EP1171154A2 - Nouvelles utilisations de la proteine ox2 de mammifere et reactifs associes - Google Patents

Nouvelles utilisations de la proteine ox2 de mammifere et reactifs associes

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Publication number
EP1171154A2
EP1171154A2 EP00923257A EP00923257A EP1171154A2 EP 1171154 A2 EP1171154 A2 EP 1171154A2 EP 00923257 A EP00923257 A EP 00923257A EP 00923257 A EP00923257 A EP 00923257A EP 1171154 A2 EP1171154 A2 EP 1171154A2
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Prior art keywords
mammalian
tissue
protein
antagonist
agonist
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English (en)
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Robert M. Hoek
Jonathan D. Sedgwick
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Merck Sharp and Dohme Corp
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Schering Corp
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    • 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
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
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    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
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    • 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 0X2 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/macrophage, 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/macrophage
  • 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 OX2.
  • 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. 27:1911-1918. 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.
  • the distribution of the 0X2 is consistent with a hypothesis that OX2 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
  • 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.
  • 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
  • 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 Peotide Synthesis Springer-Verlag, New York; Bodanszky (1984) The Principles of Peotide 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 0X2 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 Enzvmologv. 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) QlAexoress: The High Level Expression & Protein Purification System QUIAGEN. Inc., Chatsworth, CA.
  • Immunogenetics 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 0X2 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 0X2 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; K ⁇ rner, 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 0X2 KO mice appeared enlarged (but not edematous) and filled with F4/80+ cells, i.e., macrophages as it should be.
  • 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 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 lipopolysaccharide (LPS) known to induce rapid macrophage activation.
  • LPS lipopolysaccharide
  • 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.

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Abstract

On décrit des compositions et des procédés dans lesquels on utilise un ligand OX2 de mammifère pour traiter une condition pathologique anormale chez un individu. Les procédés consistent à administrer une quantité thérapeutiquement efficace de OX2 seul ou bien en combinaison avec d'autres réactifs thérapeutiques; ou un antagoniste OX2.
EP00923257A 1999-04-13 2000-04-12 Nouvelles utilisations de la proteine ox2 de mammifere et reactifs associes Withdrawn EP1171154A2 (fr)

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US5753218A (en) * 1996-05-03 1998-05-19 Schering Corporation Method for treating inflammation
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MXPA01010480A (es) 2002-03-27
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WO2000061171A2 (fr) 2000-10-19
AU4341300A (en) 2000-11-14

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