MXPA01010480A - Novel uses of mammalian ox2 protein and related reagents. - Google Patents

Abstract

Compositions and methods for using mammalian ligand OX2 to treat an abnormal physiological condition in an individual. The methods comprise administering a therapeutically effective amount of OX2 alone, or in combination with other therapeutic reagents; or an OX2 antagonist.

Description

NEW USES OF MAMMERIAN OX2 PROTEIN AND RELATED REAGENTS FIELD OF THE INVENTION The present invention relates to methods for the use of proteins that function in the control of the physiology, development and differentiation of mammalian cells, for example, cells of a neural or mammalian immune system. In particular, it provides methods for the use of proteins and mimetics that regulate the physiology, development and cellular differentiation, or the function of various cell types, including hematopoietic or neural cells.

BACKGROUND OF THE INVENTION The immune system of vertebrates consists of a number of organs and several different types of cells. Two types of major cells include the myeloid and lymphoid lineages. Among the lineage of lymphoid cells are B cells, which were originally characterized as differentiation in fetal liver or adult bone marrow, and T cells, which were originally characterized as differentiation in the thymus. Another type of cell is the mononuclear phagocyte, a cell line widely distributed in most tissues. Phagocytes play a role in inflammation, defenses against hosts and reaction against a variety of materials derived from themselves and strangers. See, for example, Paul (ed. 1997) Fundamental Immunoloqy (4th ed.) Raven Press, New York. In many aspects of the development or regulation of an immune response or cellular differentiation, soluble or membrane proteins play an important role in the regulation of cellular interactions. These proteins also intervene in cellular activities in many ways. It has been shown, in many cases, that they modulate the proliferation, growth and differentiation of hematopoietic stem cells in the vast number of progenitors that make up the lineages responsible for an immune response. Others are important mediators of intercellular signaling, often as receptors or ligands. They are also quite important in immune responses and physiology. However, cellular molecules that are expressed by different stages of cell development in the maturation pathways are still incompletely identified. Likewise, the roles and mechanisms of action of the signaling molecules that induce, sustain or modulate the various physiological, developmental or proliferative states of these cells are poorly understood. Clearly, the immune system and its response to various types of stress have relevance to medicine, for example, evacuation of cellular materials or other materials after injury, infectious diseases, cancer-related responses and treatment, and transplant rejection responses. and allergic. See, for example, Thorn, et al. Harrison's Principles of Intemal Medicine McGraw Hill, New York- Ziegler, et al. (ed. 1997) Growth Factors and Wound Healing: Basic Science and Potential Clinical Applications Springer Verlag, Clark (ed.1996) The Molecular and Cellular Bioloqy of Wound Repar Plenum; and Peacock (1984) Wound Repair Saunders. Medical science is based, in large part, on the renewal or appropriate suppression of the immune system in effecting cures for insufficient or inadequate physiological responses to environmental factors. However, the lack of understanding as to how the immune system is regulated or differentiated has blocked the ability to advantageously modulate the immunological mechanisms to biological challenges, ie, response to biological injury. Medical conditions characterized by abnormal or inadequate regulation of the development or physiology of relevant cells therefore remain unmanageable. The discovery and characterization of specific regulatory pathways and their physiological effects will contribute to the development of therapies for a wide range of degenerative conditions or other conditions that affect the biological system, immune cells, as well as other types of cells. The present invention provides solutions to some of these problems and many others.

BRIEF DESCRIPTION OF THE INVENTION The present invention is based, in part, on the discovery of the physiological role of the OX2 ligand, also referred to herein as the OX2 protein, in various models of immune response. In particular, the role of the ligand OX2 has been elucidated in routes involved in infectious disease, hematopoietic development and viral infection. The present invention provides methods for the modulation of trafficking or activation of a leukocyte in an animal, the methods comprising contacting cells of myeloid lineage, eg, monocyte / macrophage, in the animal with a therapeutic amount of a protein agonist. Mammalian OX2 or an antagonist of a mammalian OX2 protein. Preferred embodiments include where: the mammalian OX2 protein is a primate protein; and / or the antagonist is an antibody that binds to mammalian OX2. Certain embodiments include where myeloid lineage cells, eg, monocyte / macrophage, include a macrophage, microglial, granulocyte, or dendritic cell, or when the animal exhibits signs or symptoms of an infectious, inflammatory, leukoproliferative, neurodegenerative or post-infectious condition. -traumatic Preferred embodiments include when the signal or symptom is in nervous tissue, lymphoid tissue, myeloid tissue, pancreas, gastrointestinal tissue, thyroid tissue, muscle or skin or collagenous tissue.

Other methods include when the modulation is to inhibit the function of the leukocyte cell; and / or when the agonist is administered. Preferably, the agonist is mammalian OX2. Certain modalities include when the animal is experiencing signs or symptoms of autoimmunity; an inflammatory condition; tissue-specific autoimmunity; degenerative autoimmunity; rheumatoid arthritis; atherosclerosis; multiple sclerosis; vasculitides; delayed hypersensitivities; skin graft; a transplant; spinal injury; cerebrovascular accident neurodegeneration or ischemia. The administration may be in combination with: an anti-inflammatory cytokine agonist or antagonist; a painkiller; an anti-inflammatory agent or a steroid. Various methods are provided where the modulation is to increase the function of the leukocyte cell, and / or the administration is the antagonist. Preferably, the antagonist is: an antibody that binds to mammalian OX2 or a mutein of mammalian OX2 that competes with mammalian OX2 for binding to an OX2 receptor, although it does not signal substantially. In several modalities, the method is applied when the animal experiences signs or symptoms of infection, wound healing or clot formation. The administration will often be in combination with: an angiogenic factor; a growth factor, including FGF or PDGF; an antibiotic or antiviral reagent, or a clotting factor. Various methods are provided, for example, for modulating the activation of a leukocyte in a tissue, the method comprising contacting cells of myeloid lineage or monocyte / macrophage in the tissue with: an agonist of a mammalian OX2 protein; or an antagonist of a mammalian OX2 protein. Often the modulation is to inhibit the leukocyte cell and the contact is with the agonist. The administration is often in combination with: an anti-inflammatory cytokine agonist or antagonist; a painkiller; an anti-inflammatory agent or a steroid. Alternatively, the modulation is to increase, and the contact is with the antagonist. The administration can be in combination with: an angiogenic factor; a growth factor, including FGF or PDGF; an antibiotic or antiviral, or a clotting factor. 1. General The OX2 antigen was first characterized in rat, using a monoclonal antibody (mAb) MRC OX2. See, for example, McMaster and Williams (1979) Eur. J. Immunol. 9: 426-433; Barclay (1981) Immunoloqy 44: 727-736; Barclay (1981) Immunoloav 42: 593-600; Bukovsky et al. (1984) Immunology 52: 631-640; and Webb and Barclay (1984) J. Neurochem. 43: 1061-1067. The use of this antibody in immunohistochemical staining (IHC) of tissue sections or cell suspensions for flow cytometry revealed that the OX2 antigen was expressed by a wide variety of cells, eg, neurons, vascular endothelium, B cells, T cells activated, follicular dendritic cells, interlaced dendritic cells, smooth muscle cells and trophoblasts. Additionally, human OX2 is known to be expressed in normal brain and B cells. McCaughan, et al. (1987) Immunoqenetics 25: 329-335. The characterization of the rat protein recognized by MRC OX2 (Clark, et al (1985) EMBO J. 4: 113-118) revealed that OX2 consists of approximately 248 amino acids comprising two extracellular domains of immunoglobulin (Ig), one domain of transmembrane and a short C-terminal cytoplasmic tail. The molecule is glycosylated through 6 N-linked glycosylation sites, three of which are present in the Ig domain of the V-terminal N type and the others reside in the Ig domain of the C2 type proximal to the membrane. This places OX2 in the Ig (IgSF) superfamily, forming a subgroup of small IgSF molecules with molecules such as CD2, CD48, CD58, CD80, CD86, CD90 and CD147. Interestingly, CD90 is also highly expressed by neurons. Williams, et al. (1977) Cold Spring Harb. Syrnp. Quant. Biol. 41 Pt 1: 51-61. Additionally, OX2 has been shown to be a structural homologue of CD80 and CD86 (Borriello, et al (1997) J. Immunol., 158: 4548-4554) and that the OX2 gene was closely linked to those encoding CD80 and CD86 on chromosome 16 in the mouse. Boriello, et al. (1998) Mamm. Genome 9: 114-118. Both CD80 and CD86 serve as ligands in a process known as co-stimulation, and therefore it is likely that OX2 acted as a ligand as well. The OX2 antigen will be referred to hereinafter as the OX2 protein or OX2 ligand. The binding partner will be referred to as the OX2 receptor, although it has not been fully characterized.

To identify the receptor for OX2 (OX2R) the Barclay group prepared a multivalent reagent using rat CD4-OX2 fusion protein bound to fluorescent beads. This reagent was shown to bind rat and mouse peritoneal macrophages, and this binding could be blocked by MRC OX88 mAb. Presten, et al. (1997) Eur. J. Immunol. 27: 1911-1918. It was shown that this mAb binds to isolated macrophages from both peritoneum and spleen and in IHC staining sections of spleen was found in areas known to contain high proportions of macrophages. The exaggerated or defective activation of macrophages contributes to the pathogenesis of a wide range of immune diseases and other diseases. See, for example, McGee, et al. (eds. 1992) Oxford Textbook of Pathology Oxford University Press, Oxford; Lewis and McGee (eds. 1992) The Macrophaqe IRL Press, Oxford; and Bock and Goode (eds. 1997) The Molecular Basis of Cellular Defense Mechanisms Wilev & Sons. The distribution of OX2 is consistent with a hypothesis that OX2 transmits a signal through OX2R to macrophages, and possibly other cells of the myeloid or monocyte-macrophage lineages. In this scenario, for example, the expression of OX2 in the neurons could establish a direct path of communication to the resident macrophages of the brain called microglia that could express OX2R, since they originate from the monocyte-macrophage lineage. Perry and Gordon (1988) Trends Neurosci. 11: 273-277. Using the MRC OX88 mAb in IHC of brain sections it has not been possible to identify the molecule in microglia. However, this negative result could be caused by the fact that MRC OX88 is an IgM, an antibody isotype of which it is known in general to have low affinity. To study the biological role of OX2, and in particular if OX2-OX2R interactions are involved in the regulation of macrophage function, a mouse OX2 genomic clone was isolated from a C57BL / 6 library. This allowed the construction of a target vector, with which knockout (KO) mice were created by the targeted interruption of the OX2 gene by homologous recombination in C57BL / 6 ES cells. The homozygous KO mice were bred and developed normally, although initial examination of the internal organs showed anatomical abnormalities in some lymphoid tissues. These included enlarged red pulp of the spleen, and weakened segregation of the mesenteric lymph nodes with enlarged marginal sinus. Both changes can be attributed to an expanded macrophage and, in the spleen at least, a population of expanded granulocytes. These results indicate that even in the stable state, OX2 can regulate the myeloid cell, for example, macrophage, numbers and its activation, presumably by means of the binding of OX2R. KO OX2 mice can now be used in studies of the function of myeloid or macrophage cells, particularly of monocyte / macrophage lineage activities, applying model systems for the activation of cells of these cell lineages. The first model system used for this purpose is a paradigm for the activation of microglia in the brain through nerve injury. Streit and Graeber (1993) Glia 7: 68-74. This model makes use of the fact that the cross section of the facial nerve, which directs the motor behavior in the facial area, produces the activation of microglia after four to seven days in the facial nucleus in the brain stem, where the neurons are located motor In the OX2 KO mouse, this activation occurs already two days after surgery, much earlier than in a normal mouse. This activation is accompanied by the expression of the activation marker DAP12, as shown by I HC. Both the results of the constant state and the cross section of the facial nerve are consistent with a hypothesis that the binding of OX2R in macrophages by OX2 gives rise to a downward regulatory signal. This hypothesis can be studied in more detail and in different model systems, as in in vivo activation of monocyte lineage cells, macrophages, for example, by intraperitoneal injections with LPS and determination of serum levels of TNF. In OX2 KO mice, the response to TNF after challenge with LPS may be stronger, and the macrophages in these mice lack a particular deregulatory mechanism. If this hypothetical role of the OX2-OX2R interaction is true, the manipulation of this interaction may have important clinical implications. In circumstances in which activation of macrophages is desired, eg, wound healing, some aspects of healing in CNS injury, etc., blocking of OX2 or use of an OX2R antagonist would be advantageous. The release of the typical suppression will result in a faster or more pronounced activation. The increased granulocyte activity would also be advantageous for the control of bacterial infection. In contrast, in situations where macrophage activation should be suppressed, for example, inflammation such as that seen in rheumatoid arthritis, the activation of OX2R by agonists, for example, a soluble recombinant OX2 in a multivalent form that can interlock the OX2R, could be useful. This would delay or prevent the release of active deletion. The descriptions that follow are directed, for exemplification purposes, to primates, for example, a human or rodent, for example, mouse or rat OX2 ligand, although they are also applicable to related modalities of other species. Accordingly, conditions that are known to be mediated by or related to macrophage functions can be regulated using these reagents.

II. Nucleic Acids The general description of nucleic acids, their manipulation and their uses (including, for example, complementary nucleic acids and antisense) are provided in the following references: NCBI Access numbers Entrez (search "MRC OX-2") X05323- 26 (human); X01785 (rat); AA924563, AF029214-216 and AH006102 (mouse); McCaughan, et al. (1987) Immunoaenetics 25: 329-335: Goodnow (1992) "Transgenic Animáis" in Roitt (ed.) Encvclopedia of Immunoloqy Academic Press, San Diego, pages 1502-1504; Travis (1992) Science 256: 1392-1394; Kuhn, et al. (1991) Science 254: 707-710; Capecchi (1989) Science 244: 1288; Robertson (ed. 1987) Teratocarcinomas and Embrvonic Stem Cells: A Practical Approach IRL Press, Oxford; Rosenberg (1992) J. Clinical Oncoloqy 10: 180-199; Cournoyer and Caskey (1993) Ann. Rev. Immunol. 11: 297-329; Wetmur and Davidson (1968) J, Mol. Biol. 31: 349-370, Weintraub (1990) Scientific American 262: 40-46; Jaroszewski and Cohen (1991) Advanced Drug Delivery Reviews 6: 235-250; Akhtar, et al. (1992) pages 133-145 in Erickson and Izant (eds.) Gene Requlation: Bioloqy of Antisense RNA and DNA Raven Press, New York; Zhao, et al. (1994) Blood 84: 3660-3666; Misquitta, et al. (1999) Proc. Nat'l Acad. Sci. USA 96: 1451-1456; and Treco WO96 / 29411, each of which is incorporated by reference. Additional aspects will be apparent to the expert having knowledge in the art in light of the teachings provided herein. lll. OX2 purified ligand protein General descriptions of proteins and polypeptides in pharmaceutical or biochemical contexts can be found, for example, in: Goodman, et al. (eds. 1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York; Lieberman, et al. (eds. 1990) Pharmaceutical Dosaqe Forms: Tablets Dekker, New York; Lieberman, et al. (eds. 1990) Pharmaceutical Dosaqe Forms: Disperse Systems Dekker, New York; Freifelder (1982) Phvsical Biochemistry (2nd ed.) W. H. Freeman; Cantor and Schimmel (1980) Biophvsical Chemistrv, parts 1-3, W. H. Freeman & Co., San Francisco. Specific descriptions of OX2 are found, for example, in WO97 / 21450 (human); NCBI Entrez access numbers (search MRC OX-2) include P41217 (human); P04218 (rat); and AAC15911 (mouse). The recombinant methods for preparing the protein are well known. The preparation of fragments by synthetic methods or by biochemical dissociation of natural or recombinant forms, are available.

IV. Preparation of the OX2 protein; Mimetics The DNA encoding the ligand OX2 protein or its fragments can be obtained by chemical synthesis, classification of cDNA libraries or by sorting genomic libraries prepared from a wide variety of cell lines or tissue samples. This DNA can be expressed in a wide variety of expression systems as described in, for example, U.S.S.N. 08 / 250,846; U.S.S.N. 08 / 177,747; U.S.S.N. 08 / 077,203; PCT / US95 / 00001; Kaufman, et al. (1985) Molec. and Cell. Biol. 5: 1750-1759; Pouwels, et al. (1985 and Supplements) Cloning Vectors: A Laboratory Manual. Elsevier, N.Y., Rodríguez, et al. (eds. 1988) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, MA; Rodríguez and Denhardt (eds.) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, Chapter 10, pages 205-236; Okayama, et al. (1985) Mol. Cell Biol. 5: 1136-1142; pMCI neo Poli-A, see Thomas, et al. (1987) CeJI 51: 503-512; O'Reilly et al (1992) Baculovirus Expression Vectors: A Laboratorv Manual Freeman and Co., CRC Press, Boca Raton, Fia; Low (1989) Biochim. Biophvs. Acta 988: 427-454; Tse, et al. (1985) Science 230: 1003-1008; and Brunner, et al. (1991) J. Cell Biol, 1 14: 1275-1283; each of which is incorporated herein by reference. Now that the various OX2 ligand proteins have been characterized, fusion polypeptides, fragments or derivatives thereof can be prepared by conventional processes to synthesize peptides. These include processes such as those described in Stewart and Young (1984) Solid Phase Peptide Svnthesis Pierce Chemical Co., Rockford, IL-1 Bodanszky and Bodansky (1984) The Practice of Peptide Svnthesis Springer-Verlag, New York; Bodanszky (1984) The Principies of Peptide Svnthesis Springer-Verlag, New York; and Merrifield, et al. (1963) in J. Am. Chem. Soc. 85: 2149-2156; each of which is incorporated herein by reference. Additional aspects will be apparent to the person skilled in the art in light of the teachings provided herein.

V. Physical variants Proteins or peptides that have substantial amino acid sequence homology to the amino acid sequence of the OX2 protein are also contemplated. Variants include species or allelic variants. Homology or sequence identity is defined in, for example, U.S.S.N. 08 / 250,846; U.S.S.N. 08 / 177,747; U.S.S.N. 08 / 077,203; PCT / US95 / 00001; Needleham, et al. (1970) J. Mol. Biol. 48: 443-453; Sankoff et al. (1983) Chapter One in Time Warps, Strinq Edits and Macromolecules: The Theory and Practice of Sequence Comparison Addison-Wesley, Reading, MA; software packages from NCBI, NIH; and University of Wisconsin Genetics Computer Group, Madison, Wl. The isolated DNA encoding an OX2 protein can be easily modified as described in, for example, Sambrook, et al. (1989); Ausubel, et al. (1987 and Supplements); Cunningham, et al. (1989) Science 243: 1330-1336; O'Dowd, et al. (1988) J. Biol. Chem. 263: 15985-15992; and Carruthers (1981) Tetra, letts. 22: 1859-1862; each of which is incorporated herein by reference. Further methods will be apparent to the person skilled in the art in light of the teachings provided herein.

SAW. Functional Variants Blocking the physiological response to ligand OX2 proteins may be the result of inhibition of ligand binding to its natural binding partner by a variant of natural OX2 or OX2 antibody. The methods for preparing that type of variant are described in, for example, Godowski, et al. (1988) Science 241: 812-816; Beaucage and Carruthers (1981) Tetra, letts. 22: 1859-1862; Sambrook, et al. (1989) Molecular Cloning: A laboratory Manual (2nd ed.) Vols. 1-3, Cold Spring Harbor laboratory, Merrifield (1963) J. Amer. Chem. Soc. 85: 2149-2156, Merrifield (1986) Science 232: 341-347; Atherton, et al. (1989) Solid Phase Peptide Svnthesis: A Practical Approach, IRL Press, Oxford; Cunningham, et al. (1989) Science 243: 1339-1336; O'Dowd, et al. (1988) J. Biol. Chem. 263: 15985-15992; and Lechleiter et al. (1990) EMBO J. 9: 4381-4390; each of which is incorporated herein by reference. Further methods will be apparent to the person skilled in the art in light of the teachings provided herein.

Vile. Antibodies Antibodies can be cultured to the ligand OX2 proteins, including allelic species or variants and their fragments, both in their natural forms and in their recombinant forms. Additionally, antibodies can be cultured to ligand OX2 proteins in their active or inactive forms. Anti-idyotypic antibodies are also contemplated. Methods for generating antibodies and binding compositions and their uses are described in, for example, Coligan (1991) Current Protocols in Immunology Wiley / Greene; Harlow and Lane (1989) Antibodies: A Laboratorv Manual Cold Spring Harbor Press; Chan (ed. 1987) Immunoassav: A Practical Guide Academic Press, Orlando, FL; Ngo (ed. 1988) Nonisotopic Immunoassav Plenum Press, NY; Price and Newman (eds. 1991) Principles and Practice of Immunoassav Stockton Press, NY; (1969) Microbioloqy Hoeber Medical Division, Harper and Row; Landsteiner (1962) Specificity of Serological Policies Reactions Publications, New York; Williams, et al. (1967) Methods in Immunoloqy and Immunochemistry, Vol. 1, Academic Press, New York; Stites, et al. (eds.) Basic and Clinical Immunoloqy (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited there; Harlow and Lane (1988) Antibodies: A Laboratorv Manual CSH Press; Goding (1986) Monoclonal Antibodies: Principies and Practice (2nd ed.) Academic Press, New York; Kohler and Milstein (1975) Nature 256: 495-497; Huse, et al. (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda" Science 246: 1275-1281; Ward, et al. (1989) Nature 341: 544-546; U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241; and Cabilly, U.S. Patent No. 4,816,567; each of these references is incorporated herein by reference. Further methods will be apparent to the person skilled in the art in light of the teachings provided herein.

VIII. Uses Mammalian OX2 reagents will have a variety of therapeutic uses for, for example, the treatment of conditions or diseases in which the function or dysfunction of macrophage or myeloid cells has been implicated. This would include, for example, wound healing, some aspects of healing in CNS injury, and inflammation as seen in rheumatoid arthritis. The administration of an effective amount of ligand OX2 will typically be at least about 100 ng per kg of body weight, generally at least about 1 μg per kg of body weight; and often less than about 1 mg per kg of body weight; or preferably less than about TO mg per kg of body weight. An effective amount will modulate the symptoms, or the time to onset of the symptom, typically by at least about 10%; generally at least about 20%; preferably at least about 30%; or more preferably at least about 50%. The present invention provides reagents that will find use in additional therapeutic and diagnostic applications as described elsewhere herein, for example, in the general description for physiological or developmental abnormalities, or later in the description of diagnostic equipment. See, for example, Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J .; Thom, et al. Harrison's Principies of Intemal Medicine McGraw-Hill, NY, Gilman, et al, (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics 8th Ed., Pergamon Press; (1990) Reminqton's Pharmaceutical Sciences (18 ed.) Mac Publishing Co., Easton, Penn; Langer (1990) Science 249: 1527-1533; Merck Index, Merck & Co., Rahway, New Jersey; Avis, et al. (eds, 1993) Pharmaceutical Dosaqe Forms: Parenteral Medications 2nd ed., Dekker, NY, Lieberman, et al. (eds. 1990) Pharmaceutical Dosaqe Forms: Tablets 2nd ed., Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosaqe Forms: Disperse Systems Dekker, NY; Fodor, et al. (1991) Science 251: 767-773, Coligan Current Protocols in Immunology; Hood, et al. Immunoloqy Benjamin / Cummings; Paul (ed.) Fundamental Immunoloqy: Methods in Enzymoloqy Academic Press; Parce, et al. (1989) Science 246: 243-247; Owicki, et al. (1990) Proc. Nat'l Acad. Sci. USA 87: 4007-4011; and Blundell and Johnson (1976) Protein Crystallographv, Academic Press, New York; each of which is incorporated herein by reference. Further uses will be apparent to the person skilled in the art in light of the descriptions provided herein.

IX. Equipment This invention also contemplates the use of ligand OX2 proteins, their fragments, peptides and their fusion products and related reagents will also be useful in a variety of diagnostic kits and methods for detecting the presence of a binding composition as described in , for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH; Pat. from United States No. 3,645,090; Pat. from United States No. 3,940,475; Rattle, et al. (1984) Clin. Chem. 30: 1457-1461: Pat. from United States No. 4,659,678; and Viallet, et al. (1989) Progress in Growth Factor Res. 1: 89-97; each of which is incorporated herein by reference. The broad scope of this invention is best understood by reference to the following examples, which are not intended to limit the invention to specific embodiments.

EXAMPLES I. General Methods Some of the conventional methods are described or referenced, for example, in Maniatis, et al. (1982) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al. (1989) Molecular Cloninq: A Laboratorv Manual (2nd ed.) Vols. 1-3, CSH Press, NY; Ausubel, et al. (1987 and Supplements) Current Protocols in Molecular Biology, Greene / Wiley, New York; or Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods and Applications Academic Press, N.Y. Methods for protein purification include methods such as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization and others. See, for example, Ausubel et al. (1987 and periodic supplements); Coligan, et al. (eds. 1995 and periodic supplements) Current Protocols in Protein Science Wiley & Sons; Deutscher (1990) "Guide to Protein Purification" in Methods in Enzvmology vol. 182, and other volumes in this series; and manufacturer's literature on the use of protein purification products, for example, Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA. The combination with recombinant techniques allows fusion to appropriate segments, for example, to a FLAG sequence or an equivalent that can be fused by means of a protease-removable sequence. See, for example, Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic Enqineerinq, Principie and Methods 12: 87-98, Plenum Press, N.Y .; and Crowe, et al. (1992) QIAexpress: The Hiqh level Expression & Protein Purification System QUIAGEN, Inc., Chatsworth, CA. FACS analyzes are described in Melamed, et al. (1990) Flow Cvtometry and Sorting Wiley-Liss, Inc., New York, NY; Shapiro (1988) Practical Flow Cytometry Liss, New York, NY; and Robinson, et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, NY.

II. Isolation of a DNA clone encoding the ligand OX2 protein The isolation of OX2 from human ligand is described in McCaughan, et al. (1987) Immunogenetics 25: 329-335. Conventional hybridization methods, or PCR primers constructed to isolate the clone, can be used. The Entrez access numbers for both the nucleotide and amino acid sequences are provided above.

Various cells are classified using an appropriate probe for high-level message expression and the expression distribution has been published. Appropriate cells are selected as sources for cDNA cloning, for example, using conventional PCR or hybridization methods. Conventional PCR techniques are used to amplify an OX2 gene sequence of genomic DNA or an OX2 or cDNA fragment derived from mRNA. Appropriate primers are selected from the described sequences, and a full-length clone is isolated. Various combinations of initiators can be prepared, of various lengths and possibly with differences in sequence. The full-length clone can be used as a hybridization probe to classify other homologous genes using severe or less severe hybridization conditions. In another method, oligonucleotides are used to classify a library. In combination with polymerase chain reaction (PCR) techniques, synthetic oligonucleotides are used in appropriate orientations as primers to select correct clones from a library. lll. Large-scale production of OX2 For in vitro or in vivo biological assays, OX2 or OX2-E label are produced, for example, in large quantities with transfected COS-7 cells grown in RPMI medium supplemented with 1% Nutridoma HU (Boehringer Mannheim, Mannheim, Germany) and subsequently purified. Adenovirus expression systems can be used. The recombinant protein can be purified using conventional procedures. Epitope tagged fusion protein affinity chromatography can be used.

IV. Preparation of antibodies specific for OX2 Inbred Balb / c mice are immunized, for example, with 1 ml of purified OX2 emulsified in Freund's complete adjuvant at day O, and in incomplete Freund's adjuvant at days 15 and 22. The mice are reinforced with 0.5 ml of purified OX2 administered intravenously. Hybridomas are created, for example, using the non-segregating myeloma cell line SP2 / 0-Ag8 and polyethylene glycol 1000 (Sigma, St. Louis, MO) as the fusion agent. The hybridoma cells are placed in a 96-well Falcon tissue culture plate (Becton Dickinson, NJ) and fed with F12 DMEM (Gibco, Gaithersburg, MD) supplemented with 80 μg / ml gentamicin, 2 mM glutamine, % horse serum (Gibco, Gaithersburg, MD), 1% ADCM (CRTS, Lyon, France) 10"5 M azaserin (Sigma, St. Louis, MO) and 5 x 10" 5 M hypoxanthine. Hybridoma supernatants are classified for production of antibodies against OX2, for example, by immunocytochemistry (ICC) using COS-7 cells transfected with OX2 fixed with acetone and / or by ELISA using OX2 purified from COS-7 supernatants as a carrier antigen. covering. Aliquots of positive cell clones are expanded for 6 days and cryopreserved as well as propagated in ascites of Balb / c mice treated with pristane (2,6,10,14-tetramethylpentadecane, Sigma, St. Louis, MO), which they had received by intraperitoneal injection of pristane 15 days before. Approximately 105 hybridoma cells in 1 ml of PBS are administered intraperitoneally, and 10 days later, ascites is collected from each mouse. After centrifugation of the ascites, the antibody fraction can be isolated by precipitation with ammonium sulfate and anion exchange chromatography on a Zephyr-D silicon column (IBF Sepracor) equilibrated with 20 mM Tris pH 8.0. The proteins are eluted with a gradient of NaCl (ranging from 0 to 1 M NACÍ). Fractions of 2 ml can be collected and assayed by ELISA to determine the presence of anti-OX2 antibody. Fractions containing specific anti-OX2 activity are pooled, dialyzed and frozen.

V. Preparation of an OX2 Deletion Mouse OX2 knockout (KO) mice were prepared essentially according to the procedure described by Galli-Taliadoros, et al. (1995) J_, Immunol. Methods 181: 1-15; Kórner, et al. (1997) Eur. J. Immunol. 27: 2600-2609; and Lemckert, et al. (1997) Nucí. Acids Res. 25: 917-918. Briefly, a C57BL / 6 genomic library was sorted using a PCR fragment from the mouse OX2 cDNA as a probe. The isolated genomic clone contained an insert of approximately 16 kB of which a Sali fragment of 9.5 kB was subcloned into pBluescript. This clone contained part of intron 1, exon 11 (coding for signal peptide), intron II, exon III (coding for the 1g domain of type V), intron lll, exon IV (coding for the Ig domain of type C2), and part of intron IV. From this clone a target construct was created by replacing a Ncol fragment encoding the C-terminal part of the Ig domain of type V with the Neomycin cassette and shortening the upstream part of the clone so that it contained only the 3 'part of the exon encoding the signal peptide. An ES cell line derived from C57BL / 6J mice (Bruce 4, see Galli-Taliadoros, et al (1995) J. Immunol.Methods 181: 1-15 and Lemckert, et al. (1997) Nucí.Aids Res. 25: 917-918) were transfected by electroporation, and G418 resistant colonies were isolated and sorted for homologous recombination by PCR and Southern blotting. A homologous recombinant of 1000 clones was isolated and used to create chimeric mice. See Lemckert, et al. (1997) Nucí. Acids Res. 25: 917-918. Male chimeras were procreated with C57BL / 6J mice of the wild type female and the result with black coat color (indicating germline transmission) were classified to determine the presence of the allele sought. F1 heterozygous mice were cross-linked to obtain homozygous knockout mice, which were used to establish a pure C57BL / 6.OX2 - / - breeding colony. C57BL / 6J wild-type mice grouped by age and sex were used as controls in all studies.

SAW. Initial observations in OX2-1- (knockout) mice The analysis of OX2 KO vs. Wild type (wt) mice involved a large analysis of organ structures. At the macroscopic level, the structures of the organs appeared normal, with the exception of mesenteric lymph nodes (MLN) that seemed to "fuse" with each other to form a long tube-like structure. In wt mice, the normal MLN structure is characterized by separate lymph nodes joined by lymphatic blood vessels in a "string of beads" configuration. The spleen was slightly enlarged, as well as the lymph nodes. The differences were more evident at the histological level after staining for a variety of leukocyte antigens. In particular, the red pulp of OX2 KO mice spleen appeared enlarged (although not edematous) and loaded with F4 / 80 + cells, ie, macrophages as it should be. The subpopulation of metallophilic macrophages surrounding the B cell follicles in the spleen (MOMA-1 +) were also increased by 2-3 fold. Gr-1 + cells, for example, granulocytes, were also more numerous in OX2 KO mouse spleen, by a factor of approximately 2-fold. The white pulp areas were of normal size. Consequently, there appeared to be a relative expansion of myeloid lineage cells, including macrophages, in the spleen that could be responsible for the increased size. The MLN 'tube' was made up of clearly demarcated individual lymph node structures, although each bound together (fused) with what appeared to be an expanded paracortical or subcapsular region and this was also positive for macrophages F4 / 80 + / MOMA- 1 +. The cells appeared enlarged and activated and were MHC II + class. Sections of the CNS of wild type (wt) and OX2 KO mice are colored for microglia, the macrophage of the resident CNS, with an antibody of Mac-1 (CD11 b). The main findings were: (i) In the OX2 KO mice there appeared to be an increase in the number of microglia in about 20% of the spinal cord compared to wt mice. (I) Small foci of microglial cells, occasionally even a structure that resembles the microglial clustering associated with neuritic amyloid plaques, were observed in the spinal cord of OX2 KO. These foci are never seen in the normal healthy wt CNS, and indicate the activation, proliferation or grouping of microglial cells. The expression levels of CD45 were generally increased with respect to the microglia in OX2 KO mice. CD45 is generally low in normal microglia, although increased activation fire. See, for example, Sedgwick, et al. (1991) Proc. Nat'l. Acad. Sci. USA 88: 7438-7442, Sedgwick, et al. (1993) J. Exp. Med. 177: 1145-1152, Ford et al. (1995) J, Immunol. 154: 4309-4321; Ford, et al. (1996) J. Exp. Med. 184: 1737-1745; Sedgwick and Hickey (1997) in Keane and Hickey (eds.) Immunology of the Nervous Svstem Oxford Press, New York; and Sedgwick, et al. (1998) J. Immunol. 160: 5320-5330.

These findings were consistent with the view that the loss of OX2 (in this case in neurons) leads to a certain degree of deregulation of resident macrophages (being microglial cells resident in the CNS). The general message of our studies in the OX2 KO mouse was that the loss of this molecule freed myeloid cells generally, and cells of macrophage lineage specifically, of normal regulation even in the steady state. Therefore, it was possible that in situations where the activation and proliferation of macrophages were increased (for example, in pathological states), the loss of OX2 can lead to an even greater or faster increase in macrophage activation. * To test this hypothesis, macrophage activation models were chosen. The first was a cross-sectional model of the facial nerve to investigate the activation of macrophages (macroglial cell) of the CNS within the descending facial nucleus that follows the cut of the facial nerve. See Streit and Graeber (1993) Glia 7: 68-74. This model is appropriate in the present case since it is known that the damaging effects of the cross-section of the nerve in the neurons (which are OX2 positive) which subsequently leads to a response by the microglial cells (which are positive OX2-R) inside the facial nucleus. This response can be examined by immunohistological evaluation of the facial nucleus. According to this hypothesis, it is predicted that in the absence of OX2 in neurons in OX2 KO mice, the response of microglial cells would be faster and of greater magnitude. In fact this was found, particularly that two days after the cross section, the activation of the microglial cells was already evident in OX2 KO but not in wt mice. Also, differences at day 4 between wt and OX2 KO mice were more evident. On day 7, the activation of microglial cells was equivalent in both types of mice. This experiment provides direct evidence that the OX2 signals of a non-macrophage lineage cell (in this case, the neuron) participate in the regulation of macrophages. In a second model, the mice were injected parenterally with lipopolysaccharide (IPS) that is known to induce rapid activation of macrophages. Within a period of 90 minutes, the quantification of serum TNF production is useful as a measure of macrophage activation. OX2 KO mice should respond to much lower doses of LPS and with increased TNF production. This correlation has been confirmed; in certain cases, TNF production in these mice was 2-4 times higher than in wild-type mice. OX2 KO mice show an earlier and more accelerated onset of EAE with respect to wild-type mice. Disease finally 'is not greater than in wild-type mice, then, analogously to microglia, the onset is rapid but ultimately does not exceed the start in wild-type control. The elimination of the interaction of OX2 with its receptor increases the response of macrophages, leading to a greater or more rapid onset of the disease. The opposite effect would typically be therapeutically desired. Therefore, when macrophages are stimulated within or outside the CNS, a negative OX2 environment leads to increased activity and function of macrophages. All references cited herein are incorporated by reference in their entirety. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention should be limited only by the terms of the appended claims, together with the full scope of equivalents to which such claims are entitled.

LIST OF SEQUENCES < 110 > Schepng Corporation < 120 > Novel Uses of Mammalian 0X2 Protein and Related Reagents < 130 > -.X 333S < 150 > ÜS 097290825 < 151 > 1999-04 -13 < 160 > 3 < 170 > Patentln Ver. 2.0 < 210 > 1 < 21I > 274 < 2i2 > PRT < 213 > primate < 00 > 1 Val He? Rg Met Prc Phe Ser His Leu Ser Thr fyr Ser Leu Val Trp 1 5 10 15 Vai Met Ala Ala Val Val Leu Cys Thr Ala Gln Val Val Val Vai Thr 20 25 30 Gln Aso Glu Arg Glu Gln Leu Tyr Thr Thr Ala Ser Leu Lys Cys Ser 35 40 45 Leu GIn Asn Wing Gln Glu Aia lie Val Thr Trp Gln Lys Lys Lys 50 55 60 Wing Val Ser Pro Glu Asn Met Val Thr Phe Ser Giu Asn His Gly Val 65 70. 75 '80 Val ila Gin Pro Ala Tyr ys Asp Lys He Asn He Thr Gin Leu Gly 85' 90 95 Leu Gln Asn Ser Thr lie Thr Phe Trp Asn He Thr Leu Glu Asp Glu 100 105 110 Gly Cys Tyr Met Cys Leu Phe Asn Thr Phe Gly Phe Gly Lys He Sar 115 120 - 125 Giy Thr Ala Cys Leu Thr Val Tyr Val Gln Pro He Va.1 Sex Leu His 130 135 140 Tyr Lys Phe Ser Glu Asp His Leu Asn He Thr Cys Ser Ala Thr Ala 145 and 150 155 16D Arg Pro Wing Pro Met Val Phe Trp Lys Val Pro Arg Ser Gly He Glu 155 170 175 Asn Ser Thr Val Thr Leu Ser His Pro Asn Gly Thr Thr Ser Val Thr 180 185. 190 Ser He -Leu. His He Lys Asp Pro Lys Asn Gln Val Giy Lys Glu Val 155 200 '205 H Cys Gip Val Leu I-is e Gly Thr Val Thr Asp Phe Lys Gin Tr 210 210"" "220 Val Asn? -ys Gly Tyr Trp Phe S.er Val Pra Leu Leu .Leu Ser I_e \ a_ 225 230 235 24C Ser Leu Val Xle Leu Leu Vai Leu He Ser lie Leu Leu Tyr Trp Lys 245 250 255 Arg His Arg Asn Gin Asp Arg Giy Giu Leu Ser Gln Gly Val Gln Lys 260 255 270 Met T r < 210 > 2 < 21i > 2781 < 212 > PRT < 2i3 > rodent < 400 > 2 Met Aia Leu Val Phe Arg Arg Pro Phe Cys Hrs Leu Ser Thr 1 5 10 15 Ser Leu He Trp Gly Met Wing Wing Val Aia Leu Ser Thr Wing Gln Val '20 25 30 Glu Val Val Thr Gln Asp Glu Arg Lys Aia Leu Hrs .hr Thr Wing Ser 35 40 45 Leu Arg Cys Ser Leu Lys Thr Ser Gir . Glu Pro Leu He Val Thr Tr: 50 60 Gin Lys Lys Lys Wing Val Ser Pro GJ Lu Asn Ket Vai Thr Tyr Ser L / s 65 70 75 SO T-nr His Gly Val Vai He Gln Pro Aia Tyr Lys Asp Arg He Asn Val 85 90 95 Thr Glu Leu Gly Leu Trp Asn Be Ser Thris Thr Phe Trp Asn Thr Thr 100 105 110 Leu Glu Asp Glu Gly Cys Tyr Mer Cys Leu Phe "-sn Thr Phe Gly Ser 115 I2Q - 125 Gln Lys Val Ser Gly Thr Ala Cys Leu Thr Leu Tyr Vai Gln Pro He 130 -. 130 -135 140 to His Leu His Tyr Asn Tyr Phe Glu Asp Kis Leu Asn He Thr Cys 145 150 155 160 Be Ala Thr Ala Arg Pro Ala Ala Ala Be Trp Lys Gly Thr Gly 165 170. 175 Thr Gly He Glu Asn Ser Thr Glu Ser Hxs Phe HLS S = r Asn Gly Thr 180 185 190 Thr Ser Val Thr Ser He Leu A.rg Val Lys Asp Pro Lys Thr Gln Val 195 200 205 Gly Lys Glu Val H e C; , s yB z yyy .ST 220 - 3 - -vs Gln Leu, í-C Gly Phe Trp Phe Ser .u Le.- 225 230 235 24C Leu Ser Tie Vai Ser Leu Val He Leu Leu Val He Ser He 245 250 255 Leu i and: Trp Lys Arg His Arg Asn Gln Arg Giy Glu Be Ser Gln 260 265 270 Gly Met Gin Arg Met Lys 275 < 210 > 3 < 2H > -278 < 212 > PRT < 213 > rodent < 4Q0 > 3 Met Gly Ser Pro Val Phe Arg Arg Pro Phe Cys His Leu Ser Thr Tyr 1 5 10 15 Be Leu Leu Trp Ala Lie Aia Ala Val Ala Leu Ser Thr Aia Gln Val 20 25 30 Glu Val Vai Thr Gin Asp Glu Arg Lys Leu Leu Hrs Thr Thr Aia Ser 35 40 '45 Leu Arg Cys Ser Leu Lys Thr Thr Gln Giu F ? ^ Leu He Val Thr Trp 50 55 60 Gin Lys Lys Lys Wing Vai Giy Pro Glu Asn Met Val Thr Tyr Ser Lys 65 70 75 80 Wing His Gly Val Vai He Gln Pro Thr Tyr Lys Asp Arg He Asn He 85 90 95 Thr Glu Leu Giy Leu Leu Asn Thr Be He Thr Phe Trp Asn Thr Thr 100 105 110 Lea. Asp Glu Gly Cys Tyr Met Cys Leu Phe Asn Met Phe Gly Ser 115 120 125 Giy Lys Val Ser Giy Thr Ala Cys Leu Thr Leu Tyr Val Gin Pro He 130 135 140- Vai His Leu Hrs Tyr Asr. Tyr Phe Glu Asp His Leu Asn He Thr Cys 145 150 _ 155 160 Ser Ala Thr Ala Arg Pro Ala Pro Ala He Ser Trp Lys Gly Thr Gly 165 170"175 Being Giy He Glu Asn Being Thr Glu Ser His Being His Being Asn Gly Thr 130 135 190 Phr Ser Val Thr Ser He Leu Arg Val Lys Asp Pro Lys Thr Gln Val 195 200 205 Lys .iu Cys Gl Vs 1. ', Le G- 210 2"Tyr Lys GTin Ser Leu Asp Lys Gly Phe Trp Pne Ser Vai Pro Leu Leu 225 230 235 240 Leu Ser He Val Ser Leu Vai He Leu Leu Vai Leu He Ser He Leu 245 250 '255 Leu Tyr Trp Lys Arg H-.3 Arg Asn Gln Glu Arg Gly Giu Ser 'Ser Gln 260 265 270 Gly Met Gln Arg Met Lys 275

Claims (22)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of a therapeutically effective amount of an agonist of a mammalian OX2 protein or antagonist of a mammalian OX2 protein, and a pharmaceutically acceptable carrier, for the manufacture of a medicament useful for modulating the movement or activation of a leukocyte in a animal contacting cells of myeloid lineage in said animal.

2. The use as claimed in claim 1, wherein said: a) mammalian OX2 protein is a primate protein; b) antagonist is an antibody that binds said mammalian OX2; or c) said cells are monocyte / macrophage lineage cells.

3. The use as claimed in claim 2, wherein said myeloid lineage cells include a monocyte, macrophage, mycroglial or dendritic cell.

4. The use as claimed in claim 1, wherein said animal exhibits signs or symptoms of an inflammatory, infectious, leukoproliferative, neurodegenerative, or post-traumatic condition.

5. The use as claimed in claim 4, wherein said sign or symptom appears in the neural tissue; lymphoid tissue; myeloid tissue; pancreas; gastrointestinal tissue; thyroid tissue; muscle tissue or skin tissue or collagen.

6. The use as claimed in claim 1, wherein said modulation is by a function of inhibition of said leukocyte cell.

7. The use as claimed in claim 1, wherein an agonist is used.

8. The use as claimed in claim 7, wherein said agonist is said mammalian OX2.

9. The use as claimed in claim 7, wherein said animal exhibits signs or symptoms of one or more conditions selected from: autoimmunity; an inflammatory condition; an infection; tissue-specific autoimmunity; degenerative autoimmunity; rheumatoid arthritis; atherosclerosis; multiple sclerosis; vasculitides; delayed hypersensitivities; skin graft; a transplant; spinal injury; apoplectic attack; neurodegeneration; or ischemia.

10. The use as claimed in claim 7, wherein said use further comprises: a) an anti-inflammatory cytokine agonist or antagonist; b) an analgesic; c) an anti-inflammatory agent; or d) a steroid.

11. The use as claimed in claim 1, wherein said modulation is by an augmenting function of said leukocyte cell.

12. The use as claimed in claim 11, wherein an antagonist is used.

13. - The use as claimed in claim 12, wherein said antagonist is: a) an antibody that binds said mammalian OX2; or b) a mutein of said mammalian OX2 competing with said mammalian OX2 at binding to an OX2 receptor, but which produces signal substantially.

14. The use as claimed in claim 12, wherein said animal exhibits signs or symptoms of wound healing or clot formation.

15. The use as claimed in claim 12, wherein said use further comprises: an angiogenic factor; b) a growth factor, including FGF or PDGF; c) an antibiotic; or d) a coagulation factor.

16. The use of a therapeutically effective amount of an agonist of a mammalian OX2 protein or an antagonist of a mammalian OX2 protein, and a pharmaceutically acceptable carrier, for the manufacture of a medicament useful for the modulation of the activation of a leukocyte in a tissue contacting cells of myeloid lineage in said tissue.

17. The use as claimed in claim 16, wherein said agonist is used, and wherein said modulation is inhibiting said leukocyte cell.

18. The use as claimed in claim 17, wherein the use further comprises: a) an anti-inflammatory cytokine agonist or antagonist; b) an analgesic; c) an anti-inflammatory agent; or d) a steroid.

19. - The use as claimed in claim 16, wherein said antagonist is used, and wherein said modulation is increasing the activation of the leukocyte.

20. The use as claimed in claim 19, wherein the use further comprises: a) an angiogenic factor; b) a growth factor, including FGF or PDGF; c) an antibiotic; or d) a coagulation factor. I

21. A pharmaceutical composition comprising a therapeutically effective amount of an agonist of a mammalian OX2 protein or an antagonist of a mammalian OX2 protein, and a pharmaceutically acceptable carrier, which is useful for the modulation of the movement or the activation of a leukocyte in an animal contacting "myeloid lineage cells in said animal"

22. A pharmaceutical composition comprising a therapeutically effective amount of an agonist of a mammalian OX2 protein or an antagonist of a mammalian OX2 protein, and a pharmaceutically carrier acceptable, which is useful for the modulation of the activation of a leukocyte in a tissue by contacting cells of myeloid lineage in said tissue.