added to a third reaction mixture, to serve as posrtive control
• the binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as desc ⁇ bed above
• the formation of a complex in the control react ⁇ on(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
• the composrtrons useful m the treatment of immune related diseases include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and nbozyme molecules, t ⁇ ple 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 molecule act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation
• ohgodeoxy ⁇ bonucleotides de ⁇ ved from the translation initiation site e g between about -10 and +10 positions of the target gene nucleotide sequence
• Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA Ribozymes act by sequence-specific hybndization 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 Brology 4, 469-471 (1994), and PCT publication No WO 97/33551 (published September 18, 1997)
• Nucleic acid molecules in tnple helix formation used to inhibit transcnption should be single- stranded and composed of deoxynucleotides
• the base composition of these oligonucleotides is designed such that it promotes t ⁇ ple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of pu ⁇ nes or py ⁇ midines on one strand of a duplex
• Hoogsteen base pairing rules which generally require sizeable stretches of pu ⁇ nes or py ⁇ midines on one strand of a duplex
• Some of the most promrsmg drug candidates according to the present invention are antibodies and antibody fragments whrch may rnhrbit (antagonists) or strmulate (agonrsts) T cell prolrferation, eosmophil infiltration, etc l Polyclonal Antibodies
• the immunizing agent may include the polypeptide of the invention 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 thyroglobuhn, and soybean trypsin inhibitor
• adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Liprd A, synthetrc trehalose dicorynomycolate)
• the immunization protocol may be selected by one
• Antibodies which recognize and bind to the polypeptides of the invention or which act as agonist therefor may, alternatively, be monoclonal antrbodres
• Monoclonal antibodies may be prepared using hyb ⁇ doma methods, such as those described by Kohler and Milstem, Nature. 256 495 ( 1975)
• a hybndoma method a mouse, hamster, or other approp ⁇ ate host ammal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro
• the immunizing agent will typically include the polypeptide of the invention or a fusion protein thereof Generally, either penpheral blood lymphocytes ("PBLs") are used if cells of human ongin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired
• PBLs penpheral 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 hybndoma cell [Godmg, Monoclonal Antibodies Principles and Practice, Academic Press, (1986) pp 59-103]
• Immortalrzed cell lrnes are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human ongm
• rat or mouse myeloma cell lines are employed
• HGPRT hypoxanthine guanrne phosphonbosyl transferase
• Preferred rmmortahzed cell lines are those that fuse efficrently, 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 rmmortahzed cell lrnes are munne myeloma lines, which can be obtained, for instance, from the Salk Instrtute Cell Distrrbutron Center. San Drego, California and the Ame ⁇ can Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lrnes also have been desc ⁇ bed for the productron of human monoclonal antrbodres [Kozbor, J Immunol.. 133.3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Ine . 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 antrbodies drrected against the polypeptrde of the mvention or having similar activity as the polypeptide of the invention.
• the binding specificity of monoclonal antibodies produced by the hybridoma cells is determmed by lmmunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked lmmunoabsorbent assay (ELISA)
• RIA radioimmunoassay
• ELISA enzyme-linked lmmunoabsorbent assay
• the brndmg affinity of the monoclonal antibody can, for example, be determmed 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 [Godmg, supra] Suitable culture media for this purpose mclude, for example, Dulbecco's Modrfied Eagle's Medium and RPMI-1640 medium Alternatively, the hyb ⁇ doma cells may be grown in vivo as ascites in a mammal
• the monoclonal antibodies secreted by the subclones may be isolated or punfied from the culture medium or ascites fluid by conventional immunoglobulin punfication procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
• the monoclonal antrbodres 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 ot munne antibodies)
• the hybndoma cells of the invention serve as a prefe ⁇ ed source of such DNA
• the DNA may be placed to expression vectors, which are then transfected mto host cells such as simian COS 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 covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
• a non- immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
• the antibodies are preferably 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 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 ammo acid residues introduced into it from a source which is non-human. These non-human ammo acid residues are often referred to as "import" residues, which are typically taken from an “import” vanable domarn. Humamzation can be essentially performed followmg the method of Winter and coworkers [Jones et al., Nature, 321.522-525 ( 1986); Riechmann et al., Nature, 332.323-327 (1988); Verhoeyen et al, Science.
• humanized antibodies are chimenc antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human vanable domain has been substituted by the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies m 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 the art, including phage display libraries [Hoogenboom and Winter, J Mol. Biol.. 227.381 (1991); Marks et al., J. Mol. Brol, 222:581 (1991)].
• the technrques 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); Boerner et al, J. Immunol. 147(0:86-95 (1991); U. S. 5,750, 373].
• 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, whrch closely resembles that seen rn humans in all respects, including gene rearrangement, assembly, and antrbody repertoire This approach is descnbed, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825, 5.625,126; 5,633,425; 5,661,016, and in the followmg screntrfic publications: Marks et al , Bio/Technology 10. 779-783 (1992), Lonberg et al .
• Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two drfferent antrgens.
• one of the brnding specificities may be for the polypeptide of the invention, the other one is for any other antigen, and preferably for a cell-surface protern or receptor or receptor subunrt.
• brspecrfic antrbodies are known in the art Tradrtronally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy-chain/hght-cham pairs, where the two heavy chains have different specificities (Milstem and
• Antibody vanable domains with the desired binding specificities can be fused to rmmunoglobulm constant domain sequences
• the fusion preferably is with an immunoglobulin heavy-chain constant domain, compnsmg at least part of the hinge, CH2, and CH3 regions It is preferred to have the first heavy-chain constant region (CH 1 ) containing the site necessary for light-chain binding present in at least one of the fusions DNAs encoding the immunoglobulin heavy-chain fusrons and, rf desired, the immunoglobulin light chain, are inserted mto separate expressron vectors, and are cotransfected into a suitable host organism
• CH 1 first heavy-chain constant region
• Heteroconjugate antibodres 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 crosslmking agents
• immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond
• suitable reagents for thrs purpose mclude iminothiolate and methyl-4- mercaptobuty ⁇ midate and those disclosed, for example, n U S Patent No 4,676,980 vr Effector function engrneenng
• the homodimenc antibody thus generated may have improved mternahzation capabrlrty and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity ( ADCC) See Caron et al , J Exp Med 176 1 191-1 195 (1992) and Shopes, B J Immunol 148 2918-2922 (1992) Homodimenc antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-lrnkers as descnbed rn Wolff et al Cancer Research 53 2560-2565 (1993) Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities See Stevenson
• the invention also pertains to immunoconjugates compnsmg an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g an enzymatically active toxin of bactenal, fungal, plant or animal ongm, or fragments thereof), or a radioactive isotope (/ e a radioconjugate)
• a cytotoxic agent such as a chemotherapeutic agent, toxin (e g an enzymatically active toxin of bactenal, fungal, plant or animal ongm, or fragments thereof), or a radioactive isotope (/ e a radioconjugate)
• Enzymatically actrve toxrns and fragments thereof which can be used include diphthena A chain, nonbindmg active fragments of diphthena toxm, exotoxm A chain (from Pseudomonas aeruginosa), ncm A chain, ab ⁇ n A chain, modeccm A chain, alpha-sarcm, Aleuntes fordn proteins, dianthm proteins.
• Phvtolaca americana proteins PAPI, PAPII, and PAP-S
• momordica charantia inhibitor curcm, crotin, sapaona ⁇ a officinahs inhibitor, gelonm, mrtogellm, restnctocm, phenomycm, enomycm and the tncothecenes
• radionuchdes are available for the production of radioconjugated antibodies Examples include 212 B ⁇ , 131 I, 131 In, 90 Y and 186 Re
• Conjugates of the antibody and cytotoxic agent are made using a va ⁇ ety of bifunctional protein coupling agents such as N-succm ⁇ m ⁇ dyl-3-(2-pyndyldrth ⁇ ol) propionate (SPDP), lm othiolane (IT), bifunctional de ⁇ vatives of lmidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccmrmrdyl suberate).
• bifunctional protein coupling agents such as N-succm ⁇ m ⁇ dyl-3-(2-pyndyldrth ⁇ ol) propionate (SPDP), lm othiolane (IT), bifunctional de ⁇ vatives of lmidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccmrmrdyl suberate).
• aldehvdes such as glutareldehyde
• bis-azido compounds such as bis (p-azidobenzoyl) hexanediamme
• bis-diazomum de ⁇ vatives such as b ⁇ s-(p- d ⁇ azomumbenzoyl)-ethylened ⁇ am ⁇ ne
• dnsocyanates such as tolyene 2,6-dnsocyanate
• bis-active fluorrne compounds such as l,5-difluoro-2,4-dimtrobenzene
• a ncm immunotoxin can be prepared as desc ⁇ bed in Vitetta et al , Science 238 1098 (1987) Carbon- 14-labeled 1- ⁇ soth ⁇ ocyanatobenzyl-3-methyld ⁇ ethylene t ⁇ aminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody See W
• the antibody may be conjugated to a "receptor” (such streptavidin) for utilization in tisue pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation usmg a clearing agent and then administration of a "ligand” (e g avidm) which is conjugated to a cytotoxic agent (e g a radionucleotide) viii Immunohposomes
• protems, antrbodres, etc disclosed herein may also be formulated as immunohposomes
• 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
• hposomes can be generated by the reverse phase evaporation method with a lipid composrtion compnsmg phosphatidylcholme.
• cholesterol and PEG-denvatized phosphatidylethanolamme (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 doxorubicm
• Gabizon et al J National Cancer Inst 81( 19) 1484 (1989) 10 Pharmaceutical Compositions
• active 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 molecule are prepared for storage by mixing the active molecule having the desired degree of punty with optional pharmaceutically acceptable earners, excipients or stabilizers (Remington 's Pharmaceutical Sciences 16th edrtron, Osol, A Ed [1980]), in the form of lyophrhzed formulations or aqueous solutions
• Acceptable earners, excrprents, or stabrhzers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, crtrate, and other organrc acrds, antroxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chlonde, hexamethonium chlo ⁇ de, benzalkonium chlo ⁇ de, benzethomum chlonde, phenol, butyl
• 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 polyvmylpyrrolrdone, ammo acrds such as glycme, glutamine, asparagme, histrdme, arginine, or lysine, monosaccha ⁇ des, disacchandes, and other carbohydrates including glucose, mannose, or dext ⁇ ns, chelating agents such as EDTA, sugars such as sucrose, mannitol.
• hydrophilic polymers such as polyvmylpyrrolrdone, ammo acrds such as glycme, glutamine, asparagme, histrdme, arginine, or lysine, monosaccha ⁇ des, disacchandes,
• trehalose or sorbitol such as sodium, metal complexes (e g , Zn- protein complexes), and/or non-iomc surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG)
• Lipofections or liposomes can also be used to deliver the polypeptide, antibody, or an antibody fragment, mto cells Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred
• peptide molecules can be designed which retain the abrlity 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,
• the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other Alternatively, or rn addition, the composition may compnse a cytotoxic agent, cytokine or growth inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended
• the active molecules may also be entrapped in microcapsules prepared, for example, by coacervation technrques or bv mterfacial polymenzation, for example, hydroxymethylcellulose or gelatm-mrcrocapsules and poly-(methylmethacylate) mrcrocapsules, respectrvely, rn colloidal drug delivery systems (for example, liposomes. albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remingto 's Pharmaceutical Sciences 16th edition, Osol. A Ed (1980)
• the formulations to be used for in vivo admrnrstration must be stenle Thrs is readily accomplished by filtration through stenle filtration membranes
• sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semipermeable matnces of solrd hydrophobic polymers contarning the antibody, which matnces are in the form of shaped articles, e g films, or microcapsules
• sustained-release matnces include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vmylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L- glutamrc acrd and ethyl-L-glutamate, non-degradable ethylene-vmyl acetate, degradable lactrc acid- glycohc acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate), and poly-D-(-)-3-hydroxybutync acid While polymers such as ethylene-
• polypeptides, antibodies and other active compounds of the present invention may be used to treat vanous immune related diseases and conditrons, such as T cell medrated drseases, including those charactenzed by infiltration of inflammatory cells mto a tissue, stimulation of T-cell prohferatron, inhibition of T-cell proliferation, mcreased or decreased vascular permeability or the inhibition thereof
• vanous immune related diseases and conditrons such as T cell medrated drseases, including those charactenzed by infiltration of inflammatory cells mto a tissue, stimulation of T-cell prohferatron, inhibition of T-cell proliferation, mcreased 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 arthntrs, juvenile chronic arthntis, osteoarth ⁇ trs, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis. polymyositis), Sjogren's syndrome systemic vascuhtis.
• sarcordosrs autormmune hemolytic anemia (immune pancytopema, paroxysmal nocturnal hemoglobinu ⁇ a), 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 (glomerulonephntrs.
• autormmune hemolytic anemia immuno pancytopema, paroxysmal nocturnal hemoglobinu ⁇ a
• autoimmune thrombocytopenia idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia
• thyroiditis Gramve's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis
• diabetes mellitus immune-mediated renal disease (glomerulonephntr
• demyehnatmg diseases of the central and penpheral nervous systems such as multiple sclerosis, idiopathic demyehnatmg polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyehnatmg polyneuropathy, hepatobihary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, p ⁇ mary biliary cirrhosis, granulomatous hepatitis, and sclerosmg cholangitis, mflammatory bowel disease (ulcerative colitis Crohn's disease), gluten- sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skm diseases including bullous skm diseases, erythema multiforme and contact dermatitis, psonasis, allergic diseases such as asthma,
• 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, cardrovascular system, gastrointestinal tract, bone marrow and blood
• Rheumatoid arthritis is a chronic systemic autoimmune mflammatory 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-antrbodres directed agamst self IgG.
• n RA Other mamfestatrons whrch can occur n RA include- pencarditis, pleu ⁇ tis, coronary arterrtis, mtestitial pneumonitis with pulmonary fbrosrs, keratoconjunctrvrtis srcca, 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 simila ⁇ ties to RA; some patients which are rhematoid factor posrtrve are classified as juvenile rheumatoid arthntrs.
• the arthntrs can be severe and is typically destructive and leads to joint ankylosis and retarded growth.
• Other manifestations can include chronic antenor uveitis and systemic amylordosrs
• Spondyloarthropathies are a group of drsorders wrth some common clrmcal features and the common association with the expression of HLA-B27 gene product.
• the disorders include ankylosmg sponyhtis, Rerter's syndrome (reactive arthntrs), arthntrs assocrated with inflammatory bowel drsease, spondyhtrs associated with psoriasis, juvenile onset spondyloarthropathy and undifferentiated spondyloarthropathy.
• Distinguishing features include sacroileitis with or without spondyhtis; inflammatory asymmetnc arthntis, assocratron 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 forergn peptrde expressed by MHC class I molecules It has been hypothesized that an epitope of HLA-B27 may mimic a bactenal or other microbial antigenic epitope and thus induce a CD8+ T cells response
• 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 m 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 rn skm lesrons suggesting that T cell interaction with these cells may have a role m the pathogenesis of the disease.
• Other organs involved include, the gastrointestinal tract" smooth muscle atrophy and fibrosis resulting in abnormal pe ⁇ stalsis/motihty, kidney concentnc subendothelial mtimal proliferation affecting small arcuate and interlobular arterres wrth resultant reduced renal cortical blood flow, results in proteinuna, azotemia and hypertension.
• Idiopathic mflammatory myopathies mcludrng dermatomyositis, polymyositis and others are disorders of chronic muscle inflammation of unknown etiology resulting m muscle weakness. Muscle injury/inflammation rs often symmet ⁇ c 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, mvolved in protein synthesis.
• Sjogren'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 agamst 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, pe ⁇ pheral or sensory neuropathy, and palpable purpura.
• Systemrc vasculrtrs are diseases rn whrch the pnmary lesion is inflammation and subsequent damage to blood vessels which results m ischemia/necrosis/degeneration to tissues supphed by the affected vessels and eventual end-organ dysfunction in some cases.
• Vascuhtides can also occur as a secondary lesron or sequelae to other rmmune-rnflammatory mediated diseases such as rheumatoid arthntis, systemic sclerosis, etc., particularly in diseases also associated with the formation of rmmune complexes.
• Mrscellaneous vasculrtrdes include: mucocutaneous lymph node syndrome (MLNS or Kawasaki's drsease), isolated CNS vascuhtis.
• vascuhtis Behet's disease, thromboangiitis oblrterans (Buerger's disease) and cutaneous necrotizing venuhtis
• the pathogenic mechanism of most of the types of vascuhtis listed is believed to be p ⁇ ma ⁇ ly due to the deposition of immunoglobulin complexes in the vessel wall and subsequent induction of an inflammatory response erther via ADCC, complement activation, or both
• Sarcoidosis is a condition of unknown etiology which is charactenzed 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 rn 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 mechanrsms
• platelet destruction/removal occurs as a result of either antibody or complement attaching to platelets and subsequent removal by complement lysis, ADCC or 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), rnducrble models immunization of animals with either thyroglobulm, thyroid microsomal antigen (thyroid peroxidase)
• Type I diabetes mellitus or sulm-dependent diabetes is the autoimmune destruction of pancreatrc islet ⁇ cells, this destruction rs medrated by auto-antrbodies and auto-reactive T cells
• Antibodies to insulin or the insulin receptor can also produce the phenotype of lnsuhn-non- responsiveness
• Immune mediated renal diseases including glomerulonephntis and tubulomterstrtral neph ⁇ tis, 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 agamst renal antigens or indirectly as a result of the deposition of antibodies and/or immune complexes in the kidney that are reactive agamst 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
• Demyehnatmg diseases of the central and penpheral nervous systems including Multiple Sclerosis, idiopathic demyehnatmg polyneuropathy or Guillain-Ba ⁇ syndrome, and Chronic Inflammatory Demyehnatmg Polyneuropathy are believed to have an autoimmune basis and result m nerve demyelination as a result of damage caused to ohgodendrocytes or to myelrn directly In MS there is evrdence to suggest that disease induction and progression is dependent on T lymphocytes
• Multiple Sclerosis is a demyehnatmg disease that is T lymphocyte-dependent and has either a relapsing-remitting course or a chronic progressive course
• the etiologj is unknown, however, viral infections, genetic predisposition, environment, and autoimmunity all contnbute Lesions contain infiltrates of predominantly T lymphocyte mediated, microghal cells and infiltrating macrophages, CD4+T lymph
• Psonasis is a T lymphocyte-mediated inflammatory disease Lesions contain infiltrates of T lymphocytes, macrophages and antigen processing cells, and some neutrophrls
• Allergrc drseases including asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, and urtrcana are T lymphocyte dependent These diseases are predommantly mediated by T lymphocyte induced inflammation, IgE mediated-mflammation or a combination of both
• Transplantation associated diseases including Graft rejection and Graft- Versus-Host-Disease (GVHD) are T lymphocyte-dependent, mhrbition of T lymphocyte functron is amelroratrve
• infectious disease including but not limited to viral infection (including but not limited to AIDS, hepatitis A, B, C, D, E and herpes) bacte ⁇ al infection, fungal infections, and protozoal and parasitic infections (molecules (or denvatives/agomsts) which stimulate the MLR can be utilized therapeutically to enhance the immune response to infectious agents), diseases of immunodeficiency (molecules/denvatives/agonists) which stimulate the MLR can be utihzed therapeutically to enhance the immune response for conditions of mhented, acquired, infectious induced (as in HIV infectron), or ratrogemc (i.e as from chemotherapy) immunodeficiency), and neoplasia
• inhibition of molecules with proinflammatory properties may have therapeutic benefit in reperfusion rnjury, stroke, myocardial infarction, atherosclerosis, acute lung injury, hemorrhagic shock; burn, sepsis/ septic shock, acute tubular necrosis, endomet ⁇ osis; degenerative joint disease and pancreatis
• the compounds of the present mventron are admrmstered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a penod of time, by intramuscular, mtrape ⁇ toneal, mtracerobrospinal, subcutaneous, lntra-articular. mtrasynovial, intrathecal, oral, topical, or inhalation (mtranasal, mtrapulmonary) routes. Intravenous or inhaled administration of polypeptides and antibodies is preferred.
• admrnrstration of an anti-cancer agent may be combined wrth the admrnrstration of the proterns, antibodies or compounds of the instant mvention.
• the patient to be treated with a the lmmunoadjuvant of the mventron may also recerve an anti-cancer agent (chemotherapeutic agent) or radiation therapy.
• Preparation and dosmg schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determmed empi ⁇ cally by the skilled practitioner Preparation and dosmg schedules for such chemotherapy are also descnbed rn Chemotherapy Service Ed., M.C. Perry, Wrlhams & Wilkins, Baltimore, MD ( 1992)
• the chemotherapeutic agent may precede, or follow administration of the lmmunoadjuvant or may be given simultaneously therewith.
• an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onap ⁇ stone (see, EP 616812) may be given in dosages known for such molecules.
• the polypeptides of the mvention are coadmmistered with a growth inhibitory agent.
• the growth inhibitory agent may be administered first, followed by a polypeptide of the mventron.
• 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 polypeptide of the invention.
• the appropnate 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 drscretion of the attending physician
• the compound is suitably administered to the patient at one time or over a senes of treatments
• about 1 ug/kg to 15 mg/kg (e g 0 l-20mg/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 ug/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 mav be useful The progress of this therapy
• an article of manufacture containing materials useful for the dragnosis or treatment of the disorders descnbed above is provided.
• the article of manufacture comp ⁇ ses a contamer and a label
• Surtable containers include, for example, bottles, vials, synnges.
• the containers may be formed from a vanety of matenals such as glass or plastic
• the container holds a composition which is effective for dragnosmg or treating the condition and may have a sterile access port (for example the contamer may be an mtravenous solutron bag or a vral havrng a stopper pierceable by a hypodermrc injection needle)
• the active agent m the composition is usually a polypeptide or an antibody of the mvention The label on.
• the container indicates that the composition is used for diagnosing or treating the condition of choice
• the article of manufacture may further compnse a second container compnsmg a pharmaceutically-acceptable buffer, such as phosphate-buffered salrne, Rmger's solution and dextrose solution It may further mclude other matenals desirable from a commercral and user standpomt, including other buffers, diluents, filters, needles, synnges, 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 treatment
• the same proteins along with secreted proteins encoded by the genes amplified in immune related disease states find additional use in the diagnosis and prognosis of these diseases
• antibodies directed agamst the protein products of genes amplified in multiple sclerosis, rheumatoid arthntis, 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, fluo ⁇ metry, or other technrques known m the art These technrques are particularly suitable, if the overexpressed gene encodes a cell surface protern
• Such binding assavs are performed essentially as dec ⁇ bed above
• In situ detection of antibodv 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 antibodv on a biological sample This procedure also allows for determining the dist ⁇ bution of the marker gene product in the tissue examined It will be apparent for those skilled in the art that a wrde varrety of hist
• the extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proterns from the Swrss-Prot publrc protern database were used to search expressed sequence tag (EST) databases
• the EST databases included publrc EST databases (e g ,
• GenBank GenBank
• LIFESEQTM Incyte Pharmaceuticals, Palo Alto
• a consensus DNA sequence encoding PR0245 was assembled relative to the other identified EST sequences, where the consensus sequence was designated herein as DNA30954, and the polypeptide showed some structural homology to transmembrane protein receptor tyrosine kinase proteins
• oligonucleotides were synthesized to identify by PCR a cDNA library that contamed the sequence of mterest and for use as probes to isolate a clone of the full-length coding sequence for PR0245
• a parr of PCR primers (forward and reverse) were synthesized forward PCR pnmer 5'-ATCGTTGTGAAGTTAGTGCCCC-3' (SEQ ID NO 15) reverse PCR p ⁇ mer 5'-ACCTGCGATATCCAACAGAATTG-3' (SEQ ID NO 16)
• a synthetic oligonucleotide hybndization probe was constructed from the consensus DNA30954 sequence whrch had the followmg nucleotrde sequence hvb ⁇ drzatron probe
• DNA from the hbranes was screened by PCR amplification with the PCR pnmer pair identified above A positive library was then used to isolate clones encoding the PR0245 gene using the probe oligonucleotide and one of the PCR pnmers
• RNA for construction of the cDNA libraries was isolated from human fetal liver tissue
• the cDNA hbranes used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invrtrogen, San Drego, CA
• the cDNA was pruned with oligo dT containing a Notl site, linked with blunt to Sail hemikmased adaptors, cleaved with Notl, sized appropnately by gel electrophoresis, and cloned in a defined onentation mto 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 Xliol and Notl sites
• Clone UNQ219 (DNA35638) contains a single open reading frame wrth an apparent translatronal initiation site at nucleotide positions 89-91 [Kozak et al , supra] and ending at the stop codon at nucleotide positions 1025-1027 (Fig. 3).
• the predicted polypeptide precursor is 312 amino acids long ( Figure. 4. PR0245; SEQ ID NO 2)
• Clone UNQ219 (DNA35638) has been deposited with ATCC on September 17, 1997 and is assigned ATCC Deposit No. 209265.
• 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., Dayhof, GenBank), and prop ⁇ etary databases (e g LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA).
• the search was performed using the computer program BLAST or BLAST2 (Altschul, SF and Gish ( 1996), Methods in Enzx'mology 266" 460-80 (1996), http://blast.wustl/edu/blast/README.html) as a companson of the ECD protein sequences to a 6 frame translatron of the EST sequences.
• Consensus DNA sequences encoding EGF-hke homologues were assembled (DNA28726, DNA28730 and DNA28760) using phrap. In some cases, the consensus DNA sequence was extended using repeated cycles of blast and phrap to extend the consensus sequence as far as possrble usmg the three sources of EST sequences listed above
• oligonucleotides were synthesized. 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to rsolate a clone of the full-length codmg sequence
• the parr of forward and reverse PCR pnmers may range from 20 to 30 nucleotides (typically 24), and are designed to give a PCR product of 100-1000 bp m length.
• the probe sequences (notated as *.p) are typically 40-55 bp (typically 50) in length
• additional oligonucleotides are synthesrzed when the consensus sequence is greater than 1-1 5 kbp.
• RNA for the construction of the cDNA libraries was isolated using standard isolation protocols, e.g., Ausubel et al, Current Protocols in Molecular Biolog ⁇ ', from tissue or cell line sources or it was purchased from commercial sources (e.g., Clontech).
• the cDNA libraries used to isolate the cDNA clones were constructed by standard methods (e.g., Ausubel et al.) using commercially available reagents (e.g., Invitrogen).
• the cDNA was primed with oligo dT containing a Noti site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoesis, and cloned in a defined orientation in a suitable cloning vector (pRK5B or pRK5D) in the unique Xhol and Notl sites.
• a cDNA clone was sequenced in its entirety.
• the entire nucleotide sequence of EGF-like homologue PR0217 is shown in Figure 5 (SEQ ID NO: 3).
• the predicted polypeptide is 379 (PR0217; Figure 6; SEQ ID NO: 4) amino acids in length with a molecule weight of approximately 41.52 kDa.
• oligonucleotide sequences used in the above procedure were the following: 28726.p (OLI500) (SEQ ID NO: 18) GGGTACACCTGCTCCTGCACCGACGGATATTGGCTTCTGGAAGGCC
• 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 EST databases (e.g., GenBank), a proprietary EST database (LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA).
• the search was performed using the computer program BLAST or BLAST2 [Altschul et al, Methods in Enzymology, 266:460-480 (1996); http://blast.wustl/edu/blast/README.html] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences.
• a consensus DNA sequence encoding DNA35936 was assembled using phrap. In some cases, the consensus DNA sequence was extended using repeated cycles of blast and phrap to extend the consensus sequence as far as possible using the three sources of EST sequences listed above.
• oligonucleotides 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 may range from 20 to 30 nucleotides (typically about 24), and are designed to give a PCR product of 100- 1000 bp in length.
• the probe sequences (notated as *.p) are typically 40-55 bp (typically about 50) in length.
• additional oligonucleotides are synthesized when the consensus sequence is greater than 1-1.5 kbp.
• 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 by the in vivo cloning procedure suing the probe oligonucleotide and one of the PCR primers.
• DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO301 gene using the probe oligonucleotide and one of the PCR primers.
• RNA for construction of the cDNA libraries was isolated from human fetal kidney.
• the cDNA libraries used to isolated the cDNA clones were constructed by standard methods using commercially available reagents (e.g., Invitrogen, San Diego, CA; Clontech, etc.)
• the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, 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 Notl sites.
• a suitable cloning vector such as pRKB or pRKD; pRK5B is
• FIG. 7 The full length nucleotide sequence of native sequence PRO301 is shown in Figure 7 (SEQ ID NO: 5).
• Clone DNA40628 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 52-54 [Kozak et al, supra] (Fig. 7).
• the predicted polypeptide (PRO301; Figure 8; SEQ ID NO: 6) is 299 amino acids long with a predicted molecular weight of 32583 daltons and pi of 8.29.
• Clone DNA40628 has been deposited with ATCC and is assigned ATCC deposit No. 209432.
• PRO301 Based on a BLAST and FastA sequence alignment analysis of the full-length sequence, PRO301 shows amino acid sequence identity to A33 antigen precursor (30%) and coxsackie and adenovirus receptor protein (29%).
• oligonucleotide sequences used in the above procedure were the following:
• the extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases.
• the EST databases included public EST databases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altshul 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 sequence.
• oligonucleotides 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 for PR0266.
• 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 l-1.5kbp. In order to screen several hbranes for a full-length clone.
• DNA from the hbranes was screened by PCR amplification, as ber Ausubel et al , Current Protocols in Molecular Biology, with the PCR pnmer pair A positive library was then used to isolate clones encodmg the gene of interest by the in vivo clongm procedure using the probe oligonucleotide and one of the pnmer pairs
• a pair of PCR p ⁇ mers ( forward and reverse) were synthesized forward PCR pnmer 5'-GTTGGATCTGGGCAACAATAAC-3' (SEQ ID NO. 33) reverse PCR pnmer 5'-ATTGTTGTGCAGGCTGAGTTTAAG-3' (SEQ ID NO 34)
• hvbrrdrzation probe 5'-GGTGGCTATACATGGATAGCAATTACCTGGACACGCTGTCCCGGG-3' (SEQ ID NO 35)
• DNA from the hbranes was screened by PCR amplification with the PCR pnmer pair identified above A positive library was then used to isolate clones encoding the PR0266 gene using the probe oligonucleotide and one of the PCR pnmers
• RNA for construction of the cDNA hbranes was isolated from human fetal bram tissue
• the cDNA hbranes 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 pruned with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined o ⁇ entation into a suitable clomng 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 Notl sites
• PR0266 may be a novel leucine rich repeat protein V Isolation of cDNA Clones Encoding Human PRQ335 PRQ331 or PRQ326
• the extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the Swrss-Prot publrc protein database were used to search expressed sequence tag (EST) databases
• the EST databases included public EST databases (e g , GenBank) and a prop ⁇ etary EST DNA database (LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA)
• the search was performed using the computer program BLAST or BLAST2 (Altshul et al , Methods m Enzymology 266 460-480 (1996)) as a companson of the ECD protern sequences to a 6 frame translation of the EST sequence Those compansons resulting rn 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, Washington, http //bozeman m
• oligonucleotides were synthesized 1 ) to identify by PCR a cDNA library that contamed the sequence of mterest, and 2) for use as probes to rsolate a clone of the full- length coding sequence for PR0335, PR0331 or PR0326
• Forward and reverse PCR pnmers 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 olrgonucleotides are synthesrzed when the consensus sequence rs greater than about 1-1 5kbp
• DNA from the hbranes was screened by PCR amplrficatron, as per Ausubel et al , Current Protocols in Molecular Biology,
• DNA from the hbranes was screened by PCR amplification with the PCR pnmer pair identified above A positiv e library was then used to isolate clones encoding the PR0335, PR0331 or PR0326 gene usmg the probe oligonucleotide and one of the PCR pnmers
• RNA for construction of the cDNA hbranes was isolated from human fetal krdney trssue (PR0335 and PR0326) and human fetal brain (PR0331)
• the cDNA hbranes used to isolate the cDNA clones were constructed by standard methods usmg commercially available reagents such as those from Invitrogen, San Diego, CA
• the cDNA was pnmed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized approp ⁇ atelv 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 X
• DNA sequencing of the clones isolated as desc ⁇ bed above gave the full-length DNA sequence for PR0335 (Frgure 1 1 SEQ ID NO 9), PR0331 ( Figure 13. SEQ ID NO 1 1 ) or PR0326 ( Figure 15. SEQ ID NO 13) and the denved protein sequence for PR0335 ( Figure 12. SEQ ID NO 10).
• PR0331 ( Figure 14, SEQ ID NO. 12) or PR0326 ( Figure 16, SEQ ID NO 14) The nucleic acid encoding PR0335 was deposited with the ATCC on 2 June 1998 and is assigned ATCC Accession No. 209927.
• nucleic acid encoding PR0331 was deposited with the ATCC on 7 November 1997 and is assigned ATCC Accession No 209439, and the nucleic acrd encoding PR0326 was deposited with the ATCC on 21 November 1997 and is assigned ATCC Accession No. 209489
• polypeptides of the mvention are active as a stimulator of the proliferation of stimulated T-lymphocytes.
• Compounds which stimulate prohferatron of lymphocytes are useful therapeutically where enhancement of an immune response is beneficial
• Compounds which inhibit prohferatron of lymphocytes are useful therapeutically where suppression of an immune response is beneficial
• a therapeutic agent may take the form of antagonists of the polypeptide of the invention, for example, mu ⁇ ne-human chimenc, humanized or human antibodies agamst the polypeptide.
• PBMC penpheral blood mononuclear cells
• mammalian individuals for example a human volunteer
• leukopheresis one donor will supply stimulator PBMCs, the other donor will supply responder PBMCs
• the cells are frozen m fetal bovine serum and DMSO after isolation. Frozen cells may be thawed overnrght in assay media (37°C, 5% CO2 ) and then washed and resuspended to 3 x 10 ⁇ cells/ml of assay media
• RPMI 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES. 1% non- essential amino acids, 1 % pyruvate
• the stimulator PBMCs are prepared by irradiating the cells (about 3000 Rads)
• the assay is prepared by plating in tnphcate wells a mixture of lOO ⁇ l of test sample diluted to 1% or to 0.1%
• PBMC cells 100 microliters of cell culture media or 100 microliter of CD4-IgG is used as the control.
• the wells are then incubated at 37°C, 5% CO2 for 4 days. On day 5 and each well is pulsed with tritiated thymidine (i.O mC/well; Amersham). After 6 hours the cells are washed 3 times and then the uptake of the label is evaluated.
• tritiated thymidine i.O mC/well; Amersham
• PBMCs are isolated from the spleens of Balb/c mice and C57B6 mice.
• the cells are teased from freshly harvested spleens in assay media (RPMI; 10% fetal bovine serum, 1 % penicillin/streptomycin. 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1 % pyruvate) and the PBMCs are isolated by overlaying these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 m for 20 minutes, collecting and washing the mononuclear cell layer in assay media and resuspending the cells to lx 10 7 cells/ml of assay media.
• assay media RPMI; 10% fetal bovine serum, 1 % penicillin/streptomycin. 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1 % pyruvate
• the assay is then conducted as described above using a sample having a PRO concentration obtained by diluting a stock solution.
• the results of this assay for compounds of the invention are shown below. Positive increases over control are considered positive with increases of greater than or equal to 180% being preferred. However, any value greater than control indicates a stimulatory effect for the test protein.
• EXAMPLE 3 Skin Vascular Permeabrlrty Assay (No. 64) This assay shows that certam polypeptides of the invention stimulate an immune response and induce inflammation by inducing mononuclear cell, eosinophil and PMN infiltration at the site of injection of the animal.
• This skm vascular permeabrlrty assay rs conducted as follows. Harrless guinea pigs weighing 350 grams or more are anesthetized with ketamme (75-80 mg/Kg) and 5 mg/Kg xylazme intramuscularly (IM).
• a sample of punfied polypeptide of the invention or a conditioned media test sample is injected intradermally onto the backs of the test ammals wrth 100 ⁇ L per injection site. It is possible to have about 10-30, preferably about 16-24, rnjection sites per animal.
• One L of Evans blue dye (1% in physiologic buffered saline) is injected mtracardially Blemishes at the injection sites are then measured (mm diameter) at lhr and 6 hr post injection. Anrmals were sac ⁇ ficed at 6 hrs after injection.
• Each skm injection site was biopsied and fixed in formalm The skins were then prepared for hrstopathalogrc evaluatron Each site was evaluated for mflammatory cell infiltration mto the skin. Sites with visible inflammatory cell mflammatron were scored as positive. Inflammatory cells can be neutrophihc, eosinophilic, monocytic or lymphocytic. The results of this test for compounds of the invention is shown below.
• EXAMPLE 4 Human Co-Stimulation Assay
• T cell activation requires a costimulatory signal.
• One costimulatory signal is generated by the interaction of B7 (CD3) with CD28.
• B7 CD3
• 96 well plates are coated with CD3 with or without CD28 and then human peripheral blood lymphocytes followed by a test protein, are added. Proliferation of the lymphocytes is determined by tritiated thymidine uptake.
• a positive assay indicates that the test protein provided a stimulatory signal for lymphocyte proliferation.
• Plates are prepared by coating 96 well flat bottom plates with anti-CD3 antibody (Amac) or anti-CD28 antibody (Biodesign) or both in Hyclone D-PBS without calcium and magnesium.
• Anti - CD3 antibody is used at 10 ng/well (50 ⁇ l of 200 ng/ml) and anti -CD28 antibody at 25 ng/well (50 ⁇ l of 0.5 ⁇ g/ml) in 100 ⁇ l total volume.
• PBLs are isolated from human donors using standard leukophoresis methods. The cell preparations are frozen in 50% fetal bovine serum and 50% DMSO until the assay is conducted. Cells are prepared by thawing and washing PBLs in media, resuspending PBLs in 25 mis of media and incubating at 37°C, 5% CO2 overnight.
• the coated plate is washed twice with PBS and the PBLs are washed and resuspended to 1 x 10" cells/ml using 100 ⁇ L /well 100 ⁇ l of a test protein or control media are added to the plate making a total volume per well of 200 ⁇ L.
• the plate is incubated for 72 hours.
• the plate is then pulsed for 6 hours with tritiated thymidine ( 1 mC/well; Amersham) and the PBLs are harvested from the plates and evaluated for uptake of the tritiated thymidine.
• In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping.
• the probe was run on a TBE/urea gel 1-3 ⁇ L of the probe or 5 ⁇ L of RNA Mrk III were added to 3 ⁇ L of loading buffer After heating on a 95C heat block for three minutes, the gel was immediately placed on ice The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes The gel was wrapped in saran wrap and exposed to XAR film with an intensifying screen in -70C freezer one hour to overnrght
• Olrgo D-200Z (SEQ ID NO 39) CTATGA AATTAA CCC TCA CTA AAG GGA TGTCTT CCA TGC CAA CCTTC
• EXAMPLE 6 In situ Hybridization in Cells and Diseased Tissues
• the in situ hybridization method of Example 5 is used to determine gene expression, analyze the tissue distribution of transcription, and follow changes in specific mRNA synthesis for the genes DNAs and the proteins of the invention in diseased tissues isolated from human individuals suffering from a specific disease. These results show more specifically where in diseased tissues the genes of the invention are expressed and are more predictive of the particular localization of the therapeutic effect of the inhibitory or stimulatory compounds of the invention (and agonists or antagonists thereof) in a disease.
• Hybridization is performed according to the method of Example 5 using one or more of the following tissue and cell samples:
• lymphocytes and antigen presenting cells (a) lymphocytes and antigen presenting cells (dendritic cells, langherhans cells, macrophages and monocytes, NK cells);
• lymphoid tissues normal and reactive lymph node, thymus, Bronchial Associated Lymphoid Tissues, (BALT), Mucosal Associated Lymphoid Tissues (MALT);
• Lung tissue including BALT and tissue lymph nodes from Chronic and acute bronchitis, pneumonia, pneumonitis, pleuritis
• Expression is observed in one or more cell or tissue samples indicating localization of the therapeutic effect of the compounds of the invention (and agonists or antagonists thereof) in the disease associated with the cell or tissue sample.
• DNA 35638 (PR0245) was found to be expressed in inflamed human tissues (psoriasis, inflammatory bowel disease (IBD), inflamed kidney, inflamed lung, hepatitis (liver block), normal tonsil, adult and chimp (multiblocks). Expression was present in the endothelium/intima of large vessels in the lung afflicted with chronic inflammation, in the superficial dermal vessels of the psoriatic skin, in arterioles in a specimen of chronic sclerosing nephritis, and in capillaries including the perifollucular sinuses of the tonsil. These results indicate that PRO 245 is immunostimulatory (enhances T lymphocyte proliferation in the MLR and costimulation) and has proinflammatory properties (induces a neutrophjil infiltrate in vivo).
• the following method describes use of a nucleotide sequence encoding PR0245, PR0217, PRO301 , PR0266, PR0335, PR0331 or PR0326 as a hybridization probe.
• DNA comprising the coding sequence of full-length or mature PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 (as shown in Figures 4, 6, 8, 10, 12, 14 and 16) is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326) in human tissue cDNA libraries or human tissue genomic libraries.
• homologous DNAs such as those encoding naturally-occurring variants of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326
• Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions.
• Hybridization of radiolabeled PR0245. PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326-derived probe to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20 hours. Washing of the filters is performed in an aqueous solution of 0. lx SSC and 0.1% SDS at 42°C.
• DNAs having a desired sequence identity with the DNA encoding full-length native sequence PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can then be identified using standard techniques known in the art.
• EXAMPLE 8 Expression ofPRQ245. PRQ217. PRO301. PRQ266. PRQ335. PRQ331 or PRQ326 _ E. coli This example illustrates preparation of an unglycosylated form of PR0245, PR0217,
• PRO301 , PR0266, PR0335, PR0331 or PR0326 by recombinant expression in E. coli.
• the DNA sequence encoding PR0245, PR0217, PRO301 , PRO266, PR0335, PR0331 or PR0326 is initially amplified using selected PCR primers.
• the primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector.
• a variety of expression vectors may be employed.
• An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al, Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance.
• the vector is digested with restriction enzyme and dephosphorylated.
• the PCR amplified sequences are then ligated into the vector.
• the vector will preferably include sequences which encode for an antibiotic resistance gene, a tip promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 coding region, lambda transcriptional terminator, and an argU gene.
• the ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al, supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.
• Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics.
• the overnight culture may subsequently be used to inoculate a larger scale culture.
• the cells are then grown to a desired optical density, during which the expression promoter is turned on.
• the cells After culturing the cells for several more hours, the cells can be harvested by centrifiigation.
• the cell pellet obtained by the centrifiigation can be solubilized using various agents known in the art, and the solubilized PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 protern can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
• PR0245, PR0217, PRO301 and PR0266 were expressed in E. coli in a poly-His tagged form, using the following procedure.
• the DNA encoding PR0245, PR0217, PRO301 and PR0266 was initially amplified using selected PCR primers.
• the primers contained restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase.
• the PCR-amplified, poly-His tagged sequences were then ligated into an expression vector, which was used to transform an E.
• Cultures were then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH ) 2 S0 , 0.71 g sodium citrate.2H20, 1.07 g KC1, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgS0 4 ) and grown for approximately 20-30 hours at 30C with shaking. Samples were removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets were frozen until purification and refolding.
• CRAP media prepared by mixing 3.57 g (NH ) 2 S0 , 0.71 g sodium citrate.2H20, 1.07 g KC1, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3,
• E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) was resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1M and 0.02 M, respectively, and the solution was stirred overnight at 4C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution was centrifuged at 40,000 ⁇ m in a Beckman Ultracentifuge for 30 min.
• the supernatant was diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. Depending the clarified extract was loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4C. Protein concentration was estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
• the proteins were refolded by diluting 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 were chosen so that the final protein concentration was between 50 to 100 micrograms/ml The refolding solution was stirred gently at 4C for 12-36 hours. The refolding reaction was quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added to 2-10% final concentration.
• 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 were chosen so that the final protein concentration
• the refolded protein was 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 were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein were pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
• This example illustrates preparation of a potentially glycosylated form of PR0245, PR0217, PRO301, PR0266. PR0335, PR0331 or PR0326 by recombinant expression in mammalian cells.
• the vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector.
• the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 DNA using ligation methods such as described in Sambrook et al, supra.
• the resulting vector is called pRK5-PR0245, PR0217. PRO301, PR0266, PR0335, PR0331 or PR0326.
• 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.
• PRO266, PR0335, PR0331 or PR0326 DNA is mixed with about 1 ug DNA encoding the VA RNA gene [Thimmappaya et al, Cell. 31:543 (1982)] and dissolved in 500 uL 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 uCi/ml 35 S-cysteine and 200 uCi/ml 35 S-methionine.
• 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 PR0245, PR0217, PRO301, PR0266. PR0335, PR0331 or PR0326 polypeptide.
• the cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
• PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 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 ug pRK5-PR0245. PR0217. PRO301, PR0266, PR0335.
• PR0331 or PR0326 DNA is added The cells are first concentrated from the spinner flask by centnfugation and washed with PBS The DNA-dextran precipitate is mcubated on the cell pellet for four hours The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re- mtroduced mto the spinner flask containing tissue culture medium. 5 ⁇ g/ml bovine insulin and 0.1 ug/ml bovine transfernn After about four days, the conditroned medra rs centrrfuged and filtered to remove cells and deb ⁇ s The sample contarmng expressed PR0245, PR0217. PRO301. PR0266, PR0335. PR0331 or PR0326 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography
• PR0245. PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can be expressed m CHO cells
• the pRK5-PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can be transfected into CHO cells using known reagents such as CaP0 4 or DEAE-dextran. As desc ⁇ bed above, the cell cultures can be mcubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 3:> S-meth ⁇ onme After determrnmg the presence of PR0245. PR0217. PRO301.
• the culture medium may be replaced with serum free medrum.
• the cultures are mcubated for about 6 days, and then the condrtioned medium is harvested.
• PR0266, PR0335, PR0331 or PR0326 can then be concentrated and pu ⁇ fred by any selected method.
• Epitope-tagged PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 may also be expressed m host CHO cells
• the PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 may be subcloned out of the pRK5 vector
• the subclone msert can undergo PCR to fuse m frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector.
• PR0331 oi PR0326 insert can then be subcloned into a SV40 dnven vector containing a selection marker such as DHFR for selection of stable clones Finally, the CHO cells can be transfected (as descnbed above) with the SV40 driven vector. Labeling may be performed, as described above, to ve ⁇ fy expression
• the culture medium containing the expressed poly-His tagged PR0245, PR0217. PRO301. PR0266. PR0335. PR0331 or PR0326 can then be concentrated and pu ⁇ fied by any selected method, such as by N ⁇ 2+ -chelate affinity chromatography
• PR0245, PR0217 and PRO301 were expressed m CHO cells by both a transient and a stable expression procedure
• the proterns were expressed as an IgG construct (lmmunoadhesm), in which the coding sequences for the soluble forms (e.g extracellular domains) of the respective proteins were 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 mterest to allow the convement shuttling of cDNAs
• the vector used expression in CHO cells is as desc ⁇ bed in Lucas et al , Nucl Acids Res 24 9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to dnve expression of the cDNA of interest and dihydrofolate reductase (DHFR) DHFR expressron permits selection for stable maintenance of the plasmid followmg transfectron
• the ampules containing the plasmid DNA were thawed by placement mto water bath and mixed by vortexmg
• the contents were pipetted into a centrifuge tube containing 10 mLs of medra and cent ⁇ fuged at 1000 ⁇ m for 5 mmutes
• the supernatant was asprrated and the cells were resuspended rn 10 mL of selectrve media (0 2 ⁇ m filtered PS20 with 5% 0 2 ⁇ m diafiltered fetal bovme serum)
• the cells were then alrquoted to a 100 mL sprnner containing 90 mL of selective media After 1-2 days, the cells were transferred into a 250 mL spmner filled with 150 mL selective growth medium and incubated at 37C After another 2-3 days, a 250 mL, 500 mL and 2000 mL spinners were seeded with 3 x 10 5 cells/mL
• the cell medra was exchanged
• the proteins were punfied using a Ni-NTA column (Qiagen) Before pu ⁇ frcation, imidazole was added to the conditioned media to a concentration of 5 mM The conditioned media was pumped onto a 6 ml Ni-NTA column equilrbrated 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/mm at 4C After loading, the column was 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 was 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 -80C.
• Immunoadhesin (Fc containing) constructs of were purified from the conditioned media as follows.
• the conditioned medium was 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 was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
• the eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ⁇ L of 1 M Tris buffer, pH 9.
• the highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
• PR0326 was also produced by transient expression in COS cells.
• the following method describes recombinant expression of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 in yeast.
• yeast expression vectors are constructed for intracellular production or secretion of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 from the ADH2/GAPDH promoter.
• DNA encoding PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326.
• DNA encoding PR0245, PR0217, PRO301, PR0266, PR0335 DNA encoding PR0245, PR0217, PRO301, PR0266, PR0335.
• PR0331 or PR0326 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 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 PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326.
• Yeast cells such as yeast strain ABI 10
• 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% trichloroacetic acid and separation by SDS- PAGE, followed by staining of the gels with Coomassie Blue stain.
• Recombinant PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 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 PR0245, PR0217, PRO301. PR0266. PR0335. PR0331 or PR0326 may further be purified using selected column chromatography resins.
• EXAMPLE 1 1 Expression of PRQ245. PRQ217, PRO301. PRQ266. PRQ335. PRQ331 or PRQ326 in Baculovirus- Infected Insect Cells
• the following method describes recombinant expression of PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 in Baculovirus-infected insect cells.
• the sequence coding for PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 is fused upstream of an epitope tag contained within a baculovirus expression vector.
• epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG).
• a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PR0245, PR0217, PRO301, PR0266, PR0335. PR0331 or PR0326 or the desired portion of the coding sequence of PR0245.
• PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers complementary to the 5' and 3' regions.
• the 5' primer may inco ⁇ orate flanking (selected) restriction enzyme sites.
• the product is then digested with those selected restriction enzymes and subcloned into the expression vector.
• Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA (Pharmingen) into Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al, Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
• PR0326 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.
• 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 um 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 2 so 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
• purification of the IgG tagged (or Fc tagged) PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
• PR0245, PRO301 and PR0266 were expressed in baculovirus infected Sf9 insect cells. While the expression was actually performed in a 0.5-2 L scale, it can be readily scaled up for larger (e.g. 8 L) preparations.
• the proteins were expressed as an IgG construct (immunoadhesin), in which the protein extracellular region was fused to an IgG 1 constant region sequence containing the hinge, CH2 and CH3 domains and or in poly-His tagged forms.
• baculovirus expression vector pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins
• vector and Baculogold baculovirus DNA were co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711), using Lipofectin (Gibco BRL).
• Sf9 Spodoptera frugiperda
• pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences.
• the cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells were incubated for 5 days at 28C. The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells were incubated for 3 days at 28C.
• MOI multiplicity of infection
• the supernatant was harvested and the expression of the constructs in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 mL of Ni-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
• the first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells were incubated for 3 days at 28C. The supernatant was harvested and filtered. Batch binding and SDS-PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed.
• the conditioned medium from the transfected cells (0.5 to 3 L) was harvested by centrifiigation to remove the cells and filtered through 0.22 micron filters.
• the protein construct were purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media were 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 4C.
• the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
• the highly purified protein was 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 -80C.
• Irnmunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows.
• the conditioned media were 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 was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
• the eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 mL of 1 M Tris buffer, pH 9.
• the highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins.
• the homogeneity of the proteins was verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.
• PR0245, PR0217, PRO301, PR0266, PR0331 and PR0326 were also expressed in baculovirus infected High-5 cells using an analogous procedure.
• This example illustrates preparation of monoclonal antibodies which can specifically bind PR0245.
• Immunogens that may be employed include purified PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326, fusion proteins containing PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326. and cells expressing recombinant PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 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 PR0245.
• PR0335, PR0331 or PR0326 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms.
• the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads.
• 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 penodrcally obtamed from the mrce by retro-orbrtal bleedmg for testing rn ELISA assays to detect antr-PR0245. PR0217, PRO301. PR0266, PR0335, PR0331 or PR0326 antibodies.
• the animals "positive" for antibodies can be injected with a final intravenous injection of PR0245, PR0217. PRO301, PR0266, PR0335, PR0331 or PR0326
• the mice are sacrificed and the spleen cells are harvested.
• the spleen cells are then fused (usmg 35% polyethylene glycol) to a selected munne myeloma cell line such as P3X63AgU.l, available from ATCC, No CRL 1597
• the fusions generate hyb ⁇ doma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine. ammopte ⁇ n. and thymidme) medium to inhibit proliferation of non- fused cells, myeloma hyb ⁇ ds, and spleen cell hybnds.
• HAT hyperxanthine. ammopte ⁇ n. and thymidme
• the hybndoma cells will be screened m an ELISA for reactivity agamst PR0245, PR0217, PRO301, PR0266. PR0335.
• PR0331 or PR0326 Determination of "positive" hybndoma cells secreting the desired monoclonal antibodies agamst PR0245, PR0217, PRO301. PR0266. PR0335.
• PR0331 oi PR0326 is within the skill in the art.
• the posrtrve hybndoma cells can be mjected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the ant ⁇ -PR0245, PR0217, PRO301, PR0266, PR0335, PR0331 or PR0326 monoclonal antibodies.
• the hyb ⁇ doma cells can be grown in tissue culture flasks or roller bottles.
• Pu ⁇ fication of the monoclonal antibodies produced m the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography Alternatively, affnrty chromatography based upon bindmg of antibody to protein A or protein G can be employed
• Max file length is 65535 (limited bv unsigned short x in the jmp struct)
• IJ dx[ ⁇ d] ljmp, if (dx[ ⁇ d] jp n[0] && ('dna
• (ndelx > MAXJMP && xx > dx[ ⁇ d] jp x[ ⁇ j]+MX)
• *ps[ ⁇ ] toupper(*ps[ ⁇ ]), po[ ⁇ ]++, ps[ ⁇ ]++
• *hne ' ⁇ '
• *py++ *px, else if ( ⁇ slower(*px))
• *py++ toupper(*px), if ( ⁇ ndex( "ATGCU”,*(py- 1 ))) natgc++, ⁇ ⁇

Landscapes

• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Applications Claiming Priority (5)

Publications (1)

Family

ID=26793864

Family Applications (1)

Country Status (1)

• 1999
  • 1999-09-15 EP EP99952930A patent/EP1114154A2/de not_active Withdrawn

Non-Patent Citations (1)

Similar Documents

Legal Events