WO2000061171A9 - Uses of mammalian ox2 protein and related reagents - Google Patents
Uses of mammalian ox2 protein and related reagentsInfo
- Publication number
- WO2000061171A9 WO2000061171A9 PCT/US2000/009719 US0009719W WO0061171A9 WO 2000061171 A9 WO2000061171 A9 WO 2000061171A9 US 0009719 W US0009719 W US 0009719W WO 0061171 A9 WO0061171 A9 WO 0061171A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- mammalian
- tissue
- protein
- antagonist
- agonist
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1891—Angiogenesic factors; Angiogenin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Definitions
- the present invention relates to methods of using proteins which function in controlling physiology, development, and differentiation of mammalian cells, e.g., cells of a mammalian immune or neural system.
- mammalian cells e.g., cells of a mammalian immune or neural system.
- it provides methods of using proteins and mimetics which regulate cellular physiology, development, differentiation, or function of various cell types, including hematopoietic or neural cells.
- the immune system of vertebrates consists of a number of organs and several different cell types.
- Two major cell types include the myeloid and lymphoid lineages.
- lymphoid cell lineage are B cells, which were originally characterized as differentiating in fetal liver or adult bone marrow, and T cells, which were originally characterized as differentiating in the thymus.
- Another cell type is the mononuclear phagocyte, a cell lineage widely distributed throughout most tissues. The phagocytes play a role in inflammation, host defenses, and reaction against a range of autologous and foreign materials. See, e.g., Paul (ed. 1997) Fundamental Immunology (4th ed.) Raven Press, New York.
- soluble or membrane proteins play a critical role in regulating cellular interactions. These proteins also mediate cellular activities in many ways. They have been shown, in many cases, to modulate proliferation, growth, and differentiation of hematopoietic stem cells into the vast number of progenitors composing 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 immunological responses and physiology.
- Medical science relies, in large degree, to appropriate recruitment or suppression of the immune system in effecting cures for insufficient or improper physiological responses to environmental factors.
- the lack of understanding of how the immune system is regulated or differentiates has blocked the ability to advantageously modulate the immunological mechanisms to biological challenges, i.e., response to biological injury.
- Medical conditions characterized by abnormal or inappropriate regulation of the development or physiology of relevant cells thus remain unmanageable.
- the discovery and characterization of specific regulatory pathways and their physiological effects will contribute to the development of therapies for a broad range of degenerative or other conditions which affect the biological system, immune cells, as well as other cell types.
- the present invention provides solutions to some of these and many other problems.
- the present invention is based, in part, upon the discovery of the physiological role of the ligand 0X2, also referred herein as the 0X2 protein, in various models of immune response.
- the role of ligand OX2 has been elucidated in pathways involved in infectious disease, hematopoietic development, and viral infection.
- the present invention provides methods of modulating the trafficking or activation of a leukocyte in an animal, the methods comprising contacting myeloid lineage cells, e.g., monocyte/ma ' crophage, in the animal with a therapeutic amount of an agonist of a mammalian 0X2 protein; or an antagonist of a mammalian 0X2 protein.
- myeloid lineage cells e.g., monocyte/ma ' crophage
- Preferred embodiments include where: the mammalian 0X2 protein is a primate protein; and/or the antagonist is an antibody which binds to the mammalian 0X2.
- the myeloid lineage cells include a macrophage, microglial, granulocyte, or a dendritic cell, or where the animal exhibits signs or symptoms of an infectious, inflammatory, leukoproliferative, neurodegenerative, or post-traumatic condition.
- the sign or symptom is in neural tissue; lymphoid tissue; myeloid tissue; pancreas; gastrointestinal tissue; thyroid tissue; muscle tissue; or skin or collagenous tissue.
- Other methods include where the modulating is inhibiting function of the leukocyte cell; and/or where the administering is the agonist.
- the agonist is the mammalian 0X2.
- Certain embodiments include where 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 grafting; a transplant; spinal injury; stroke; neurodegeneration; or ischemia.
- the administering may be in combination with: an anti-inflammatory cytokine agonist or antagonist; an analgesic; an anti-inflammatory agent; or a steroid.
- the modulating is enhancing function of the leukocyte cell, and/or the administering is the antagonist.
- the antagonist is: an antibody which binds to the mammalian 0X2; or a mutein of the mammalian 0X2 which competes with the mammalian OX2 in binding to an 0X2 receptor, but does not substantially signal.
- the method is applied where the animal experiences signs or symptoms of infection, wound healing, or clot formation.
- the administering 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.
- Different methods are provided, e.g., of modulating the activation of a leukocyte in a tissue, the method comprising contacting myeloid or monocyte/macrophage lineage cells in the tissue with: an agonist of a mammalian 0X2 protein; or an antagonist of a mammalian 0X2 protein.
- the modulating is inhibiting the leukocyte cell, and the contacting is with the agonist.
- the administering is often in combination with: an anti-inflammatory cytokine agonist or antagonist; an analgesic; an anti-inflammatory agent; or a steroid.
- the modulating is enhancing, and the contacting is with the antagonist.
- the administering may be in combination with: an angiogenic factor; a growth factor, including FGF or PDGF; an antibiotic or antiviral; or a clotting factor.
- the 0X2 antigen was first characterized in rat, using a monoclonal antibody (mAb) MRC 0X2.
- mAb monoclonal antibody
- 0X2 consists of about 248 amino acids comprising two extracellular immunoglobulin (Ig) domains, a transmembrane domain and a short C-terminal cytoplasmic tail.
- the molecule is glycosylated through 6 N-linked glycosylation sites, three of which are present in the N-terminal V-like Ig domain and the others reside in the membrane proximal C2-like Ig domain.
- IgSF Ig superfamily
- CD90 is also highly expressed by neurons. Williams, et al. (1977) Cold Spring Harb. Svmp. Quant. Biol. 41 Pt 1 :51-61.
- 0X2 was a structural homologue of CD80 and CD86 (Borriello, et al. (1997) J. Immunol. 158:4548-4554) and that the 0X2 gene was closely linked to those coding for CD80 and CD86 on chromosome 16 in the mouse. Borriello, et al.
- CD80 and CD86 serve as ligands in a process known as co-stimulation, and therefore it is likely that 0X2 would act as a ligand as well.
- the 0X2 antigen will be referred hereafter as the 0X2 protein or ligand 0X2.
- the binding partner will be referred to as the 0X2 receptor, though it has not been fully characterized.
- the group of Barclay prepared a multivalent reagent using rat 0X2-rat CD4 fusion protein bound to fluorescent beads.
- This reagent was shown to bind to mouse and rat peritoneal macrophages, and this binding could be blocked by the mAb MRC 0X88. Preston, et al. (1997) Eur. J. Immunol. 27:191 1-1918. This mAb was shown to bind to macrophages isolated from both peritoneum and spleen and in IHC on spleen sections staining was found in areas known to contain high proportions of macrophages.
- 0X2 The distribution of the 0X2 is consistent with a hypothesis that 0X2 relays a signal through the 0X2R to macrophages, and possibly other cells of the myeloid or monocyte-macrophage lineages.
- expression of 0X2 on neurons could establish a direct way of communication to the resident macrophages of the brain called microglia that might express 0X2R, since they originate from the monocyte-macrophage lineage.
- microglia that might express 0X2R, since they originate from the monocyte-macrophage lineage.
- mice The homozygous KO mice bred and developed normally, although initial examination of the internal organs showed anatomical anomalies in some lymphoid tissue. These included enlarged red pulp of the spleen, and failed segregation of the mesenteric lymph nodes with enlarged marginal sinus. Both these changes are attributable to an expanded macrophage and, in the spleen at least, an expanded granulocyte population. These results indicate that even in the steady state, 0X2 may regulate myeloid cell, e.g., macrophage, numbers and their activation, presumably via ligation of 0X2R.
- myeloid cell e.g., macrophage
- the 0X2 KO mice can now be used in studies of myeloid cell or macrophage function, particularly of monocyte/macrophage lineage activities, by applying model systems for activation of cells of these cell lineages.
- the first model system used for this purpose is a paradigm for microglia activation in the brain through nerve injury. Streit and Graeber (1993) Glia 7:68-74. This model makes use of the fact that transection of the facial nerve, that directs motor behavior in the facial area, elicits microglia activation after four to seven days in the facial nucleus in the brainstem, where the motor neurons are located.
- this activation occurs already 2 days after surgery, much earlier than in a normal mouse. This activation is accompanied by expression of the activation marker DAP12, as shown by IHC.
- macrophage activation In settings where macrophage activation is desired, e.g., wound healing, some aspects of healing in CNS injury, etc., blocking of 0X2 or using an OX2R antagonist would be beneficial. Release from the typical suppression will result in quicker or more pronounced activation. Enhanced granulocyte activity would also be beneficial for control of bacterial infection. Conversely, in situations where macrophage activation should be suppressed, e.g., inflammation such as seen in rheumatoid arthritis, activation of the OX2R by agonists, e.g., a recombinant soluble 0X2 in a multivalent form that can cross-link the OX2R, could be useful. This would delay or prevent release from active suppression.
- agonists e.g., a recombinant soluble 0X2 in a multivalent form that can cross-link the OX2R
- DNA which encodes the ligand OX2 protein or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or by screening 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, e.g., 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., Rodriguez, et al. (eds. 1988) Vectors: A Survey of Molecular Cloning Vectors and Their Uses.
- fusion polypeptides, fragments, or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young (1984) Solid Phase Peptide Synthesis Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis Springer-Verlag, New York; Bodanszky (1984) The Principles of Peptide Synthesis Springer-Verlag, New York; and Merrifield, et al. (1963) in J. Am. Chem. Soc.
- Proteins or peptides having substantial amino acid sequence homology with the amino acid sequence of the 0X2 protein are also contemplated.
- the variants include species or allelic variants. Homology, or sequence identity, is defined in, e.g., 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. String Edits, and Macromolecules: The Theory and Practice of
- the blocking of physiological response to ligand OX2 proteins may result from the inhibition of binding of the ligand to its natural binding partner by a variant of natural OX2 or antibody to 0X2.
- Methods for making such a variant are described in, e.g., 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 (2d ed.) Vols. 1-3, Cold Spring Harbor Laboratory; Merrifield (1963) J. Amer. Chem. Soc.
- Antibodies can be raised to the various ligand OX2 proteins, including species or allelic variants, and fragments thereof, both in their naturally occurring forms and in their recombinant forms. Additionally, antibodies can be raised to ligand 0X2 proteins in either their active forms or in inactive forms. Anti-idiotypic antibodies are also contemplated. Methods for generating antibodies and binding compositions and their uses are described in, e.g., Coligan (1991 ) Current Protocols in Immunology Wiley/Greene; Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press; Chan (ed. 1987) Immunoassav: A Practical Guide Academic Press, Orlando, FL; Ngo (ed.
- Mammalian OX2 reagents will have a variety of therapeutic uses for, e.g., the treatment of conditions or diseases in which myeloid or macrophage cell function or dysfunction has been implicated. These would include, e.g., wound healing, some aspects of healing in CNS injury, and inflammation such as seen in rheumatoid arthritis.
- Administration of an effective amount of ligand OX2 will typically be at least about 100 ng per kg of body weight; usually at least about 1 ⁇ g per kg of body weight; and often less than about 1 mg per kg of body weight; or preferably less than about 10 mg per kg of body weight.
- An effective amount will modulate the symptoms, or time to onset of symptom, typically by at least about 10%; usually by at least about 20%; preferably at least about 30%; or more preferably at least about 50%.
- the present invention provides reagents which will find use in additional diagnostic and therapeutic applications as described elsewhere herein, e.g., in the general description for physiological or developmental abnormalities, or below in the description of kits for diagnosis. See, e.g., Berkow (ed.) The Merck Manual of Diagnosis and Therapy. Merck & Co., Rahway, N.J.; Thorn, et al. Harrison's Principles of Internal Medicine McGraw-Hill, NY; Gilman, et al. (eds.
- This invention also contemplates use of ligand OX2 proteins, fragments thereof, 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, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH; U.S. Pat. No. 3,645,090; U.S. Pat. No. 3,940,475; Rattle, et al. (1984) Clin. Chem. 30:1457-1461 ; U.S. Pat. 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.
- Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., 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 use of protein purification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA.
- Combination with recombinant techniques allow fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence.
- appropriate segments e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence.
- Hochuli (1990) Purification of Recombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering. Principle and Methods 12:87- 98, Plenum Press, N.Y.; and Crowe, et al. (1992) QIAexpress: The High Level Expression & Protein Purification System QUIAGEN, Inc., Chatsworth, CA.
- Immunooenetics 25:329-335 Standard hybridization methods can be used, or PCR primers constructed to isolate the clone. Entrez accession numbers for both the nucleotide and amino acid sequences are provided above.
- Various cells are screened using an appropriate probe for high level message expression, and expression distribution has been published. Appropriate cells are selected as sources for cDNA cloning, e.g., using standard methods of PCR or hybridization.
- Standard PCR techniques are used to amplify an OX2 gene sequence from genomic DNA or an OX2 or fragment from cDNA derived from mRNA. Appropriate primers are selected from the sequences described, and a full length clone is isolated.
- primers of various lengths and possibly with differences in sequence, may be prepared.
- the full length clone can be used as a hybridization probe to screen for other homologous genes using stringent or less stringent hybridization conditions.
- oligonucleotides are used to screen a library.
- synthetic oligonucleotides in appropriate orientations are used as primers to select correct clones from a library.
- 0X2 or OX2-E-tag are produced, e.g., in large amounts with transfected COS-7 cells grown in RPMI medium supplemented with 1 % Nutridoma HU (Boehringer Mannheim, Mannheim, Germany) and subsequently purified.
- Adenovirus expression systems may be used.
- Recombinant protein may be purified using standard procedures. Affinity chromatography of epitope tagged fusion protein may be utilized.
- Inbred Balb/c mice are immunized, e.g., with 1 ml of purified 0X2 emulsified in Freund's complete adjuvant on day 0, and in Freund's incomplete adjuvant on days 15 and 22. The mice are boosted with 0.5 ml of purified OX2 administered intravenously. Hybridomas are created, e.g., using the non-secreting myeloma cells line SP2/0-Ag8 and polyethylene glycol 1000 (Sigma, St. Louis, MO) as the fusing agent.
- Hybridoma cells are placed in a 96-well Falcon tissue culture plate (Becton Dickinson, NJ) and fed with DMEM F12 (Gibco, Gaithersburg, MD) supplemented with 80 ⁇ g/ml gentamycin, 2 mM glutamine, 10% horse serum (Gibco, Gaithersburg, MD), 1% ADCM (CRTS, Lyon, France) 10" 5 M azaserine (Sigma, St. Louis, MO) and 5 x 10 " 5 M hypoxanthine.
- Hybridoma supernatants are screened for antibody production against OX2, e.g., by immunocytochemistry (ICC) using acetone fixed OX2 transfected COS-7 cells and/or by ELISA using OX2 purified from COS-7 supernatants as a coating antigen.
- Aliquots of positive cell clones are expanded for 6 days and cryopreserved as well as propagated in ascites from pristane (2,6,10,14- tetramethylpentadecane, Sigma, St. Louis, MO) treated Balb/c mice who had received on intraperitoneal injection of pristane 15 days before. About 10 ⁇ hybridoma cells in 1 ml of PBS are given intraperitoneally, and 10 days later, ascites are collected from each mouse.
- the antibody fraction may be isolated by ammonium sulfate precipitation and anion-exchange chromatography on a Zephyr-D silicium column (IBF Sepracor) equilibrated with 20 mM Tris pH 8.0. Proteins are eluted with a NaCI gradient (ranging from 0 to 1 M NaCI). 2 ml fractions may be collected and tested by ELISA for the presence of anti-OX2 antibody. The fractions containing specific anti-OX2 activity are pooled, dialyzed, and frozen. V. Preparation of an 0X2 deletion mouse.
- 0X2 knockout (KO) mice were made essentially according to the procedure described by Galli-Taliadoros, et al. (1995) J. Immunol. Methods 181 :1-15; Korner, et al. (1997) Eur. J. Immunol. 27:2600-2609; and Lemckert, et al. (1997) Nucl. Acids Res. 25:917-918.
- a C57BL/6 genomic library was screened using a PCR fragment of the mouse OX2 cDNA as a probe.
- the isolated genomic clone contained an insert of about 16 kB from which a 9.5 kB Sail fragment was sub-cloned into pBluescript.
- This clone contained part of intron I, exon II (encoding the signal peptide), intron II, exon III (encoding the V-like Ig domain), intron III, exon IV (encoding the C2-like Ig domain), and part of intron IV. From this clone a targeting construct was created by replacing an Ncol fragment encoding the C-terminal part of the V-like Ig domain 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.
- Heterozygous F1 mice were inter-crossed to obtain homozygous knockout mice, which were used to establish a pure C57BL/6.0X2-/- breeding colony. Age a sex-matched wild type C57BL/6J mice were used as controls in all studies.
- mice involved a gross analysis of organ structures. At the macroscopic level, organ structures appeared normal, with the exception of mesenteric lymph nodes (MLN) that appeared "fused" together into one long tube-like structure. In wt mice, the normal MLN structure is characterized by separate lymph nodes joined by lymphatic vessels in a 'string of pearls' configuration. The spleen was slightly enlarged, as were some lymph nodes. Differences were more apparent at the histological level upon staining for a variety of leukocyte antigens.
- MDL mesenteric lymph nodes
- the red pulp of the spleen of OX2 KO mice appeared enlarged (but not edematous) and filled with F4/80+ cells, i.e., macrophages as it should be.
- the subpopulation of metallophilic macrophages surrounding the B cell follicles in spleen (MOMA-1 +) were also increased by 2-3 fold.
- Gr-1 + cells e.g., granulocytes, were also more numerous in 0X2 KO mouse spleen, by a factor of about 2 fold.
- White pulp areas were of normal size.
- the MLN "tube” consisted of clearly demarcated individual lymph node structures, but each attached together (fused) with what appeared to be an expanded paracortical or subcapsular region and this was positive also for F4/80+/MOMA-1 + macrophages. Cells appeared enlarged and activated and were MHC class II+.
- This model is appropriate in the present case as it is known that it is the damaging effects of nerve transection on neurons (which are OX2-positive) that leads subsequently to a response by microglial cells (which are OX2-R-positive) within the facial nucleus. This response can be examined by immunohistological assessment of the facial nucleus.
- microglial cell response in the absence of 0X2 on neurons in 0X2 KO mice, the microglial cell response would be more rapid and of greater magnitude. Indeed this was found, particularly that two days after transection, microglial cell activation was already evident in 0X2 KO but not wt mice. Moreover, the differences at day 4 between wt and 0X2 KO mice were more apparent. By day 7, microglial cell activation was equivalent in both types of mice.
- This experiment provides direct evidence that 0X2 signals from a non- macrophage-lineage cell (in this case, the neuron) participate in macrophage regulation.
- mice were injected parenterally with lipopoiysaccharide (LPS) known to induce rapid macrophage activation.
- LPS lipopoiysaccharide
- quantitation of serum TNF production is useful as a measure of macrophage activation.
- the 0X2 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 fold higher than in wild type mice.
- 0X2 KO mice show an earlier and more accelerated onset of EAE relative to wt mice. The disease ultimately is not greater than wt mice, so, analogous to the microglia, the onset is fast but it ultimately does not exceed that in the wt control.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Neurology (AREA)
- Biomedical Technology (AREA)
- Neurosurgery (AREA)
- Rheumatology (AREA)
- Physical Education & Sports Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
- Pulmonology (AREA)
- Dermatology (AREA)
- Psychiatry (AREA)
- Urology & Nephrology (AREA)
- Hospice & Palliative Care (AREA)
- Communicable Diseases (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Oncology (AREA)
- Cell Biology (AREA)
- Pain & Pain Management (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000610503A JP2002541210A (en) | 1999-04-13 | 2000-04-12 | Novel use of mammalian OX2 protein and related reagents |
CA002369454A CA2369454A1 (en) | 1999-04-13 | 2000-04-12 | Uses of mammalian ox2 protein and related reagents |
AU43413/00A AU4341300A (en) | 1999-04-13 | 2000-04-12 | Novel uses of mammalian ox2 protein and related reagents |
MXPA01010480A MXPA01010480A (en) | 1999-04-13 | 2000-04-12 | Novel uses of mammalian ox2 protein and related reagents. |
EP00923257A EP1171154A2 (en) | 1999-04-13 | 2000-04-12 | Uses of mammalian ox2 protein and related reagents |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US29082599A | 1999-04-13 | 1999-04-13 | |
US09/290,825 | 1999-04-13 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2000061171A2 WO2000061171A2 (en) | 2000-10-19 |
WO2000061171A3 WO2000061171A3 (en) | 2001-01-25 |
WO2000061171A9 true WO2000061171A9 (en) | 2002-01-03 |
Family
ID=23117713
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/009719 WO2000061171A2 (en) | 1999-04-13 | 2000-04-12 | Uses of mammalian ox2 protein and related reagents |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP1171154A2 (en) |
JP (1) | JP2002541210A (en) |
AR (1) | AR023464A1 (en) |
AU (1) | AU4341300A (en) |
CA (1) | CA2369454A1 (en) |
MX (1) | MXPA01010480A (en) |
WO (1) | WO2000061171A2 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2316485A1 (en) | 2001-10-12 | 2011-05-04 | Schering Corporation | Use of bispecific antibodies to regulate immune responses |
GB201608197D0 (en) | 2016-05-10 | 2016-06-22 | Ducentis Biotherapeutics Ltd | Novel proteins |
GB202115803D0 (en) | 2021-11-03 | 2021-12-15 | Ducentis Biotherapeutics Ltd | Novel proteins |
WO2023214388A1 (en) | 2022-05-06 | 2023-11-09 | Ducentis Biotherapeutics Limited | Novel cd200 fusion proteins |
WO2023214387A1 (en) | 2022-05-06 | 2023-11-09 | Ducentis Biotherapeutics Limited | Novel cd200 fusion proteins |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5859208A (en) * | 1988-07-06 | 1999-01-12 | Fiddes; John C. | Human basic fibroblast growth factor analog |
AU1144097A (en) * | 1995-12-08 | 1997-07-03 | Brigham And Women's Hospital | Ox-2 costimulatory molecule |
US5753218A (en) * | 1996-05-03 | 1998-05-19 | Schering Corporation | Method for treating inflammation |
EP1032662B1 (en) * | 1997-11-07 | 2006-03-08 | Trillium Therapeutics Inc. | Methods and compositions for immunomodulation |
-
2000
- 2000-04-12 CA CA002369454A patent/CA2369454A1/en not_active Abandoned
- 2000-04-12 WO PCT/US2000/009719 patent/WO2000061171A2/en not_active Application Discontinuation
- 2000-04-12 MX MXPA01010480A patent/MXPA01010480A/en unknown
- 2000-04-12 EP EP00923257A patent/EP1171154A2/en not_active Withdrawn
- 2000-04-12 AR ARP000101685A patent/AR023464A1/en unknown
- 2000-04-12 JP JP2000610503A patent/JP2002541210A/en not_active Withdrawn
- 2000-04-12 AU AU43413/00A patent/AU4341300A/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
EP1171154A2 (en) | 2002-01-16 |
AU4341300A (en) | 2000-11-14 |
JP2002541210A (en) | 2002-12-03 |
MXPA01010480A (en) | 2002-03-27 |
CA2369454A1 (en) | 2000-10-19 |
WO2000061171A3 (en) | 2001-01-25 |
WO2000061171A2 (en) | 2000-10-19 |
AR023464A1 (en) | 2002-09-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20020192215A1 (en) | Novel uses of mammalian OX2 protein and related reagents | |
ES2149149T3 (en) | PROTEINAS DE UNION A INTERLEUQUINA-18, ITS PREPARATION AND USE. | |
Yamamoto et al. | Cloning and expression of myelin-associated oligodendrocytic basic protein. A novel basic protein constituting the central nervous system myelin. | |
US20120141456A1 (en) | Use of DR6 and p75 Antagonists to Promote Survival of Cells of the Nervous System | |
EP2073827A2 (en) | Multiple sclerosis therapy | |
WO2007098283A2 (en) | Use of antagonists of maif receptor complex molecules and neurotrophic factors for treatment of neurological diseases and disorders | |
EP1326974B1 (en) | Methods of using a human il-17-related polypeptide to treat disease | |
US20020138860A1 (en) | Novel uses of mammalian CCR6 receptors and related reagents | |
WO2002070002A2 (en) | Methods for regulation of immune responses to conditions involving mediator-induced pathology | |
WO2000061171A9 (en) | Uses of mammalian ox2 protein and related reagents | |
CA2235951C (en) | Novel uses of mammalian ctla-8 and related reagents | |
WO2014106104A1 (en) | Use of dr6 antagonists to improve motor neuron disease | |
AU735317B2 (en) | Rath genes and polypeptides and methods for the treatment and diagnosis of immune disorders | |
KR20000075732A (en) | Lymphocyte activation inhibitors | |
Hasegawa et al. | Monoclonal antibodies to epitope of CD45R (B220) inhibit interleukin 4-mediated B cell proliferation and differentiation | |
AU705281B2 (en) | Secreted human fas antigen | |
WO1996020206A9 (en) | Secreted human fas antigen | |
US6063372A (en) | Uses of mammalian CTLA-8 and related reagents | |
WO2001058484A2 (en) | Novel uses of mammalian ccr8 receptors and related reagents | |
WO1995002611A1 (en) | Cell surface protein present on nk (natural killer cells) named dx1 | |
US20030148369A1 (en) | Novel uses of mammalian CCR8 receptors and related reagents | |
US20040241804A1 (en) | Novel polypeptide, a cDNA encoding the same, and use of it | |
Sytwu | T cell activation, apoptosis, and autoimmune disease | |
US20060165689A1 (en) | Compositions and methods for modulation of effects on phagocyte and lymphoid cell populations employing tirc7 | |
US20030082184A1 (en) | Uses of mammalian cytokine; related reagents |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CZ DE DK DM DZ EE ES FI GB GD GE HR HU ID IL IN IS JP KG KR KZ LC LK LR LT LU LV MA MD MG MK MN MX NO NZ PL PT RO RU SE SG SI SK SL TJ TM TR TT TZ UA UZ VN YU ZA |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CZ DE DK DM DZ EE ES FI GB GD GE HR HU ID IL IN IS JP KG KR KZ LC LK LR LT LU LV MA MD MG MK MN MX NO NZ PL PT RO RU SE SG SI SK SL TJ TM TR TT TZ UA UZ VN YU ZA |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
ENP | Entry into the national phase in: |
Ref document number: 2369454 Country of ref document: CA Ref country code: CA Ref document number: 2369454 Kind code of ref document: A Format of ref document f/p: F |
|
ENP | Entry into the national phase in: |
Ref country code: JP Ref document number: 2000 610503 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: PA/a/2001/010480 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2000923257 Country of ref document: EP |
|
AK | Designated states |
Kind code of ref document: C2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CZ DE DK DM DZ EE ES FI GB GD GE HR HU ID IL IN IS JP KG KR KZ LC LK LR LT LU LV MA MD MG MK MN MX NO NZ PL PT RO RU SE SG SI SK SL TJ TM TR TT TZ UA UZ VN YU ZA |
|
AL | Designated countries for regional patents |
Kind code of ref document: C2 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
COP | Corrected version of pamphlet |
Free format text: PAGES 1-4, SEQUENCE LISTING, ADDED |
|
WWP | Wipo information: published in national office |
Ref document number: 2000923257 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2000923257 Country of ref document: EP |