WO1997021450A1 - Molecule costimulante ox-2 - Google Patents

Molecule costimulante ox-2 Download PDF

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Publication number
WO1997021450A1
WO1997021450A1 PCT/US1996/019189 US9619189W WO9721450A1 WO 1997021450 A1 WO1997021450 A1 WO 1997021450A1 US 9619189 W US9619189 W US 9619189W WO 9721450 A1 WO9721450 A1 WO 9721450A1
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cell
cells
protein
domain
nucleic acid
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PCT/US1996/019189
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Francescopaolo Borriello
Arlene H. Sharpe
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Brigham And Women's Hospital, Inc.
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Priority to AU11440/97A priority Critical patent/AU1144097A/en
Publication of WO1997021450A1 publication Critical patent/WO1997021450A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to methods and compositions for modulating a T cell mediated immune response in a subject.
  • the methods and compositions involve administering to the subject the OX-2 protein or a T cell costimulatory molecule related to the OX-2 protein.
  • Background of the Invention For CD4+ T lymphocyte activation to occur, two distinct signals must be delivered by antigen presenting cells to resting T lymphocytes (Schwartz, R.1I. (1990) Science 248: 1349- 1356; Williams, I.R. and Unanue, E.R. (1991 ) J Immunol. 147:3752-3760: Mueller. D.L. et al., (1989) J Immunol. 142:2617-2628).
  • the first, or primary, activation signal is mediated physiologically by the interaction ofthe T cell receptor/CD3 complex (TcR/CD3) with MHC class Il-associated antigenic peptide and gives specificity to the immune response.
  • the second signal the costimulatory signal, regulates the T cell proliferative response and induction of effector functions.
  • Costimulatory signals are pivotal in determining the functional outcome of T cell activation since delivery of an antigen-specific signal to a T cell in the absence of a costimulatory signal results in functional inactivation of mature T cells, leading to a state of tolerance (Schwartz, R.H. (1990) Science 248:1349-1356).
  • T cell costimulatory molecules include murine B7-1 (mB7-l ; Freeman, G.J. et al., (1991) J Exp. Med. 174:625-631 ), and the more recently identified murine B7-2 (mB7-2; Freeman. G.J. et l, f l 993 J Exp. Med. 178:2185-2192).
  • murine B7-1 mB7-l
  • Freeman. G.J. et l Freeman. G.J. et l, f l 993 J Exp. Med. 178:2185-2192
  • Human counte ⁇ arts to the murine B7-1 and B7-2 molecules have also been described (human B7- 1 (hB7-l ) Freedman, A.S. et al., (1987) J Immunol.
  • B7-1 and B7-2 are described in international application PCT/US95/02576, which claims priority to U.S. Serial No. 08/205,697.
  • the B7-1 and B7-2 genes are members of the immunoglobulin gene superfamily; the B7-1 and B7-2 proteins each contain a two immunoglobulin domain extracellular structure comprised of IgV-like and IgC-like domains.
  • Bolh B7-1 and B7-2 are counter-receptors for two ligands.
  • CD28 and CTLA4 expressed on T lymphocytes (Linsley, P.S. et al., (1990) Proc. Natl. Acad Sci. USA 87:5031-5035; Linsley, P.S. et al., (1 91) J Exp. Med. 174:561 -569).
  • CD28 is constitutively expressed on T cells and, after ligation by a costimulatory molecule, induces IL-2 secretion and T cell proliferation (June, CH. el al. (1990) Immunol. Today 1 1 :21 1-216).
  • CTLA4 is homologous to CD28 (Linsley, P.S., Protein Science 3: 1341-1343 (1994)) and appears on T cells after activation (Freeman, G.J. et al. (1992) J Immunol. 149:3795-3801 ). Although CTLA4 has a significantly higher affinity for B7- 1 than does CD28, its role in T cell activation appear to be that of negative regulation (Tivol et al. (1995) Immunology. 3:541 -547) remains to be determined. It has been shown that antigen presentation to T cells in the absence of the B7-1/CD28 costimulatory signal results in T cell anergy in vitro (Gimmi, CD. et al. (1993) Proc.
  • T cell costimulatory molecules such as B7-1 and B7-2 to bind to CD28 and/or CTLA4 on T cells and trigger a costimulatory signal in the T cells provides a functional role for these molecules in T cell activation.
  • OX-2 is a member of the immunoglobulin gene superfamily; the
  • OX-2 protein contains a two domain extracellular structure comprised of IgV-like and IgC-like domains.
  • the rat OX-2 (alternatively referred to herein as rOX-2) protein was first defined using antibodies developed in mice against Type Ia giycoproteins purified from rat thymocytes by lectin chromatography (Barclay et al., Immunogenetics 25(2): 133-135 (1987)).
  • the rat OX-2 protein is expressed on a variety of cells, including, for example, thymocytes, neurons, endothelium, B cells and follicular dendritic cells.
  • the rat OX-2 cDNA was cloned by Clark et al., EMBO 4(1): 113- 1 18, 1985 and shown to encode a type I transmembrane protein with a leader, an extracellular IgV-like and IgC-like domain and a transmembranc/cytoplasmic domain.
  • the inclusion of OX-2 into the immunoglobulin supergene family and the relationship of the OX-2 primary structure to the T cell receptor (TCR)-beta chain and Ig light chains also have been reported.
  • the human OX-2 homologue (hOX-2) was cloned by McCaughan ct al., Immunogenetics 25:329-335 (1987) and shown to be encoded by at least four exons spread over > 10 kb.
  • Cell hybrid analysis was used to genetically map the human OX-2 homologue to chromosome 3. Throughout the period between the cloning of the rat and human OX-2 cDNAs to the present, no function has been ascribed to the OX-2 protein.
  • the sequence homology between the Alzheimer amyloid (770) protein and the OX-2 antigen was reported not to be predictive of a function for the OX-2 protein (Richard, S. J. et al., (1995) Brain Res. Bull. 38(3):305-306). Summary of the Invention
  • a functional in vivo activity for the OX-2 protein is disclosed herein, namely, the OX-2 protein serves as a T cell costimulatory molecule to modulate a T cell mediated immune response to an antigen i vivo.
  • the T cell costimulatory activity is believed to be mediated by binding of the OX-2 protein to a receptor (the OX-2 receptor) on the target cell surface (e.g., a T cell).
  • a receptor the OX-2 receptor
  • Such functional variants include agonists (or antagonists) ofthe OX-2 protein which mimic (or inhibit) the T cell costimulatory activity ofthe OX-2 protein.
  • the invention embraces compositions containing the functional variants of the OX-2 protein, nucleic acids encoding the functional variants, vectors containing these nucleic acids, and host cells including the vectors.
  • a method for modulating a T cell mediated immune response in a subject is provided.
  • the subject is a mammal that has been diagnosed as having a condition that is treatable by modulation ofthe subject's T cell mediated immune response.
  • the method involves administering to the subject a therapeutically effective amount of an OX-2 therapeutic agent to modulate the T cell mediated immune response and thereby treat the condition.
  • OX-2 therapeutic agents refers to the OX-2 protein, an OX-2 agonist, an OX-2 antagonist, and other related molecules (e.g., nucleic acids encoding the foregoing proteins, antibodies that react with OX-2 and affect its ability to bind to the OX-2 receptor) that are useful for modulating a T cell mediated immune response.
  • Conditions that are treatable by up regulation of a T cell mediated immune response include immunosuppressive disorders (e.g., AIDS), neoplastic states and infections using, e.g.. vaccine strategies for infectious agents and anti-tumor gene therapy approaches.
  • Conditions that are treatable by down regulation of a T cell mediated immune response include autoimmune diseases (such as rheumatoid arthritis and lupus), graft versus host disease and transplant rejection.
  • an OX-2 therapeutic agent e.g., the OX-2 protein, an OX-2 agonist, or an OX-2 antagonist
  • the method involves contacting a biological sample ofthe subject with an OX-2 diagnostic agent to detect the presence of OX-2 (or a nucleic acid encoding OX-2) in the biological sample.
  • OX-2 diagnostic agents include, for example, antibodies which specifically recognize the OX-2 protein, as well as nucleic acids which specifically hybridize to an OX-2 transcript under stringent conditions in vitro and/or which specifically hybridize to an OX-2 transcript in vivo.
  • the diagnostic kits further contain instructions for using the OX-2 diagnostic agents to determine the presence or absence of OX-2 or an OX-2 related molecule (e.g., a nucleic acid encoding OX-2) in a biological sample.
  • diagnostic agents e.g., an OX-2-immunoglobulin fusion protein
  • can be provided for detecting the presence of an OX-2 receptor e.g., for diagnosing a condition that is treatable by administration of an OX-2 receptor by, for example, gene therapy for delivery of the receptor in situ.
  • the biological sample can be located in vivo (e.g., the biological sample can be a lymphatic tissue), in which instance the OX-2 diagnostic agent is used to detect OX-2 expression in vivo (e.g., for imaging purposes).
  • the biological sample can be located in vitro.
  • the biological sample can be a cell-containing sample, e.g., peripheral blood or biopsy material, in which the cells are, for example, T cells, B cells, thymocytes. endothelial cells, neurons, and dendritic cells, and the presence of OX-2 in the cell-containing sample is determined to diagnose the condition.
  • Yet another aspect ofthe invention is directed to a method for enhancing the effectiveness of a vaccine by coadministering OX-2, an OX-2 agonist (or a nucleic acid encoding OX-2 or an OX-2 agonist that can be expressed in vivo) to the recipient of the vaccine.
  • enhancing the effectiveness of a vaccine to elicit an immune response to an antigen involves coadministering a therapeutically effective dose of an OX-2 therapeutic agent to the subject to up regulate the T cell mediated immune response of the subject to the antigen.
  • the OX-2 therapeutic agent can be administered simultaneously or sequentially with the vaccine.
  • the vaccine contains the antigen, as well as the OX-2 therapeutic agent.
  • a vaccinia virus genetically engineered to express the antigen, together with the OX-2 therapeutic agent on its surface, can be administered to a subject to elicit an enhanced immune response to the antigen.
  • the vaccinia virus can be genetically engineered to further express a major histocompatibility complex molecule (MHC molecule) to obtain a vaccine that (i) expresses the antigen, (ii) presents the antigen on its surface in the context of the MHC molecule, and (iii) delivers the OX- 2 therapeutic agent to up regulate the immune response of the subject to the presented antigen.
  • MHC molecule major histocompatibility complex molecule
  • the vaccinia virus further is genetically engineered to additionally express other costimulatory molecules and/or cytokines (e.g., IL-12).
  • cancer cells which express cancer specific (surface) antigens can be genetically engineered to coexpress an OX-2 therapeutic agent to enhance the T cell mediated immune response ofthe subject to cancer specific antigens.
  • Novel compositions containing the OX-2 protein, functional variants of the OX-2 protein which bind to the OX-2 receptor and/or which exhibit an OX-2 T cell costimulatory activity, nucleic acids containing the same, vectors containing the nucleic acids and host cells including the vectors also are provided.
  • Antibodies which specifically recognize the OX-2 protein or its functional variants and/or which inhibit the T cell costimulatory activity of the OX-2 protein or its functional variants also are provided.
  • the invention embraces both soluble and insoluble forms of the OX-2 protein.
  • Soluble forms of the OX-2 protein include, for example, chimeric OX-2 molecules in which one or both ofthe OX-2 extracellular (immunoglobulin) domain(s) is covalently coupled to yet another soluble protein (e.g., an immunoglobulin heavy chain).
  • the invention also embraces liposome-associated forms of the OX-2 protein or its functional variants in which the OX-2 protein/liposomc structure is constructed and arranged to permit contact of the OX-2 extracellular domain(s) with OX-2 receptors that are located on OX-2 targeted cells.
  • a chimeric OX-2 molecule is provided.
  • a chimeric OX-2 molecule refers to protein chimeras such as fusion proteins, as well as nucleic acid chimeras which, e.g., encode the protein chimeras.
  • the chimeric OX-2 molecules are useful as OX-2 therapeutic agents and/or diagnostic agents.
  • T cell costimulatory molecules it is believed that one or both of the OX-2 extracellular immunoglobulin domains interact with an OX-2 receptor that is present on a target cell (e.g., a T cell) to modulate a T cell mediated immune response.
  • a particularly preferred embodiment of the invention is a chimeric OX-2 molecule in which the extracellular IgV-like domain and/or the extracellular IgC-like domain ofthe OX-2 protein are covalently coupled at their C-terminus to the N-terminus of an immunoglobulin constant region.
  • I he chimeric OX-2 protein binds to the OX-2 receptor and exhibits an OX-2 T cell costimulatory activity.
  • the chimeric OX-2 protein, as well as the other functional variants of OX-2 do not bind to CTL ⁇ 4 and CD28.
  • Exemplary OX-2 T cell costimulatory activities include the ability to costimulate CD4+ T cells and/or the ability to costimulate thymocytes in an antigen dependent context (see, e.g., the Examples).
  • novel OX-2 therapeutic agents e.g., chimeric OX-2 proteins
  • OX-2 T cell costimulatory molecules i.e., functional variants of OX-2
  • isolated novel proteins encoded by the OX-2 gene are provided.
  • the proteins correspond to alternative forms ofthe OX-2 protein which bind to an OX-2 receptor and exhibit a T cell costimulatory activity.
  • the alternative forms correspond to naturally-occurring, alternatively-spliced forms of the OX-2 protein or to functional variants ofthe alternatively-spliced forms that can be produced using recombinant DNA techniques.
  • alternatively-spliced forms ofthe OX-2 T cell costimulatory molecules exist in nature.
  • Exemplary novel functional variants ofthe OX-2 protein contain an alternative structural domain (i.e., a structural domain having an amino acid sequence which differs from that reported for the OX-2 protein, e.g., a novel signal sequence) or have a structural domain deleted (e.g., the IgC-like domain deleted).
  • Exemplary novel functional variants of the OX-2 nucleic acids e.g., an OX-2 cDNA, an OX-2 mRNA
  • OX-2 functional variants which bind to the OX-2 receptor and exhibit an OX-2 T cell costimulatory activity and which further contain a novel signal peptide domain are provided.
  • the disclosure herein of a function for the OX-2 protein permits the identification of novel OX-2 T cell costimulatory molecule genes which contain exons encoding different signal peptide domains that can be used in an alternate manner.
  • alternative splicing of the mRNA transcripts of the OX-2 gene results in the generation of naturally-occurring OX-2 T cell costimulatory molecules having different signal peptide domains. Confirmation of the existence of alternative signal peptide domain forms ofthe OX-2 protein will further support alternative functional roles for the signal peptide of the OX-2 protein.
  • Still another aspect of the invention is directed to novel OX-2 protein functional variants that bind to the OX-2 receptor and exhibit an OX-2 T cell costimulatory activity and in which at least one structural domain of the known OX-2 proteins is deleted.
  • Isolated nucleic acids encoding the foregoing OX-2 protein functional variants also are provided.
  • the OX-2 protein has an IgC-like domain deleted (i.e., an immunoglobulin variable-like domain is linked directly to a transmembrane/cytoplasmic domain).
  • the OX-2 protein has an IgV-like domain deleted (i.e., a signal peptide domain is linked directly to an immunoglobulin constant-like domain).
  • An isolated nucleic acid molecule of the invention can be incorporated into a recombinant expression vector and transfected or otherwise delivered into a host cell to express a novel structural form of the OX-2 protein.
  • the isolated nucleic acids o the invention can further be used to create transgenic and homologous recombinant non-human animals.
  • novel OX-2 T cell costimulatory molecules proteins and nucleic acids
  • the novel OX-2 T cell costimulatory molecules can be used (1 ) to modulate a costimulatory signal in a T lymphocyte; (2) to raise antibodies against novel structural domains (or exons) of the OX-2 protein (or nucleic acid); (3) to identify a receptor for the OX-2 protein; (4) to identify agents which stimulate the expression of alternative forms ofthe OX-2 T cell costimulatory molecules; (5) to identify components of the signal transduction pathway induced in a cell expressing a costimulatory molecule in response to an interaction between the costimulatory molecule and its receptor on a T lymphocyte or other cell type and/or (6) to create mouse models of OX-2 to further elucidate OX-2 function.
  • Figure 1 shows the FACS analysis of the CHO cell transfectants. The histograms confirm the expected phenotype of the CHO cell transfectant used in the study. The symbols refer to strong positive stain (++), weak positive stain (+), and negative stain (-)
  • Figure 2A shows the costimulation of T cell proliferation using Balb/c responder T cells and anti-CD3. Paraformaldehyde fixed CHO cells were used to provide costimulation to CD4+ splenic T cells from "old” (52 weeks) or "young" mice (6-8 weeks) which had been activated with suboptimal concentrations of anti-CD3 (al 1 ⁇ g/ml).
  • Figure 2B shows the specificity of costimulation of T cell proliferation by ratOX-2.
  • Paraformaldehyde fixed CHO-IAd-ratOX-2 cells were used to provide costimulation to CD4+ splenic T cells from young mice (6-8 weeks) which had been activated with anti-CD3 at 1 ⁇ g/ml.
  • CHO cells were preincubated with the indicated blocking antibody reagents for 30 minutes prior to the addition of T cells and anti-CD3. Proliferation was measured at day 3 with tritium inco ⁇ oration. Note that the I Ad is irrelevant for pu ⁇ oses of this experiment.
  • Figure 2C shows the costimulation of T cell proliferation using T cells from the T cell receptor (TCR) transgenic strain that contains rearranged TCR ⁇ and ⁇ in the germ line DNA that encode a TCR specific for chicken ovalbumin 323-339 bound to the 1-Ad class II MHC molecule.
  • TCR T cell receptor
  • Paraformaldehyde fixed CHO cells were used to provide costimulation to CD4+ splcnic T cells from DO.l 1 mice which had been activated with OVA-peptide at 10 ⁇ g/ml.
  • Both CHO-IAd-B7-l and OX-2 showed costimulatory activity in an antigen-dependent fashion.
  • a direct comparison between the costimulatory potential of B7-1 and ratOX-2 is not implied by these proliferation data as that would require further information regarding antigen density on the CHO cells as well as receptor density on the T cell.
  • Figure 3 shows the costimulation of thymocytes.
  • Paraformaldehyde fixed CHO cells were used to provide costimulation to thymocytes from DO. l 1 mice which had been activated with nothing, anti-CD3 at 1 ⁇ g/ml or OVA-peptide at 10 ⁇ g/ml.
  • CHO-IAd-ratOX-2 showed the costimulatory activity in both instances.
  • Methods and compositions for modulating (up regulating or down regulating) a T cell mediated immune response to an antigen in a subject in which the subject has a condition that is treatable by modulation of the subject's T cell mediated immune response are described herein.
  • Exemplary conditions that are treatable by up regulation of a T cell mediated immune response include immunosuppressive disorders, (e.g., AIDS), neoplastic states and infections using, e.g., vaccine strategies for infectious agents and anti-tumor gene therapy approaches.
  • Exemplary conditions that are treatable by down regulation of a T cell mediated immune response include autoimmune diseases (such as rheumatoid arthritis and lupus), graft versus host disease and transplant rejection.
  • the invention can be used to augment a weak immune response to an antigen (e.g., to enhance the effectiveness of a vaccine) or to suppress a harmful (e.g., autoimmune) immune response in a subject.
  • OX-2 therapeutic agent refers to the OX-2 protein, OX-2 agonists, OX-2 antagonists, and related molecules (e.g., nucleic acids encoding the foregoing proteins) that are useful for modulating a T cell mediated immune response in vivo.
  • OX-2 agonists or OX-2 antagonist
  • Screening assays which can be used to measure the functional activity of a putative OX-2 agonist or OX-2 antagonist are provided in the Examples.
  • the screening assays measure the ability of OX-2 or an OX-2 agonist to (1 ) costimulate CD4+ T and/or CD8+ T cells and/or (2) costimulate thymocytes in an antigen dependent context.
  • the screening assays measure the ability of a putative OX-2 antagonist to inhibit the above-described OX-2 (or OX-2 agonist) costimulatory activity.
  • the OX-2 agonists and OX-2 antagonists do not bind to CTLA4 and CD28.
  • OX-2 agonists are structural variants of OX-2 which mimic the functional activity of the naturally-occurring OX-2 protein, i.e., the OX-2 agonists are functional and structural variants of the naturally-occurring OX-2 protein.
  • the OX-2 agonists costimulate T cells by interacting with an OX-2 receptor that is expressed on the surface of the OX-2 targeted cell (e.g., a T cell).
  • the most preferred OX-2 agonists are represented by soluble functional variants o the OX-2 protein.
  • Exemplary soluble OX-2 agonists include ( 1) functional variants of the OX-2 protein in which one or more structural domains of the OX-2 protein have been deleted, (2) functional variants of the OX-2 protein containing only the OX-2 extracellular domain (or portions thereof which bind to the OX-2 receptor), and (3) chimeric OX-2 proteins in which one or more extracellular domains ofthe OX-2 protein are covalently coupled to a soluble polypeptide (e.g., an immunoglobulin constant region).
  • a soluble polypeptide e.g., an immunoglobulin constant region
  • the OX-2 agonist is a chimeric OX-2 protein that includes the IgV-like domain (denoted domain "B" herein) and/or the IgC-like domain (denoted domain “C” herein) coupled to an immunoglobulin constant region amino acid sequence.
  • the more preferred OX-2 agonists include the entire IgV- like and/or IgC-like domains.
  • Alternative preferred OX-2 agonists are or include (i.e., if a chimeric molecule) functionally active portions of the IgV-like and/or IgC-like domain, i.e., the portions are of a sufficient size and composition to costimulate T cells in the above-noted screening assays.
  • the OX-2 agonist includes the IgV ("B") domain and may include one or more other portions of the OX-2 protein (e.g., the IgC-like domain, the signal sequence, the transmembrane and cytoplasmic domain).
  • the portions of the IgV-like and IgC-like domain that can costimulate T cells arc "unique fragments.”
  • a "unique fragment" of a protein or nucleic acid sequence is a fragment which is not currently known to occur elsewhere in nature (except in allelic or allelomo ⁇ hic variants). Unique fragments act as a "signature" of the gene or protein from which they are derived. A unique fragment will generally exceed 15 nucleotides or 5 amino acids in length.
  • One of ordinary skill in the art can readily identify unique fragments of the OX-2 protein or nucleic acid by searching available computer databases of nucleic acid and protein sequences such as Genbank, (Los Alamos National Laboratories, USA), EMBL, or SWISS-PROT.
  • a unique fragment is particularly useful, for example, in generating monoclonal antibodies or in screening genomic DNA or cDNA libraries.
  • the invention also embraces insoluble OX-2 agonists and antagonists.
  • a representative insoluble OX-2 agonist is a liposomal formulation in which the OX-2 protein (or a functional variant thereof)/liposome is constructed and arranged to permit contact of the OX-2 extracellular domain(s) (or functionally active portions thereof) with, e.g., an OX-2 receptor located on an OX-2 targeted cell.
  • Exemplary protocols for liposome formulations, e.g., for in vivo delivery, are described in U.S. Patent Nos.
  • the OX-2 agonist is a chimeric OX-2 protein (i.e., a fusion protein) that is encoded by a chimeric OX-2 nucleic acid.
  • the chimeric OX-2 agonist is a chimeric immunoglobulin polypeptide which includes (a) at least one amino acid sequence selected from the group consisting of the IgV-like domain (domain "B") and the IgC-like domain (domain "C") of OX-2 and (b) an immunoglobulin constant region amino acid sequence.
  • the OX-2 IgV-like and/or IgC-like domain is coupled at its C-tcrminus to the N-terminus ofthe immunoglobulin constant region amino acid sequence.
  • Exemplar ' immunoglobulin constant region amino acid sequences that are suitable for forming chimeric OX-2 agonists (and antagonists) are described in U.S. Patent No. 5,428,130, issued to Capon et al.
  • OX-2 fusion proteins are useful as reagents for identifying the predicted OX-2 receptor on a variety of cell types.
  • soluble OX-2 fusion proteins containing one or more extracellular domains (or functionally active portions thereof) ofthe OX-2 protein can be labeled with a detectable label (e.g., a radioactive, colorimetric, fluorometric label) and used to detect the presence of the predicted OX- 2 receptor on cells in vitro or in vivo.
  • a detectable label e.g., a radioactive, colorimetric, fluorometric label
  • the chimeric OX-2 proteins can be used to screen different populations of T cells to determine which cell populations express a receptor for OX-2 and thereafter, to clone the receptor from the selected T cell populations. See Examples.
  • OX-2 antagonists arc agents which neutralize or otherwise impede (e.g.. competitively inhibit) the functional activity of the OX-2 protein.
  • OX-2 antagonists include OX-2 protein antagonists, OX-2 oligonucleotide antagonists and OX-2 antibody antagonists.
  • the first class of OX-2 antagonists are structural variants ofthe OX-2 protein ("OX-2 protein antagonists") which inhibit the functional activity of OX-2 by, e.g., competitiveh inhibiting the binding of the protein (or OX-2 agonists) to the OX-2 receptor.
  • the most preferred OX-2 protein antagonists are soluble forms of the OX-2 protein which have been modified so that the antagonist binds to the OX-2 receptor but does not exhibit a T cell activity.
  • receptors for T cell costimulatory molecules are activated to exhibit a f cell costimulatory activity by crosslinking to a multivalent ligand (OX-2).
  • OX-2 antagonist can be a single chain OX-2 fusion protein in which OX-2 is coupled to an immunoglobulin heavy chain, wherein the OX-2 portion of the chimeric molecule has been modified to prevent crosslinking ofthe chimeric molecule to the OX-2 receptor.
  • an OX-2 antagonist is OX-2 or an OX-2 agonist which has been modified to prevent crosslinking ofthe OX-2 receptor. Such modifications include, but are not limited to.
  • modifications embraced by the instant invention are modifications that are intended to convert a multivalent OX-2 ligand to a monovending OX-2 ligand and thereby prevent crosslinking of the OX-2 receptor upon OX-2 engagement.
  • OX-2 protein antagonists include fragments of the OX-2 protein extracellular domain which interact with the OX-2 receptor (as assessed in, e.g., a receptor binding assay) but which do not exhibit a T cell costimulatory activity (as assessed in, e.g..
  • OX-2 antagonists are useful for down regulating a T cell mediated immune response, for example, in treating an autoimmune condition.
  • the second class of OX-2 antagonists (“OX-2 oligonucleotide antagonists") specifically inhibit the transcription or translation of the OX-2 protein (e.g., antisense sequences that specifically hybridize to an OX-2 DNA or OX-2 mRNA, ribozymes that specifically cleave the OX-2 transcript).
  • OX-2 oligonucleotide antagonists are initially identified in in vitro screening assays ("pre-screening" assays) which measure the ability ofthe putative oligonucleotide antagonists to specifically hybridize to OX-2 mRNA under stringent conditions or to cleave the OX-2 mRNA under ribozyme cleavage conditions (see, e.g., U.S. Patent No. 4,987,071 (issued to Cech et al.) and PCT/US94/06316 (applicant Ribozyme Pharmaceuticals, Inc.).
  • the OX-2 oligonucleotide antagonists which hybridize to the OX-2 mRNA contain between about six and 100 bases, preferably between about 30 and 50 bases, and have a nucleotide sequence which is complementary to the nucleic acid sequence encoding, e.g., unique portions of the extracellular domain of the OX-2 protein or its signal sequence.
  • the OX-2 oligonucleotide antagonists have a nucleotide sequence that is complementary to the nucleotide sequence encoding amino acids -19 to -13 (Clark et al., EMBO 4(1): 1 13-1 1 , 1985, numbering scheme) of the OX-2 protein.
  • nucleotide sequence 5'-CAG GCT GTA GGT GGA CAG ATG-3' (SEQ ID NO: 13), which is one hundred percent complementary to the nucleotide sequence encoding the OX-2 signal sequence can be used to inhibit the translation o the OX-2 mRNA.
  • the putative OX-2 oligonucleotide antagonists are screened in functional activity assays to identify antagonists which inhibit OX-2 (or OX-2 agonist) mediated T cell costimulation in, for example, an animal model, in which the ability of OX-2 to costimulate T cells in vivo is determined or in an in vitro assay which measures the ability ofthe putative antagonists to inhibit OX-2 expression on cells which normally express OX-2 in vitro.
  • isolated in reference to an oligonucleotide, means an RNA or DNA polymer, portion of genomic nucleic acid, cDNA or synthetic nucleic acid which, by virtue of its origin or manipulation: (a) is not associated with all of a nucleic acid with which it is associated in nature (e.g., is present in a host cell as a portion of an expression vector); or (b) is linked to a nucleic acid or other chemical moiety other than that to which it is linked in nature; or (c) does not occur in nature.
  • isolated it is further meant a nucleic acid sequence: (i) amplified in vitro by.
  • PCR polymerase chain reaction
  • oligonucleotide sequences which encode SEQ ID NO: 2. Accordingly, the invention embraces oligonucleotides which encode the extracellular domain (as well as its fragments) but which have nucleotide sequences which differ from the sequences of the naturally-occurring OX-2 gene.
  • hybridizing under stringent conditions is a term of art which refers to the conditions of temperature and buffer components which will permit hybridization of a particular oligonucleotide or nucleic acid to its complementary sequence and not to non- complementary sequences.
  • the exact conditions which constitute “stringent” conditions depend upon the length ofthe nucleic acid sequence and the frequency of occurrence of subsets of that sequence within other non-identical sequences.
  • Stringent hybridization conditions may include hybridization conditions of from 30 to 60°C and from 5x to 0.1 x SSC. Highly stringent hybridization conditions may include hybridization at 45°C and 0.1 SSC. Less than stringent conditions are employed to isolate nucleic acid sequences which are substantially similar, allelic or homologous to any given sequence. In a particular!
  • the isolated oligonucleotide is an antisense oligonucleotide that is 100% complementary to the above-identified nucleotide sequence encoding amino acids -1 to -13 (Clark et al, ibid.) of the OX-2 protein (i.e., the shared signal sequence).
  • Exemplary high stringency hybridization conditions are provided in U.S. Patent Application Serial No. 08/199.776, the contents of which are inco ⁇ orated herein by reference.
  • the isolated oligonucleotide is an antisense oligonucleotide (e.g., SEQ. ID NOS: 16 or 17) that is capable of hybridizing under stringent conditions to a unique fragment of the OX-2 extracellular domain, e.g., a unique fragment of the IgV-like domain.
  • the preferred antisense nucleic acids hybridize under stringent conditions to a unique fragment of the IgV-like domain.
  • Exemplary antisense oligonucleotides which hybridize under stringent conditions to the IgV-like domain of OX-2 include:
  • the phrase "unique fragment” refers to a nucleic acid sequence having less than 25% sequence homology with previously identified nucleic acid sequences. More preferably, the unique fragments have less than 10%> sequence homology with known nucleic acid sequences. Such unique fragments can be identified by searching the Genbank, PIR and/or Swiss-Prot data bases using the Eugene program available through the Harvard Molecular Biology Core Research Resource, Cambridge, MA. The unique fragments are useful, for example, as probes and primers in nucleic acid hybridization assays and in amplification reactions, respectively .
  • a third class of OX-2 agonists arc antibodies to the OX-2 protein ("OX-2 antibody antagonists").
  • the antibodies recognize the extracellular domain of the OX-2 protein and are capable of specifically inhibiting the binding of OX-2 or of an OX-2 agonist to the OX-2 receptor and thereby inhibiting the functional activity of OX-2 or the OX-2 agonist.
  • Exemplar ⁇ ' OX-2 antibody antagonists are described in the Examples. Despite the existence of commercially available antibodies to OX-2. the use of such antibodies to modulate a T cell mediated immune response previously has not been possible because of the lack of understanding of a functional role for OX-2.
  • the instant disclosure of a functional role for OX-2 permits the design and construction of antibodies which specifically bind to the extracellular domain of OX-2 in a manner to modulate an OX-2 T cell mediated immune response.
  • the instant disclosure permits the design of antibody fragments. as well as humanized monoclonal antibodies, for modulating an OX-2 T cell mediated immune response. Absent a knowledge of a functional role for OX-2, one skilled in the art would not have been motivated to design and construct antibodies which function as OX-2 antibody antagonists, and in particular, one skilled in the art would not have been motivated to make humanized antibodies to the OX-2 protein for human therapeutic applications.
  • OX-2 specific antibodies are useful for diagnosing condition(s) that are treatable by administration of an OX-2 therapeutic agent.
  • the antibodies to OX-2 also are useful in combination with FACS analysis for the identification of functionally important subpopulations of T cells for which only "surrogate" markers may be available. Accordingly, the ability to use antibodies to OX-2 as possible memory T cell markers permits one skilled in the art to diagnose a condition that is manifested by a defect in the ability to make memory T cells for modulation of a T cell mediated immune response.
  • the OX-2 therapeutic agent is administered to the subject to treat a condition that is treatable by modulation ofthe subject's T cell mediated immune response.
  • a therapeutically effective amount of the OX-2 therapeutic agent is administered to the subject in accordance with standard medical practice for administering a protein or nucleic acid agent.
  • Exemplary methods for administration of a therapeutic agent including calculation of dosages and the preparation of various formulations are provided in Remington's Pharmaceutical Sciences. 18th edition, 1 90. (See, also, U.S. Patent No. 5,428,130).
  • Exemplary methods for delivering a nucleic acid to a somatic cell for human gene therapy are provided in U.S. Patent No. 5,399,346. issued to Anderson et al. and PCT Application No.PCT/US94/06809 (WO95/00654).
  • a "therapeutically effective amount" of an OX-2 therapeutic agent of the invention is a dosage that is large enough to produce the desired effect in which the symptoms of the condition are ameliorated or prevented but is not so large as to cause adverse side effects.
  • a therapeutically effective amount varies with the subject's age, condition, and sex. as well as with the extent ofthe disease in the subject and can be determined by one of skill in the art. The dosage may be adjusted by the individual physician or veterinarian in the event of any complication.
  • a therapeutically effective amount is between about 0.01 mg/kg to about 500 mg/kg, preferably between about 0.1 mg/kg to about 200 mg/kg, most preferably between about 0.2 mg/kg to about 20 mg/kg.
  • the OX-2 therapeutic agents of the invention can be administered by, for example, injection or by gradual infusion over time.
  • the administration ofthe monoclonal antibodies of the invention may, for example, be intravenous, intraperitoneal, intramuscular, intra cavity, subcutaneous, or transdermal.
  • a pharmaceutically acceptable carrier e.g., normal saline
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol. polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution. Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the therapeutically effective amount is between about 1 ⁇ g and about 100 mg/kg.
  • the preferred amount can be determined by one of ordinary skill in the art in accordance with standard practice for determining optimum dosage levels of the agent.
  • the OX-2 therapeutic agents are formulated into a pharmaceutical composition by combination with an appropriate pharmaceutically acceptable carrier.
  • OX-2, the OX-2 agonists or OX-2 antagonists may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • the OX-2 agonists or antagonists may be formulated into preparations in solid, semisolid, or liquid form such as tablets, capsules, powders, granules, ointments, solutions, suppositories and injections, in usual ways for oral, parenteral, or surgical administration.
  • Exemplary pharmaceutically acceptable carriers are described in U.S. 5,21 1 ,657. the entire contents of which patent are incorporated herein by reference.
  • the invention also includes locally administering the composition as an implant.
  • a method for enhancing, in a subject, the effectiveness of a vaccine is provided.
  • the effectiveness of a vaccine is enhanced by coadministering to the subject a therapeutically effective dose of an OX-2 therapeutic agent (OX-2 or an OX-2 agonist) to up regulate the T cell mediated immune response of the subject to an antigen.
  • OX-2 therapeutic agent OX-2 or an OX-2 agonist
  • the vaccine includes the antigen together with the OX-2 therapeutic agent.
  • the vaccinia virus can be genetically engineered to express the antigen, together with the OX-2 therapeutic agent on its surface, and can be used as a vaccine to induce an immune response to the antigen.
  • the vaccinia virus can be genetically engineered to further express a major histocompatibility complex molecule (MHC molecule) or other costimulatory molecule and/or cytokine (e.g., IL- 12) on its surface to result in a vaccine that is capable of (i) expressing the antigen, (ii) presenting the antigen on its surface in the context of the MHC molecule, and (iii) delivering the OX-2 therapeutic agent to up regulate the immune response of the subject to the presented antigen.
  • MHC molecule major histocompatibility complex molecule
  • cytokine e.g., IL- 12
  • the invention also provides a vaccine which contains the OX-2 protein and/or its functional equivalent (e.g., an OX-2 agonist).
  • methods for diagnosing in a subject, a condition that is treatable by administration of an OX-2 therapeutic agent involve contacting a biological sample of the subject with an OX-2 diagnostic agent to detect the presence of OX-2 in the biological sample.
  • contacting means placing the biological sample in sufficient proximity to the OX-2 diagnostic agent and under the appropriate conditions of, e.g., concentration, temperature, time, ionic strength, to allow the specific interaction between the diagnostic agent and OX-2 proteins (or nucleic acids encoding said proteins) that are present in the biological sample.
  • the conditions for contacting the diagnostic agent with the biological sample are conditions known by those of ordinary skill in the art to facilitate a specific interaction between a molecule and its cognate (e.g., a protein and its receptor cognate, an antibody and its protein antigen cognate, a nucleic acid and its complementary sequence cognate) in a biological sample.
  • a molecule and its cognate e.g., a protein and its receptor cognate, an antibody and its protein antigen cognate, a nucleic acid and its complementary sequence cognate
  • Exemplary conditions for facilitating a specific interaction between a molecule and its cognate are described in U.S. Patent No. 5,108,921 , issued to Low et al.
  • the biological sample can be located in vivo or in vitro.
  • the biological sample can be a lymphatic tissue in vivo and the OX-2 diagnostic agent can be used to detect the presence of OX-2 in the lymphatic tissue (e.g., for imaging portions of the lymphatic tissue that express the OX-2 protein).
  • the biological sample can be located in vitro (e.g., a blood sample, tissue biopsy, tissue extract).
  • the biological sample can be a cell-containing sample, more preferably a sample containing T cells (e.g., a peripheral blood sample), endothelial cells, B cells, dendritic cells or neurons.
  • the OX-2 diagnostic agents include many oi ' the same types of agents which are embraced by the phrase "OX-2 therapeutic agents".
  • the OX-2 therapeutic agents further include a detectable label to detect the presence of the naturally-occurring OX-2 protein or nucleic acid encoding the naturally-occurring OX-2 protein.
  • the OX-2 diagnostic agent can be a labeled oligonucleotide which hybridizes under stringent conditions to an OX-2 mRNA.
  • Exemplary stringent conditions for in vitro assays are known in the art.
  • stringent hybridization conditions is a term of art understood by those of ordinary skill in the art.
  • stringent hybridization conditions are those conditions of temperature and buffer solution which will permit hybridization of that nucleic acid sequence to its complementary sequence and not to substantially different sequences.
  • the exact conditions which constitute "stringent” conditions depend upon the length of the nucleic acid sequence and the frequency of occurrence of subsets of that sequence within other non-identical sequences.
  • Stringent hybridization conditions may include hybridization conditions of 30°C-65 °C and from 5X to 0.1X SSPC. Less than stringent hybridization conditions arc employed to isolate nucleic acid sequences which are substantially similar, allelic or homologous to any given sequence. Novel Structural Embodiments
  • OX-2 functional variants functional variants of an OX-2 T cell costimulatory molecule
  • the functional variants have a novel structural form that is related to the structure of the naturally-occurring OX-2 protein or nucleic acid.
  • the OX-2 functional variants have an OX-2 structural domain deleted.
  • the OX-2 functional variant has a structure which corresponds to that of a naturally-occurring, alternatively-spliced form (“isoform") of the OX-2 protein.
  • the alternatively-spliced OX-2 protein is isolated from a natural source or alternatively, is produced using standard recombinant DNA techniques. Functional variants of the OX-2 proteins share common structural and functional properties with the OX-2 protein. Thus, the OX-2 functional variants bind to the OX-2 receptor and exhibit an OX-2 T cell costimulatory activity.
  • An OX-2 T cell costimulatory activity is determined in a screening assay by measuring the ability of, for example, a putative OX-2 agonist to costimulate CD4+ T cells and/or to costimulate thymocytes in an antigen dependent context.
  • Putative OX-2 antagonists are identified in the screening assays by measuring the ability of the putative OX-2 antagonist to inhibit the costimulatory activity of OX-2 or of a known OX-2 agonist.
  • Exemplary screening assays are provided in the Examples and can be performed by one of ordinary skill in the art using no more than routine experimentation.
  • an OX-2 T cell costimulatory molecule of the invention includes the following structural domains: a signal peptide domain (e.g., exon 1), an immunoglobulin variable region ⁇ like domain (IgV-like) (e.g., exon 2), an immunoglobulin constant region-like domain (IgC-like) (e.g. exon 3), and a transmembrane/cytoplasmic domain (e.g., exon 4).
  • a signal peptide domain e.g., exon 1
  • an immunoglobulin variable region ⁇ like domain IgV-like
  • IgC-like domain immunoglobulin constant region-like domain
  • transmembrane/cytoplasmic domain e.g., exon 4
  • the OX-2 T cell costimulatory molecule gene is a member of the immunoglobulin gene superfamily and accordingly, the terms "immunoglobulin variable region-like domain” and “immunoglobulin constant region-like domain” as used herein have their art-recognized meanings.
  • the terms "IgV-like” and “IgC-like” domains refer to protein domains which are homologous in sequence to an immunoglobulin variable region or an immunoglobulin constant region, respectively.
  • Each structural domain of a protein usually is encoded in genomic DNA by at least one exon.
  • Selected aspects ofthe invention are based, at least in part, on the premise that, to a certain extent, the OX-2 protein gene structure parallels that of the B7 T cell costimulatory molecules described in international application PCT/US95/02576, and the prediction (based upon similarities to the B7 molecules) ofthe existence of alternative splicing in the OX-2 T cell costimulatory molecule gene which results in novel, naturally-occurring forms o the OX-2 protein.
  • exons encoding different forms (isoforms) of a structural domain of a B7 T cell costimulatory molecule can be assembled in an alternative manner by alternative splicing of the B7 primary mRNA transcripts.
  • Alternative-splicing is an art-recognized term which refers to the mechanism by which prima , mRNA transcripts of a gene are processed to produce different mature mRNA transcripts encoding different proteins.
  • Alternative-splicing can result in the excision of different exonic sequences from different primary transcripts.
  • mature mRNA transcripts can be produced from a single gene that contains different exonic sequences to produce proteins having different amino acid sequences.
  • the terms "alternative forms,” “isoforms” or “novel forms” of the OX-2 T cell costimulatory molecule refer to the gene products of a single gene which differ in nucleotide or amino acid sequence from previously disclosed forms of the OX-2 protein produced from the same gene.
  • the invention embraces these alternative nucleic acids, isolated from a natural source or prepared using recombinant techniques, as well as the OX-2 proteins encoded thereby. The various aspects of this invention are described in detail in the following subsections.
  • SEQ ID NO: 1 is the nucleotide sequence containing exon 1 ofthe genomic human OX-2 DNA. as described in McCaughan et al., Immunogenetics 25:329-335, 1987.
  • SEQ ID NO:2 is the amino acid sequence of human OX-2 protein, encoded by exons 1 -2-3-4. as described in McCaughan et al.. Immunogenetics 25:329-335. 1987.
  • SEQ ID NO:3 is the nucleotide sequence of rat OX-2 cDNA, exons 1-2-3-4, as described in
  • SEQ ID NO:4 is the amino acid sequence of rat OX-2 protein, encoded by exons 1-2-3-4, as described in Clark et al., EMBO 4(1): 1 13- 1 18, 1985.
  • SEQ ID NO:5 is the nucleotide sequence of mouse OX-2 cDNA, exons 1-2-3-4 (partial sequence).
  • SEQ ID NO:6 is the amino acid sequence of mouse OX-2 protein encoded by exons 1-2-3-4
  • SEQ ID NO: 7 is the PCR primer 0-228.
  • SEQ ID NO:8 is the PCR primer 0-229.
  • SEQ ID NO:9 is the amino acid sequence of mB7-1 as disclosed in Freeman et al., J.
  • SEQ ID NO: 10 is the amino acid sequence of mB7-2 as disclosed in Freeman et al., J. Exp. Me k 178:2185-2192, 1993.
  • SEQ ID NO: 1 1 is the nucleotide sequence containing exon 2 ofthe genomic human OX-2
  • SEQ ID NO: 12 is the nucleotide sequence containing exon 3 ofthe genomic human OX-2 DNA. as described in McCaughan et al., Immunogenetics 25:329-335, 1 87.
  • SEQ ID NO: 13 is the nucleotide sequence (antisense) that is complementary to the nucleic acid encoding the OX-2 signal sequence (5'-C ⁇ G GCT GTA GGT GGA CAG ATG-
  • SEQ ID NO: 14 is Primer 1 of Example 3 (5'- GAA GTG GTG ACC CAG GAT GAA -3').
  • SEQ ID NO: 15 is Primer 2 of Example 3 (5'- GTA TAA TAA GAT GGA GAT CAA -3').
  • SEQ ID NO: 16 is the nucleotide sequence (antisense) that is complementary to a unique fragment ofthe IgV-like domain of OX-2 (5'- TTG TTC ATC CTG GGT CAC
  • SEQ ID NO: 17 is another nucleotide sequence (antisense) that is complementary to a unique fragment of the IgV-like domain of OX-2 (5'- CTG GGT CAC CAC TTC CAC
  • SEQ ID NO: 18 is the amino acid sequence of a chicken ovalbumin peptide, OVA-p (cOV A
  • SEQ ID NO: 19 is the nucleotide sequence containing exon 4 of the genomic human OX-2
  • SEQ ID NO:20 is the PCR primer 309 of Example 4.
  • SEQ ID NO:21 is the PCR primer 226 of Example 4.
  • SEQ ID NO:22 is the PCR primer FB23 of Example 4.
  • SEQ ID NO:23 is the amino acid sequence at the hOX-2:lg junction of the hOX-2-lg fusion protein of Example 4.
  • the invention provides an isolated nucleic acid molecule encoding a novel structural form of an OX-2 T cell costimulatory molecule.
  • the novel OX-2 nucleic acid molecules disclosed herein can be isolated from a natural source or produced using recombinant methods.
  • isolated as used herein in reference to a nucleic acid refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA tcchniques, or chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid is also free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the organism from which the nucleic acid is derived.
  • nucleic acid is intended to include DNA and RNA and can be either double stranded or single stranded.
  • the isolated nucleic acid molecule is a cDNA.
  • the invention embraces isolated nucleic acids which encode proteins which bind to the OX-2 receptor, exhibit a T cell costimulatory activity, and comprise contiguous nucleotide sequences derived from at least one OX-2 T cell costimulatory molecule gene.
  • These nucleotide sequences can be represented by a formula A-B-C-D, wherein
  • A comprises a nucleotide sequence of at least one first exon of an OX-2 T cell costimulatory molecule gene, wherein the at least one first exon encodes a signal peptide domain,
  • B comprises a nucleotide sequence of at least one second exon of an OX-2 T cell costimulatory molecule gene, wherein the at least one second exon encodes an immunoglobulin variable region-like domain,
  • C comprises a nucleotide sequence of at least one third exon of an OX-2 T cell costimulatory molecule gene, wherein the at least one third exon encodes an immunoglobulin constant region-like domain, and
  • D which may or may not be present, comprises a nucleotide sequence of at least one fourth exon of an OX-2 T cell costimulatory molecule gene, wherein the at least one fourth exon encodes a transmembrane domain and a cytoplasmic domain, with the proviso that A does not comprise a nucleotide sequence encoding a known OX-2 protein signal peptide domain.
  • A, B, C and D are contiguous nucleotide sequences iinked by phosphodiester bonds in a 5' to 3' orientation from A to D.
  • D which comprises the nucleotide sequence of a transmembrane/cytoplasmic domain
  • D optionally is not present or is replaced by a soluble (hydrophilic) amino acid sequence.
  • the isolated nucleic acid encodes a protein which (1 ) binds to the OX-2 protein, (2) is encoded by an OX-2 T cell costimulatory molecule gene having at least one exon encoding a first signal peptide domain and at least one other exon encoding a second signal peptide domain, and (3) exhibits an OX-2 protein T cell costimulatory activity.
  • the at least one exon encoding the first signal peptide domain is a nucleotide sequence encoding the signal peptide domain of the human or rat OX-2 proteins.
  • the isolated nucleic acid includes a nucleotide sequence encoding at least one second signal peptide domain in addition to or in place of the exon encoding the first signal peptide domain.
  • T cell costimulatory molecules having structural domains which have been deleted are based, at least in part, on the existence of alternatively-spliced forms of B7 mRNA transcripts in which an exon encoding a structural domain has been excised (sec, international application PCT/US95/02576).
  • the exon encoding the IgV-like domain is deleted and the signal peptide domain exon is linked directly to the IgC-like domain exon to comprise a contiguous nucleotide sequence derived from at least one OX-2 T cell costimulatory molecule gene, the nucleotide sequence represented by a formula A-C-D, wherein
  • A comprises a nucleotide sequence of at least one first exon of an OX-2 T cell costimulatory molecule gene, wherein the at least one first exon encodes a signal peptide domain,
  • C comprises a nucleotide sequence of at least one second exon of an OX-2 T cell costimulatory molecule gene, wherein the at least one second exon encodes an immunoglobulin constant region-like domain
  • D comprises a nucleotide sequence of at least one third exon of an OX-2 T cell costimulatory molecule gene, wherein the at least one third exon encodes a transmembrane/cytoplasmic domain.
  • A, C and D are contiguous nucleotide sequences linked by phosphodiester bonds in a 5' to 3' orientation from A to C to D.
  • Naturally-occurring mRNA transcripts encoding murine B7-1 have been detected in which the exon encoding the IgV-Like domain has been excised and the exon encoding the signal peptide domain is spliced to the exon encoding the IgC-like domain.
  • one embodiment of the present invention embraces the above-described nucleic acid ⁇ -C-D. This embodiment encodes an alternatively-spliced form of the OX-2 protein in which the IgV-Like domain exon has been deleted.
  • the exon encoding the IgC-like domain is deleted and the IgV- like domain exon is linked directly to the transmembrane/cytoplasmic domain exon.
  • This embodiment encodes a protein comprising a contiguous nucleotide sequence derived from at least one OX-2 T cell costimulatory molecule gene, the nucleotide sequence represented by a formula A-B-D, wherein
  • A comprises a nucleotide sequence of at least one first exon of an OX-2 T cell costimulatory molecule gene, wherein the at least one first exon encodes a signal peptide domain,
  • B comprises a nucleotide sequence of at least one second exon of an OX-2 T cell costimulatory molecule gene, wherein the at least one second exon encodes an immunoglobulin variable region-like domain
  • D comprises a nucleotide sequence of at least one third exon of an OX-2 T cell costimulatory molecule gene, wherein the at least one third exon encodes a transmembrane/cytoplasmic domain.
  • A, B and D are contiguous nucleotide sequences linked by phosphodiester bonds in a 5' to 3' orientation from A to B to D.
  • Isolated nucleic acids encoding alternatively-spliced forms of murine B7- 1 in which an IgC-like domain exon has been deleted are described in international application PCT/US95/02576.
  • Naturally-occurring mRNA transcripts encoding murine B7- 1 have been detected in which the exon encoding the IgC-like domain has been excised and the exon encoding the IgV-like domain is spliced to the exon encoding the transmembrane domain.
  • the IgV-like isoform of mB7-l When expressed in a host cell, the IgV-like isoform of mB7-l reportedly was capable of binding to both mouse CTLA4 and mouse CD28 and triggering a costimulatory signal in a T cell.
  • An isolated nucleic acid having a nucleotide sequence disclosed herein can be obtained by standard molecular biology techniques.
  • oligonucleotide primers suitable for use in the polymerase chain reaction (PCR) can be prepared based upon the nucleotide sequences disclosed herein and the nucleic acid molecule can be amplified from cDNA and isolated. At least one oligonucleotide primer should be complementary to a nucleotide sequence encoding an alternative structural domain.
  • an oligonucleotide primer complementary to a nucleotide sequence encoding the predicted altemative structural domain can be used to screen a cDNA library to isolate and identify a nucleic acid of the invention.
  • Isolated nucleic acid molecules having nucleotide sequences other than those specifically disclosed herein are also encompassed by the invention.
  • novel structural forms of the OX-2 protein from species other than mouse, rat and human are within the scope of the invention.
  • additional alternatively-spliced forms ofthe OX-2 protein can be identified using the techniques described herein. These alternatively-spliced forms can be identified and isolated by one of ordinary skill in the art using no more than routine experimentation.
  • An isolated nucleic acid encoding a novel structural form of an OX-2 protein T cell costimulatory molecule can be obtained by isolating and analyzing cDNA clones encoding the OX-2 protein T cell costimulatory molecule by standard techniques (see, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory press (1989) or other laboratory handbook).
  • cDNAs encoding the OX-2 protein or a functional variant thereof can be amplified by reverse transcriptasc-polymerase chain reaction (RT-PCR) using oligonucleotide primers specific for the OX-2 T cell costimulatory molecule gene.
  • cDNAs then can be subcloned into a plasmid vector and sequenced by standard methods. Oligonucleotide primers for RT-PCR can be designed based upon previously disclosed nucleotide sequences of the OX-2 protein, as well as on the nucleotide sequences disclosed herein.
  • cDNA can be prepared using a 5' or 3' "RACE" procedure (rapid amplification of cDNA ends) as described in the Examples (see also international application PCT US95/02576).
  • a cDNA library can be prepared from a cell line which expresses the OX-2 T cell costimulatory molecule by screening the library with a probe containing all or a portion of the nucleotide sequence encoding the OX-2 T cell costimulatory molecule.
  • the screening procedure is performed under highly stringent conditions in order to selectively identify those sequences having substantial sequence homology to the nucleic acid encoding the OX-2 protein, and in particular, to the nucleic acid encoding the IgV-like and/or IgC-like domains.
  • Individual isolated cDNA clones encoding an OX-2 T cell costimulatory molecule then can be sequenced by standard techniques, such as dideoxy sequencing or Maxam-Gilbert sequencing, to identify a cDNA clone encoding an OX-2 T cell costimulatory molecule having a novel structural domain.
  • a novel structural domain can be identified by comparing the sequence of the cDNA clone to the previously disclosed nucleotide sequences encoding OX-2 T cell costimulatory molecules (SEQ ID NOS: 1 and 3). Once an alternative structural domain has been identified, the nucleotide sequence encoding the domain can be mapped in genomic DNA to determine whether the domain is encoded by a novel exon. This type of approach provides the most extensive information about alternatively-spliced forms of mRNAs encoding the OX-2 costimulatory molecules.
  • a novel structural domain for an OX-2 T cell costimulatory molecule can be identified in genomic DNA by identifying a novel exon in the gene encoding the OX-2 T cell costimulatory molecule.
  • a novel exon can be identified as an open reading frame flanked by splice acceptor and splice donor sequences.
  • Genomic clones encoding an OX-2 T cell costimulatory molecule can be isolated by screening a genomic DNA library with a probe encompassing all or a portion of a nucleotide sequence encoding the OX-2 T cell costimulatory molecule (e.g., having all or a portion of a nucleotide sequence shown in SEQ ID NOS: 1 , 3 or 5).
  • a chromosome-specific library rather than a total genomic DNA library can be used.
  • the OX-2 gene has been mapped to human chromosome 3.
  • Genomic clones can be sequenced by conventional techniques and novel exons identified. A probe corresponding to a novel exon then can be used to detect the nucleotide sequence of this exon in mRNA transcripts encoding the OX-2 T cell costimulatory molecule (e.g., by screening a cDNA library or by PCR).
  • Exon trapping is a technique that has been used successfully to identify and isolate novel exons (see e.g. Duyk, G.M. ct al. (1990) Proc. Natl. Acad. Sci. USA 21 :8995-8999: Auch. D. and Reth, M. (1990) Nucleic Acids Res. 1 1 :6743-6744; Hamaguchi, M. ct al. (1992) Proc. Natl. Acad. Sci. USA 12:9779-9783; and Krizman, D.B and Berget, S.M. (1993) Nucleic Acids Res.
  • exon trapping can be applied to identify and isolate exons encoding novel structural domains of OX-2 T cell costimulatory molecules.
  • nucleic acids encoding naturally-occurring alternatively- spliced forms of the OX-2 T cell costimulatory molecules it will be appreciated by those skilled in the art that nucleic acids encoding alternative variant forms, which may or may not occur naturally, can be obtained using standard recombinant DNA techniques.
  • alternative variant forms or “variants” as used herein refer to novel combinations of exon sequences which can be created using recombinant DNA techniques.
  • novel or known exons encoding structural domains of OX-2 T cell costimulatory molecules can be "spliced", using standard recombinant DNA techniques, to other exons encoding other structural domains of the OX-2 costimulatory molecule, regardless of whether the particular combination of exons has been observed in nature.
  • novel combinations of exons can be linked in vitro to create alternative variant forms of nucleic acids encoding novel OX-2 T cell costimulatory molecules.
  • a structural form of murine B7-1 which has a signal peptide domain directly joined to the IgC-like domain has been observed in nature in combination with the cytoplasmic domain encoded by the B7-1 exon 5.
  • analogous structural forms ofthe OX-2 nucleic acid can be created in which the IgV- like domain is deleted and the OX-2 signal peptide domain is directly joined to the IgC-like domain.
  • an exon encoding a structural domain of one OX-2 T cell costimulatory molecule can be transferred to another T cell costimulatory molecule by standard techniques.
  • the amino acid residues encompassing the different "domains" i.e., signal (S), IgV-like (V), IgC-like (C), transmembrane/cytoplasmic (TM/Cyf)
  • S signal
  • IgV-like (V) IgC-like (C)
  • TM/Cyf transmembrane/cytoplasmic
  • rat OX-2 as shown in SEQ ID NO: 4: (-30M-1 ) (S), (l )-(l 1 1 ) (V), (1 12)-(203) (C), (203)-(248) (TM/Cyt).
  • the amino acid numbering is based upon that used by Clark et al., EMBO 4(1): 1 13-1 18, 1985, in reference to rat OX-2.
  • the rat, human and mouse OX-2 have the identical structural organization. It will be appreciated by the skilled artisan that regions slightly longer or shorter than these amino acid domains (i.e., a few amino acid residues more or less at either the amino-terminal or carboxy- terminal end) may be equally suitable for use as signal, IgV-like, IgC-like and/or transmembrane/cytoplasmic domains in the proteins ofthe invention (i.e., there is some flexibility in the junctions between different domains within the proteins ofthe invention as compared to the domain junctions delineated above for OX-2 proteins).
  • proteins comprising signal, IgV-like, IgC-like and/or transmembrane/cytoplasmic domains having essentially the same amino acid sequences as those regions delineated above but which differ from the above-delineated junctions merely by a few amino acid residues, either longer or shorter, at either the amino- or carboxy-terminal end of the domain are intended to be encompassed by the invention.
  • Nucleic acid segments encoding any of the domains delineated above can be obtained by standard techniques, e.g., by PCR amplification using oligonucleotide primers based on the nucleotide sequences disclosed herein, and can be ligated together to create nucleic acid molecules encoding recombinant forms of the proteins of the invention.
  • nucleic acids which have a nucleotide sequence different from those disclosed herein due to degeneracy of the genetic code may be isolated from biological sources.
  • Such nucleic acids encode functionally equivalent proteins (e.g., a protein having an OX-2 T cell costimulatory activity as described above) to those described herein. For example, a number of amino acids are designated by more than one triplet codon.
  • Codons that specify the same amino acid, or synonyms may occur in isolated nucleic acids from different biological sources or can be introduced into an isolated nucleic acid by standard recombinant DNA techniques without changing the protein encoded by the nucleic acid.
  • Isolated nucleic acids encoding alternatively- spliced forms of OX-2 T cell costimulatory molecules having a nucleotide sequence which differs from those provided herein due to the degeneracy of the genetic code are considered to be within the scope of the invention.
  • the invention also embraces nucleic acids which encode functionally equivalent protein variants (OX-2 functional variants) in which the variant differs from the naturally-occurring OX-2 protein by including conservative amino acid substitutions.
  • conservative amino acid substitution refers to an amino acid substitution which does not alter the relative charge or size characteristics of the peptide in which the amino acid substitution is made.
  • Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) MILV; (b) FYW; (c) KRH; (d) AG; (e) ST; (0 QN; and (g) ED.
  • the invention in addition to isolated nucleic acids encoding alternative forms of OX-2 protein T cell costimulatory molecules, the invention also discloses the previously undescribed nucleotide sequences of the murine OX-2 gene and the encoded OX-2 protein (see SEQ ID NOS: 5 and 6).
  • the isolated nucleic acids of the invention are useful for constructing nucleotide probes for detecting nucleotide sequences in biological materials, such as cell extracts, or directly in cells (e.g., by in situ hybridization).
  • a nucleotide probe can be labeled with a radioactive element which provides for an adequate signal as a means for detection and has sufficient half- life to be useful for detection, such as 32 P, 3 H, l4 C or the like.
  • Other materials which can be used to label the probe include antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes and chemiluminescent compounds.
  • An appropriate label can be selected with regard to the rate of hybridization and binding of the probe to the nucleotide sequence to be detected and the amount of nucleotide available for hybridization.
  • the isolated nucleic acids of the invention, or unique fragments thereof, can be used as suitable probes for a variety of hybridization procedures well known to those skilled in the art.
  • the isolated nucleic acids of the invention enable one to determine whether a cell expresses an alternatively-spliced form of an OX-2 T cell costimulatory molecule.
  • mRNA can be prepared from a sample of cells to be examined and the mRNA can be hybridized to an isolated nucleic acid encompassing a nucleotide sequence encoding all or a portion of an alternative cytoplasmic domain of an OX- 2 protein T cell costimulatory molecule to detect the expression ofthe alternative cytoplasmic domain form of the OX-2 costimulatory molecule in the cells.
  • the isolated nucleic acids of the invention can be used to design oligonucleotide primers, e.g. PCR primers, which allow one to detect the expression of an alternatively-spliced form of an OX-2 T cell costimulatory molecule.
  • this oligonucleotide primer spans a novel exon junction created by altemative splicing and thus can only amplify cDNAs encoding this altcrnatively- spliced form.
  • an oligonucleotide primer which spans exon 4 and exon 6 of murine B7-1 reportedly can be used to distinguish between the expression of a first cytoplasmic domain form of mB7- l and expression of an alternative second cytoplasmic domain form of the B7-1 costimulatory molecule (e.g., see international application PCT/US95/02576, Example 2).
  • Novel, alternatively-spliced forms of the OX-2 T cell costimulatory molecule can be detected by designing and using PCR primers in an analogous manner to that described in international application PCT/US95/02576 in reference to discovering novel, alternatively-spliced forms of the B7 T cell costimulatory molecules.
  • the probes of the invention can be used to detect an alteration in the expression of an alternatively-spliced form of an OX-2 T cell costimulatory molecule which may occur, for example, in a disease state.
  • detection of a defect in the expression of an alternatively-spliced form of an OX-2 T cell costimulatory molecule that is associated with an immunodeficiency disorder can be used to diagnose the disorder (i.e., the probes of the invention can be used for diagnostic pu ⁇ oses) and to identify conditions that are treatable by administration of an OX-2 therapeutic agent.
  • Many congenital immunodeficiency diseases result from lack of expression of a cell-surface antigen that is important for interactions between T cells and antigen presenting cells.
  • the bare lymphocyte syndrome results from lack of expression of MHC class II antigens (see e.g., Rijkers, G.T. ct al. (1987) J Clin. Immunol 7:98- 106; Hume, CR. et al. (1989) Hum. Immunol. 25.T - l 1 )) and X-linked hyperglobulinemia results from defective expression of the ligand for CD40 (gp39) (see e.g. Korthauer, U et al. ( 1993) Nature 361 :541 ; Aruffo, A. et al. (1993) Ceil 72:291-300).
  • An immunodeficiency disorder which results from the lack (or significantly reduced) expression of OX-2 or of an alternatively- spliced form of OX-2 can be diagnosed using a probe of the invention.
  • a disorder resulting from the lack or reduced expression of the OX-2 protein can be diagnosed in a patient based upon the inability of a probe which detects this OX-2 nucleic acid to hybridize to mRNA in cells from the patient (e.g., by RT-PCR or by Northern blotting).
  • the probes of the invention also can be used for in vivo imaging.
  • An isolated nucleic acid of the invention can be inco ⁇ orated into an expression vector (i.e., a recombinant expression vector) to direct expression of a novel structural form of an OX-2 T cell costimulatory molecule encoded by the nucleic acid.
  • the recombinant expression vectors are suitable for transformation of a host cell, and include a nucleic acid (or fragment thereof) of the invention and a regulatory sequence, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid. Operatively linked is intended to mean that the nucleic acid is linked to a regulatory sequence in a manner which allows expression of the nucleic acid.
  • regulatory sequence includes promoters, enhancers and other expression control elements.
  • Such regulatory sequences are known to those skilled in the art or are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1 90). It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transfected and/or the type of protein desired to be expressed. Such expression vectors can be used to transfect cells to thereby produce proteins or peptides encoded by nucleic acids as described herein.
  • the recombinant expression vectors of the invention can be designed for expression of encoded proteins in prokaryotic or eukaryotic cells.
  • proteins can be expressed in bacterial cells such as E coli. insect cells (using bacculovirus), yeast cells or mammalian cells.
  • Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • Expression in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids usually to the amino terminus of the expressed target gene.
  • Such fusion vectors typically serve three pu ⁇ oses: 1 ) to increase expression of recombinant protein; 2 ) to increase the solubility of the target recombinant protein; and 3) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the target recombinant protein to enable separation of the target recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, NM) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • Inducible non-fusion prokaryotic expression vectors include pTrc (Amann et al.,
  • target gene expression relies on host RNA polymerase transcription from a hybrid t ⁇ -lac fusion promoter.
  • expression of inserted target genes relies on transcription from the T7 gnl 0-lac 0 fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21 (DE3) or HMS 174(DE3) from a resident prophage harboring a T7 gn 1 under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacterial strain with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185 Academic Press, San Diego, California (1990) 1 19-128).
  • Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector (e.g., a nucleic acid of the invention) so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E coli proteins (Wada ct al., (1992) Nuc. Acids Res. 20:21 1 1 -21 18).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques and are encompassed by the invention.
  • yeast S. cerivisae examples include pYepSec I (Baldari. et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54: 1 13-123), and pYES2 (Invitrogen Co ⁇ oration, San
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the p ⁇ c series (Smith el al., ( 1983) Mol Cell Biol 2:2156-2165) and the pVL series (Lucklow, V. ⁇ ., and Summers, M.D., (1989 ⁇ Virology 170:31 -39).
  • mammalian expression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J 6:187-195, U.S. Patent No. 5,449.614, issued to Danos et al.).
  • control functions ofthe expression vectors often are provided by viral material.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the recombinant expression vector can be designed such that expression of the nucleic acid occurs preferentially in a particular cell type.
  • the expression vector's control functions are provided by regulatory sequences which allow for preferential expression of a nucleic acid contained in the vector in a particular cell type, thereby allowing for tissue or cell specific expression of an encoded protein.
  • the recombinant expression vectors of the invention can be a plasmid or virus, or viral portion which allows for expression of a nucleic acid introduced into the viral nucleic acid.
  • replication defective retroviruses. adenoviruses and adeno-associated viruses can be used.
  • the recombinant expression vectors can be introduced into a host cell, e.g.
  • a host cell line can be used to express a protein ofthe invention.
  • introduction of a recombinant expression vector of the invention into a host cell can be used for therapeutic purposes when the host cell is defective in expressing the novel structural form ofthe OX-2 T cell costimulatory molecule.
  • a recombinant expression vector ofthe invention can be used for gene therapy pu ⁇ oses in a patient with an immunodeficiency disorder which results from inadequate expression of OX-2 or of a novel structural form of the OX-2 T cell costimulatory molecule. See, e.g., U.S. Patent No. 5,399,346. issued to Anderson et al., and PCT application No. PCT/US94/06809 (WO95/00654), for exemplary gene therapy protocols and related compositions.
  • the invention further provides a host cell transfected with a recombinant expression vector of the invention.
  • host cell is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector of the invention can be introduced.
  • the terms "transformed with”, “transfected with”, “transformation” and “transfection” are intended to encompass the introduction of nucleic acid (e.g., a vector) into a cell by one of a number of possible techniques known in the art.
  • Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated transformation.
  • Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate co- precipitation, DE ⁇ E-dextram-mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory handbooks.
  • the number of host cells transfected with a recombinant expression vector ofthe invention by techniques such as those described above will depend upon the type of recombinant expression vector used and the type of transfection technique used.
  • plasmid vectors introduced into mammalian cells are integrated into host cell DNA at only a low frequency.
  • a gene that contains a selectable marker i.e., resistance to antibiotics
  • Preferred selectable markers include those which confer resistance to certain drugs, such as geneticin (G418) and hygromycin.
  • Selectable markers can be introduced on a separate vector (e.g., plasmid) from the nucleic acid of interest or, preferably, are introduced on the same vector (e.g.. plasmid).
  • Host cells transformed with one or more recombinant expression vectors containing a nucleic acid of the invention and a gene for a selectable marker can be identified by selecting for cells using the selectable marker. For example, if the selectable marker encoded a gene conferring neomycin resistance, transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die.
  • the host cell transfected with a recombinant expression vector encoding a novel structural form of an OX-2 T cell costimulatory molecule is a tumor cell.
  • Expression of the Cyt-I form of murine B7-1 on the surface of B7-1 negative murine tumor cells has been shown to induce T cell mediated specific immunity against the tumor cells.
  • the induced immunity was accompanied by tumor rejection and prolonged protection to tumor challenge in mice (see Chen, L., et al. (1992) Cell 71 , 1093 -1 102; Townsend. S.E. and Allison, J.P. (1993) Science 259:368-370; Baskar, S., et al. (1993) Proc. Natl. Acad. Sci.
  • OX-2 functional variants expression of functionally-active, novel structural forms of OX-2 T cell costimulatory molecules ("OX-2 functional variants") on the surface of a tumor cell should be useful for increasing the immunogenicity of the tumor cell.
  • tumor cells obtained from a patient can be transfected ex vivo with a recombinant expression vector of the invention, e.g., encoding an alternative cytoplasmic domain form of an OX-2 costimulatory molecule, and the transfected tumor cells then can be returned to the patient.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • the tumor cell can also be transfected with recombinant expression vectors encoding other proteins to be expressed on the tumor cell surface to increase the immunogenicity ofthe tumor cell.
  • Intemational application PCT/US95/02576 describes the expression of the Cyt-1 form of B7-1 , B7-2, MFIC molecules (e.g., class I and/or class II) and/or adhesion molecules on tumor cells in conjunction with the Cyt-II form of B7-1 to enhance an immune response to tumor cell antigens.
  • MFIC molecules e.g., class I and/or class II
  • adhesion molecules e.g., class I and/or class II
  • the isolated nucleic acid molecules of the invention can also be used to design antisense nucleic acid molecules, or oligonucleotide fragments thereof, that can be used to modulate the expression of OX -2, as well as alternative, naturally-occurring forms ofthe OX-2 T cell costimulatory molecule.
  • An antisense nucleic acid comprises a nucleotide sequence which is complementary to a coding strand of a nucleic acid, e.g. complementary to an mRNA sequence according to the rules of Watson and Crick base pairing, and can hydrogen bond to the coding strand of a target nucleic acid (e.g., the OX-2 mRNA).
  • an antisense nucleic acid molecule can be designed which is complementary to a nucleotide sequence encoding a novel structural domain of an OX-2 T cell costimulatory molecule to inhibit production of that particular structural form ofthe OX-2 T cell costimulatory molecule.
  • the preferred oligonucleotide is an antisense oligonucleotide between about 10 and about 100 nucleotides in length.
  • the antisense oligonucleotide is capable of hybridizing under high stringency conditions to unique fragments of the extracellular domain of SEQ ID NO:l .
  • antisense oligonucleotide refers to an oligonucleotide (DNA, RNA and/or oligonucleotides containing non-naturally-occurring nucleotides which retain the ability to base pair with a nucleic acid target) that is capable of hybridizing to the naturally-occurring DNA or mRNA encoding the known OX-2 protein or naturally-occurring structural and functional variants, thereof.
  • the antisense oligonucleotide is capable of hybridizing in vivo to a unique portion of the nucleic acid encoding the signal sequence, the IgV-like and/or IgC-like domains, or the transcription products of the nucleic acid encoding these domains.
  • Base-pairing of the antisense oligonucleotide with the DNA (or RNA) encoding the signal sequence or the extracellular domains of OX-2 in vivo prevents costimulation of T cells by preventing transcription (or translation) of OX-2 in vivo.
  • An antisense nucleic acid molecule, or oligonucleotide fragment thereof can be constructed by chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the antisense nucleic acid or oligonucleotide can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the anti-sense nucleic acids and oligonucleotides can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e. nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • the antisense expression vector is introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids arc produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • a transgenic animal is an animal having cells that contain a transgene, wherein the transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic, stage.
  • a transgene is a DNA molecule which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • the invention provides a non-human transgenic animal which contains cells transfected to express an altemative form of an OX-2 T cell costimulatory molecule.
  • the non-human animal is a mouse.
  • a transgenic animal can be created, for example, by introducing a nucleic acid encoding the protein (typically linked to appropriate regulatory elements, such as a tissue-specific enhancer) into the male pronuciei of a fertilized oocyte, e.g., by microinjection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • a transgenic animal e.g., a mouse
  • which expresses an OX-2 protein can be made using the isolated nucleic acid shown in SEQ ID NOs:3 or 5, or the isolated hOX-2 nucleic acid represented in SEQ ID Nos: l , 1 1. 12 and 19.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • These isolated nucleic acids can be linked to regulatory sequences which direct the expression of the encoded protein in one or more particular cell types. Methods for generating transgenic animals, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos 4,736,866 and 4,870,009 and Hogan, B. et al., (1986) A Laboratory Manual. Cold Spring Harbor. New York, Cold Spring Harbor Laboratory. A transgenic founder animal can be used to breed additional animals carrying the transgene.
  • the isolated nucleic acids of the invention can be used to create a non-human homologous recombinant animal.
  • homologous recombinant animal as used herein is intended to describe an animal containing a gene which has been modified by homologous recombination.
  • the homologous recombination event may completely disrupt the gene such that a functional gene product can no longer be produced (often referred to as a "knock-out" animal) or the homologous recombination event may modify the gene such that an altered, although still functional, gene product is produced.
  • the non-human animal is a mouse.
  • an isolated nucleic acid of the invention can be used to create a homologous recombinant mouse in which a recombination event has occurred in the OX-2 gene at an exon encoding a cytoplasmic domain such that this exon is altered.
  • the invention provides a non-human knock-out animal which contains a gene encoding an OX-2 protein wherein, for example, an exon encoding a naturally-occurring known or a novel cytoplasmic domain is disrupted or altered.
  • a vector which contains the DNA sequences which are to replace the endogenous DNA sequences, flanked by DNA sequences homologous to flanking endogenous DNA sequences (see for example Thomas, K.R. and Capecchi, M. R. (1987) Cell 51 :503).
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see for example Li, E. et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see for example Bradley, A. in Tcratocarcinomas and Embryonic Stem Cells: A Practical Approach. E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 1 13- 152).
  • a chimeric embryo then can be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • the invention further provides isolated OX-2 T cell costimulatory molecules encoded by the nucleic acids of the invention. These molecules have a novel structural form, either containing a novel structural domain or having a structural domain deleted.
  • isolated in reference to a protein, refers to an OX-2 T cell costimulatory molecule that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • OX-2 T cell costimulatory molecules which include at least one novel signal peptide domain arc provided.
  • the isolated OX-2 T cell costimulatory molecule binds to the OX-2 receptor and has an amino acid sequence derived from the amino acid sequences encoded by at least one OX-2 T cell costimulatory molecule gene.
  • the OX-2 T cell costimulatory molecule comprises a contiguous amino acid sequence represented by a formula A-B-C-D, wherein
  • A comprises an amino acid sequence of a signal peptide domain encoded by at least one exon of an OX-2 T cell costimulatory molecule gene
  • B comprises an amino acid sequence of an immunoglobulin variable region-like domain encoded by at least one exon of an OX-2 T cell costimulatory molecule gene
  • C comprises an amino acid sequence of an immunoglobulin constant region-like domain encoded by at least one exon of an OX-2 T cell costimulatory molecule gene
  • D which may or may not be present, comprises an amino acid sequence of a transmembrane/cytoplasmic domain encoded by at least one exon of an OX-2 T cell costimulatory molecule gene with the proviso that A does not comprise an amino acid sequence of an OX-2 signal peptide domain of the prior art.
  • A, B, C, and D are contiguous amino acid residues linked by amide bonds from an N-terminus to a C-terminus.
  • a soluble form of the OX-2 T cell costimulatory molecule comprises the above-described molecule with the exception that D (the transmembrane/cytoplasmic domain) is absent.
  • the structural form has at least one IgV-like domain deleted.
  • the isolated protein has an amino acid sequence derived from amino acid sequences encoded by at least one OX-2 T cell costimulatory molecule gene and comprises a contiguous amino acid sequence represented by a formula A-C-D, wherein A, which may or may not be present, comprises an amino acid sequence of a signal peptide domain encoded by at least one exon of an OX-2 T cell costimulatory molecule gene,
  • C comprises an amino acid sequence of an immunoglobulin constant region-like domain encoded by at least one exon of an OX-2 T cell costimulatory molecule gene
  • D comprises an amino acid sequence of a transmembrane/cytoplasmic domain encoded by at least one exon of an OX-2 T cell costimulatory molecule gene.
  • A, C and D arc contiguous amino acid residues linked by amide bonds from an N-terminus to a C-terminus.
  • the structural form of the OX-2 T cell costimulatory molecule has at least one IgC-like domain deleted.
  • the isolated protein has an amino acid sequence derived from amino acid sequences encoded by at least one OX-2 T cell costimulatory molecule gene and comprises a contiguous amino acid sequence represented by a formula A-B-D, wherein
  • A which may or may not be present, comprises an amino acid sequence of a signal peptide domain encoded by at least one exon of an OX-2 T cell costimulatory molecule gene,
  • B comprises an amino acid sequence of an immunoglobulin variable region-like domain encoded by at least one exon of a T cell costimulatory molecule gene
  • D comprises an amino acid sequence of a transmembrane/cytoplasmic domain encoded by at least one exon of an OX-2 T cell costimulatory molecule gene.
  • A, B and D are contiguous amino acid residues linked by amide bonds from an N-terminus to a C-terminus.
  • the proteins of the invention can be isolated by expression of the molecules (e.g., proteins or peptide fragments thereof) in a suitable host cell using techniques known in the art.
  • Suitable host cells include prokaryotic or eukaryotic organisms or cell lines, for example, yeast, E. coli and insect cells.
  • the recombinant expression vectors of the invention, described above, can be used to express a costimulatory molecule in a host cell in order to isolate the protein.
  • the invention provides a method of preparing an isolated protein of the invention comprising introducing into a host cell a recombinant expression vector encoding the protein, allowing the protein to be expressed in the host cell and isolating the protein.
  • Proteins can be isolated from a host cell expressing the protein according to standard procedures of the art, including ammonium sulfate precipitation, fractionation, column chromatography (e.g. ion exchange, gel filtration, electrophoresis, affinity chromatography, etc.) and ultimately, crystallization (see generally, 'Enzyme Purification and Related Techniques', Methods in Enzymology. 22:233-577 (1971 )).
  • the costimulatory molecules of the invention can be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogeneous solution (Houbenweyl, 1987. Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 1 and II, Thieme, Stuttgart).
  • the OX-2 T cell costimulatory molecules of the invention can be used to trigger a costimulatory signal in T cells.
  • an OX-2 T cell costimulatory molecule can trigger a costimulatory signal in a T cell by allowing the costimulatory molecule to interact with its receptor on T cells (the OX-2 receptor) in the presence of a primary activation signal.
  • An OX-2 T cell costimulatory molecule ofthe invention can be obtained in membrane-bound form by expressing the molecule in a host cell (e.g., by transfecting the host cell with a recombinant expression vector encoding the molecule).
  • the OX-2 T cell costimulatory molecule should include extracellular domains (i.e., signal peptide, which may or may not be present in the mature protein, IgV-like and IgC-like domains), and a transmembrane/cytoplasmic domain.
  • extracellular domains i.e., signal peptide, which may or may not be present in the mature protein, IgV-like and IgC-like domains
  • a transmembrane/cytoplasmic domain i.e., signal peptide, which may or may not be present in the mature protein, IgV-like and IgC-like domains
  • a transmembrane/cytoplasmic domain e.g., signal peptide, which may or may not be present in the mature protein, IgV-like and IgC-like domains
  • a transmembrane/cytoplasmic domain e.g., signal peptide, which may or may not be present in the mature protein, IgV-like and
  • the preferred oligonucleotide is an antisense oligonucleotide between about 10 and about 100 nucleotides in length.
  • the antisense oligonucleotide is capable of hybridizing under high stringency conditions to unique fragments of the extracellular domains of SEQ ID NO: 1.
  • antisense oligonucleotide refers to an oligonucleotide (DNA and/or RNA) that is capable of hybridizing to the naturally- occurring DNA or mRNA encoding the human OX-2 under stringent conditions (e.g.
  • antisense molecules which specifically hybridize to the OX-2 signal sequence of extracellular domains.
  • the antisense oligonucleotide is capable of hybridizing in vivo to the nucleotide sequence encoding amino acids -19 to -13 (Clark et al. , ibid- . numbering scheme) of the OX-2 protein or its transcription product. Base-pairing of the antisense oligonucleotide with the DNA (or RNA) encoding the signal sequence or extracellular domains of OX-2 in vivo, prevents costimulation by preventing transcription (or translation) of OX-2.
  • Example 1 describes a screening assay for identifying OX-2 agonists and OX-2 antagonists that is based upon the ability of these agents to costimulate (or inhibit costimulation of) (1 ) CD4 + T cells and/or (2) thymocytes in an antigen dependent context.
  • the OX-2 T cell costimulatory molecules of the invention can also be used to inhibit or block a costimulatory signal in T cells.
  • a soluble form of an OX-2 T cell costimulatory molecule which binds to the OX-2 receptor but which does not exhibit a T cell costimulatory activity can be used to competitively inhibit the interaction of membrane-bound costimulatory molecules with the OX-2 receptor on T cells.
  • Such soluble OX-2 antagonists can be identified in the above-noted screening assays.
  • a soluble form of an OX-2 T cell costimulatory molecule can be expressed in a host cell line such that it is secreted by the host cell line and can be purified.
  • the soluble costimulatory molecule contains extracellular domains (signal peptide, which may or may not be present in the mature protein, IgV-like and IgC-like domains) but does not contain a transmembrane/cytoplasmic domain.
  • the soluble form ofthe OX-2 T cell costimulatory molecule can be in the form of a fusion protein, e.g. an immunoglobulin fusion protein wherein the extracellular portion ofthe costimulatory molecule is fused to an immunoglobulin constant region.
  • Soluble forms of the OX-2 protein can be evaluated for T cell costimulatory activity in the above-noted screening assays to identify soluble OX-2 agonists and soluble OX-2 antagonists for use in accordance with the methods of the invention.
  • a soluble form of an OX-2 T cell costimulatory molecule can be used to inhibit a costimulatory signal in T cells by contacting the T cells with the soluble molecule and allowing the antagonist to competitively inhibit binding of the in vivo generated OX-2 protein to its receptor in vivo.
  • the OX-2 T cell costimulatory molecules of the invention can be used to produce antibodies directed against the costimulatory molecule, and, in particular, to produce antibodies which specifically interact with particular domains of the OX-2 T cell costimulatory molecule to inhibit OX-2 T cell costimulatory activity.
  • Conventional methods can be used to prepare the antibodies.
  • a mammal e.g., a mouse, hamster, or rabbit
  • a costimulatory molecule e.g., a mouse, hamster, or rabbit
  • Techniques for conferring immunogenicity on a protein include conjugation to carriers or other techniques well known in the art.
  • the protein can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies.
  • antisera can be obtained and, if desired, polyclonal antibodies isolated from the serum.
  • the OX-2 costimulatory molecules of the invention can be used to raise monoclonal antibodies.
  • antibody producing cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures, thus immortalizing these cells and yielding hybridoma cells.
  • myeloma cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures, thus immortalizing these cells and yielding hybridoma cells.
  • Such techniques arc well known in the art. For example, the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the protein or portion thereof and monoclonal antibodies isolated.
  • the term antibody as used herein is intended to include fragments thereof which are also specifically reactive with the OX-2 costimulatory molecule.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab') 2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab') fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
  • Chimeric and humanized antibodies also are within the scope of the invention. It is expected that chimeric and humanized antibodies would be less immunogenic in a human subject than the corresponding non-chimeric antibodies.
  • a variety of approaches for making chimeric antibodies, comprising for example a non-human variable region and a human constant region, have been described. See, for example, Morrison et al., Proc. Natl. Acad. Sci, USA 81 :6851 (1985); Takeda et al.. Nature 314, 452 (1985), Cabilly et al., U.S. Patent No. 4,816.567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP 171496;
  • a chimeric antibody can be further "humanized" such that parts of the variable regions, especially the conserved framework regions ofthe antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin.
  • Such altered immunoglobulin molecules may be made by any of several techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. USA. 80:7308-7312 (1983); Kozbor et al., Immunology Today. 4:7279 (1983); Olsson et al., Meth. Enzymol. 92.
  • Humanized antibodies can be commercially produced by, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.
  • Another method of generating specific antibodies, or antibody fragments, reactive against a novel OX-2 protein or nucleic acid of the invention is to screen phage expression libraries encoding immunoglobulin genes, or portions thereof, with proteins produced from the nucleic acid molecules ofthe present invention or with the nucleic acids themselves.
  • complete Fab fragments, Vh regions and Fv regions can be expressed in bacteria using phage expression libraries. See for example Ward ct al., Nature 341 :544-546: (1989); Huse et al.. Science 246: 1275-1281 (1989); and McCafferty et al, Nature 348:552-554 (1990).
  • the invention provides an antibody which specifically binds to a novel structural domain of an OX-2 T cell costimulatory molecule provided by the invention or to the nucleic acid encoding the novel structural domain.
  • the preferred antibodies of the invention do not cross-react with other members of the immunoglobulin superfamily, such as the B7 T cell costimulatory molecules. Such antibodies, and uses therefor, are described in greater detail below in subsection VI, part B. C. Screening Assays
  • An OX-2 T cell costimulatory molecule ofthe invention can be used in a screening assay to identify components ofthe intracellular signal transduction pathway induced in antigen presenting cells upon binding of the OX-2 T cell costimulatory molecule to its receptor on a T cell or other target cell.
  • engagement of the OX-2 costimulatory molecule with a receptor on T cells is likely to deliver distinct signals to the antigen presenting cell (the cell expressing the OX-2 T cell costimulatory molecule), e.g. through the cytoplasmic domain.
  • OX-2 T cell costimulatory molecules By analogy to the B7 family of costimulatory molecules, it is believed that signals delivered through a novel OX-2 cytoplasmic domain may be of particular importance in the thymus, e.g.. during positive selection of T cells during development, since structural forms ofthe analogous B7 costimulatory molecules comprising a novel cytoplasmic domain are known to be preferentially expressed in the thymus.
  • host cells expressing OX-2 T cell costimulatory molecules can be stimulated by crosslinking the OX-2 costimulatory molecule on the cell surface with an antibody and identifying intracellular signals and/or other cellular changes that are induced in the host cell in response to this crosslinking. In this manner, additional proteins or other cellular agents which play a role in the intracellular signal transduction pathway can be identified.
  • an isolated OX-2 T cell costimulatory molecule of the invention can be used in methods for identifying other molecules (e.g., proteins) which interact with (bind to) the costimulatory molecule using standard in vitro assays (e.g., incubating the isolated costimulatory molecule with a cellular extract and determining by immunoprecipitation if any molecules present in the cellular extract bind to the costimulatory molecule). It is of particular interest to identify molecules which can interact with the cytoplasmic domain since such molecules may also be involved in intracellular signaling. For example, it is known that the cytoplasmic domains of many cell surface receptors can interact intracellularly with other members of the signal transduction machinery, e.g., tyrosine kinases.
  • the invention further provides a method for screening agents to identify a regulatory agent which up regulates or down regulates expression of an OX-2 T cell costimulatory molecule in vivo.
  • the method involves contacting a cell which expresses or can be induced to express an OX-2 T cell costimulatory molecule with a putative regulatory agent and observing changes in the expression levels of a known or novel form of the OX-2 T cell costimulatory molecule in the cell.
  • the term "up regulates” encompasses inducing the expression of a known or novel form of an OX-2 T cell costimulatory molecule by a cell which does not constitutively express such a molecule or increasing, by a statistically significant amount, the level of expression of a known or novel form of an OX-2 T cell costimulatory molecule by a cell which already expresses such a molecule.
  • the term “down regulates” encompasses decreasing (to a statistically significant amount) or eliminating expression of a known or novel form of an OX-2 T cell costimulatory molecule by a cell which already expresses such a molecule.
  • regulatory agent is intended to include molecules which trigger an up regulatory or down regulatory response in a cell.
  • an agent can be a small organic molecule, a biological response modifier (e.g., a cytokine) or a molecule which can crosslink surface structures on the cell (e.g., an antibody).
  • Expression levels of a known or novel OX-2 T cell costimulatory molecule(s) by the cell can be determined by, for example, detecting an mRNA transcript encoding the known or novel form of the OX-2 T cell costimulatory molecule in the cell.
  • OX-2 e.g., an alternatively-spliced form
  • cell types can be contacted with the putative regulatory agents and screened according to the above-noted screening methods of the invention to identify a regulatory agent which up regulates or down regulates expression of the novel form of OX-2.
  • Antibodies and Peptide Antibodies and Peptide.
  • Anti-ratOX-2 was purchased from Harlan Bioproducts for Science, Indianapolis, IN.
  • Anti-B7-1 was purchased from Pharmingen, San Diego, C ⁇ .
  • Murine CD28Ig and murine CTLA41g reagents were provided by Dr. T. Strom, Beth Israel Hospital, Boston, MA.
  • Anti-CD3 (2C-1 1) was provided by Dr. J. Bluestone, University of Chicago,
  • Chicken ovalbumin peptide, OVA-p (cOVA 323-339) is an IAd restricted peptide antigen (NH 2 -ISQAVHAAFIAEINEAGR-COOH; SEQ ID NO: 18) which was synthesized at Stanford University.
  • Cytokines Measurements. In order to measure cytokine secretion from the cultured T cells, culture supernatants were collected at 24 hrs (to measure IL-2) and 48 hrs (to measure IFN- ⁇ and IL-4) and assayed by ELISA (IL-2, IFN- ⁇ and IL-4 mAbs, Pharmingen, San Diego, CA) according to the manufacturer's directions. Cytokine concentrations were determined using the linear portion of the standard curve.
  • CHO cell transfection was carried out by electroporation. In brief, 4 x 10 6 cells in 0.4 ml, were pulsed at 960 ⁇ F and 120 volts using a Biorad Gene Pulser (Biorad, Hercules. C ⁇ ). The OX-2 expression plasmid was cotransfected with a plasmid encoding puromycin resistance at a ratio of 100: 1 , respectively in CHO cells or CHO cells previously transfected with the MHC Class II antigen, I-A ci (CI 10-IAd, provided by Dr. Hans Reiser, Dana Farber Cancer Institute, Boston, MA). Transfected cells were selected in 12 ⁇ g/ml puromycin (Sigma, St.
  • CHO cell transfectants were detached from tissue culture flasks with 2 mM EDTA in PBS and subsequently washed 3 times with DMEM. Cells were stained with anti- OX-2 (I larlan Bioproducts, Indianapolis), anti-B7-l and anti-I-A d antibodies (Pharmingen, San Diego), or with soluble receptor fusion proteins containing either murine CD28 or CTLA-4 linked to the murine IgGl (CD28-lg and CTLA-4-Ig, provided by Drs. Terry Strom and Peter Nickerson , Beth Israel Hospital, Boston, MA). Hamster IgG, mouse IgGl and mouse IgG2a were used as isotype matched controls (Sigma, St.
  • T cells were prepared from single cell suspensions of spleen cells by nylon wool fractionation followed by purification by treatment with the M5-1 14 antibody (anti-class II. ATCC, Rockville. MD) and rabbit complement (Cedarlane Laboratories Limited, Ontario), as previously described (Freeman et al.. Science 262:907-909, 1993). resulting in a preparation of approximately 85-95% purity.
  • CD4+ T ceils were prepared as above by antibody-complement depletion of CD8 ' and MI ICIF cells using the antibodies ⁇ DH4 and M51 14, respectively.
  • Thymocytes were prepared by mechanical dissociation of 4-5 week old thymus to make single cell suspensions, and then washing the cell suspensions three times in CI O lymphocyte plating media.
  • CHO cells were grown to confluency and harvested with PBS containing 2mM EDTA, resuspended in PBS at 10 7 cells/ml and fixed in 0.4%) paraformaldehyde for five minutes at room temperature. The fixation was stopped by adding an equal volume of 0.2M lysine in PBS followed by two times in C IO prior to use.
  • T cells (2 x 10 s ) and paraformaldehyde treated CHO (5 x IO 4 ) cell transfectants were cocultured in CI O supplemented with either anti-CD3 (2C 1 1 ) at 1 ⁇ g/ml for 3 days or with ovalbumin peptide (323-339) at 1 ⁇ g/ml.
  • Blocking antibodies (1 : 10 dilution of supernatant), fusion protein (2 ⁇ g/ml) and isotype matched control (2 ⁇ g/ml) were preincubated with the CHO cell transfectants for 30 minutes prior to the addition of the T cells.
  • Primer 0-228 (SEQ ID NO:7) contains a Kozak translational start sequence immediately upstream of the initiation methionine.
  • Primer 0-229 (SEQ ID NO:8) extends through the termination codon. Both primers have restriction sites placed at their ends for cloning pu ⁇ oses, e.g. for directional cloning into the pBK eukaryotic expression vector (Stratagene, La Jolla, CA).
  • RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
  • PCR reactions were performed in 25 ⁇ l volumes using a manual "hot start", wherein l Ox deoxynucleotide triphosphates (dNTPs) were added to the samples at 80 °C
  • Final reaction conditions were: 60 mM Tris-HCl, pH 8.5, 15 mM (NH 4 ) 2 SO 4 , 2.5 M MgCL, 200 ⁇ M dNTPs, and 2 ⁇ g/ml each of the specific primers. Cycling conditions for all amplifications were 94 °C, 4 minutes prior to 35 cycles of 94 °C for 45 seconds, 58°C for 45 seconds, and 72 °C for 3 minutes, followed by a final extension at 72 °C for 7 minutes.
  • the template for primary PCR was 2 ⁇ l of the RT reaction product and the template for secondary nested PCR was 1 ⁇ l of the primary PCR reaction product.
  • Rapid Amplification of cDNA Ends Procedure.
  • Polyadcnylated RNA purified by two cycles of oligo-dT selection is obtained from CFH B lymphoma cells, which express high levels of OX-2.
  • Primers designed to the most 5' end of the cDNA are employed with the 5' RACE Kit (Gibco BRL, Gaithersburg, MD) according to the manufacturer's instructions.
  • RNA is reverse transcribed with a gene-specific oligonucleotide, the cDNA purified, and a poly-dCTP tail is added with terminal deoxynucleotide transferase.
  • PCR is performed using a nested primer and an oligonucleotide complementary to the poly-dCTP tail. PCR bands are cloned, sequenced, and correlated with the genomic sequences.
  • Oligonucleotide hybridization Oligonucleotide(s) arc 5' end-labeled with polynucleotide kinase and ⁇ 32 P-ATP. Hybridizations are carried out in 5X SSC and 5% SDS at 55 °C overnight and subsequently are washed 3 times for 15 minutes with 2X SSC at 55 °C Blots are exposed to Kodak XAR-5 film with an intensifying screen at -80°C B. Results
  • This sequence change could be due to a PCR artifact or alternatively to a strain difference between the Lewis rat used in this study and the Sprague-Dawley rat used by Clark et al.
  • the cDNA was inserted into the pBK eukaryotic expression vector (Stratagene, LaJolla, CA) and cotransfected into either CHO or CHO-I ⁇ d cells with a puromycin resistance gene-containing plasmid. CHO cells were placed under selection of 25 ⁇ g/ml puromycin and reached conflucncy in two weeks.
  • CHO cells (5 x I O 4 ) were added to the purified T cells (2 x I O 5 ) prior to the addition of anti-CD3.
  • the assay was plated in a well triplicate format and proliferation was measured by tritiated thymidine uptake during the last 20 hours of a 3 day incubation. Costimulation by CHO-B7-1 was clearly observed, at approximately equal amounts. for both the responding T cells from young as well as old mice. Interestingly. CI IO-ratOX-2 also costimulatcd both populations of responding T cells but was greater than 2 fold more pronounced when older T cells were used as the target cells. As expected, untransfected CHO cells did not costimulate the CD4 + T cells.
  • OX-2 were used, despite the fact that the degree of T cell proliferation for both was comparable.
  • Antigen specific costimulatory activity We next appraised the ability of these
  • Thymocyte costimulation We appraised ratOX-2 and mB7-l for their ability to costimulate thymocytes (Figure 3). Thymocytes were obtained from Balb/c mice (4-5 week old) and washed three times without further purification. Using the same cell ratios as in the previous studies, cells were given a primary stimulus of either anti-CD3 or OV ⁇ -p, followed by exposure to CHO. CHO-IAd-B7-l or CHO-IAd-ratOX-2 transfectants.
  • OX-2 was also examined for its ability to provide costimulation to thymocytes from the DOl 1 TCR transgenic mice using CHO-IAd-OX-2 and CHO-I ⁇ d-B7-l cells. Both CHO-IAd- OX-2 and CHO-l ⁇ d-B7-l cells were capable of providing a costimulatory signal which led to an enhanced proliferation compared with the control CFlO-IAd cells. This was true irrespective of whether anti-CD3 or ovalbumin peptide was used to provide the TCR signaling. Experiments using OX-2 and B7-1 transfectants in the absence of cotransfected I- ⁇ d , to costimulate Balb/c thymocytes in the presence of anti-CD3, produced similar results.
  • Table II Amino acid sequence comparison between the MRC OX-2 grasp and murine B7-1 and B7-2 proteins.
  • the transmembrane (Tm) and cytoplasmic (Cyt) domains show remarkable conservation and suggest a functional constraint during evolution.
  • the conservation in the transmembrane domain suggests that OX-2 may have proceeded so as to preserve a critical function for that domain which may include the capacity to associate with other membrane proteins.
  • the four conserved polar serine residues in the transmembrane also argue for association of OX-2 with other proteins.
  • the conserved cytoplasmic tail suggests either a conserved signaling function or an associative function with other proteins. This is in marked contrast to the cross species comparison of the B7- 1 and B7-2 costimulatory proteins which, while maintaining a distinct homology in the extracellular domains, have little if any homology in the transmembrane or cytoplasmic domains.
  • the hOX-2 locus defines a "hot spot'" to search for additional costimulatory molecules in this class. ⁇ similar genomic hotspot is observed for the TNF-NGFR family of costimulators.
  • the receptor for OX-2 possesses a similar structure to CD28 and CTLA4 (a single IgV-like extracellular domain) and may also map to the genetic locus defined by CD28 and CTLA4.
  • ratOX-2 is a costimulatory protein for T cell activation. While ratOX-2 shares sequence homology to the well studied B7-1 and B7-2 costimulatory proteins, it failed to crossreact (stain) with either CTLA4Ig or CD28Ig. Furthermore, several features of the OX-2 costimulatory activity toward T cells and thymocytes are quite distinct from those observed in connection with B7 costimulation. These data defme ratOX-2 as a novel costimulatory protein which functions through a B7:CD28/CTLA4 independent pathway.
  • ratOX-2 protein has been reported to be present on some of the important antigen presenting cells in the immune system including B cells and endothelium. as well as on follicular dendritic cells.
  • the presence of OX-2 on thymocytes also indicated to us that OX-2 may play a role in the development of thymocytes and possibly in the maturation of the TCR repertoire.
  • the ratOX-2 protein can be used to identify distinct functional parameters in T cell populations.
  • Thymocytes T cells
  • Thymocytes were stimulated either with anti-CD3 at 1 ⁇ g/ml or OVA-peptide at 10 ⁇ g/ml.
  • CHO-B7-1 costimulated proliferation in either case.
  • CD4' spleen cells from the DO.] 1 mouse can be costimulated by CHO-ratOX-2 and anti-CD3. This suggests that a developmental difference between the TCR transduction pathway in immature thymocytes versus that found in mature splenocytes.
  • OX-2 is part of a T cell costimulatory pathway functionally distinct from the B7/CD28 pathway.
  • OX-2 expression on the endothelium suggests that OX-2 may be involved in the events by which T cells are activated at sites of inflammation, by their interaction with the endothelium. that lead to transmigration and downstream effector functions.
  • Strong expression on the follicular dendritic cells also suggests a possible role for OX-2 in the interaction of B-lymphocytes and professional antigen presenting cells.
  • EXAMPLE 2 Identification of a Novel OX-2 Signal Peptide Domain cDNA fragments corresponding to the 5' and 3' ends of naturally-occurring human, rat or mouse OX-2 mRNA transcripts were prepared by using a commercially available kit (Marathon RACE Kit, CLONTECH Laboratories, Palo Alto, CA) according to the manufacturer's instructions. Using this procedure, polymo ⁇ hisms at both the 5' and 3' ends of the OX-2 cDNAs were identified. ⁇ polymorphism at the 3' end involves the 3' untranslated region and may be relevant to post-transcriptional control of OX-2 expression.
  • a polymo ⁇ hism at the 5' end involves the use of an altemative signal sequence which is approximately 75 base pairs longer than the known signal exon. I he additional amino acids at the amino terminus of the protein may have functional ramifications for the interaction of this OX-2 variant with its receptor.
  • EXAMPLE 3 Identification of alternatively-spliced forms of OX-2 having a structural domain deleted. Reverse-transcriptase polymerase chain reaction is used to amplify human, rat or mouse cDNA fragments derived from human tumor cells, peripheral blood and spleen cell RNA.
  • Oligonucleotide primers useful for PCR are as follows.
  • An exemplary primer pair which bridges the IgV to the transmembrane/cytoplasmic domain and hence is useful for detecting an alternatively spliced OX-2 which lacks IgC domain is:
  • Primer 1 sense, IgV exon, spans amino acids 3-9 (Clark et al., ibid.. numbering scheme)
  • Primer 2 5'- GAA GTG GTG ACC CAG GAT GAA -3' (SEQ ID NO: 14) Primer 2, antisense, transmembrane exon. spans amino acids 222-228 (Clark et al., ibid., numbering scheme)
  • a cDNA fragment is detected which comprises a nucleotide sequence encoding a human OX-2 molecule in which the signal peptide domain is spliced directly to the IgC-like domain (i.e., the IgV-like domain is deleted).
  • another cDNA fragment is detected with comprises a nucleotide sequence encoding a human OX-2 molecule in which the IgV-like domain is spliced directly to the transmembrane/cytoplasmic domain (i.e., the IgC-like domain is deleted).
  • This protein is referred to herein as an IgV-like isoform of OX-2 or as a "pOX-2" ("p" is for "partial" OX-2 molecule).
  • CHO Chinese Hamster Ovary
  • IgV-like isoform of hOX-2 on the surface of the CHO-pOX-2 cells is confirmed by F ⁇ CS analysis using an OX-2 receptor or anti-OX-2 antibody as the primary staining reagent. This reagent stains the CHO-pOX-2 cells. Positive staining of CHO-pOX-2 with the OX-2 receptor indicates that the IgV-like isoform of hOX-2 is capable of interacting with the OX-2 receptor.
  • T cells that receive a primary activation signal are stimulated to produce IL-2 when incubated with either CHO-OX-2 cells or CHO-pOX-2 cells but not when incubated with untransfected CHO cells. Similar results are observed when T cell proliferation is assayed as an indicator of T cell costimulation.
  • EXAMPLE 4 Preparation and characterization of OX-2 antibodies and fusion proteins
  • Monoclonal antibodies to murine OX-2 were prepared by standard methodologies using a mOX-2Ig fusion protein as an antigen.
  • a panel often monoclonal antibodies which bind specifically to mOX-2 were characterized for utility in FACS analysis of cells expressing mOX- 2.
  • Two of the ten monoclonal antibodies stained murine splenocytes sufficiently to permit detection by FACS.
  • the other 8 monoclonal antibodies were not as useful in FACS analysis but can be used for immunoprecipitation or Western blot analysis.
  • a monoclonal antibody also was developed against rat OX-2 using standard methodologies and rOX-2-Ig fusion protein as an immunogen. This antibody is structurally and functionally distinct from the commercially available rat OX-2 antibody.
  • OX-2 fusion proteins Soluble variants (OX-2-Ig fusion proteins) of murine, rat and human OX-2 molecules were prepared (mOX-2-Ig, rOX-2-Ig and hOX-2-Ig, respectively). These molecules are analogous to CTLA4-Ig soluble costimulatory molecule prepared by Brian Seed (Zettlmeissl et al. (1990) DNA and Cell Biol. 9(5):347-353) and used to block costimulation of T cells.
  • the following primers were used to generate PCR products from which BamHI fragments having (GGATCC) ends could be formed and spliced into an lg expression vector available from Dr. Brian Seed (Massachusetts General Hospital, Boston, MA).
  • the protein sequences produced in this manner have only slight amino acid differences from the composite native proteins at the junction formed by BamHI ligations.
  • the amino acid sequence of the mOX-2 or rOX-2 portion is as listed (SEQ ID NOs:4 and 6) except that after the DK motif at the end of the IgC domain, there is the amino acid sequence DPR, unrelated to mOX-2, rOX-2 or Ig. followed by the human IgGl heavy chain hinge region and the CH2 and CH3 domains.
  • the amino acid sequence of the hOX-2 portion is as listed (SEQ ID NOs:4 and 6) except that after the DK motif at the end of the IgC domain, there is the amino acid sequence DPR, unrelated to mOX-2, rOX-2 or Ig. followed by the human IgGl heavy chain hinge region and the CH2 and CH3 domains.
  • the amino acid sequence of the hOX-2 portion is as listed (SEQ ID NOs:4 and 6) except that after the DK motif at the end of the IgC domain, there is the amino acid sequence DPR, unrelated to mOX-2
  • the mOX-2Ig prepared as described above was found to inhibit the function of lymphocytes in several in vitro assays, including an allogeneic mixed lymphocyte reaction (allo- MLR) and anti CD3 induced proliferation of mouse splenocytes.
  • allo- MLR allogeneic mixed lymphocyte reaction
  • unadulterated lymphocytes of one genetic background are cultured with inactivated cells of a different genetic background. After several days of incubation the unadulterated cells will respond against the foreign cells by proliferating, producing cytokines and developing specific cytotoxic effector functions.
  • mOX-2-lg When mOX-2-lg was added to an allo-MLR at 25 ⁇ g/ml, proliferation ofthe unadulterated cells was reduced greater than 5 fold compared to an allo-MLR to which a control lg was added. Addition of mOX-2-Ig to an anti-CD3 splenocyte proliferation assay reduces proliferation of splenocytes to approximately the same extent as does mCTL ⁇ 4Ig.
  • mOX-2Ig inhibited an allo-MLR induced proliferation in a human system, i.e., the murine OX-2 fusion protein cross-reacts with the human receptor for OX-2.
  • the mOX- 2 Ig also can be used to detect OX-2 receptors by FACS analysis as described in Example 1 and can be used in adhesion assays to determine whether the OX-2 receptor is expressed on certain cell types.
  • the murine OX-2 fusion protein has been used to demonstrate that Con ⁇ anti-CD3 activated but not resting splenocytes adhere to mOX-2-lg coated plastic wells. Thus at least one receptor for OX-2 appears to be an activation induced surface molecule.
  • EXAMPLE 5 - OX-2 specifically stimulates Thl T cells. An adhesion assay was performed to assess the specificity of T cell stimulation by OX-2.
  • the fusion protein (mOX-2-Ig) is purified from supernatants ofthe stable CHO cell transfectants using a Protein ⁇ sepharose column. The supernatants arc passed over the column, the column is washed with phosphate buffered saline (PBS) and the fusion protein is eluted in a O.l M glycine buffer at pH 3.5. The fusion protein elutes in the first 3ml and the eluate is rapidly neutralized with Tris base.
  • PBS phosphate buffered saline
  • This material is standardized for IgGl content using standard curves of human IgGl protein.
  • This material is then plated onto a polystyrene surface which binds proteins. The incubation proceeds for 1 hour at 37°C and the surface is then washed and blocked with ⁇ % bovine serum albumin (BSA) in PBS. The cells to be tested are then placed on the plastic and allowed to settle to the surface for 1 hour at 37 C C The surface is gently washed to remove cells which arc not bound while leaving the bound cells undisturbed. This step is empirically established and dependent on the strength of the interaction being tested. Bound cells arc then counted and reported either by cell number per unit area, or percent input cells bound. A control fusion protein is always used to control for nonspecific binding of which there is none in the assays performed to date.
  • BSA bovine serum albumin
  • an adhesion assay indicated that a Thl T cell clone, but not a Th2 T cell clone, constitutively expressed the receptor for OX-2. Unstimulated spleen cells did not bind to the murine OX-2 fusion protein. Thus, the receptor for OX-2 may represent a costimulatory receptor which is differentially used by the Thl and Th2 helper T cell subsets.
  • rOX-2 transgenic mouse strain In support of the Th l - specific nature of OX-2 costimulation, rOX-2 transgenic mouse strain was developed. The overexpression of a costimulatory protein is essentially a pharmacalogic experiment to address the question of what effect an overdose of the costimulatory signal may have in vivo.
  • a rOX-2 cDNA was cloned by PCR amplification as described above, BamHI digestion of the PCR product and ligation into an expression vector.
  • the vector contains the immmunoglobulin promoter-enhancer which drives expression of the cDNA in B cells and/or T cells.
  • the vector also contains growth hormone 3' untranslated sequences (including poly A sequence) to stabilize the mRNA; these sequences are thought to promote higher expression levels of the cDNA cloned into the vector.
  • the mouse was generated using standard transgenic techniques involving the injection of the rOX-2 expression vector DNA into fertilized eggs.
  • Transgenic mice were selected by PCR using oligos 0-228 and 0-229. PCR with these oligonucleotides produced a 0.9kb rOX-2 cDNA amplification product which is not present in wildtype mice.
  • the transgenic mice and their wildtype littermates were then examined for histology. One physical finding is that the thymuses of the transgenic mice were half the size of the the thymuses of the wildtype mice A rOX-2 transgenic mouse was immunized with a haptenated model protein antigen called TNP-KLII.
  • TNP trinitrophenol
  • KLH keyhole limpet hemocyanin
  • IgG2a:IgGl was increased 3-8 fold in the 7 days into a primary response of the transgenic mice. This result is consistent with an increase in the ratio of Thl :Th2 activity in the immune response. as Thl activity promotes IgG2a immunoglobulin isotype switching while Th2 promotes IgG l switching. This result is consistent with the result that OX-2 interacts preferentially with a receptor on Th 1 cells.
  • EXAMPLE 6 Isolation of mouse OX-2 genomic clones. To isolate genomic DNA clones corresponding to the OX-2 cDNA, an OX-2 probe was hybridized to a mouse genomic library according to standard procedures. Clones were isolated and sequenced according to standard procedures to confirm the identity of the genomic clones with OX-2 cDN ⁇ . EXAMPLE 7 - Preparation of antibodies to the OX-2 receptor and characterization of the OX-2 receptor.
  • Monoclonal antibodies to the OX-2 receptor are developed using the following exemplary procedure.
  • Hamsters are immunized with a Thl clone (e.g., ⁇ E7) that constitutively expresses the receptor.
  • Spleen cells from the immunized hamster are fused with myeloma cells to create hybridoma fusion cells using standard techniques.
  • the hybridoma cells are cultured and supernatants of the hybridoma cultures are screened for the ability to specifically inhibit the adhesion reaction of Thl cells to the mOX-2-Ig coated plastic wells. All inhibiting antibodies must be directed against either mOX-2-Ig on the substrate or OX-2 receptor(s) on the surface of the Thl clone.
  • mOX-2-Ig As mOX-2-Ig is plated in excess, it is improbable that all of the mOX-2-Ig can be blocked by hybridoma supernatant. In contrast, the use of a limited number of Thl cells and preincubation of the Thl cells with the hybridoma supernatant permits the limited number of receptor(s) to be specifically blocked, thus inhibiting the adhesion to the mOX-2-Ig coated wells.
  • anti-OX-2 receptor monoclonal antibodies permits characterization of the receptor(s), both at the biochemical level by immunoprecipitation and western blotting, and at the functional level using in vitro and in vivo blocking experiments.
  • the development of anti-OX-2 receptor monoclonal antibodies also permits cloning of the OX- 2 receptor itself.
  • the cloning of the OX-2 receptor is achieved by expression cloning from a cDNA library made from the mRNA of activated spleen cells or the Thl cells used previously in generating the anti-OX-2 receptor monoclonal antibodies.
  • the OX-2 receptor is also cloned by purification of the antigen recognized by the anti-OX-2 receptor monoclonal antibodies, followed by microsequencing of the antigen and PCR amplification of the cDNA using degenerate oligonucleotides as PCR primers.
  • the mOX-2-Ig fusion protein may interact sufficiently tightly with the receptor to directly permit an expression cloning strategy.
  • ADDRESSEE Wolf, Greenfield & Sacks, P.C.
  • GAAAATTTGC CATGTCTGAG ATCTGATGGA ATTTTAGGAA GAAAACTGAA GAGCGATCAA 1320
  • GAGAGCAAAA GACCAGACNC
  • GAGCCTGTGC GGGATTTGAC TTTTTGCTGT TGTCCCAGGT 960
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic) (ill) HYPOTHETICAL: NO (ix) FEATURE:
  • CAAAACTCAA CCATCACCTT CTGGAATATC ACCCTGGAGG ATGAAGGGTG TTACATGTGT 480 CTCTTCAATA CCTTTGGTTT TGGGAAGATC TCAGGAACGG CCTGCCTCAC CGTCTATGGT 540
  • CCCTTCACTA CAAATTCTCT GAAGACCACC TAAATATCAC TTGCTCTGCC ACTGCCCGCC 120
  • TGTCTCACCC AAATGGGACC ACGTCTGTTA CCAGCATCCT CCATATCAAA GACCCTAAGA 240 ATCAGGTGGG GAAGGAGGTG ATCTGCCAGG TGCTGCACCT GGGGACTGTG ACCGACTTTA 300
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • SEQUENCE DESCRIPTION SEQ ID NO:23: Gly Asp Pro Arg 1

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  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des procédés et des compositions servant à utiliser la protéine OX-2 pour moduler une réaction immunitaire à médiation par les lymphocytes T. L'invention concerne aussi de nouvelles formes structurelles des molécules costimulantes OX-2 des lymphocytes T. Ces formes structurelles comprennent un nouveau domaine structurel ou l'un de leurs domaines structurels est supprimé. Les formes structurelles correspondent à des formes de molécules costimulantes OX-2 de lymphocytes T existant dans la nature, épissées différemment, ou à des variantes de ces molécules, qui peuvent être produites grâce à des techniques classiques d'ADN recombinant. Les nouvelles formes structurelles des molécules costimulantes OX-2 de lymphocytes T peuvent être utilisées pour identifier des agents qui stimulent l'expression de formes différentes de molécules costimulantes et pour identifier des constituants du processus de transduction de signaux qui a pour effet la costimulation de lymphocytes T.
PCT/US1996/019189 1995-12-08 1996-11-27 Molecule costimulante ox-2 WO1997021450A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU11440/97A AU1144097A (en) 1995-12-08 1996-11-27 Ox-2 costimulatory molecule

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US875495P 1995-12-08 1995-12-08
US60/008,754 1995-12-08

Publications (1)

Publication Number Publication Date
WO1997021450A1 true WO1997021450A1 (fr) 1997-06-19

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Cited By (14)

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WO1999024565A1 (fr) * 1997-11-07 1999-05-20 Transplantation Technologies Inc. Procedes et compositions pour immunomodulation
WO2000061171A2 (fr) * 1999-04-13 2000-10-19 Schering Corporation Nouvelles utilisations de la proteine ox2 de mammifere et reactifs associes
US6955811B2 (en) 1997-11-07 2005-10-18 Trillium Therapeutics Inc. Methods of inhibiting immune response suppression by administering antibodies to OX-2
US7223729B2 (en) * 1997-11-07 2007-05-29 Trillium Therapeutics Inc. Methods of treating allergy by administering a CD200 protein
US7408041B2 (en) 2000-12-08 2008-08-05 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
US7427665B2 (en) 2000-12-08 2008-09-23 Alexion Pharmaceuticals, Inc. Chronic lymphocytic leukemia cell line
WO2009014744A1 (fr) * 2007-07-25 2009-01-29 Alexion Pharmaceutical, Inc. Procédés et compositions pour traiter une maladie auto-immune
US7915000B2 (en) 2000-12-08 2011-03-29 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
US8075884B2 (en) 2006-01-12 2011-12-13 Alexion Pharmaceuticals, Inc. Antibodies to OX-2/CD200 and uses thereof
US9085623B2 (en) 2010-02-11 2015-07-21 Alexion Pharmaceuticals, Inc. Therapeutic methods using anti-CD200 antibodies
US9180186B2 (en) 2010-01-11 2015-11-10 Alexion Pharmaceuticals, Inc. Biomarkers of immunomodulatory effects in humans treated with anti-CD200 antibodies
US9249229B2 (en) 2000-12-08 2016-02-02 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
US11761963B2 (en) 2017-09-27 2023-09-19 Alexion Pharmaceuticals, Inc. Biomarker signature for predicting tumor response to anti-CD200 therapy
US11802154B2 (en) 2017-12-20 2023-10-31 Alexion Pharmaceuticals, Inc. Humanized anti-CD200 antibodies and uses thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. SOC. SYMP., 1985, Vol. 51, BARCLAY et al., "Neuronal/Lymphoid Membrane Glycoprotein MRC OX-2 is a Member of the Immunoglobulin Superfamily With a Light-Chain-Like Structure", pages 149-157. *
EUR. J. IMMUNOL., 1990, Vol. 20, MJAALAND et al., "Modulation of Immune Responses With Monoclonal Antibodies I. Effects on Regional Lymph Node Morphology and on Anti-hapten Responses to Haptenized Monoclonal Antibodies", pages 1457-1461. *
IMMUNOGENETICS, 1987, Vol. 25, McCAUGHAN et al., "Characterization of the Human Homolog of the Rat MRC OX-2 Membrane Glycoprotein", pages 329-335. *
MOLECULAR IMMUNOLOGY, 1987, Vol. 24, No. 12, PATERSON et al., "Antigens of Activated Rat T Lymphocytes Including a Molecule of 50,000 Mr Detected Only on CD4 Positive T Blasts", pages 1281-1290. *

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US7223729B2 (en) * 1997-11-07 2007-05-29 Trillium Therapeutics Inc. Methods of treating allergy by administering a CD200 protein
WO1999024565A1 (fr) * 1997-11-07 1999-05-20 Transplantation Technologies Inc. Procedes et compositions pour immunomodulation
AU748587B2 (en) * 1997-11-07 2002-06-06 Trillium Therapeutics Inc Methods and compositions for immunomodulation
US6652858B2 (en) 1997-11-07 2003-11-25 Trillium Therapeutics Inc. Use of OX-2 to suppress an immune response
US6749854B2 (en) 1997-11-07 2004-06-15 Trillium Therapeutics Inc. Use of OX-2 to inhibit fetal loss
US7452536B2 (en) 1997-11-07 2008-11-18 Trillium Therapeutics Inc. Methods of treating cancer by administering antibodies to CD200
US6984625B2 (en) 1997-11-07 2006-01-10 Trillium Therapeutics Inc. Methods and compositions for modulating autoimmunity
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US7238352B2 (en) 1997-11-07 2007-07-03 Trillium Therapeutics Inc. Methods and compositions for modulating tumor growth
US8187598B2 (en) 1997-11-07 2012-05-29 Trillium Therapeutics Inc. Methods of treating cancer by administering antibodies to CD200
US7902151B2 (en) 1997-11-07 2011-03-08 Trillium Therapeutics Inc. Methods and compositions for modulating immunity
US7887798B2 (en) 1997-11-07 2011-02-15 Trillium Therapeutics Inc. Methods of treating cancer by administering antibodies to CD200
WO2000061171A2 (fr) * 1999-04-13 2000-10-19 Schering Corporation Nouvelles utilisations de la proteine ox2 de mammifere et reactifs associes
WO2000061171A3 (fr) * 1999-04-13 2001-01-25 Schering Corp Nouvelles utilisations de la proteine ox2 de mammifere et reactifs associes
US9249229B2 (en) 2000-12-08 2016-02-02 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
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US8999328B2 (en) 2000-12-08 2015-04-07 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic Leukemia cells and uses thereof
US7435412B2 (en) 2000-12-08 2008-10-14 Alexion Pharmaceuticals, Inc. Chronic lymphocytic leukemia cell line
US7427665B2 (en) 2000-12-08 2008-09-23 Alexion Pharmaceuticals, Inc. Chronic lymphocytic leukemia cell line
US7915000B2 (en) 2000-12-08 2011-03-29 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
US8840885B2 (en) 2000-12-08 2014-09-23 Alexion Pharmaceuticals, Inc. Methods for treating chronic lymphocytic leukemia
US8114403B2 (en) 2000-12-08 2012-02-14 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
US7408041B2 (en) 2000-12-08 2008-08-05 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
US7598353B2 (en) 2000-12-08 2009-10-06 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
US9150661B2 (en) 2000-12-08 2015-10-06 Alexion Pharmaceuticals, Inc. Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof
US8709415B2 (en) 2006-01-12 2014-04-29 Alexion Pharmaceuticals, Inc. Antibodies to OX-2/CD200 and uses thereof
US8075884B2 (en) 2006-01-12 2011-12-13 Alexion Pharmaceuticals, Inc. Antibodies to OX-2/CD200 and uses thereof
US9000133B2 (en) 2006-01-12 2015-04-07 Alexion Pharmaceuticals, Inc. Antibodies to OX-2/CD200 and uses thereof
JP2010534242A (ja) * 2007-07-25 2010-11-04 アレクシオン ファーマシューティカルズ， インコーポレイテッド 自己免疫疾患を治療するための方法および組成物
US8986684B2 (en) 2007-07-25 2015-03-24 Alexion Pharmaceuticals, Inc. Methods and compositions for treating autoimmune disease
AU2008279618B2 (en) * 2007-07-25 2014-05-22 Alexion Pharmaceuticals, Inc. Methods and compositions for treating autoimmune disease
WO2009014744A1 (fr) * 2007-07-25 2009-01-29 Alexion Pharmaceutical, Inc. Procédés et compositions pour traiter une maladie auto-immune
US9180186B2 (en) 2010-01-11 2015-11-10 Alexion Pharmaceuticals, Inc. Biomarkers of immunomodulatory effects in humans treated with anti-CD200 antibodies
US9085623B2 (en) 2010-02-11 2015-07-21 Alexion Pharmaceuticals, Inc. Therapeutic methods using anti-CD200 antibodies
US9862767B2 (en) 2010-02-11 2018-01-09 Alexion Pharmaceuticals, Inc. Therapeutic methods using anti-CD200 antibodies
US11761963B2 (en) 2017-09-27 2023-09-19 Alexion Pharmaceuticals, Inc. Biomarker signature for predicting tumor response to anti-CD200 therapy
US11802154B2 (en) 2017-12-20 2023-10-31 Alexion Pharmaceuticals, Inc. Humanized anti-CD200 antibodies and uses thereof

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