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WO1997017360A2 - Procedes de modulation des reponses des lymphocytes t par manipulation d'une chaine gamma commune de recepteurs de cytokine - Google Patents

Procedes de modulation des reponses des lymphocytes t par manipulation d'une chaine gamma commune de recepteurs de cytokine Download PDF

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Publication number
WO1997017360A2
WO1997017360A2 PCT/US1996/017927 US9617927W WO9717360A2 WO 1997017360 A2 WO1997017360 A2 WO 1997017360A2 US 9617927 W US9617927 W US 9617927W WO 9717360 A2 WO9717360 A2 WO 9717360A2
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Prior art keywords
cell
agent
chain
cytokine receptor
antigen
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PCT/US1996/017927
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English (en)
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Vassiliki A. Boussiotis
Lee M. Nadler
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Dana-Farber Cancer Institute
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Priority to AU10506/97A priority Critical patent/AU1050697A/en
Publication of WO1997017360A2 publication Critical patent/WO1997017360A2/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2026—IL-4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2046—IL-7
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells

Definitions

  • TCR T cell receptor
  • MHC major histocompatibility complex
  • TCR signaling can induce a state of anergy in the T cell.
  • a costimulatory signal can be triggered in a T cell through a T cell surface receptor, such as CD28.
  • a T cell surface receptor such as CD28.
  • sub optimal polyclonal stimulation of T cells e.g. by anti-CD3 antibodies or phorbol ester, either of which can provide a primary activation signal
  • CD28 can be potentiated by cross linking of CD28 with anti- CD28 antibodies
  • CD28 ligands include members of the B7 family of proteins, such as B7-1(CD80) and B7-2 (B70) (Freedman. A.S. et al. ( 1987) J. Immunol. 117:3260-3267; Freeman, G.J. et al. (1989) J. Immunol. 143:2714- 2722; Freeman. G.J. et al. (1991 ) J. Exp. Med.
  • CTLA4Ig fusion protein which binds to both B7-1 and B7-2 and blocks their interaction with CD28/CTLA4, has been used to inhibit rejection of allogeneic and xenogeneic grafts (see e.g., Turka, L.A. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1 1 102-1 1 105; Lenschow, D.J. et al.
  • T cell surface receptors which bind costimulatory molecules such as B7-1 and B7-2 are desirable targets for manipulating immune responses. Delivery of an antigen specific signal to a T cell in the absence of a costimulatory signal does not induce a T cell response, but rather has been found to induce a state of T cell unresponsiveness or anergy (see Schwartz, R.H. (1990) Science 248:1349; Jenkins, M.K. et al. (1988) J Immunol. 140:3324). In a number of clinical situations it is desirable to inhibit T cell responses (e.g., in transplantation or autoimmune disorders).
  • CTLA4Ig fusion protein which binds both B7-1 and B7-2, has been used to inhibit rejection of allogeneic and xenogeneic grafts (see e.g., Turka, L.A. et al. (1992) Proc. Natl. Acad Sci. USA 89, 1 1 102- 1 1 105; Lenschow, D.J. et al. (1992) Science 257, 789-792).
  • antibodies reactive with B7-1 and/or B7-2 have been used to inhibit T cell proliferation and IL-2 production in vitro and inhibit primary immune responses to antigen in vivo (Hathcock K.S. et al. (1993) Science 262. 905-907; Azuma, M. et al. (1993) Nature 366:76-79; Powers, G.D. et al. (1994) Cell. Immunol. 153, 298-31 1 ; Chen C. et al. (1994) J. Immunol. 152. 2105-21 14).
  • T cells When stimulated through the T cell receptor(TCR)/CD3 complex without requisite costimulation through the CD28/B7 interaction. T cells enter a state of antigen specific unresponsiveness or anergy.
  • This invention is based, at least in part, on the discovery that signaling though a common cytokine receptor ⁇ chain (e.g., interleukin-2 receptor, interleukin-4 receptor, interleukin-7 receptor, interleukin 15 receptor) prevents the induction of T cell anergy.
  • a common cytokine receptor ⁇ chain e.g., interleukin-2 receptor, interleukin-4 receptor, interleukin-7 receptor, interleukin 15 receptor
  • This ⁇ chain has been found to be associated with a JAK kinase having a molecular weight of about 116 kD (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) and signaling through the ⁇ chain induces phosphorylation of the JAK kinase.
  • This 1 16kD kinase has been identified as JAK3.
  • one embodiment of this invention pertains to methods for stimulating proliferation by a T cell which expresses a cytokine receptor ⁇ chain and which has received a primary activation signal under conditions which normally result in unresponsiveness in the T cell (i.e., lack of costimulation).
  • T cell unresponsiveness or anergy is prevented by contacting T cells with an agent which binds to the cytokine receptor ⁇ chain and stimulates an intracellular signal in the T cell resulting in T cell proliferation.
  • the agent is an anti- ⁇ chain antibody capable of cross linking the receptor or a soluble form of natural ligand which binds to the ⁇ chain, such as interleukin-4 or interleukin-7.
  • T cells can be contacted with an agent which acts intracellularly to stimulate phosphorylation ofthe JAK3 kinase.
  • an agent which acts intracellularly to stimulate phosphorylation ofthe JAK3 kinase to induce an immune response against a pathogen, such as a virus, bacteria or parasite in vivo the pathogen or component thereof can be administered in conjunction with an agent which binds to the cytokine receptor ⁇ chain and stimulates an intracellular signal in the T cell.
  • tumor immunity can be can be induced in a tumor bearing host in vivo or ex vivo by contacting T cells of the subject in the presence of tumor cells expressing tumor antigens with a ⁇ chain stimulatory agent (e.g., a cross linking anti- ⁇ chain antibody).
  • a ⁇ chain stimulatory agent e.g., a cross linking anti- ⁇ chain antibody
  • Another embodiment ofthe invention pertains to methods for inducing unresponsiveness to an antigen in a T cell which expresses a cytokine receptor ⁇ chain.
  • T cells are contacted in vivo or ex vivo in the presence of an antigen with an agent which inhibits delivery of a signal through the cytokine receptor ⁇ chain resulting in T cell unresponsiveness to the antigen.
  • Such agents can act extracellularly to inhibit delivery of a signal through the ⁇ chain, such as an inhibitory or blocking anti- ⁇ chain antibody or an agent which binds a natural ligand of the ⁇ chain to inhibit binding ofthe ligand to the ⁇ chain (e.g., an anti-interleukin-2 antibody, an anti-interleukin-4 antibody or an anti- interleukin-7 antibody).
  • the agent can act intracellularly to inhibit delivery of a signal through the cytokine receptor ⁇ chain, such as an agent which inhibits association ofthe ⁇ chain with the JAK3 kinase or inhibits phosphorylation ofthe ⁇ chain or the JAK3 kinase or both.
  • Methods for inducing T cell unresponsiveness are particularly useful for inhibiting transplant rejection and graft-versus-host disease and for treating auto immune diseases.
  • Figure IA-B are graphic representations ofthe proliferation of DR7-specific T cells upon challenge with LBL-DR7, demonstrating that IL-2, IL-4 and IL-7 can prevent in the induction of T cell anergy.
  • the T cells were given an anergic signal by stimulation with antigen (LBL-DR7) while blocking costimulation with CTLA4Ig.
  • the T cells were given an anergic signal by stimulation with antigen alone (t-DR7) in the absence of a costimulatory signal.
  • FIGS. 2A-B are graphic representations ofthe proliferation of DR7-specific T cells upon challenge with LBL-DR7, demonstrating that cross linking ofthe common ⁇ -chain of the IL-2, IL-4 and IL-7 receptors prevents in the induction of T cell anergy.
  • the T cells were given an anergic signal by stimulation with antigen (LBL-DR7) while blocking costimulation with CTLA4Ig.
  • the T cells were given an anergic signal by stimulation with antigen alone (t-DR7) in the absence of a costimulatory signal.
  • Figure 3A-E are photographs of immunoprecipitation filters, depicting the association and phosphorylation of ⁇ c and a 1 16 kD JAK kinase upon stimulation of T cells with IL-2. IL-4 or IL-7.
  • Panel A depicts coimmunoprecipitation of the 1 16 kD JAK kinase with ⁇ c by an anti-IL-2R ⁇ antibody and phosphorylation of both ⁇ c and the 1 16 kD JAK kinase by binding of an anti-phosphotyrosine antibody (4G10).
  • Figure 4 A is a photograph of an immunoprecipitation filter, depicting phosphorylation of the 1 16 kD JAK kinase upon stimulation of DR7-specific T cells with an antigenic signal and a costimulatory signal (.-DR7/B7-1) but not an antigenic signal alone (t-DR7).
  • Figure 4B is a photograph of an immunoprecipitation filter, depicting phosphorylation ofthe 1 16 kD JAK kinase upon stimulation of DR7-specific T cells with an antigenic signal (t-DR7) and either IL-2, IL-4 or IL-7.
  • Figure 5 are graphic representations of the proliferation of DR7-specific T cells upon challenge with LBL-DR7. following culture in the presence of NIH-3T3 cells transfected with DR7, (t-DR7), NIH-3T3 cells transfected with DR7 and B7-1 (t-DR7/B7- 1 ). t-DR7 cells and IL-13, and t-DR7 cells and IL-15. demonstrating that IL-15 can prevent in the induction of T cell anergy.
  • Figure 6 is a photograph of an immunoprecipitation filter which identifies the 1 16kD protein as JAK3 based on the binding of anti-JAK3 antibodies. Hybridization was detected using a horseradish peroxidase-conjugated antibody to rabbit IgG.
  • Figure 7 is a photograph of an immunoprecipitation filter which shows the association of IL-2R ⁇ with JAK1 under all conditions tested, but with JAK3 only in the presence of IL-2.
  • Figure 8 is a photograph of an immounprecipitation filter which demonstrates the constitutive association of JAK3 with ⁇ c .
  • Figure 9 A is a graphic representation of the proliferation of cells upon re-challenge with irradiated cells from the original donor.
  • Primary cultures contained anti-B7-l and anti-B7-2 monoclonal antibodies along with the indicated concentrations of recombinant IL-2.
  • Figure 9B is a graphic representation of the proliferation of cells upon re-challenge with irradiated cells from the original donor.
  • Primary cultures contained anti-B7-l and anti-B 7-2 monoclonal antibodies along with the indicated concentrations of recombinant IL-4.
  • Figure 9C is a graphic representation of the proliferation of cells upon secondary culture with irradiated cells from the original donor.
  • Primary cultures contained anti- ICAM-1 monoclonal antibodies along with the indicated concentrations of recombinant IL- 2.
  • Figure 10 shows the peak levels of IL-2 and IL-4 produced in primary cultures of cells in the presence ofthe reagents shown in column 1.
  • Figure II depicts the level or IL-2 or IL-4 mRNA in cells after primary culture as detected by RT PCR.
  • a common cytokine receptor gamma chain or " ⁇ c " as used herein refers to a polypeptide subunit that is shared by certain cytokine receptors, including the interleukin-2 receptor (IL-2), the interleukin-4 receptor (IL-4) the interleukin-7 receptor (IL-7) and the interleukin- 15 receptor (IL-15).
  • the gamma chain is present in the intermediate affinity ( ⁇ subunits) and high affinity ( ⁇ subunit) IL-2 receptors.
  • ⁇ c is a polypeptide encoded by a nucleotide sequence disclosed in Takeshita, T. et al.
  • ⁇ c is a polypeptide of about 64 kD.
  • Cytokines that can stimulate through ⁇ c include IL-2, IL-4, IL-7, and IL-15. Other cytokines which bind to a receptor that utilizes ⁇ c can also be used to stimulate through ⁇ c . Cytokines described herein are commercially available. For example. IL-2, IL-4 and IL-7, and IL-15 can be obtained from Genzyme Corp.
  • a stimulatory form of an antibody, or fragment thereof, which binds to ⁇ c can be used to stimulate through ⁇ c .
  • a "stimulatory form" of an anti- ⁇ c antibody refers to a form ofthe antibody which induces an intracellular signal through ⁇ c upon binding to ⁇ c .
  • the stimulatory form of anti- ⁇ c antibody is a soluble antibody that is cross linked, e.g., by a secondary antibody.
  • the stimulatory form of anti- ⁇ c is an immobilized form of an antibody, e.g., an antibody bound to a solid support, such as a culture plate or bead.
  • the stimulatory antibody can be polyclonal antisera or a monoclonal antibody.
  • Antibodies that bind ⁇ c can be prepared by standard techniques known in the art. Animals can be immunized with a ⁇ c "immunogen".
  • the term "immunogen" is used herein to describe a composition containing a ⁇ c peptide or protein as an active ingredient used for the preparation of antibodies against ⁇ c . Both soluble and membrane bound CTLA4 protein or peptide fragments are suitable for use as an immunogen.
  • the ⁇ c immunogen can be a cell which expresses a cytokine receptor utilizing ⁇ c (e.g., a cell line expressing a ⁇ c -containing form of IL-2R, IL-4R, IL-7R. or IL-15R).
  • ⁇ c e.g., a cell line expressing a ⁇ c -containing form of IL-2R, IL-4R, IL-7R. or IL-15R.
  • a preferred cell for use as an immunogen is a T cell, which constitutively expresses ⁇ c .
  • the immunogen can be a purified ⁇ c protein or a ⁇ c peptide fragment.
  • a ⁇ c protein can be purified from cells by standard techniques or produced recombinantly by expression in a host cell of a nucleic acid encoding ⁇ c (e.g., a nucleic acid having a nucleotide sequence disclosed in Takeshita, T. et al. (1992) Science 257:379-382).
  • a ⁇ c peptide fragment can be chemically synthesized based upon the predicted amino acid sequence of a ⁇ c protein (e.g., as disclosed in Takeshita, cited supra).
  • An isolated form of ⁇ c protein or peptide can itself be directly used as an immunogen. or alternatively, can be linked to a suitable carrier protein by conventional techniques, including by chemical coupling.
  • the isolated ⁇ c protein can also be covalently or noncovalently modified with non-proteinaceous materials such as lipids or carbohydrates to enhance immunogenecity or solubility.
  • an isolated ⁇ c protein can be coupled with or incorporated into a viral particle, a replicating virus, or other microorganism in order to enhance immunogenicity.
  • nucleic acid e.g., DNA
  • immunogen is also intended to include nucleic acid encoding a protein or peptide against which antibodies are to be raised.
  • an expression vector construct containing nucleic acid encoding the protein of interest (e.g, ⁇ c or a peptide thereof) is delivered intracellularly into the skin of an animal (e.g., mouse) by coating particles (e.g., gold particles) with the construct and injecting the particles into the skin.
  • particles e.g., gold particles
  • This results in antigen production in the skin and development of a specific antibody response see e.g.. Tang, D.C. et al. (1992) Nature 356: 152-154; Eisenbraun. M.D. et al. (1993) DNA Cell Biol. 12:791-797; Wang. B. et al. (1993) DNA Cell Biol. 12:799-805).
  • Polycolonal antibodies to ⁇ c can generally be raised in animals by standard methods. Animals can be boosted until the anti- ⁇ c titer plateaus. Also, aggregating agents such as alum can be used to enhance the immune response. The antibody molecules can then be collected from the mammal (e.g., from the blood) and isolated by well known techniques, such as protein A chromatography. to obtain the IgG fraction. To enhance the specificity ofthe antibody, the antibodies may be purified by immunoaffinity chromatography using solid phase-affixed immunogen. The antibody is contacted with the solid phase-affixed immunogen for a period of time sufficient for the immunogen to immunoreact with the antibody molecules to form a solid phase-affixed immunocomplex. The bound antibodies are separated from the complex by standard techniques.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular protein with which it immunoreacts.
  • the monoclonal antibody used in the subject method is further characterized as immunoreacting with a ⁇ c protein derived from humans.
  • Monoclonal antibodies useful in the compositions and methods ofthe invention are directed to an epitope ofa ⁇ c
  • a monoclonal antibody to an epitope of ⁇ c can be prepared by using a technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497). and the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today
  • the antibody preparation applied in the subject method is a monoclonal antibody produced by a hybridoma cell line.
  • Hybridoma fusion techniques were first introduced by Kohler and Milstein (Kohler et al. Nature (1975) 256:495-97: Brown et al.
  • the monoclonal antibody compositions of the present invention can be produced by the following method, which comprises the steps of:
  • a rodent mammal such as a rabbit, rat or mouse is used.
  • the mammal is then maintained for a time period sufficient for the mammal to produce cells secreting antibody molecules that immunoreact with the ⁇ c immunogen.
  • Such immunoreaction is detected by screening the antibody molecules so produced for immunoreactivity with a preparation of the immunogen protein.
  • an assay e.g., a membrane-associated form of ⁇ c .
  • Antibody-producing cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals. Spleen cells are preferred, and can be mechanically separated into individual cells in a physiologically tolerable medium using methods well known in the art. Mouse lymphocytes give a higher percentage of stable fusions with the mouse myelomas described below. Rat, rabbit and frog somatic cells can also be used.
  • the spleen cell chromosomes encoding desired immunoglobulins are immortalized by fusing the spleen cells with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • myeloma cell lines may be used as a fusion partner according to standard techniques; for example, the P3-NSl/l-Ag4-l . P3-x63- Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md.
  • ATCC American Type Culture Collection
  • the resulting cells which include the desired hybridomas, are then grown in a selective medium, such as HAT medium, in which unfused parental myeloma or lymphocyte cells eventually die. Only the hybridoma cells survive and can be grown under limiting dilution conditions to obtain isolated clones.
  • the supernatants ofthe hybridomas are screened for the presence of antibody ofthe desired specificity, e.g., by immunoassay techniques using the antigen that has been used for immunization. Positive clones can then be subcloned under limiting dilution conditions and the monoclonal antibody produced can be isolated.
  • a selective medium such as HAT medium
  • Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques known in the art.
  • the individual cell line may be propagated in vitro, for example in laboratory culture vessels, and the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation.
  • the yield of monoclonal antibody can be enhanced by injecting a sample of the hybridoma into a histocompatible animal ofthe type used to provide the somatic and myeloma cells for the original fusion. Tumors secreting the specific monoclonal antibody produced by the fused cell hybrid develop in the injected animal.
  • the body fluids of the animal such as ascites fluid or serum, provide monoclonal antibodies in high concentrations.
  • human hybridomas or EBV-hybridomas are used.
  • Immunodeficient or nude mice may be used or the hybridoma may be passaged first into irradiated nude mice as a solid subcutaneous tumor, cultured in vitro and then injected intraperitoneally into pristane primed, irradiated nude mice which develop ascites tumors secreting large amounts of specific human monoclonal antibodies.
  • DMEM Dulbecco's minimal essential medium
  • antibodies produced in non-human subjects are used therapeutically in humans, they are recognized to varying degrees as foreign and an immune response may be generated in the patient.
  • One approach for minimizing or eliminating this problem, which is preferable to general immunosuppression, is to produce chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region.
  • Such antibodies are the equivalents of the monoclonal and polyclonal antibodies described above, but may be less immunogenic when administered to humans. and therefore more likely to be tolerated by the patient.
  • Chimeric mouse-human monoclonal antibodies reactive with ⁇ c can be produced by recombinant DNA techniques known in the art.
  • a gene encoding the constant region of a murine (or other species) anti-human ⁇ c antibody molecule is substituted with a gene encoding a human constant region, (see Robinson et al., Internationai Patent Publication PCT US86/02269; Akira, et al.. European Patent
  • a chimeric antibody can be further "humanized” by replacing portions of the variable region not involved in antigen binding with equivalent portions from human variable regions.
  • General reviews of "humanized” chimeric antibodies are provided by Morrison. S. L. (1985) Science 229: 1202-1207 and by Oi et al. (1986) BioTechniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of an immunoglobulin variable region from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from an anti- ⁇ c antibody producing hybridoma.
  • cDNA encoding the chimeric antibody, or fragment thereof can then be cloned into an appropriate expression vector.
  • Suitable "humanized” antibodies can be alternatively produced by CDR or CEA substitution (see U.S. Patent 5.225.539 to Winter; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141 :4053-4060).
  • a human mAb directed against a human protein can be generated.
  • Transgenic mice carrying human antibody repertoires have been created which can be immunized with human a ⁇ c protein or a human cell expressing ⁇ c .
  • Splenocytes from these immunized transgenic mice can then be used to create hybridomas that secrete human mAbs specifically reactive with human ⁇ c (see, e.g.. Wood et al. PCT publication WO 91/00906.
  • Monoclonal antibody compositions of the invention can also be produced by other methods well known to those skilled in the art of recombinant DNA technology.
  • An alternative method referred to as the "combinatorial antibody display” method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal anti- ⁇ c antibodies (for descriptions of combinatorial antibody display see e.g.. Sastry et al. (1989) PNAS 86:5728; Huse et al. (1989) Science 246:1275; and Orlandi et al. (1989) PNAS 86:3833).
  • the antibody repertoire of the resulting B- cell pool is cloned.
  • Methods are generally known for directly obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR.
  • mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification ofthe heavy and light chain variable regions from a number of murine antibodies (Larrick et al. (1991) Biotechniques U . : 152-156).
  • a similar strategy can also been used to amplify human heavy and light chain variable regions from human antibodies (Larrick et al. (1991) Methods: Companion to Methods in Enzymoiogy 2:106-1 10).
  • RNA is isolated from activated B cells from, for example, peripheral blood cells, bone marrow, or spleen preparations, using standard protocols (e.g.. U.S. Patent No. 4,683.202; Orlandi, et al. .PN ⁇ .S (1989) 86:3833-3837: Sastry et al., PNAS (1989) 86:5728-5732; and Huse et al. (1989) Science 246:1275-1281.) First-strand cDNA is synthesized using primers specific for the constant region of the heavy chain(s) and each of the K and ⁇ light chains, as well as primers for the signal sequence.
  • variable region PCR primers the variable regions of both heavy and light chains are amplified, each alone or in combinantion, and ligated into appropriate vectors for further manipulation in generating the display packages.
  • Oligonucleotide primers useful in amplification protocols may be unique or degenerate or inco ⁇ orate inosine at degenerate positions. Restriction endonuclease recognition sequences may also be incorporated into the primers to allow for the cloning ofthe amplified fragment into a vector in a predetermined reading frame for expression.
  • the V-gene library cloned from the immunization-derived antibody repertoire can be expressed by a population of display packages, preferably derived from filamentous phage, to form an antibody display library.
  • the display package comprises a system that allows the sampling of very large and diverse antibody display libraries, rapid sorting after each affinity separation round, and easy isolation of the antibody gene from purified display packages.
  • kits for generating phage display libraries e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene Sur ZAP ⁇ phage display kit. catalog no. 240612).
  • the V region domains of heavy and light chains can be expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFV gene subsequently cloned into the desired expression vector or phage genome.
  • a flexible linker As generally described in McCafferty et al.. Nature (1990) 348:552-554, complete Vj-j and VL domains of an antibody, joined by a flexible (Gly4-Ser)3 linker can be used to produce a single chain antibody which can render the display package separable based on antigen affinity. Isolated scFV antibodies immunoreactive with ⁇ c can subsequently be formulated into a pharmaceutical preparation for use in the subject method.
  • the antibody library is screened with a ⁇ c protein, or peptide fragment thereof, to identify and isolate packages that express an antibody having specificity for ⁇ c .
  • Nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques.
  • Peptide fragments or modified forms of natural ligands for the common gamma chain of cytokine receptors that stimulate through the gamma chain are also encompassed by the invention.
  • a peptide fragment or modified form of IL-2, IL-4, IL-7. or IL-15 that retains the ability to stimulate through ⁇ c can be used.
  • peptide mimetics and other small molecules e.g., drugs
  • a modified cytokine, peptide fragment, peptide mimetic or small molecule that stimulates through ⁇ c can be identified by screening substances using screening assays as described herein.
  • nucleic acid encoding a stimulatory ligand for ⁇ c .
  • nucleic acid e.g., DNA
  • an anti- ⁇ c antibody or fragment thereof
  • cytokine that binds a receptor containing ⁇ c
  • IL-2, IL-4, IL-7. IL-15 can be introduced into cells in vitro, or administered to a subject in vivo, as gene therapy to stimulate T cells responses and prevent the induction of anergy.
  • Recombinant expression vectors for expressing proteins or peptides in cells e.g., recombinant viral vectors
  • nucleic acid delivery mechanisms suitable for gene therapy in vitro or in vivo are well known in the art.
  • An expression vector encoding a soluble, secreted form of anti- ⁇ c antibody, or a cytokine can be used to produce within cells a ⁇ c - ligand which is then secreted from the cells and binds to a ⁇ c -containing surface cytokine receptor on activated T cells (e.g., in culture or in vivo) to prevent induction of anergy.
  • ⁇ c stimulatory agent for is one which acts intracellularly to trigger a signal mediated by ⁇ c .
  • this agent does not bind to an extracellular portion of ⁇ c or a receptor containing ⁇ c . but rather mimics or induces an intracellular signal (e.g., second messenger) associated with ligation of ⁇ c .
  • the agent that acts intracellularly to trigger a signal mediated by ⁇ c stimulates phosphorylation of ⁇ c .
  • the agent stimulates phosphorylation of a JAK3 kinase.
  • a inhibitory, or blocking, form of an antibody, or fragment thereof, which binds to ⁇ c but does not stimulate through ⁇ c can be used to inhibit signalling through ⁇ c .
  • inhibitory form of an anti- ⁇ c antibody refers to a form ofthe antibody which binds to ⁇ c but does not induces an intracellular signal through ⁇ c upon binding.
  • the inhibitory form of anti- ⁇ c antibody preferably inhibits or prevents interaction of ⁇ c with its natural ligands, e.g., inhibits or prevents signalling through ⁇ c by IL-2, IL-4, IL-7. or IL-15.
  • the inhibitory form of anti- ⁇ c antibody is a soluble antibody that does not crosslink ⁇ c .
  • the inhibitory form of anti- ⁇ c is an antibody fragment, such as a Fab or Fv fragment, that binds to ⁇ c but does not induce a signal through ⁇ c .
  • Inhibitory anti- ⁇ c antibodies, and fragments thereof, can be prepared using standard methodologies, as described above.
  • a signal through ⁇ c can also be inhibited using an antibody, or fragment thereof, which neutralizes a cytokine that binds to a receptor containing gc (e.g., a neutralizing antibody against IL-2, IL-4, IL-7, or IL-15).
  • a neutralizing antibody refers to an antibody which binds to the cytokine and inhibits or prevents its interaction with its receptor on a T cell.
  • Antibodies against cytokines such as IL-2, IL-4, IL-7, or IL-15 are commercially available or can be prepared using standard methodologies, as described above.
  • a modified cytokine, peptide fragment, peptide mimetic or small molecule that inhibits intracellular signalling ⁇ c can be identified by screening substances using screening assays as described herein.
  • rational drug design can be used to design a molecule that blocks binding of natural cytokine ligands (e.g., IL-2, IL-4, IL-7, or IL-15) with ⁇ c .
  • inhibitory agent contemplated by the invention is a nucleic acid that is antisense to a nucleic acid encoding ⁇ c (e.g., antisense to a coding or regulatory region of a ⁇ c gene).
  • an antisense nucleic acid e.g., DNA
  • the antisense nucleic acid can be an oligonucleotide or a recombinant expression vector containing a ⁇ c cDNA or gene, or portion thereof, in an orientation that leads to expression of ⁇ c antisense nucleic acid.
  • Antisense nucleic acid can be introduced into T cells in vitro or in vivo by a delivery mechanism suitable for gene therapy in vitro or in vivo that are known in the art.
  • ⁇ c inhibitory agent for is one which acts intracellularly to inhibit a signal mediated by ⁇ c .
  • this agent does not block binding of a natural cytokine ligand to the extracellular portion of a ⁇ c -containing receptor, but rather inhibits an intracellular signal (e.g., second messenger) associated with ligation of ⁇ c .
  • the agent that acts intracellularly to inhibit a signal mediated by ⁇ c inhibits phosphorylation of ⁇ c .
  • the agent inhibits phosphorylation ofa JAK3 kinase.
  • the agent inhibits an interaction, or association. between ⁇ c and the JAK3 kinase.
  • An agent that stimulates an intracellular signal through ⁇ c can be used to stimulate a T cell response to an antigen by preventing induction of antigen specific anergy in the T cell and stimulating proliferation ofthe T cell. Stimulation through ⁇ c may be therapeutically useful for enhancing, prolonging and/or maintaining immune responses in which antigen presentation to a T cell occurs under conditions that naturally may induce T cell anergy.
  • T cells specific for tumor antigens may be susceptible to becoming anergized by stimulation of the T cell with tumor antigens on the surface of tumor cells in the absence of a costimulatory signal (e.g., tumors cells that do not express costimulatory molecules such as B7-1 or B7-2 may anergize T cells, thereby downmodulating anti -tumor responses).
  • anti-tumor responses may be enhanced by stimulating tumor antigen-specific T cells through ⁇ c in the presence of a tumor antigen-specific signal.
  • a ⁇ c stimulatory agent as described above can be administered to a tumor-bearing subject.
  • T cells from a tumor-bearing subject can be contacted in vitro with tumor cells and a ⁇ c stimulatory agent and then readministered to the subject.
  • T cell responses to pathogens may be enhanced and prolonged by administering to a subject harboring the pathogen a ⁇ c stimulatory agent described herein.
  • the efficacy of vaccination may also be increased by stimulating T cells through ⁇ c .
  • the vaccine can be administered together with a ⁇ c stimulatory agent to enhance to the immune response against the vaccinating material.
  • the ⁇ c inhibitory agents ofthe invention can be used to inhibit a T cell response to an antigen and, moreover, to induce antigen specific T cell anergy such that the T cell will not respond to the antigen upon rechallenge.
  • a T cell is contacted with a ⁇ c inhibitory agent in the presence of an antigen specific signal.
  • the responsiveness of a T cell to an antigen can be inhibited according to the methods ofthe invention either in vitro or in vivo.
  • T cells are contacted with a ⁇ c inhibitory agent together with a cell presenting antigen to the T cell (e.g., an allogeneic cell to inhibit alloantigen specific responses).
  • a ⁇ c inhibitory agent is administered to a subject.
  • T cells receive the required antigen stimulation through the TCR/CD3 complex by an endogeneous stimulus in vivo (e.g., an autoantigen or foreign antigen presented by antigen presenting cells in vivo).
  • an antigenic stimulus can be coadministered with the ⁇ c inhibitory agent (e.g.. to induce allergen-specific anergy. the allergen can be coadministered with the ⁇ c inhibitory agent).
  • T cell responses can be inhibited non-specifically by delivering a signal through the TCR/CD3 complex with a non-specific reagent, such as an anti-CD3 antibody together with a ⁇ c inhibitory agent.
  • T cell responses and induce anergy in a subject it may also be beneficial to inhibit or prevent T cells from receiving a costimulatory signal in vivo, such as the costimulatory signal mediated by the interaction of CD28 with either B7-1 or B7-2.
  • a costimulatory signal such as the costimulatory signal mediated by the interaction of CD28 with either B7-1 or B7-2.
  • the T cell in addition to contacting a T cell with a ⁇ c inhibitory agent, the T cell can also be contacted with another agent which inhibits generation of a costimulatory signal in T cells, such as a blocking molecule which binds to CD28. B7-1 or B7-2.
  • suitable blocking molecules include an anti-CD28 Fab fragment, anti-B7-l or anti-B7-2 blocking antibodies (i.e..
  • CTLA4Ig fusion protein e.g., a CTLA4Ig fusion protein
  • blocking molecules e.g. an anti-B7-l antibody and an anti-B7-2 antibody may be used.
  • the methods ofthe invention for inhibiting a T cell response to an antigen and inducing antigen specific anergy are applicable to a variety of clinical situations where it is desirable to downmodulate T cell responses, as described in greater detail in the subsections below.
  • A. Organ Transplantation/GVHD Induction of T cell anergy is useful in situations of cellular, tissue, skin and organ transplantation and in bone marrow transplantation (e.g., to inhibit graft- versus-host disease (GVHD)).
  • GVHD graft- versus-host disease
  • anergization of alloreactive T cells may result in reduced tissue destruction in tissue transplantation and long-term graft acceptance without the need for generalized immunosuppression.
  • rejection ofthe graft is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the graft.
  • a ⁇ c inhibitory agent can be administered to a transplant recipient together with the transplanted cells to induce alloantigen specific T cell unresponsiveness.
  • An agent that inhibits a costimulatory signal through CD28/CTLA4Ig. such as CTLA4Ig can be coadministered with the ⁇ c inhibitory agent.
  • Donor bone marrow can be incubated prior to transplantation in vitro with cells from the recipient (e.g., hematopoietic cells) and a ⁇ c inhibitory agent. Additional agents that inhibit the generation of a costimulatory signal in the T cells (e.g., anti-B7-l and/or anti-B7-2 antibodies. CTLA4Ig, etc.) can be included in the incubation.
  • the treated bone marrow is then administered to the recipient, who may further be treated in vivo with a ⁇ c inhibitory agent alone or in combination with an agent that inhibits a costimulatory signal.
  • ⁇ c inhibitory agent in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al.. Science, 257: 789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci. USA, 89: 1 1 102-1 1 105 (1992).
  • murine models of GVHD see Paul ed.. Fundamental Immunology, Raven Press, New York. 1989, pp. 846-847) can be used to determine the effect of inducing T cell unresponsiveness using a ⁇ c inhibitory agent on the development of that disease.
  • Induction of antigen specific T cell unresponsiveness by the methods of the invention may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue (i.e.. reactive against autoantigens) and which promote the production of cytokines and autoantibodies involved in the pathology ofthe diseases. Preventing the activation of autoreactive T cells thus may reduce or eliminate disease symptoms.
  • Administration of a ⁇ c inhibitory agent can be used to inhibit T cell responses to autoantigens and, moreover, to induce autoantigen specific anergy.
  • a ⁇ c inhibitory agent is administered to a subject in need of treatment.
  • the autoantigen can be coadministered to the subject with the inhibitory agent.
  • This method can be used to treat a variety of autoimmune diseases and disorders having an autoimmune component, including diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis.
  • Sj ⁇ gren's Syndrome including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions. Crohn's disease, aphthous ulcer, ulceris, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions.
  • leprosy reversal reactions erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease. Graves ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.
  • ⁇ c crosslinking agents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis. systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology. Raven Press. New York. 1989, pp. 840-856).
  • the IgE antibody response in atopic allergy is highly T cell dependent and, thus, inhibition of allergen specific T cell responses and induction of allergan specific anergy may be useful therapeutically in the treatment of allergy and allergic reactions.
  • a ⁇ c inhibitory agent can be administered to an allergic subject exposed to an allergen to induce apoptosis in allergen specific T cells, thereby downmodulating allergic responses in the subject.
  • Administration of a ⁇ c inhibitory agent to an allergic subject may be accompanied by enviromental exposure to the allergen or by coadministration ofthe allergen to the subject.
  • Allergic reactions may be systemic or local in nature, depending on the route of entry ofthe allergen and the pattern of deposition of IgE on mast cells or basophils.
  • a ⁇ c inhibitory agent and an allergen are coadminstered subcutaneously to an allergic subject.
  • a ⁇ c inhibitory agent and an allergen are coadminstered subcutaneously to an allergic subject.
  • D. Induction of Antigen-Specific Anergy The methods ofthe invention for inducing T cell unresponsiveness can essentially be applied to any antigen (e.g., protein) to anergize T cells to that antigen in a subject.
  • an antigen of interest to which T cells are to be anergized can be administered to a subject together with a ⁇ c inhibitory agent.
  • the antigen may be administered in a soluble form or attached to a carrier or support (e.g., a bead).
  • a carrier or support e.g., a bead
  • This basic approach has widespread application as an adjunct to therapies which utilize a potentially immunogenic molecule for therapeutic purposes.
  • an increasing number of therapeutic approaches utilize a proteinaceous molecule, such as an antibody, fusion protein or the like, for treatment of a clinical disorder.
  • a limitation to the use of such molecules therapeutically is that they can elicit an immune response directed against the therapeutic molecule in the subject being treated (e.g., the efficacy of murine monoclonal antibodies in human subjects is hindered by the induction of an immune response against the antobodies in the human subject).
  • the method ofthe invention for inducing antigen specific T cell unresponsiveness can be applied to these therapeutic situations to enable long term usage of the therapeutic molecule in the subject without elicitation of an immune response.
  • a therapeutic antibody e.g., a murine mAb which typically activates T cells specific for the antibody in a human subject
  • the therapeutic antibody is administered to a subject (e.g.. human) together with a ⁇ c inhibitory agent.
  • the method may additionally involve administration of an agent that inhibits a CD28/CTLA4-mediated costimulatory signal, such as CTLA4Ig.
  • the agents of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo to stimulate or inhibit T cell responses.
  • biologically compatible form suitable for administration in vivo is meant a form of the agent to be administered in which any toxic effects are outweighed by the therapeutic effects ofthe agent.
  • subject is intended to include living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, and transgenic species thereof.
  • an agent ofthe invention as described herein can be in any pharmacological form including a therapeutically active amount of ⁇ c stimulatory or inhibitory agent alone or in combination with another therapeutic molecule (e.g., an agent which stimulates or inhibits a signal through a receptor (e.g., CD28/CTLA4) for a costimulatory molecule (e.g., B7-1 and/or B7-2), such as stimulatory or blocking antibodies to CD28, B7-1 or B7-2 blocking antibodies, CTLA4Ig etc.) and a pharmaceutically acceptable carrier.
  • Administration of a therapeutically active amount of the therapeutic compositions ofthe present invention is defined as an amount effective, at dosages and for periods of time necessay to achieve the desired result.
  • a therapeutically active amount of an ⁇ c stimulatory or inhibitory agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies ofthe therapeutic situation.
  • the active compound may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.). oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • an agent may be administered to an individual in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
  • Liposomes include water-in-oil-in- water emulsions as well as conventional liposomes (Strejan et aL, (1984) J.
  • the active compound may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol. liquid polyethylene glycols. and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol.
  • phenol asorbic acid, thimerosal. and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol. sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the compound When the active compound is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • a method for identifying an agent that inhibits signalling through ⁇ c involves: contacting a T cell that experesses a cytokine receptor containg ⁇ c (e.g., IL-2R. IL-4R. IL-7R, IL-15R) with an first agent that stimulates a primary activation signal (e.g.. an anti-CD3 antibody or an antigen presented by an antigen presenting cell) and a second agent that stimulates a signal through ⁇ c (e.g., a cytokine such as IL-2, IL-4.
  • a primary activation signal e.g. an anti-CD3 antibody or an antigen presented by an antigen presenting cell
  • a second agent that stimulates a signal through ⁇ c e.g., a cytokine such as IL-2, IL-4.
  • T cell proliferation can be measured by standard assays, such as tritiated thymidine inco ⁇ oration.
  • an intracellular response can be measured, such as the association between ⁇ c and the JAK3 kinase, phosphorylation of ⁇ c or phosphorylation ofthe JAK3 kinase.
  • a substance that inhibits signalling through ⁇ c can be identified based upon the ability ofthe substance to inhibit an association between ⁇ c and the JAK3 kinase, phosphorylation of ⁇ c or phosphorylation ofthe JAK3 kinase.
  • the association between ⁇ c and the JAK3 kinase can be measured by coimmunoprecipitation assays, as described in Example 3.
  • the phosphorylation of ⁇ c and the JAK3 kinase can be assayed using anti- phosphotyrosine antibodies, as described in Example 3.
  • screening assays can be used to identify agents that stimulate an intracellular signal through gc.
  • a screening assays involves contacting a T cell that expresses contacting a T cell that experesses a cytokine receptor containg ⁇ c (e.g., IL-2R, IL-4R. IL-7R. IL-15R) with an agent that stimulates a primary activation signal (e.g., an anti-CD3 antibody or an antigen presented by an antigen presenting cell) without inducing a costimulatory signal through CD28/CTLA4, in the presence and absence of a substance to be tested, followed by measurement of T cell proliferation.
  • a primary activation signal e.g., an anti-CD3 antibody or an antigen presented by an antigen presenting cell
  • Stimulation of the T cell only the agent that stimulates a primary activation signal will result in induction of anergy in the T cell and a lack of T cell proliferation.
  • a substance which stimulates a signal through ⁇ c can be identified based upon its ability to prevent induction of anergy in the T cell. That is, in the presence of the stimulatory substance, the T cell will proliferate and will respond to antigen upon rechallenge. Alternatively, an intracellular response, such as phosphorylation of ⁇ c or the JAK3 kinase can be measured.
  • An agent that stimulates through ⁇ c can be identified based upon the ability ofthe substance to induce phosphorylation of ⁇ c or the JAK3 kinase.
  • a two-hybrid assay system such as that described in U.S. Patent No. 5,283,173 and PCT application WO 94/10300 is used to identify agents that inhibit an interaction between ⁇ c and a JAK3 kinase.
  • Kits for performing the two-hybrid assay system are commercially available from Clontech. Palo Alto, CA.
  • glutathione-S-transferase fusion proteins of ⁇ c and/or the JAK3 kinase can be prepared and used to identify agents that inhibit an interaction between ⁇ c and a JAK3 kinase.
  • a GST fusion of one protein is made, incubated with a labeled preparation of the other protein, in the presence and absence of a substance to be tested, and the ⁇ c- JAK3 kinase complex precipitated with glutathione-agarose.
  • a substance which inhibits an interaction between ⁇ c and the JAK3 kinase can be identified based upon the ability ofthe substance to reduce the amount of labeled protein that is precipitated with the GST fusion protein.
  • EXAMPLE 1 IL-2, IL-4 and IL-7 Prevent the Induction of Anergy in T Cells
  • HLA-DR7 alloantigen-specific T-cell clones TC-3 and TC-4 (CD4 + . CD8 ⁇ CD28 + . B7 ' ) were generated using standard methodology.
  • the DR7-specific T cell clones were cultured with a DR7 + lymphoblastoid cell line (LBL-DR7) or NIH-3T3 cells transfected to express DR-7 alone (t-DR7) or DR-7 and B7-1 (t-DR7/B7-l).
  • LBL- DR7 is an EBV transformed lymphoblastoid B-cell line, which is homozygous for HLA- DR?
  • NIH-3T3 cell transfectants are described in Gimmi. CD. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6586.
  • different cytokines or antibodies were added to the culture. Before each experiment, T-cell clones were rested for 10 to 14 days in IL-2 without alloantigen re-stimulation. Prior to use. cells were cultured overnight without stimulus or IL-2.
  • alloantigen-specific (i.e.. DR7-specific) T cell clones were incubated in primary culture with 1) LBL-DR7, 2) LBL-DR7 plus CTLA4-Ig, either in media or in the presence of various cytokines, 3) t-DR7/B7-l or 4) t-DR7 either in media or in the presence of various cytokines.
  • cytokine used in the experiments are as follows: IL-2 (50 U/ml); IL-4 (5 ng/ml) (Genzyme, Cambridge, MA); IL-6 (30 ng/ml) (Genzyme); IL-7 (10 ng/ml) (Genzyme); IL- 12 (10 U/ml) (Genetics Institute, Cambridge, MA); TNF ⁇ (500 U/ml) (Genzyme); IFN ⁇ (500 U/ml) (Biogen. Cambridge, MA).
  • IL-2 50 U/ml
  • IL-4 5 ng/ml
  • IL-6 (30 ng/ml)
  • IL-7 (10 ng/ml) (Genzyme)
  • IL- 12 (10 U/ml) (Genetics Institute, Cambridge, MA)
  • TNF ⁇ 500 U/ml
  • IFN ⁇ 500 U/ml)
  • T cell clones were cultured in a primary culture for 24 hours. Following primary culture, T cells were separated from LBL-DR7 by Ficoll and from NIH 3T3 transfectants by Percoll, and recultured in media without IL-2 for 12 hours. Each population was subsequently rechallenged with LBL-DR7 stimulators in secondary culture. Samples were cultured and proliferation was measured by [ J H]-thymidine (1 ⁇ Ci) inco ⁇ oration. The results for T cells stimulated with LBL-DR7 are shown in Figure 1. panel A. The results for T cells stimulated with NIH-3T3 transfectants are shown in Figure 1 , panel B. Results represent response in rechallenge and are expressed as the means of triplicate cultures.
  • HLA-DR7 homozygous lymphoblastoid cell line
  • t-DR7/B7-l transfectants expressing HLA-DR7 and the B7-1 costimulatory molecule
  • HLA-DR7-specific alloreactive T cell clones significantly proliferated to secondary rechallenge with LBL-DR7 cells.
  • primary culture ofthe T cell clones was with either LBL-DR7 cells in the presence of CTLA4-Ig. to block B7 family mediated costimulation. or with transfectants expressing HLA-DR7 alone (t-DR7).
  • EXAMPLE 2 Stimulation of the Common ⁇ -Chain of the IL-2, IL-4 and IL-7
  • IL-4, and IL-7 receptors ( ⁇ c ).
  • Primary culture of T cell clones was with either LBL-DR7 plus CTLA4-Ig or t-DR7, together with each ofthe above mAbs cross linked with rabbit anti-mouse Ig (RaM). Primary culture and rechallenge were performed as described in Example 1.
  • Antibodies against IL-2R ⁇ (IgG2a) (D. A. Fox, et al. (1984) J. Immunol. 133:1250) (Coulter), IL-2R ⁇ (IgG) (M.
  • T cell clones were cultured with either IL-2, IL-4, or IL-7 and cell lysates immunoprecipitated with anti- ⁇ c mAb. Alloantigen- specific human helper T cell clones were incubated in D-MEM serum-free media without IL-2 for 12 hours and subsequently stimulated for 15 min with media, IL-2, IL-4, IL-7, TNF ⁇ , or IL-12.
  • Cells were lysed with lysis buffer containing 10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate. 50 mM NaFl, 1 mM sodium orthovanadate.
  • blots were incubated with 4G10 anti-phosphotyrosine monoclonal antibody (1 :2000) for 60 min at room temperature. The blots were washed three times with wash buffer (50 mM Tris-HCl, pH 7.6, 200 mM NaCl, 0.1 % Tween-20), followed by 60 min incubation with horseradise peroxidase conjugated-sheep anti-mouse IgG (1 :5000) (Amersham, Arlington Heights, IL).
  • wash buffer 50 mM Tris-HCl, pH 7.6, 200 mM NaCl, 0.1 % Tween-20
  • the blots were washed three times with wash buffer followed by incubation with the enhanced chemiluminescence substrate (Amersham). exposed to X-ray film and developed. Following ECL immunodeplefion, the immunoblot was stripped by incubation in 62.5 mM Tris-HCl (pH 6.8), 3% w/v SDS and 100 mM ⁇ -mercaptoethanol at 50 °C for lhr.
  • membranes were blocked and reprobed with either anti-JAK (R80)(l : 1000) antibody (panel B), or peptide specific mAbs for JAK1 (Upstate Biotechnology, 1 :2000) shown in panel C, JAK2 (panel D) and Tyk2 (panel E). washed and detected as described above.
  • FIG. 3 panel A, shows that the 64kD band of ⁇ c is co-precipitated with a band of 1 16kD.
  • Western blotting with anti-phosphotyrosine mAb demonstrated phosphorylation of both the 64kD and 1 16kD bands on a tyrosine residue(s).
  • T cell clones were cultured with either media, t-DR7 or t-DR7/B7-l for 24 hrs. Following culture, T cells were separated from transfectants by Percoll gradient, lysed. and then immunoprecipitated with R80. Cell lysates were prepared, immunoprecipitated with the common JAK (R80) antibody and immunoblot analysis with 4G10 anti-phosphotyrosine monoclonal antibody (1 :2000) was performed as described above. t-DR7/B7-l culture (non-anergizing conditions) resulted in significant phosphorylation of 116kD protein ( Figure 4, panel A).
  • EXAMPLE 4 IL-15 prevents the induction of anergy in T cells
  • IL-15 which is capable of signalling through a common ⁇ chain, prevents the induction of anergy in T cells.
  • HLA-DR7 alloantigen specific T-cells were incubated for 24 hours in a primary culture with 1 ) NIH- 3T3 cells transfected with DR7 and B7-1 (t-DR7/B7-l); 2) NIH-3T3 cells transfected with DR7 (t-DR7); 3) t-DR7 and IL-13 at 50 u/ml (Genzyme); and 4) t-DR7 and IL-15 at 50 u/ml (Genzyme). T cells were then separated from the t-DR7 cells and recultured in media without IL-2 for 12 hours. Each population of cells was subsequently rechallenged with LBL-DR7 stimulators in secondary culture. Proliferation was measured by [ J H]-thymidine (1 ⁇ Ci) inco ⁇ oration.
  • IL-7, IL-15 is also capable of preventing the induction of anergy in T cells.
  • IL-15 is another cytokine which is capable of preventing anergy in T cells through phosphorylation of a JAK3 kinase associated with the common ⁇ chain of the receptor.
  • EXAMPLE 5 Identification Of The 116 kD Protein As JAK3
  • JAK3 The association of JAK3 with IL2R ⁇ and ⁇ c was investigated. Immunoprecipitations were performed with a monoclonal antibody to IL-2R ⁇ . and blots were probed with antibody to phosphotyrosine (4G10). Blots were stripped and reprobed with antibody to common JAK(R80), JAK 1. or JAK 3. Results presented in Figure 7 show that IL-2 R ⁇ was associated with JAK1. which was tyrosine-phosphorylated in the presence of IL-2 and IL-4; only in the presence of IL-2, however, did IL-2R ⁇ become associated with JAK3.
  • T cell clones were incubated overnight in serum-free media without IL-2 and subsequently stimulated for 15 minutes with media, IL-2, IL-4, or IL-7 and then immunoprecipitated with either anti- ⁇ c or anti-JAK3.
  • the results of such an experiment are shown in Figure 8. Immune complexes were resolved by SDS-PAGE, transferred on nitrocellulose membranes, and blotted with antiserum to JAK3. Immunoblots were then stripped and reprobed with JAK1 antiserum or monoclonal antibody to phosphotyrosine. This experiment demonstrated that JAK3, but not JAK1 was coprecipitated with ⁇ c under all ofthe above culture conditions. However, immunoblots with a monoclonal antibody to phosphotyrosine revealed that JAK3 was phosphorylated to a much greater extent in the presence ofthe cytokines.
  • ⁇ c signaling prevented the induction of anergy in a human alloantigen system using T cell clones (see Example 1 ), it was determined whether ⁇ c signaling would prevent the induction of anergy using a fully mismatched mixed lymphocyte reaction (MLR) as a model system for GVHD.
  • MLR mixed lymphocyte reaction
  • Cells were treated ex vivo with antibodies which anergize cells in this model system and the role of cytokines in preventing anergy was tested. Briefly, responder peripheral blood lymphocytes (PBL) or bone marrow mononuclear cells (BMMC) were isolated using standard methods and stimulated with fully mismatched, irradiated (2500cGy) donor peripheral blood mononuclear cells (PBMC).
  • PBL responder peripheral blood lymphocytes
  • BMMC bone marrow mononuclear cells
  • IL-2 and IL-4 levels were measured under the blocking conditions.
  • Donor and irradiated responder peripheral blood mononuclear cells were cultured in triplicate at a cell ratio of 1 : 1 in the presence of agents blocking antigen recognition (anti-Class II or cyclosporin A), adhesion (anti-ICAM-1 monoclonal antibody) or costimulation (CTLA4Ig).
  • Complete blockade of B7 mediated costimulation was accomplished by using CTLA4-Ig or anti-B7-l and B7-2 monoclonal antibodies or with blockade of B7-1 or B7-2 alone.
  • Figure 10 shows the peak IL-2 and IL-4 levels for these experiments.
  • Blockade of allorecognition either by anti- ICAM-1. anti-class-II or cyclosporin A. resulted in undetectable or near undetectable levels of IL-2 and IL-4. demonstrating the requirement for a TCR mediated signal for the production of these cytokines.
  • Complete blockade of B7 mediated costimulation also resulted in undetectable IL-2 or IL-4 accumulation.
  • significant IL-2 and IL-4 levels were still detected when only B7-1 or B7-2 signaling was inhibited.
  • Figure 10 shows the levels of IL-2 and IL-4 detected during the primary MLR when only B7-1 or B7-2 signaling was inhibited.

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Abstract

Lorsqu'ils sont stimulés par le complexe récepteur de lymphocytes T (TCR)/CD3 sans la costimulation nécessaire par interaction de CD28/B7, les lymphocytes T entrent dans un état de non réponse ou anergie spécifique à certains antigènes. La présente invention se fonde, au moins en partie, sur la constatation selon laquelle une signalisation par l'intermédiaire d'une chaîne η de récepteurs de cytokine commune (par exemple d'un récepteur d'interleukine-2, -4, -7 ou -15) prévient l'induction de l'anergie des lymphocytes T. Il a été constaté que cette chaîne η est associée à une JAK3 kinase présentant un poids moléculaire d'environ 116 kD (tel que déterminé par électrophorèse sur gel polyacrylamide au dodécyl sulfate de sodium) et que la signalisation par l'intermédiaire de la chaîne η induit la phosphorylation de la JAK3 kinase. L'invention porte, en conséquence, sur des procédés de stimulation ou d'inhibition de la prolifération d'un lymphocyte T qui exprime une chaîne η de récepteurs de cytokine.
PCT/US1996/017927 1995-11-09 1996-11-12 Procedes de modulation des reponses des lymphocytes t par manipulation d'une chaine gamma commune de recepteurs de cytokine WO1997017360A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1093457A1 (fr) * 1998-03-19 2001-04-25 Human Genome Sciences Analogue de chaine gamma commune de recepteur de cytokine
WO2001052892A2 (fr) * 2000-01-24 2001-07-26 Genzyme Corporation Inhibiteurs du trajet de jak/stat et leurs utilisations
US6861227B2 (en) 1998-03-19 2005-03-01 Human Genome Sciences, Inc. Antibodies to cytokine receptor common gamma chain like
US6982320B2 (en) 1998-03-19 2006-01-03 Human Genome Sciences, Inc. Cytokine receptor common gamma chain like
EP1875900A2 (fr) * 2000-01-24 2008-01-09 Genzyme Corporation Inhibiteurs du trajet de JAK/STAT et leur utilisation pour le traitement de l'ostéoarthrite
US11629195B2 (en) 2019-02-01 2023-04-18 Regeneron Pharmaceuticals, Inc. Anti-IL2 receptor gamma antigen-binding proteins

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6982320B2 (en) 1998-03-19 2006-01-03 Human Genome Sciences, Inc. Cytokine receptor common gamma chain like
EP1093457A4 (fr) * 1998-03-19 2003-01-02 Human Genome Sciences Analogue de chaine gamma commune de recepteur de cytokine
US6861227B2 (en) 1998-03-19 2005-03-01 Human Genome Sciences, Inc. Antibodies to cytokine receptor common gamma chain like
EP1093457A1 (fr) * 1998-03-19 2001-04-25 Human Genome Sciences Analogue de chaine gamma commune de recepteur de cytokine
EP1982990A1 (fr) * 1998-03-19 2008-10-22 Human Genome Sciences, Inc. Récepteur de cytokine et chaîne de gamma commune similaire
US7504105B2 (en) 1998-03-19 2009-03-17 Human Genome Sciences, Inc. Treatment using antibodies to cytokine receptor common gamma chain like
WO2001052892A2 (fr) * 2000-01-24 2001-07-26 Genzyme Corporation Inhibiteurs du trajet de jak/stat et leurs utilisations
WO2001052892A3 (fr) * 2000-01-24 2002-01-24 Genzyme Corp Inhibiteurs du trajet de jak/stat et leurs utilisations
JP2004500376A (ja) * 2000-01-24 2004-01-08 ジェンザイム・コーポレーション Ｊａｋ／ｓｔａｔ経路阻害剤およびその使用
EP1875900A2 (fr) * 2000-01-24 2008-01-09 Genzyme Corporation Inhibiteurs du trajet de JAK/STAT et leur utilisation pour le traitement de l'ostéoarthrite
EP1875900A3 (fr) * 2000-01-24 2010-07-21 Genzyme Corporation Inhibiteurs du trajet de JAK/STAT et leur utilisation pour le traitement de l'ostéoarthrite
JP2012041354A (ja) * 2000-01-24 2012-03-01 Genzyme Corp Ｊａｋ／ｓｔａｔ経路阻害剤およびその使用
US11629195B2 (en) 2019-02-01 2023-04-18 Regeneron Pharmaceuticals, Inc. Anti-IL2 receptor gamma antigen-binding proteins

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