TITLE
GITR LIGAND AND GITR LIGAND-RELATED MOLECULES AND ANTIBODIES AND USES THEREOF
[0001] This apphcation claims the benefit of US Provisional Application Ser No 60/472,844, filed May 23,2003, and U S Provisional Apphcation Ser No 60/547,975, filed February 26, 2004, both of which are incorporated herein by reference in then: entireties
[0002] This invention was made with Government support under NTH Intramural Research Proj ect #Z01 -AI-000224 The Government has certain rights in the invention
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present mvention is directed to novel methods for diagnosing, prognosing, monitoring the progress of, and treating disorders arising from disregulation of the immune system (e g, autoimmune disorders, inflammatory diseases, and transplant rejection, and cancer and infectious diseases) related to glucocorticoid-induced TNF receptor (GITR) and the ligand associated with GITR (GITRL) and modulators related thereto The present mvention is further directed to novel therapeutics and therapeutic targets, and to methods of screening and
assessing test compounds for the intervention (treatment) and prevention of disorders ansmg from disregulation of the immune system, as related to GITR and GITRL
Related Background Art
[0004] Generally, T lymphocytes are responsible for cell-mediated immunity and play a regulatory role by enhancing or suppressmg the responses of other white blood cells The notion that T lymphocytes play a role in suppression of the immune response is well known (see, e g, Gershon et al (1970) Immunology 18 723-35) However, the target antigens for these suppressor cells and the mechanisms controlling their function are still subjects of study
[0005] One population of regulatory T cells that is generated in the thymus is distinguishable from effector T cells by the expression of unique membrane antigens These regulatory T cells make up a subpopulation of CD4+ T cells (l e, T cells that express the CD4 antigen) that coexpress the CD25 antigen CD25 is also known as the mterleukin-2 receptor (IL-2R) cc-chain Cotransfer of, or reconstitution with, CD25+ T cells is associated with prevention of both inflammatory lesions and autoimmunity in various animal models (see Shevach (2000) Ann Rev Immunol 18 423-49, and references therein) CD4+CD25+T cells have also been associated with inhibition of T cell activation in vitro, and adoptive suppression of CD4+CD25" T cells in coculture (Shevach, supra)
[0006] More than two decades ago it was demonstrated that some self-reactive T cells escape mechanisms of central tolerance and exist in the periphery under the control of thymic-derrved regulatory T cells In 1995, Sakaguchi and colleagues demonstrated that a small population of CD4+ T cells that naturally express the a-cham of EL-2R (l e, CD25) are mvolved in the control of organ-specific autoreactive T cells (Sakaguchi et al (1995) / Immunol 155 1151-64) Specifically, they demonstrated that transfer of CD4+CD25" T cells to immunodeficient hosts led to a spectrum of autoimmune diseases, which could be prevented by cotransfer of CD4+CD25+ T cells (Sakaguchi et al, supra) Subsequent studies have implicated CD4+CD25+ regulatory T cells in the suppression of immune responses to viral, bacterial and protozoal infections (Aseffaetal (2002) J Immunol 169 3232-41, Belkaidetal (2002) Nature
420 502-07, Hisaeda et al (2004) Nat Med 10 29-30, Kursar et al (2002) J Exp Med 196 1585-92, Lundgren et al (2003)Infect Immun 71 1755-62, Maloy et al (2003) J Exp Med 197 111-19) Together, these studies provided evidence that removal of CD4+CD25+ T cells enhanced the immune response Many attempts have been made to define the activation of, and suppression by, these CD4+CD25+ T cells These cells represent a unique lineage of thymic-denved cells that potently suppress both in vitro and in vivo effector T cell function
[0007] Several in vitro studies revealed that CD4+CD25+ cells suppress proliferation of CD4+ T cells in response to both mitogens and antigens by turning off transcription of IL-2 (e g, Thornton and Shevach (1998) J Exp Med 188 287-96, Takahashi et al (1998) Int Immunol 10 1969-80) Cotransfer of CD4+CD25+ T cells in vivo with autoreactive CD4+ T cells is sufficient to suppress both the induction and effector phase of organ-specific autoimmunity (Sun-Payer et al (1999) Eur J Immunol 29 669-77, Sun-Payer et al (1998) J Immunol 160 1212-18) Other properties of the CD4+CD25+ T cells include hyporesponsiveness to T cell receptor (TCR) stimulation in the absence of exogenous IL-2, immunosuppression via cell-cell interaction, and a requirement for TCR signaling to induce then* suppressive phenotype (once they have been activated, however, their suppressive function is mdependent of antigenic stimulus) It has also been demonstrated that the mere acquisition of CD25 expression, as can be achieved by stimulation of CD4+CD25" T cells, does not induce the suppressive phenotype These CD4+CD25+ T cells are known to exist in humans (Shevach (2001) J Exp Med 193 F1-F6)
[0008] One study demonstrated that altered thymic selection is required for generation of regulatory CD4+CD25+ T cells (Jordan et al (2001) Nat Immunol 2 301-06) In addition, studies with knockout mice demonstrated that molecules involved in IL-2 synthesis and responsiveness are required for generation of these cells, mice genetically deficient for IL2 or IL2Rp, or B7 1 (CD80) and B7 2 (CD86), or CD28 all have severe reduction in CD4+CD25+ cells, with resulting lymphadenopathy and hyperprohferation in the penphery of some of these mice (Papiermk et al (1998) Int Immunol 10 371-78, Salomon et al (2000) Immunity 12 431-40, Kumanogoh et al (2001) J Immunol 166 353-60)
[0009] Until recently, the art had failed to determine the mechanisms involved in CD4+CD25+-mediated suppression of the immune system, e g , the antigen specificity, the molecules involved in acquisition of suppression, and the cell surface molecules or short acting cytokines involved in the effector phase of suppression, the molecular targets of CD25+ T cells in modulating autoimmunity remained largely unknown as well It has now been demonstrated, by examining differential expression of genes through the use of gene chip analyses on CD4+CD25+ and CD4+CD25~ T cells, that several CD25+ differential genes exist (McHugh et al (2002) Immunity 16 311-23, see also U S Patent Application 10/194,754, incorporated herein by reference in its entirety) These genes, determined to be preferentially expressed on the CD4+CD25+ T cells, can serve as targets for therapeutic intervention and screening methods for autoimmune disorders, inflammatory diseases and transplant rejection, as well as for cancer and infectious diseases
[0010] Significantly, one of the genes determined to be differentially expressed in CD25+ cells is glucocorticoid-induced TNF receptor (GITR) (McHugh et al, supra) GITR, a cell-surface, transmembrane protein receptor, is a member of the tumor necrosis factor receptor (TNFR) superfamily GITR has been demonstrated to be constitutively present on nonactivated T cells (Gavin et al (2002) Nat Immunol 3 33-41, McHugh et al, supra, Shimizu et al (2002) Nat Immunol 3 135-42) GITR binds to another transmembrane protein referred to as GITR Ligand (GrTRL) Agomstic antibodies to GITR have been shown to abrogate the suppressor activity of CD4+CD25+ T cells, demonstrating a functional role for GITR in regulating the activity of these cells (McHugh et al, supra) Another study confirmed that stimulation of GITR with a specific monoclonal antibody abrogated CD4+CD25+ T cell-mediated suppression, thereby inducing autoimmunity (Shimizu et al, supra) These studies have led to the proposal that GITR is a more faithful marker of CD4+CD25+ T cells (Uraushihara et al (2003) J Immunol 171 708-16), however, GITR expression alone does not exclusively distinguish this subset, as upregulation of GITR also occurs following activation of CD4+CD25" T cells (McHugh et al , supra, Shimizu et al, supra)
[0011] Because GITR has been shown to be important in the regulation of suppressor activity of CD4+CD25+ T cells on CD4+CD25" T cells, it is desirable to
ldentify and characterize novel molecules that interact with GITR Such novel molecules that interact with GITR are disclosed herein Additionally, modulators of these molecules are provided
SUMMARY OF THE INVENTION [0012] The present invention provides the nucleotide and amino acid sequences of a novel mouse homolog of human GITRL The present invention also provides antibodies to mouse GITRL The present invention also provides methods both to reverse immune suppression by inducing agonistic GITR-GITRL bindmg, and to restore or enhance immune suppression by antagonizing GITR-GITRL bindmg, e g, through the use of neutralizing antibodies that inhibit GITRL activity (e g, that block the interaction between GITR and GITRL) Such reversal, or restoration/enhancement, of immune suppression is beneficial in the treatment of varied disorders resulting from disegulated immune responses, such as autoimmune disorders, inflammatory diseases and transplant rejection, and cancer and infectious diseases The methods of the present invention are directed to manipulation of GITRL and GITR, including, but not limited to, mouse GITRL and GITR and their homologs, specifically included among these homologs is human GITRL and human GITR
[0013] The present invention provides novel isolated and purified polynucleotides and polypeptides related to a novel ligand for GITR (GITRL) The invention also provides antibodies to GITRL, as well as methods for treating, diagnosing, prognosing, and monitoring the progress of autoimmune disorders, inflammatory diseases, and transplant rejection, and cancers and infectious diseases In one embodiment of the invention, the disclosed methods and molecules can be used to manipulate the outcome of an immune response during the treatment of a disease or disorder, including autoimmune disorders, inflammatory diseases, and transplant rejection, as well as cancers and infectious diseases In another embodiment, disclosed polynucleotides and polypeptides of the invention that block or inhibit the interaction between GITR and GITRL, for example by downregulating the expression or activity of GITRL or by binding to GITRL, but do not induce GITR signaling, can be used to restore or enhance suppression of the immune system In another embodiment, the interaction between GITR and
GITRL can be blocked or inhibited by a small molecule It will be appreciated by one of skill in the art that these types of regulation (1 e, these embodiments) will be most beneficial in the treatment of autoimmune disorders and some inflammatory diseases, and similar or related disorders, as well as in the treatment of transplant rejection In another embodiment, disclosed polynucleotides and polypeptides of the mvention that induce GITR signaling, for example by upregulating the expression or activity of GITRL or by agonistic binding to GITR, can be used to reverse, block, or abrogate suppression of the immune system In another embodiment, the interaction between GITR and GITRL can be enhanced or mimicked by a small molecule It will be appreciated by one of skill in the art that these types of regulation will be most beneficial in the treatment of cancers and like diseases, as well as infectious diseases One of skill in the art would also be aware of the likely benefits of combining these novel therapies with established and other therapies
[0014] In one embodiment, the invention provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO 1 or SEQ ID NO 3 In another embodiment, the nucleic acid molecule is operably linked to at least one expression control sequence In another embodiment, a host cell transformed or transfected with the nucleic acid molecule is provided
[0015] In another embodiment, the mvention provides an isolated allele of SEQ ID NO 1 or SEQ ID NO 3 In another embodiment, the mvention provides an isolated gene comprising the nucleotide sequence of SEQ ID NO 3
[0016] In another embodiment, the invention provides an isolated nucleic acid molecule that specifically hybridizes under highly stringent conditions to the nucleotide sequence set forth in SEQ ID NO 1 or SEQ ID NO 3, or the complement thereto
[0017] In another embodiment, the mvention provides an isolated nucleic acid molecule that encodes a protein comprising the amino acid sequence of SEQ ID NO 2, or a fragment thereof that encodes an active fragment of the protein In another embodiment, the nucleic acid molecule, or fragment thereof, is operably linked to at least one expression control sequence In another embodiment, a host cell transformed or transfected with the isolated nucleic acid molecule, or fragment
thereof, operably linked to at least one expression control sequence is provided In another embodiment, the invention provides a nonhuman transgenic animal in which the somatic and germ cells contain the isolated nucleic acid molecule, or fragment thereof In another embodiment, the invention provides a nonhuman transgenic animal ra which the somatic and germ cells contain DNA comprising the nucleotide sequence of SEQ ED NO 1 or SEQ ID NO 3
[0018] In another embodiment, the invention provides an isolated protein comprising the ammo acid sequence encoded for by an isolated nucleic acid that specifically hybridizes under highly stnngent conditions to the nucleotide sequence set forth in SEQ ID NO 1 or SEQ ID NO 3, or the complement thereto In another embodiment, the invention provides an isolated protein comprising the ammo acid sequence of SEQ ED NO 2, or an active fragment thereof
[0019] In another embodiment, the invention provides an isolated nucleic acid molecule composing a nucleotide sequence complementary to the nucleotide sequence of SEQ ED NO 1 or SEQ ED NO 3, or a fragment thereof, wherein expression of the nucleic acid molecule in a cell results in decreased production of GITRL In another embodiment, the nucleic acid molecule, or fragment thereof, is operably linked to at least one expression control sequence In another embodiment, a host cell transformed or transfected with the isolated nucleic acid molecule, or fragment thereof, operably linked to at least one expression control sequence is provided In another embodiment, the invention provides a nonhuman transgenic animal in which the somatic and germ cells contain the isolated nucleic acid molecule, or fragment thereof
[0020] In another embodiment, the invention provides an antisense ohgonucleotide complementary to a mRNA corresponding to a nucleic acid molecule comprising the nucleotide sequence of SEQ ED NO 1 or SEQ ED NO 3, or a fragment thereof, wherein the oligonucleotide inhibits expression of GITRL In another embodiment, the invention provides a siRNA molecule corresponding to a nucleic acid molecule comprising the nucleotide sequence of SEQ ED NO 1 or SEQ ED NO 3, or a fragment thereof, wherein the siRNA molecule inhibits expression of GITRL
[0021] In another embodiment, the invention provides an isolated antibody capable of specifically binding to an isolated protein compnsmg the ammo acid sequence encoded for by an isolated nucleic acid that specifically hybridizes under highly stringent conditions to the nucleotide sequence set forth in SEQ ID NO 1 or SEQ ID NO 3, or the complement thereto In another embodiment, the antibody neutralizes GITRL activity In another embodiment, the antibody is 5F1, having ATCC number PTA-5336, or 10F12, having ATCC number PTA-5337 In another embodiment, the antibody comprises the antigen binding fragments of 5F1 or 10F12 In another embodiment, the mvention provides an isolated antibody capable of specifically bmdmg to an isolated protein compnsmg the ammo acid sequence of SEQ ID NO 2, or an active fragment thereof In another embodiment, the antibody neutralizes GITRL activity In another embodiment, the antibody is 5F1, having ATCC number PTA-5336, or 10F12, having ATCC number PTA-5337 In another embodiment, the antibody compnses the antigen binding fragments of 5F1 or 10F 12
[0022] In another embodiment, the mvention provides a method of screening for test compounds capable of inhibiting or blocking the interaction of GITRL with GITR compnsmg the steps of contacting a sample containing GITRL and GITR with a test compound and determining whether the interaction of GITRL with GITR in the sample is decreased relative to the interaction of GITRL with GITR in a sample not contacted with the compound, whereby a decrease in the interaction of GITRL with GITR in the sample contacted with the compound identifies the compound as one that inhibits or blocks the mteraction of GITRL with GITR In another embodiment, the identified compound is used in a method of treating a subject at nsk for, or diagnosed with, an autoimmune disorder, an inflammatory disease, or transplant rejection, the method compnsmg the steps of isolating T cells from the subject, treating the isolated T cells with the identified compound, and transferring the treated T cells back into the subject In another embodiment, the identified compound is used in a method of treating a subject at nsk for, or diagnosed with, an autoimmune disorder, an inflammatory disease, or transplant rejection, the method compnsmg admimstenng to the subject the identified compound In another embodiment, the mvention provides a method for assessmg the efficacy of the identified compound in a subject compnsmg the steps of
detecting a first number of effector T cells from the subject prior to admimstration of the compound to the subject, detecting a second number of effector T cells from the subject after admimstration of the compound to the subject, and comparmg the first number and the second number, whereby a significant decrease in the number of effector T cells in the second number as compared to the first number mdicates that the compound is efficacious in treating an autoimmune disorder, an inflammatory disease, or transplant rejection in the subject In another embodiment, the effector T cells are CD4+ T cells or CD8+ T cells
[0023] In another embodiment, the invention provides a method of screening for test compounds capable of enhancing or mimicking the mteraction of GITRL with GITR comprising the steps of contacting a sample containing GITRL and GITR with a test compound and determining whether the interaction of GITRL with GITR in the sample is increased relative to the interaction of GITRL with GITR in a sample not contacted with the compound, whereby an increase in the mteraction of GITRL with GITR in the sample contacted with the compound identifies the compound as one that enhances or mimics the mteraction of GITRL with GITR In another embodiment, the identified compound is used in a method of treating a subject at risk for, or diagnosed with, cancer or an infectious disease, the method comprising the steps of isolating T cells from the subject, treating the isolated T cells with the identified compound, and transferring the treated T cells back into the subject In another embodiment, the identified compound is used in a method of treating a subject at risk for, or diagnosed with, cancer or an infectious disease, the method comprising administering to the subject the identified compound In another embodiment, the mvention provides a method for assessing the efficacy of the identified compound in a subject compnsmg the steps of detecting a first number of effector T cells from the subject prior to admimstration of the compound to the subject, detecting a second number of effector T cells from the subject after admimstration of the compound to the subject, and comparmg the first number and the second number, whereby a significant increase in the number of effector T cells in the second number as compared to the first number indicates that the compound is efficacious in treating cancer or an infectious disease in the subject In another embodiment, the effector T cells are CD4+ T cells or CD8+ T cells
[0024] In another embodiment, the invention provides a method for diagnosing an autoimmune disorder, an inflammatory disease, or transplant rejection in a subject comprising the steps of detecting a test amount of a GITRL gene product in a sample from the subject, and comparing the test amount with a normal amount of the GITRL gene product in a control sample, whereby a test amount sigmficantly above the normal amount provides a positive indication in the diagnosis of an autoimmune disorder, an inflammatory disease, or transplant rejection In another embodunent, the invention provides a method for diagnosing cancer or an infectious disease in a subject comprising the steps of detecting a test amount of a GITRL gene product in a sample from the subject, and comparing the test amount with a normal amount of the GITRL gene product in a control sample, whereby a test amount sigmficantly below the normal amount provides a positive indication in the diagnosis of cancer or an infectious disease
[0025] In another embodunent, the mvention provides a method of treating a subject at risk for, or diagnosed with, an autoimmune disorder, inflammatory disease, or transplant rejection comprising admmistenng to the subject a GITR antagonist In another embodunent, the method compnses admmistenng the GITR antagonist such that the susceptibility of the effector T cells in the subject to suppression by CD4+CD25+ regulatory T cells is maintained (e g, in an amount effective to maintain such susceptibility) In another embodunent, the GITR antagonist is selected from the group consisting of a neutralizing anti-GITRL antibody, a neutralizing anti-GITR antibody, a fusion protein containing GITR, a fusion protein containing an active fragment of GITR, an antagonistic small molecule, an antisense GITRL nucleic acid molecule, and a siRNA GITRL nucleic acid molecule In another embodunent, the autoimmune disorder or inflammatory disease is selected from the group consisting of rheumatoid arthntis, encephalomyelitis, osteoarthntis, multiple sclerosis, autoimmune gastntis, systemic lupus erythematosus, psonasis and other inflammatory dermatoses, type I diabetes, asthma, allergy, and inflammatory bowel diseases, including Crohn's disease and ulcerative colitis
[0026] In another embodiment, the mvention provides a method of treating a subject at nsk for, or diagnosed with, cancer or an mfectious disease compnsmg admmistenng to the subject a GITR agomst In another embodunent, the method
compnses admimstenng the GITR agonist such that GITR agomst provides a costimulatory signal to effector T cells in the subject and renders them less susceptible to suppression by CD4+CD25+ regulatory T cells in the subject (e g, in an amount effective to provide such a signal) In another embodiment, the GITR agomst is selected from the group consisting of GITRL, an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containing an active fragment of GITRL, and an agomstic GITR antibody
[0027J In another embodiment, the invention provides a method of inducmg proliferation of a cell population containmg effector T cells comprising admimstenng a GITR agomst to the cell population In another embodiment, the GITR agomst is selected from the group consisting of GITRL, an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containing an active fragment of GITRL, and an agomstic GITR antibody In another embodiment, the effector T cells are CD4+ T cells or CD8+ T cells
[0028] In another embodiment, the invention provides a method of inhibiting proliferation of a cell population containing effector T cells comprising admimstenng a GITR antagonist to the cell population In another embodiment, the GITR antagonist is selected from the group consisting of a neutralizing anti-GITRL antibody, a neutralizing anti-GITR antibody, a fusion protein containmg GITR, a fusion protein containing an active fragment of GITR, an antagonistic small molecule, an antisense GITRL nucleic acid molecule, and a siRNA GITRL nucleic acid molecule In another embodiment, the effector T cells are CD4+ T cells or CD8+ T cells In another embodiment, the GITR antagonist is 5F1 or 10F12
[0029] In another embodiment, the invention provides a method of inhibiting or blocking suppression of a cell population compnsmg effector T cells in the presence of CD4+CD25+ regulatory T cells compnsmg administering a GITR agomst to the cell population In another embodiment, the method compnses administenng the GITR agonist such that the GITR agomst provides a costimulatory signal to the effector T cells and renders them less susceptible to suppression by the CD4+CD25+ regulatory T cells (e g, in an amount effective to provide such a signal) In another embodiment, the GITR agomst is selected from the group consisting of GITRL, an active fragment of GITRL, a fusion protein
contammg GITRL, a fusion protein contaimng an active fragment of GITRL, and an agonistic GITR antibody In another embodiment, the effector T cells are CD4+ T cells or CD8+T cells
[0030] In another embodiment, the invention provides a method of suppressmg a cell population compnsmg effector T cells in the presence of CD4+CD25+ regulatory T cells comprising administering a GITR antagonist to the cell population In another embodiment, the method comprises administering the GITR antagonist such that the susceptibility of the effector T cells to suppression by the CD4+CD25+ regulatory T cells is maintained (e g, in an amount effective to maintain such susceptibility) In another embodiment, the GITR antagonist is selected from the group consisting of a neutralizing anti-GITRL antibody, a neutralizing anti-GITR antibody, a fusion protein containing GITR, a fusion protein containing an active fragment of GITR, an antagonistic small molecule, an antisense GITRL nucleic acid molecule, and a siRNA GITRL nucleic acid molecule In another embodiment, the effector T cells are CD4+ T cells or CD8+ T cells In another embodiment, the GITR antagonist is 5F1 or 10F12
[0031] In another embodiment, the mvention provides a method of inhibiting the expression of GITRL in a cell population composing treating the cell population with an isolated nucleic acid molecule compnsmg a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO 1 or SEQ ID NO 3, or a fragment thereof, wherein expression of the nucleic acid molecule in a cell results in decreased production of GITRL In another embodiment, the mvention provides a method of inhibiting the expression of GITRL in a cell population compnsmg treating the cell population with an antisense oligonucleotide complementary to a mRNA corresponding to a nucleic acid molecule compnsmg the nucleotide sequence of SEQ ID NO 1 or SEQ ED NO 3, or a fragment thereof, wherein the oligonucleotide inhibits expression of GITRL
[0032] In another embodiment, the mvention provides a method of inhibiting the expression of GITRL in a cell population compnsmg treating the cell population with a siRNA molecule targeted to a mRNA corresponding to an isolated nucleic acid molecule compnsmg the nucleotide sequence of SEQ ID NO 1 or SEQ ID NO 3 In another embodiment, the mvention provides a method of inhibiting the expression of GITRL in a cell population compnsmg treating the cell population
with a siRNA molecule targeted to a mRNA corresponding to an isolated nucleic acid molecule comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO 1 or SEQ ID NO 3
[0033] In another embodiment, the invention provides a method of inhibiting the expression of GITRL in a cell population comprising treating the cell population with an antisense oligonucleotide to a nucleic acid molecule encodmg GITRL In another embodiment, the invention provides a method of inhibiting the expression of GITRL in a cell population comprising treating the cell population with a siRNA molecule targeted to a mRNA encoding GITRL
[0034] In another embodiment, the invention provides a method of inducmg the expression of GITRL in a cell population comprising treating the cell population by transforming or transfecting the cell population with an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO 1 or SEQ ID NO 3, or with an isolated nucleic acid molecule that encodes a protein comprising the ammo acid sequence of SEQ ID NO 2, or a fragment thereof that encodes an active fragment of the protein, wherein the nucleic acid molecule is operably linked to at least one expression control sequence
[0035] In another embodiment, the invention provides a population of effector T cells that have been contacted in vitro or ex vivo with a GITR agomst In another embodiment, the GITR agomst is selected from the group consisting of GITRL, or an active fragment of GITRL, a fusion protein contammg GITRL, a fusion protein containing an active fragment of GITRL, an agonistic small molecule, and an agomstic anti-GITR antibody In another embodiment, the effector T cells are CD4+ T cells or CD8+ T cells
[0036] In another embodiment, the mvention provides a method of treating cancer or an infectious disease in a subject, the method comprising the steps of obtaining a population of effector T cells, treating the population with a GITR agomst, and administering the treated population to the subject afflicted with cancer or an infectious disease In another embodiment, the GITR agomst is selected from the group consisting of GITRL, an active fragment of GITRL, a fusion protein contammg GITRL, a fusion protein contammg an active fragment of GITRL, an agomstic small molecule, and an agomstic anti-GITR antibody In another
embodiment, the subject is afflicted with cancer and the treated population is used as a tumor vaccine
[0037] In another embodiment, the invention provides a pharmaceutical composition compnsmg a GITR agomst and a pharmaceutically acceptable earner In another embodiment, the GITR agomst is selected from the group consisting of GITRL, an active fragment of GITRL, a fusion protein containmg GITRL, a fusion protein containing an active fragment of GITRL, an agomstic small molecule, and an agomstic anti-GITR antibody
[0038] In another embodiment, the invention provides a pharmaceutical composition compnsmg a GITR antagonist and a pharmaceutically acceptable earner In another embodiment, the GITR antagonist is selected from the group consisting of a neutralizing anti-GITRL antibody, a neutralizing anti-GITR antibody, a fusion protein containing GITR, a fusion protein containmg an active fragment of GITR, an antagonistic small molecule, an antisense GITRL nucleic acid molecule, and a siRNA GITRL nucleic acid molecule In another embodiment, the antibody compnses the antigen binding fragments of 5F1 or 10F12
[0039] In another embodiment, the mvention provides a vaccme adjuvant compnsmg a GITR agonist and an antigen selected from the group consisting of a viral antigen, a bactenal antigen, a fungal antigen, a parasitic antigen, a cancer antigen, a tumor-associated antigen, and fragments thereof In another embodiment, the GITR agomst is selected from the group consisting of GITRL, or an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containmg an active fragment of GITRL, an agomstic small molecule, and an agomstic anti-GITR antibody
[0040] In another embodiment, the invention provides a vaccine adjuvant compnsmg a GITR antagonist and an antigen selected from the group consisting of an autoantigen, amyloid peptide protein, an alloantigen, a transplant antigen, an allergen, and fragments thereof In another embodiment, the GITR antagonist is selected from the group consisting of a neutralizing anti-GITRL antibody, a neutralizing anti-GITR antibody, a fusion protein containing GITR, a fusion protein containing an active fragment of GITR, an antagonistic small molecule, an
antisense GITRL nucleic acid molecule, and a siRNA GITRL nucleic acid molecule In another embodiment, the antibody composes the antigen binding fragments of 5F1 or 10F12
[0041] In another embodiment, the invention provides a method of screening for test compounds capable of neutralizing GITRL activity comprising the steps of contacting a sample contaimng GITRL and a neutralizing antibody with the compound, and determining whether the interaction of GITRL with the neutralizing antibody in the sample is decreased relative to the interaction of GITRL with the neutralizing antibody in a sample not contacted with the compound, whereby a decrease in the mteraction of GITRL with the neutralizing antibody in the sample contacted with the compound identifies the compound as one that inhibits or blocks the mteraction of GITRL with the neutralizing antibody In another embodiment, the antibody is 5F1 or 10F12
[0042] In another embodiment, the invention provides a method of providmg a costrmulatory signal to a cell population comprising effector T cells, the method comprising administering a GITR agomst In another embodiment, the GITR agomst is protein selected from the group consisting of selected from the group consisting of GITRL, or an active fragment of GITRL, a fusion protein contaimng GITRL, a fusion protein containing an active fragment of GITR, and an agonistic anu-GITR antibody In another embodiment, the effector T cells are CD4+ T cells or CD8+ T cells
BRIEF DESCRIPTION OF THE DRAWINGS [0043] Figure 1 shows the alignment (based on BLOSUM62 amino acid substitution matrix, see Hernkoff and HenikofF (1992) Proc Natl Acad Set USA 89 10915-19) of the ammo acid sequences for mouse (m) and human (h) GITRL
[0044] Figure 2 shows the results of experiments on the effects of GITRL GITR bmdmg on prohferation of CD4+CD25+ T cells Thymidine incorporation is measured (CPM) as a means of assessmg cellular proliferation Figure 2A shows that an agonistic anti-GITR antibody stimulated the proliferation of CD4+CD25+ T cells, but not CD4+CD25" T cells Figure 2B shows that YB2/0 cells expressing GITRL stimulated the proliferation of CD4+CD25+ cells
[00451 Figure 3A shows that GITRL expressed by YB2/0 cells (-50,000), as well as agomstic anti-GITR antibody (2 Hg/ml), reversed the suppression (l e , negative percent suppression) produced by freshly isolated CD4+CD25+ suppressor T cells (# suppressors) Figure 3B shows that GITRL-YB2/0 cells in numbers less than 50,000 (I e, -3,000-25,000) were able to partially reverse suppression in a dose-dependent manner
[0046] Figure 4 shows that GITRL-YB2/0 cells (-50,000) did not reverse suppression when activated CD4+CD25+ T cells were used in place of freshly isolated CD4+CD25+ T cells, similar results were obtained with agomstic anti-GITR antibody
[0047] Figures 5 A and 5B show that the GITRL-induced reversal of the suppression produced by freshly isolated CD4+CD25+ T cells can itself be reversed (l e, restoration of suppression) in the presence of anti-GrTRL antibody ("anti-GITRL" = 5F1 antibody) Figure 5B includes an additional experiment showing that a control antibody ("control Ig") did not restore suppression
[0048] Figure 6 shows that anti-GITRL antibody can only enhance suppression in the presence of CD4+CD25+ T cells Figure 6 A shows the suppression of the proliferation of lymph node cells in the presence of anti-GITRL antibody (5F1) Figure 6B shows the lack of the suppressive activity of anti-GITRL antibody when the lymph node cell population was depleted of CD4+CD25+ T cells
[0049] Figure 7 shows the distribution of GITRL expressmg cells in lymphoid tissues Figure 7A Flow cytometric analysis was performed on CD1 lc+ cells, enriched from the spleen of BALB/c mice with magnetic beads, by staining with anti-CD4, anti-CD8 and anti-GITRL antibodies GITRL expression was determined by comparing the fluorescence intensity of CD4+, CD8+, and CD4" CD8" -gated subsets (top, middle, and bottom histogram panels, respectively) stained with anti-GITRL antibody (filled histograms) to the fluorescence intensity of these cells stained with an isotype control antibody (unfilled histograms) Figure 7B Expression of GITRL by splemc dendritic cells (DCs) and B-l B cells was determined by staining freshly isolated BALB/c CD1 lc+ splemc DCs (top histogram panel) and CDllclowB220+ plasmacytoid DCs (bottom histogram panel) with anti-GITRL mAb (filled histograms) or an isotype control antibody (unfilled
histograms) and performmg flow cytometric analysis Figure 7C (top histogram panel) The fluorescence intensities of B220+ B cells among total splenocytes (filled histogram), and CD1 lb+B220+-gated peritoneal (perC) B-l B cells (solid line unfilled histogram) stained with anti-GITRL antibody were compared with the fluorescence intensity of cells stamed with an isotype control (broken line unfilled histogram) Figure 7C (bottom histogram panel) A comparison of the fluorescence intensity of GITRL-antibody stamed (filled histogram) and isotype antibody stamed (unfilled histogram) perC macrophages (CD1 lb+B220~ cells) is shown Figure 7D Thymocytes were stamed for expression of CD4, CD8, and either GITRL or an isotype control The fluorescence intensities of CD4+CD8~ (top left quadrant), CD4+CD8+ (top right quadrant) and CD4"CD8+ (bottom right quadrant) cells stamed with anti-GITRL antibody (filled histogram) were compared with the fluorescence intensities of these cells stamed with an isotype control antibody (unfilled histogram) Figure 7E Expression of GITRL by gated CD44+CD25"(R1), CD44+CD25+(R2), CD44"CD25+ (R3) orCD44-CD25(R4) thymic precursors was determined by comparing the fluorescence of these cells stamed with anti-GITRL antibody (filled histogram) with the fluorescence of these cells stamed with isotype control antibody (unfilled histogram) Figure 7F Unstimulated lymph node cells were stained with anti-CD4, anti-CD8, anti-CD25 and/or anti-GITRL antibodies CD4+CD8" cells (top left quadrant), and not CD4" CD8+ cells (bottom right quadrant), were further delineated with respect to the expression of CD25 by these cells Expression of GITRL by CD4+CD8"CD25" (top right histogram panel), CD4+CD8'CD25+ (lower right histogram panel) or CD4"CD8+ (bottom (middle) histogram panel) -gated lymph node cells was determined by comparing the fluorescence intensity of these cells stamed with anti-GITRL antibody (filled histograms) with the fluorescence of these cells stained with an isotype control antibody (unfilled histograms) Results are representative of five mdependent experiments
[0050] Figure 8 shows the downregulation by APCs of GITRL following stunulation Figure 8 A Expression of GITRL by punfied splemc B220+ B cells or total peritoneal (PerC) B220+CD1 lb+ B-l B cells was determined for different tune points following treatment with polyl C (10|j.g/ml), LPS (0 5^ig/ml), CpGs (ODN 1826, l^iM), anti-CD40 and IL-4 (10p.g/ml and 20ng/ml, respectively) and
anti-IgM (F(ab')2 fragment of goat-anti-IgM |j.-chain, lug/ml) The fluorescence intensities of anti-GITRL-stained stimulated cells (filled histograms), anti-GITRL-stained unstimulated (medium) cells (solid line unfilled histograms) and isotype control antibody stained cells (broken lme unfilled histograms) are presented Figure 8B Expression of GITRL by B220+ B cells (filled histogram) present among total splenocytes treated with anti-CD3 mAb (0 5ug/ml) after a 48-hour culture period was compared with expression of GITRL by unstimulated B220+ B cells (solid lme unfilled histogram) and B220+ B cells stained with an isotype control antibody (broken line unfilled histogram) Figure 8C Expression of GITRL (top histogram panels) and B7 2 (1 e, CD86) (bottom histogram panels) by purified CDllc+ DCs following culture with or without LPS (0 5ng/ml) at the mdicated time pomts Figure 8D Expression of GITRL by total splenocytes gated on CD4+orCD8+ -expressing cells after a 48-hour culture period in the absence or presence of soluble anti-CD3 mAb (0 5^g/ml) Graphs are representative of two to four independent experiments, all experiments were earned out with tissues isolated from BALB/c mice
[0051] Figure 9 demonstrates the effects of blocking GITR/GITRL interactions on inhibition of lymphocyte proliferation For Figures 9 A and 9B, bars indicate the s d values Figure 9 A Proliferation (y-axis) of lymph node (LN, lxl 05) and spleen cells (Sp, 0 5xl05) with or without CD25+ cells (Total or A25, respectively) was determined after 72-hour culture with different concentrations of soluble anti-CD3 (x-axis) Cells were mcubated either in the presence of purified anti-GrTRL mAb (lOfig/ml, closed circles) or a rat IgG2a isotype control (lOug/ml, open circles) Results are representative of three mdependent experiments Figure 9B CD4XD25- or CD8+ T cells were cultured in the presence of 5x104 irradiated (3000R) T-depleted APCs and 5x104 irradiated (8000R) YB2/0-GITRL (open circles) or control YB2/0 (closed circles) cells Cultures were activated with different concentrations of soluble anti-CD3 mAb (x-axis), and proliferation (y-axis) was measured after a 72-hour culture period Figure 9C Mean fluorescence (x-axis) of purified CD4+CD25" T cells stained with anti-GITR antibody was determined at different time pomts following activation with soluble anti-CD3 (0 5(ig/ml) in the presence of irradiated (3000R) T-depleted splenocytes Results are representative of at least two mdependent experiments
[0052] Figure 10 demonstrates that GITR expression by CD25" T cells is required to reverse suppression Figure 10A Proliferation of cocultures of CD4+CD25~T cells (5xl04) from vanous knockout mice, and vanable numbers of CD4+CD25+ T cells (x-axis) from vanous knockout mice [(Aa) CD4XD25- GITR+/+, CD4+CD25+ GITR+/+, (Ab) CD4+CD25" GITR+/+, CD4+CD25+ GITR.'-, (Ac) CD4+CD25" GITR'-, CD4+CD25+ GTTR+/+, and (Ad) CD4+CD25" GITR'', CD4+CD25+ GITR-'], mcubated with irradiated APCs from wild type mice (5xl04) and with soluble anu-CD3 (0 5ug/ml) and 2\ig/ml of either anti-GITR antibody (filled circles) or an isotype control antibody (open circles) was determined by measuring 3H-thymidine uptake (cpm, y-axis) Figure 10B Proliferation of cocultures was performed as above (Fig 10 A) with vanable numbers of mouse CD4+CD25+ T cells (x-axis) and either (Ba) mouse CD4+CD25" T cells or (Bb) rat CD4+CD25' T cells in the presence of irradiated (3000R) rat APCs Cultures were stimulated with a cocktail of antibodies against both rat and mouse anti-CD3 (0 25jjg/ml of each), and were treated with 2fig/ml of either an isotype control (Rat IgG, open circles) or anti-GITR (DTA-1, filled circles) antibody Bars indicate the s d values calculated from proliferation in tnphcate cultures Figure 10C Fluorescence (x-axis) of CFSE-stained mouse CD4XD25" (top panels) and rat CD4+CD25" T cells (bottom panels) cocultured at a 1 8 suppressor to responder ratio with isotype control (Rat IgG, left panels) or anti-GITR antibody (DTA-1, nght panels) is depicted Mouse and rat T cell subsets were distinguished by staining with specific anti-CD4 antibodies Results are representative of two to four independent experiments
[00531 Figure 11 demonstrates that GITR signals are required to overcome suppression mediated by endogenous regulatory T cells Figure 11A CFSE-labeled lymph node (LN) cells (5xl04) from B6 (wild type), G/ZK+A, CD2$'~ and GITR'' mice were cultured for 72 hours with different concentrations of soluble anti-CD3 mAb (x-axis) Total LN cells were cultured without (Aa) or with (Ac) exogenous of IL-2 (50U/ml) LN cells depleted of CD25+ cells (LNA25) were cultured without (Ab) or with (Ad) exogenous IL-2 (50U/ml) Bars indicating the sd values were omitted for clanty Figure 1 IB Flow cytometnc assessment of CFSE dilution by CD4+ and CD8+-gated lymph node T cells isolated from CD2^' , GITR'-, GITR+/+ or GITR+A animals was performed after 72-hour culture The
results correspond to the 0 63u.g/ml concentration of soluble anti-CD3 (as in Fig 11 A) Figure 11C Flow cytometnc analysis of CD25 expression was performed on H-2Db positive CD4+CD25' cells that remained unstimulated (broken line unfilled histograms), were obtained from GITR'A mice (solid line unfilled histograms), or were obtained from GITR+/+ mice (filled histograms) CD25 expression by CD4+CD25" cells obtained from GITR''" or GJTR+/+ mice was determined after 24-hour culture with LN APCs from wild type mice, in the presence of anti-CD3 (0 5ug/ml), and in the absence (left histogram panels) or presence (nght histogram panels) of CD4+CD25+ cells from BALB/c mice at a 1 2 suppressor to responder ratio CD25 expression was also determined in the absence (top histogram panels) or presence (bottom histogram panels) of 50U/ml rhIL-2 Results above are representative of three independent experiments
[0054] Figure 12 demonstrates that CD28-dependent costimulation enhances GITR expression and responsiveness Figure 12 A Flow cytometnc analysis of GITR expression by punfied CD4+CD25" or CD8+ T cells (2 5x104) after 72-hour culture with plate-bound anti-CD3 and 2(ag/ml of either plate-bound hamster lsotype ("aCD3") or plate-bound anti-CD28 ("aCD3+aCD28") Figure 12B (left histogram panel) Anti-GITR staining of CD4+CD25' T cells cultured in the presence of irradiated, T cell-depleted splenocytes and soluble anti-CD3 (0 5p.g/ml) with or without a cocktail of anti-CD80/86 (10µg/ml of each) antibodies (I e, anti-B7 1/7 2 antibodies) for 72 hours Figure 12B (nght histogram panel) Anti-GITR staining of CD4+CD25' T cells cultured in the presence of irradiated, T cell-depleted splenocytes and soluble anti-CD3 (0 5ng/ml) with or without a cocktail of antibodies against IL-2 and IL-2Ra Figure 12C Proliferation was assessed in the presence or absence of anti-CD80/86 mAbs (lOug/ml of each, "aB7") with the addition of either anti-GITR mAb (2ng/ml, "DTA-1") or an lsotype control antibody (2ug/ml, "Rat IgG") Bars indicate the s d values Results are representative of two to three independent experiments
[0055] Figure 13 demonstrates that GITRL binding to GITR provides a costimulatory signal to effector T cells Figure 13 A Prohferation of effector G1TR+/TCR+ HT-2 T cells (4xl04) alone (white bars) or cocultured with 1x104
control YB2/0 cells (cross-hatch bars) or GITRL-expressing YB2/0 cells (filled bars), in the absence or presence of one or two anti-CD3 beads per HT-2 cell, was determined by measunng H-thyrmdine uptake (cpm, y-axis) Figure 13B Proliferation of 4x104 HT-2 cells cocultured with two anti-CD3 beads per cell, lxlO4 GITRL-expressing YB2/0 cells, and mcreasing concentrations (ng/ml, x-axis) of an anti-GITRL antibody (5F1 1, filled circles) or an isotype control antibody (rlgGl, open circles) was determined by measunng H-thymidine uptake (cpm, y-axis) Figure 13C Proliferation of 4xl04 HT-2 cells cocultured with two anti-CD3 beads per cell, lxlO4 GITRL-expressing YB2/0 cells, and mcreasing concentrations (ng/ml, x-axis) of four different anti-GITRL antibodies 5F1 1 (filledcircles), MGLT-10 (filled squares), MGTL-15 (open squares) or a polyclonal antibody (open circles) was determined by measunng H-thymidine uptake (cpm, y-axis)
[0056] Figure 14 demonstrates that blocking GITR-GITRL bmdmg with an anti-GITRL antibody prevents adoptive transfer of PLP-induced experimental autoimmune encephalomyelitis (EAE) The mcidence of EAE in mice was assessed Mice were mjected with 5xl06 splenocytes that were isolated from female SLJ mice immunized with 150 fig PLP peptide and restimulated ex vivo for 3 days in three different conditions 10 ug/ml PLP alone (open circles), 10 ug/ml PLP and 10 jig/ml of an isotype control antibody (CKOl, filled circles), or 10 jig/ml PLP and anti-GITRL antibody (5F1 1, filled squares) The mcidence of EAE was mointored for 52 days (x-axis) and scored on a scale of 0 to 5 (y-axis)
DETAILED DESCRIPTION OF THE INVENTION [0057] As the antibodies to GITR that produce a reversal of suppressive activity appear to produce an agonistic signal, it was predicted that engagement of GITR by GITRL should also inhibit the suppressive activity of regulatory T cells The lack of suitable reagents previously has precluded a detailed functional analysis of GITR/GITRL interactions under more physiological conditions Here, the mouse ortholog of GITRL has been identified, and antagonistic antibodies that specifically bind to the mouse ortholog of GITRL, l e, do not cross-react with human GITRL, have been generated
[0058] Using this reagent, the tissue distribution and regulation of GITRL were examined hi addition, the abihty of GITR/GITRL interactions to regulate T cell suppression was investigated using GITK'~ mice As both CD25" and CD25+ T cells express GITR, albeit to varying degrees, the previous studies demonstrating an inhibition of suppressor function upon treatment of cocultures with an agonistic anti-GITR antibody yielded equivocal results regarding the cellular target of engagement of GITR Here, using combinations of CD4+CD25+ and CD4+CD25" T cells from wild type and GITR"'' mice in coculture expenments, it was found that ligation of GITR on the CD4+CD25" responder T cells, not the CD4+CD25+ suppressor T cells, was required to abrogate suppression In the absence of CD4+CD25+ T cells, GFTK1' T cells mounted proliferative responses similar to those of wild type animals, although they were totally suppressed in the presence of physiological numbers of CD4+CD25+ T cells These results suggest, for the first time, that GITR/GITRL engagement provides a previously undefined signal that renders effector T cells resistant to the inhibitory effects of CD4+CD25+ T cells Thus, the downregulation of GITRL expression subsequent to secondary inflammatory signals may facilitate CD4+CD25+-mediated suppression and prevent the deleterious consequences of an exuberant effector cell response
[0059] This research has shed new light on the mechanisms underlying the interaction of GITR and its ligand, GITRL, especially regarding the effects on CD4+CD25" T cells, the cells traditionally understood to be the target of suppressive activity In summary, using GJTR'1' mice, the capacity of anti-GITR mAb (the agonistic mouse antibody to GITR) to abrogate suppression was demonstrated to be mediated by its action on CD4+CD25", not CD4+CD25+ T cells (as previously proposed by several studies) APCs Xantigen presenting cells) constitutively express GITRL, which is downregulated following Toll-like receptor signaling Although GITR'A mouse CD4+CD25" T cells were capable of mounting proliferative responses, they were mcapable of proliferation in the presence of physiological numbers of CD4+CD25+ T cells Thus, GITRL provides an important signal for CD4+CD25" T cells, and other effector T cells (e g, CD8+ T cells), rendering them resistant to CD4+CD25+-mediated regulation at the initiation of the immune response The downregulation of GITRL by inflammatory stimuli may enhance the susceptibility of effector T cells (e g,
CD4+CD25" T cells) to suppressor activity during the course of, e g, cancer or an infectious msult
[0060] To this end, the present invention provides the nucleotide and ammo acid sequences of a novel mouse homolog of human GITRL Human GITRL has been identified (Kwon et al (1999) J Biol Chem 274 6056-61, Gurneyetal (1999) Curr Biol 9 215-18), in addition, several groups very recently also reported the cloning of the murine GITR ligand (Kim et al, 2003, Tone et al, 2003, Yu et al, 2003)
[0061] In one aspect, the present mvention provides nucleotide sequences, and amino acid sequences, and active fragments and/or fusion proteins thereof, of a novel mouse homolog of human GITRL GITRL polynucleotides of the mvention include polynucleotides that modulate expression of GITRL, e g, expression vectors comprising GITRL polynucleotides that may upregulate expression of GITRL, and/or antisense and/or RNAi GITRL polynucleotides that downregulate the expression of GITRL Use of such polynucleotides to modulate the expression of GITRL in cells and/or animals are also provided In addition to GITRL polypeptides, the invention also provides other agonistic polypeptides, e g, active fragments of GITRL and/or GITRL fusion proteins that are capable of mimicking GITRL, I e, inducing GITR activity in effector T cells Transformed host cells and transgenic animals containing GITRL polynucleotides are also within the scope of the mvention
[0062] In another aspect, antibodies that specifically bmd to the novel murine GITRL polypeptides of the invention (l e, do not bmd to human GITRL) are provided In particular, neutralizing antibodies that inhibit the activity of GITRL (e g, antibodies that prevent GITRL from binding GITR) are provided, these antibodies can be said to neutralize the activity of GITRL (l e, render GITRL ineffective) Neutralizing antibodies of the mvention include nonhuman and human antibodies to GITRL that inhibit GITRL activity, as well as chrmenzed and/or humanized versions of nonhuman antibodies of the mvention that inhibit GITRL activity Also included within the scope of the mvention are antagonistic antibodies that may have one or more mutations, which may function to increase the half-life, stability or affinity of the antibody, or may function to modify the effector function of the antibody
[0063] Another aspect of the invention provides screening assays in which the GITRL polynucleotides and polypeptides, including but not limited to human homo logs thereof, are used to identify compounds capable of modulating the activity of GITR in a cell, organism or subject The invention also provides methods to assess the efficacy of identified compounds whereby the number of T cells in a patient is determined before and after administration of the identified compound Additionally, the invention provides methods of treating patients or subjects using the identified compounds
[0064] In addition to providing methods of screening test compounds capable of modulating GITR activity, e g, GITR agonists or GITR antagonists, the invention provides methods for diagnosing, prognosing and monitoring the progress of disorders related to disregulation of the immune system, e g, autoimmune diseases, inflammatory diseases and transplant rejection, and cancer and infectious diseases
[0065] Methods for using GITRL and related molecules of the invention are also disclosed herein, including agonistic GITR molecules (I e, GITRL polynucleotides, GITRL polypeptides, active fragments thereof and/or fusion proteins thereof, agonistic small molecules, and agonistic GITR antibodies), and antagomstic GITR molecules (l e, GITRL inhibitory polynucleotides, neutralizmg GITR antibodies, neutralizmg GITRL antibodies, antagomstic small molecules, and GITR fusions proteins), for the therapeutic treatment of disorders related to disregulation of the immune system For example, methods for treating a subject at nsk for, or diagnosed with, an autoimmune disorder, transplant rejection, and/or other inflammatory diseases comprising administering GITR antagonists, e g, a neutralizing anti-GITRL antibody to the subject are provided, also, methods of treating a subject at nsk for, or diagnosed with, cancer or infectious diseases compnsing administering GITR agonists, e g, GITRL, or an agonistic fusion protein thereof, are provided Alternatively, methods of inducmg or inhibiting proliferation of T cells via the admmistration of GITR agonists, e g, GITRL (including agonistic fusion proteins thereof), or GITR antagonists, e g, neutralizmg anti-GITRL antibodies or antagomstic GITR fusion proteins, respectively, are provided Similarly, methods of blocking or enhancing suppression of T cells in the presence of CD4+CD25+ T cells compnsing
admmistration of GITR agonists, e g, GITRL (including agomstic fusion proteins thereof), or GITR antagonists, e g, neutralizing anti-GITRL antibodies, respectively, are also provided T cell populations treated with GITRL polypeptides and related molecules (including agomstic fusion proteins thereof) are within the scope of the invention, and may be administered to a subject in a method of treating cancer or an infectious disease Other methods of treatment are provided, including a method of treating a subject at nsk for, or diagnosed with, an autoimmune disorder, an inflammatory disease, or transplant rejection with an antagonistic compound that decreases GITR activity, and methods of treating a subject at nsk for, or diagnosed with, cancer or an infectious disease with an agomstic compound that increases GITR activity Pharmaceutical compositions, e g, vaccine adjuvants, compnsing GITRL polynucleotides, polypeptides and related molecules (including agomstic GITRL fusion proteins and antagonistic anti-GITRL antibodies) of the invention are also within the scope of the invention The methods of the present mvention are directed to GITRL and GITR, including, but not limited to, mouse GITRL and GITR and their homologs, specifically included among these homologs is human GITRL and GITR
GITRL Polynucleotides and Polypeptides
[0066] The present invention provides novel isolated and punfied polynucleotides and polypeptides related to a novel ligand for GITR (GITRL) The genes, polynucleotides, proteins, and polypeptides of the present mvention include, but are not limited to, mouse GITRL and its homologs
[0067] For example, the mvention provides punfied and isolated polynucleotides encoding munne GITRL Preferred DNA sequences of the invention include genomic, cDNA and chemically synthesized DNA sequences
[0068] The nucleotide sequence of a cDNA encodmg this novel ligand, designated mouse GITRL cDNA, is set forth in SEQ ID NO 1 Polynucleotides of the present mvention also include polynucleotides that hybndize under stringent conditions to SEQ ID NO 1, or its complement, and/or encode polypeptides that retain substantial biological activity (1 e, active fragments) of full-length mouse GITRL Polynucleotides of the present invention also include contmuous portions of the sequence set forth in SEQ ID NO 1 compnsing at least 21 consecutive nucleotides
[0069] The nucleotide sequence of a genomic DNA encoding this novel ligand, designated mouse GITRL genomic DNA, is set forth in SEQ ID NO 3 Polynucleotides of the present mvention also include polynucleotides that hybridize under stringent conditions to SEQ ID NO 3, or its complement, and/or encode polypeptides that retain substantial biological activity of full-length mouse GITRL Polynucleotides of the present mvention also include continuous portions of the sequence set forth in SEQ ID NO 3 comprising at least 21 consecutive nucleotides
[0070] The ammo acid sequence of mouse GITRL is set forth in SEQ ID NO 2 Polypeptides of the present mvention also include continuous portions of the sequence set forth in SEQ ID NO 2 comprising at least 7 consecutive ammo acids A preferred polypeptide of the present mvention includes any contmuous portion of the sequence set forth in SEQ ID NO 2 that retams substantial biological activity of full-length mouse GITRL Polynucleotides of the present mvention also include, in addition to those polynucleotides of murine origin described above, polynucleotides that encode the ammo acid sequence set forth in SEQ ID NO 2 or a continuous portion thereof, and that differ from the polynucleotides described above only due to the well-known degeneracy of the genetic code
[0071] The isolated polynucleotides of the present mvention may be used as hybridization probes and primers to identify and isolate nucleic acids having sequences identical to or similar to those encodmg the disclosed polynucleotides Hybridization methods for identifying and isolating nucleic acids include polymerase chain reaction (PCR), Southern hybridizations, in situ hybridization and Northern hybridization, and are well known to those skilled in the art
[0072] Hybridization reactions can be performed under conditions of different stnngency The stnngency of a hybndization reaction includes the difficulty with which any two nucleic acid molecules will hybndize to one another Preferably, each hybndizing polynucleotide hybndizes to its corresponding polynucleotide under reduced stnngency conditions, more preferably stnngent conditions, and most preferably highly stnngent conditions Examples of stnngency conditions are shown in Table 1 below highly stnngent conditions are those that are at least as stnngent as, for example, conditions A-F, stnngent conditions are at least as
stnngent as, for example, conditions G-L, and reduced stnngency conditions are at least as stnngent as, for example, conditions M-R
TABLE 1
(Table Removed)
[0073] The isolated polynucleotides of the present invention may be used as hybridization probes and primers to identify and isolate DNA having sequences encodmg allelic vanants of the disclosed polynucleotides Allelic vanants are naturally occurring alternative forms of the disclosed polynucleotides that encode polypeptides that are identical to or have significant sirmlanty to the polypeptides encoded by the disclosed polynucleotides Preferably, allelic vanants have at least 90% sequence identity (more preferably, at least 95% identity, most preferably, at least 99% identity) with the disclosed polynucleotides
[0074] The isolated polynucleotides of the present invention may also be used as hybndization probes and primers to identify and isolate DNAs having sequences encodmg polypeptides homologous to the disclosed polynucleotides These homologs are polynucleotides and polypeptides isolated from a different species than that of the disclosed polypeptides and polynucleotides, or within the same species, but with significant sequence similanty to the disclosed polynucleotides and polypeptides Preferably, polynucleotide homologs have at least 50% sequence identity (more preferably, at least 75% identity, most preferably, at least 90% identity) with the disclosed polynucleotides, whereas polypeptide homologs have at least 30% sequence identity (more preferably, at least 45% identity, most preferably, at least 60% identity) with the disclosed polypeptides Preferably, homologs of the disclosed polynucleotides and polypeptides are those isolated from mammalian species
[0075] The isolated polynucleotides of the present invention may also be used as hybndization probes and primers to identify cells and tissues that express the polypeptides of the present invention and the conditions under which they are expressed
[0076] Additionally, the isolated polynucleotides of the present invention may be used to alter (l e, enhance, reduce, or modify) the expression of the genes corresponding to the polynucleotides of the present invention in a cell or orgamsm These corresponding genes are the genomic DNA sequences of the present invention (e g, SEQ ID NO 3) that are transcnbed to produce the mRNAs from which the cDNA polynucleotides of the present invention (e g, SEQ ID NO 1) are denved
[0077] Altered expression of the genes of the present invention, including but not limited to mouse GITRL and its homologs, may be achieved in a cell or organism through the use of various inhibitory polynucleotides, such as antisense polynucleotides (e g, antisense GITRL nucleic acid molecules) and nbozymes that bind and/or cleave the mRNA transcribed from the genes of the invention (see, e g, Galdensi et al (1999) J Cell Physiol 181 251-57, Sioud (2001) Curr Mol Med 1 575-88) Such inhibitory polynucleotides may be useful in preventing or treating autoimmune disorders, inflammatory diseases, transplant rejection, and similar or related disorders
[0078] The antisense polynucleotides or nbozymes of the mvention can be complementary to an entire codmg strand of a gene of the mvention, or to only a portion thereof Alternatively, antisense polynucleotides or nbozymes can be complementary to a noncoding region of the coding strand of a gene of the mvention The antisense polynucleotides or nbozymes can be constructed using chemical synthesis and enzymatic ligation reactions using procedures well known in the art The nucleoside linkages of chemically synthesized polynucleotides can be modified to enhance then ability to resist nuclease-mediated degradation, as well as to mcrease their sequence specificity Such linkage modifications include, but are not limited to, phosphorothioate, methylphosphonate, phosphoroamidate, boranophosphate, morpholino, and peptide nucleic acid (PNA) linkages (Galdensi et al, supra, Heasman (2002) Dev Biol 243 209-14, Micklefield (2001) Curr Med Chem 8 1157-79) Alternatively, these molecules can be produced biologically using an expression vector into which a polynucleotide of the present invention has been subcloned in an antisense (I e, reverse) onentation
[0079] The inhibitory polynucleotides of the present mvention also include tnplex-forming oligonucleotides (TFOs) that bind in the major groove of duplex DNA with high specificity and affinity (Knauert and Glazer (2001) Hum Mol Genet 10 2243-51) Expression of the genes of the present mvention can be inhibited by targeting TFOs complementary to the regulatory regions of the genes (l e, the promoter and/or enhancer sequences) to form tnple helical structures that prevent transcnption of the genes
[0080] In one embodiment of the mvention, the inhibitory polynucleotides of the present mvention are short interfering RNA (siRNA) molecules (e g, siRNA
GITRL nucleic acid molecules) These siRNA molecules are short (preferably 19-25 nucleotides, most preferably 19 or 21 nucleotides), double-stranded RNA molecules that cause sequence-specific degradation of target mRNA This degradation is known as RNA interference (RNAi) (e g, Bass (2001) Nature 411 428-29) Originally identified in lower organisms, RNAi has been effectively applied to mammalian cells and has recently been shown to prevent fulminant hepatitis in mice treated with siRNA molecules targeted to Fas mRNA (Song et al (2003) Nature Med 9 347-51) In addition, intrathecaily delivered siRNA has recently been reported to block pain responses in two models (agonist-induced pain model and neuropathic pam model) in the rat (Dora et al (2004) Nucleic Acids Res 32(5) e49)
[0081] The siRNA molecules of the present invention can be generated by annealing two complementary single-stranded RNA molecules together (one of which matches a portion of the target mRNA) (Fire et al, U S Patent No 6,506,559) or through the use of a single hairpin RNA molecule that folds back on itself to produce the requisite double-stranded portion (Yu et al (2002) Proc Natl Acad Sci USA 99 6047-52) The siRNA molecules can be chemically synthesized (Elbashir et al (2001) Nature 411 494-98) or produced by in vitro transcription using single-stranded DNA templates (Yu et al, supra) Alternatively, the siRNA molecules can be produced biologically, either transiently (Yu et al, supra, Sui et al (2002) Proc Natl Acad Sci USA 99 5515-20) or stably (Paddison et al (2002) Proc Natl Acad Sci USA 99 1443-48), using an expression vectors) containing the sense and antisense siRNA sequences Recently, reduction of levels of target mRNA in primary human cells, in an efficient and sequence-specific manner, was demonstrated using adenoviral vectors that express hairpin RNAs, which are further processed into siRNAs (Arts et al (2003) Genome Res 13 2325-32)
[0082] The siRNA molecules targeted to the polynucleotides of the present invention can be designed based on criteria well known in the art (e g, Elbashir et al (2001) EMBO J 20 6877-88) For example, the target segment of the target mRNA preferably should begm with AA (most preferred), TA, GA, or CA, the GC ratio of the siRNA molecule preferably should be 45-55%, the siRNA molecule preferably should not contain three of the same nucleotides in a row, the siRNA molecule preferably should not contain seven mixed G/Cs in a row, and the target
segment preferably should be in the ORF region of the target mRNA and preferably should be at least 75 bp after the initiation ATG and at least 75 bp before the stop codon Based on these criteria, or on other known criteria (e g, Reynolds et al (2004) Nature Biotechnol 22 326-30), siRNA molecules of the present mvention, targeted to the mRNA polynucleotides of the present invention, can be designed by one of ordinary skill in the art
[0083] Altered expression of the genes of the present mvention in an organism may also be achieved through the creation of nonhuman transgenic animals into whose genomes polynucleotides of the present mvention have been introduced Such transgenic animals include animals that have multiple copies of a gene (l e, the transgene) of the present mvention A tissue-specific regulatory sequence(s) may be operably linked to the transgene to direct expression of a polypeptide of the present mvention to particular cells or a particular developmental stage Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional and are well known ' in the art (e g, Bockamp et al, Physiol Genomics, 111 15-32 (2002))
[0084] Altered expression of the genes of the present mvention in an organism may also be achieved through the creation of animals whose endogenous genes corresponding to the polynucleotides of the present mvention have been disrupted through insertion of extraneous polynucleotide sequences (I e, a knockout animal) The coding region of the endogenous gene maybe disrupted, thereby generating a nonfunctional protein Alternatively, the upstream regulatory region of the endogenous gene may be disrupted or replaced with different regulatory elements, resulting in the altered expression of the stall-functional protein Methods for generating knockout animals include homologous recombination and are well known in the art (e g, Wolfer et al, Trends Neurosci, 25 336-40 (2002))
[0085] The isolated polynucleotides of the present mvention may be operably linked to an expression control sequence and/or hgated into an expression vector for recombinant production of the polypeptides of the present mvention General methods of expressmg recombinant proteins are well known in the art Such recombinant proteins may be expressed in soluble form for use in treatment of disorders resulting from disregulation of the immune system, such disorders include, for example, cancers and infectious diseases, and autoimmune disorders
and mflammatory diseases, and transplant rejection Autoimmune disorders and inflammatory diseases include, but are not limited to, rheumatoid arthritis, encephalomyelitis, osteoarthritis, multiple sclerosis, autoimmune gastritis, systemic lupus erythematosus, psonasis and other inflammatory dermatoses, type I diabetes, asthma, allergy, and mflammatory bowel diseases, including Crohn's disease and ulcerative colitis
(0086] An expression vector, as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked One type of vector is a plasmid, which refers to a circular double stranded DNA loop into which additional DNA segments may be hgated Another type of vector is a viral vector, wherein additional DNA segments may be hgated into the viral genome Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e g, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) Other vectors (e g, nonepisomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked Such vectors are referred to herein as recombinant expression vectors (or simply, expression vectors) In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids In the present specification, plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector However, the mvention is intended to include other forms of expression vectors, such as viral vectors (e g, replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions
[0087] In one embodunent, the polynucleotides of the present mvention are used to create GITR agomsts, e g, GITRL polypeptides, mcludmg active fragments and/or fusion polypeptides thereof, which are also within the scope of the mvention For example, a GITRL polypeptide or active fragments thereof may be fused to a second moiety, e g, an immunoglobulm or a fragment thereof (e g, an Fc binding fragment thereof) In some embodiments, the first polypeptide includes full-length GITRL polypeptide Alternatively, the first polypeptide may comprise less than the full-length GITRL polypeptide Additionally, soluble forms of GITRL may be
fused through "lmker" sequences to the Fc portion of an immunoglobulin Other fusions proteins, such as those with glutathione-iS'-transferase (GST), Lex-A, thioredoxin (TRX) or maltose-binding protein (MBP), may also be used
[0088] The fusion proteins may additionally include a linker sequence joining the GITRL or GITRL fragment to the second moiety Use of such linker sequences are well known in the art For example, the fusion protein can include a peptide linker, e g, a peptide linker of about 2 to 20, more preferably less than 10, ammo acids in length In one embodiment, the peptide linker may be 2 amino acids in length
[0089] In another embodiment, the fusion protein includes a heterologous signal sequence (l e, a polypeptide sequence that is not present in a polypeptide encoded by a GITRL nucleic acid) at its N-termmus For example, a signal sequence from another protein may be fused with a GITRL polypeptide, including active fragments and/or fusion proteins thereof In certain host cells (e g, mammalian host cells), expression and/or secretion of GITRL can be mcreased through use of a heterologous signal sequence
[0090] A signal peptide that can be included in the fusion protein is MKFLVNVALVFMWYISYIYA (SEQ ID NO 11) If desired, one or more ammo acids can additionally be inserted between the first polypeptide moiety comprising the GITRL moiety and the second polypeptide moiety The second polypeptide is preferably soluble In some embodiments, the second polypeptide enhances the half-life, (e g, the serum half-hfe) of the linked polypeptide In some embodiments, the second polypeptide includes a sequence that facilitates association of the fusion polypeptide with a second GITRL polypeptide In preferred embodiments, the second polypeptide includes at least a region of an immunoglobulin polypeptide Immunoglobulin fusion polypeptide are known in the art and are described in e g, US Patent Nos 5,516,964, 5,225,538,5,428,130, 5,514,582, 5,714,147, and 5,455,165, all of which are hereby incorporated by reference
[0091] A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques For example, DNA fragments codmg for the different polypeptide sequences are hgated together m-frame in accordance with
conventional techmques, e g, by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation In another embodiment, the fusion gene can be synthesized by conventional techmques including automated DNA synthesizers Alternatively, PCR amplification of gene fragments can be earned out using anchor primers that give nse to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamphfied to generate a chimeric gene sequence (see, for example, Ausubel et al (Eds) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992) Moreover, many expression vectors are commercially available that encode a fusion moiety (e g, an Fc region of an immunoglobulin heavy chain) A GITRL-encodmg nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the lmmunoglobulm protein In some embodiments, GITRL fusion polypeptides exist as oligomers, such as dimers or trimers
[0092] A number of cell lmes may act as suitable host cells for recombinant expression of the polypeptides of the present mvention Mammalian host cell lmes include, for example, COS cells, CHO cells, 293T cells, A431 cells, 3T3 cells, CV-1 cells, HeLa cells, L cells, BHK21 cells, HL-60 cells, U937 cells, HaK cells, Jurkat cells, as well as cell strains derived from in vitro culture of primary tissue and primary explants
[0093] Alternatively, it may be possible to recombinantly produce the polypeptides of the present invention in lower eukaryotes such as yeast or in prokaryotes Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomycespom.be, Kluyveromyces strains, and Candida strains Potentially suitable bactenal strains include Escherichia coli, Bacillus subtilis, and Salmonella typhimunum If the polypeptides of the present mvention are made in yeast or bactena, it may be necessary to modify them by, for example, phosphorylation or glycosylation of appropnate sites, in order to obtain functionality Such covalent attachments may be accomplished using well-known chemical or enzymatic methods
[0094] Expression in bacteria may result in formation of inclusion bodies incorporating the recombinant protein Thus, refolding of the recombinant protein may be required in order to produce active or more active material Several methods for obtaining correctly folded heterologous proteins from bacterial inclusion bodies are known in the art These methods generally mvolve solubihzing the protein from the inclusion bodies, then denaturing the protein completely using a chaotropic agent When cysteine residues are present in the pnmary ammo acid sequence of the protein, it is often necessary to accomplish the refolding in an environment that allows correct formation of disulfide bonds (a redox system) General methods of refolding are disclosed in Kohno (1990) Meth Enzymol 185 187-95 EP 0433225, and patent application U S Ser No 08/163,877 describe other appropriate methods
[0095] The polypeptides of the present invention may also be recombinantly produced by operably linking the isolated polynucleotides of the present invention to suitable control sequences in one or more insect expression vectors, such as baculovirus vectors, and employing an insect cell expression system Materials and methods for baculovirus/Sf9 expression systems are commercially available in kit form (e g, the MaxBac® kit, Invitrogen, Carlsbad, CA)
[0096] GITR agonists, e g, GITRL protein, active fragments and/or fusion protein thereof, may be prepared by growing a culture transformed host cells under culture conditions necessary to express the desired protein Following recombinant expression in the appropriate host cells, the polypeptides of the present invention may then be purified from culture medium or cell extracts using known purification processes, such as gel filtration and ion exchange chromatography Soluble forms of GITR agonists, e g, GITRL protein, active fragments and/or fusion protein thereof, can be purified from conditioned media Membrane-bound forms of, e g, a GITRL protein of the invention can be purified by preparing a total membrane fraction from the expressing cell and extracting the membranes with a nomomc detergent such as Triton X-l 00 Purification may also include affinity chromatography with agents known to bind the polypeptides of the present invention These purification processes may also be used to punfy the polypeptides of the present mvention from other sources, mcludmg natural sources As previously described, GITR agomsts, e g, GITRL protein, active fragments
and/or fusion protein thereof, may also be expressed as a product of transgenic animals, e g, as a component of the milk of transgenic cows, goats, pigs, or sheep, which are characterized by somatic or germ cells containing a polynucleotide sequence encodmg the GITR agomsts
[0097] The methods that may be used to punfy GITR agomsts, e g, GITRL protein, active fragments and/or fusion protein thereof, are known to those skilled in the art For example, a GITRL protein of the invention may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Milbpore Pellicon ultrafiltration unit Following the concentration step, the concentrate can be applied to a punfication matrix such as a gel filtration medium Alternatively, an anion exchange resm can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) or polyetheyleneimme (PEI) groups The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein punfication Alternatively, a cation exchange step can be employed Suitable cation exchangers include vanous insoluble matnces compnsing sulfopropyl or carboxymethyl groups Sulfopropyl groups are preferred (e g, S-Sepharose® columns) The punfication of GITR agomsts, e g, GITRL protein, active fragments and/or fusion protein thereof, from culture supernatant may also include one or more column steps over such affinity resms as concanavahn A-agarose, hepann-toyopearl® or Cibacrom blue 3GA Sepharose®, or by hydrophobic interaction chromatography using such resms as phenyl ether, butyl ether, or propyl ether, or by lmmunoaffimty chromatography Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e g, silica gel having pendant methyl or other aliphatic groups, can be employed to further punfy the GITRL protein Affinity columns including antibodies to the GITRL protein can also be used in punfication in accordance with known methods Some or all of the foregomg punfication steps, in vanous combinations or with other known methods, can also be employed to provide a substantially punfied isolated recombinant protein Preferably, the isolated GITRL protein is punfied so that it is substantially free of other mammalian proteins
[0098] Alternatively, GITR agomsts, e g, GITRL protein, active fragments and/or fusion protein thereof, may also be recombmantly expressed in a form that
facihtates punfication For example, the polypeptides may be expressed as fusions with proteins such as maltose-binding protein (MBP), glutathione-5-transferase (GST), or thioredoxm (TRX) Kits for expression and punfication of such fusion proteins are commercially available from New England BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ), and Invitrogen, respectively GITR agomsts, e g, GITRL protein, active fragments and/or fusion protein thereof, can also be tagged with a small epitope and subsequently identified or punfied using a specific antibody to the epitope A preferred epitope is the FLAG epitope, which is commercially available from Eastman Kodak (New Haven, CT)
[0099] GITR agomsts, e g, GITRL protein, active fragments and/or fusion protein thereof, may also be produced by known conventional chemical synthesis Methods for chemically synthesizing such polypeptides are well known to those skilled in the art Such chemically synthetic polypeptides may possess biological properties in common with the natural, punfied polypeptides, and thus may be employed as biologically active or immunological substitutes for the natural polypeptides
[0100] GITR agomsts, e g, GITRL protein, active fragments and/or fusion protein thereof, also encompass molecules that are structurally different from the disclosed polypeptides (e g, which have a slightly altered sequence), but which have substantially the same biochemical properties as the disclosed polypeptides (e g, are changed only in functionally nonessential ammo acid residues) Such molecules include naturally occurnng allelic vanants and deliberately engineered vanants containing alterations, substitutions, replacements, insertions, or deletions Techniques for such alterations, substitutions, replacements, insertions, or deletions are well known to those skilled in the art In some embodiments, the GITRL polypeptide moiety is provided as a vanant GITRL polypeptide having mutations in the naturally occurnng GITRL sequence (wild type) that results in a GITRL sequence more resistant to proteolysis (relative to the nonmutated sequence)
[0101] The methods disclosed herein for the generation of GITR agonists, e g, GITRL, active fragments thereof and/or fusion proteins thereof may be used to generate GITR antagonists, especially soluble GITR proteins, active fragments thereof and/or fusion proteins thereof One of skill in the art will recognize that
to generate GITR antagonists, e g, soluble GITR, active fragments thereof, and/or fusion proteins thereof, all that would be required is the nucleic acid sequence or ammo acid sequence of GITR, both of which are known Using these sequences, GITR antagonists, e g, soluble GITR, active fragments thereof, and/or fusion proteins thereof, may be generated using recombinant DNA techniques and/or chemical synthesis, as described above
Anti-GITRL Antibodies
[0102] In other embodiments, the invention provides GITR antagonists as antibodies, or antigen bindmg fragments thereof, that specifically bmd to GITRL, preferably, mammalian (e g, murine) GITRL, and neutralize GITR activity
[0103] One of skill in the art will recognize that as used herein, the term "antibody" refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) cham variable regions (abbreviated herein as VL) The VH and VL regions can be further subdivided into regions of hypervanabihty, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FR") The extent of the FRs and CDRs has been precisely defined (see, Kabat, E A, et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U S Department of Health and Human Services, NIH Publication No 91-3242, and Chothia, C et al (1987) J Mol Biol 196 901-917, which are hereby incorporated by reference) Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4
[0104] The antibody can further include a heavy and light cham constant region to thereby form a heavy and light immunoglobulin chain, respectively In one embodiment, the antibody is a tetramer of two heavy immunoglobulm chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are mter-connected by, e g, disulfide bonds The heavy cham constant region is comprised of three domains, CHI, CH2 and CH3 The light chain constant region is comprised of one domain, CL The variable region of the heavy and light chams contains a binding domain that interacts with an antigen
The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e g, effector cells) and the first component (Clq) of the classical complement system
[0105] Immunoglobulm refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulm genes The recognized human immunoglobulm genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes Full-length immunoglobulm "light chains" (about 25 Kd, or 214 ammo acids) are encoded by a variable region gene at the NH2-termmus (about 110 ammo acids) and a kappa or lambda constant region gene at the COOH-terminus Full-length immunoglobulin "heavy chains" (about 50 Kd, or 446 ammo acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e g, gamma (encodmg about 330 ammo acids) The immunoglobulin heavy chain constant region genes encode for the antibody class, l e, isotype (e g, IgM or IgGl) The antigen binding fragment of an antibody (or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to an antigen (e g, CD3) Examples of binding fragments encompassed within the term "antigen bmdmg fragment" of an antibody include (I) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, (n) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (in) a Fd fragment consisting of the VH and CHI domains, (IV) a Fv fragment consisting of the VL and VH domams of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 544-46), which consists of a VH domain, and (vi) an isolated complementarity determining region (CDR) Furthermore, although the two domams of the Fv fragment, VL and VH, are coded for by separate genes, they can be jomed, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pan- to form monovalent molecules (known as single chain Fv (scFv), see e g, Bird et al (1988) Science 242 423-26, and Huston et al (1988) Proc Natl Acad Sci USA
85 5879-83) Such single chain antibodies are also intended to be encompassed within the term "antigen bmding fragment" of an antibody These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utihty in the same manner as are mtact antibodies
[0106] One of skill in the art will recognize that the methods disclosed herein for generation of antibody molecules to the polypeptides of the present mvention, e g, murine GITRL, may also be used to generate antibody molecules to other proteins, e g, GITR or human GITRL Consequently, the methods for generating antibody molecules apply not only to the polypeptides of the present mvention as disclosed, but also to, for example, GITR or human GITRL
[0107] Antibody molecules to the polypeptides of the present invention, e g, neutralizing antibodies to murine GITRL, mcludmg but not limited to mouse GITRL and its homologs, may be useful in preventing or treating autoimmune disorders, inflammatory diseases, transplant rejections, and similar or related disorders Other antibody molecules e g, agomstic GITR antibodies, may be useful in the methods of the mvention for treating cancer, infectious diseases, and similar and related disorders Such antibody molecules may be produced by methods well known to those skilled in the art For example, monoclonal antibodies can be produced by generation of hybndomas in accordance with known methods Hybndomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELIS A), to identify one or more hybndomas that produce an antibody that specifically bmds with the polypeptides of the present mvention For example, GITRL proteins of the mvention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies that specifically react with the GITRL protein and which may inhibit bmding of ligands to the receptor, I e, GITR Similarly, GITR proteins may also be used to obtain polyclonal and monoclonal antibodies that specifically react with GITR The peptide lmmunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH) Additional peptide immunogens may be generated by replacing tyrosine residues with sulfated tyrosine residues Methods for synthesizing such peptides are known in the art, for example, as in Mernfield
(1963)/ Amer Chem Soc 85 2149-54, Krstenanskyetal (1987) FEBS Lett 21110 A full-length polypeptide of the present invention may be used as the immunogen, or, alternatively, antigenic peptide fragments of the polypeptides may be used An antigenic peptide of a polypeptide of the present invention comprises at least 7 continuous ammo acid residues and encompasses an epitope such that an antibody raised against the peptide forms a specific immune complex with the polypeptide Preferably, the antigenic peptide comprises at least 10 ammo acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 ammo acid residues, and most preferably at least 30 ammo acid residues
[0108] Monoclonal antibodies may be generated by other methods known to those skilled in the art of recombinant DNA technology As an alternative to preparing monoclonal antibody-secreting hybndomas, a monoclonal antibody to a polypeptide of the present invention may be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e g, an antibody phage display library) with a polypeptide of the present invention (e g, GITRL) or with GITR, to thereby isolate immunoglobulin library members that bmd to the polypeptides of the present mvention, or to GITR, respectively Techniques and commercially available kits for generating and screening phage display libraries are well known to those skilled in the art Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in the literature For example, 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 antibodies (for descriptions of combinatorial antibody display, see, e g, Sastry et al (1989) Proc Natl Acad Sci USA 86 5728, Huse et al (1989) Science 246 1275, Orlandi et al 1989 Proc Natl Acad Sci USA 86 3833) After immunizing an animal with an immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned Methods are generally known for obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as
pnmers to a conserved 3' constant region can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies (Lamck et al (1991) Biotechniques 11 152-56) A similar strategy can also been used to amplify human heavy and light cham variable regions from human antibodies (Larnck et al (1991) Methods Companion to Methods in Enzymology 2 106-10)
[0109] Polyclonal sera and antibodies may be produced by immunizing a suitable subject with a polypeptide of the present invention The antibody titer in the immunized subject may be mointored over time by standard techniques, such as with ELISA using immobilized marker protein If desired, the antibody molecules directed against a polypeptide of the present invention may be isolated from the subject or culture media and further purified by well-known techniques, such as protein A chromatography, to obtain an IgG fraction
[0110] Fragments of antibodies to the polypeptides of the present invention may be produced by cleavage of the antibodies in accordance with methods well known in the art For example, immunologically active Fab and F(ab')2 fragments may be generated by treating the antibodies with an enzyme such as pepsm
[0111] Additionally, chimeric, humanized, and single-cham antibodies to the polypeptides of the present mvention, comprising both human and nonhuman portions, may be produced using standard recombinant DNA techniques and/or a recombinant combinatorial immunoglobulin library Humanized antibodies may also be produced using transgemc mice which are incapable of expressmg endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light cham genes For example, human monoclonal antibodies (mAbs) directed against GITRL may be generated using transgemc mice carrymg the human immunoglobulin genes rather than murine immunoglobulin genes Splenocytes from these transgemc mice immunized with the antigen of mterest may then be used to produce hybndomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e g, Wood et al, WO 91/00906, Kucherlapati et al, WO 91/10741, Lonberg et al WO 92/03918, Kay et al, WO 92/03917, Lonberg et al (1994) Nature 368 856-59, Green et al (1994) Nat Genet 7 13-21, Morrison etal (1994) Proc Natl Acad Sci USA
81 6851-55, Braggeman (1993) Year Immunol 7 33-40, Tuaillon et al (1993) Proc Natl Acad Sci USA 90 3720-24, Bruggeman et al (1991) Eur J Immunol 21 1323-26)
[0112] Chimeric antibodies, including chimeric immunoglobulin chains, can be produced by recombinant DNA techniques known in the art For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al, International Patent Publication PCT/US86/02269, Akira, et al, European Patent Application 184,187, Tamguchi, European Patent Application 171,496, Morrison et al, European Patent Apphcation 173,494, Neuberger et al, WO 86/01533, Cabilly et al,US Patent No 4,816,567, Cabilly etal, European Patent Apphcation 125,023, Better etal (1988) Science 240 1041-43, Liu etal (1987) Proc Natl Acad Sa USA 84 3439-43, Liu et al (1987) J Immunol 139 3521-26, Sun etal (1987) Proc Natl Acad Sa USA 84 214-18, Nishimura et al (1987) Cancer Res 47 999-1005, Wood et al (1985) Nature 314 446-49, and Shaw etal (1988) J Natl Cancer Inst 80 1553-59)
[01131 An antibody or an immunoglobulin chain may be humanized by methods known in the art Humanized antibodies, including humanized immunoglobulin chains, may be generated by replacing sequences of the Fv variable region that are not directly mvolved in antigen binding with equivalent sequences from human Fv variable regions General methods for generating humanized antibodies are provided by Morrison (1985) Science 229 1202-07, Oi et al (19S6) BioTechmques 4 214, Queen etal, US Patent Nos 5,585,089, 5,693,761, 5,693,762, the contents of all of which are hereby incorporated by reference Those methods include isolating, manipulating, and expressmg the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain Sources of such nucleic acid sequences are well known to those skilled in the art and, for example, may be obtained from a hybndoma producing an antibody against a predetermined target The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector
[0114] Humamzed or CDR-grafted antibody molecules or immunoglobulins may be produced by CDR grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced See, eg, US Patent No 5,225,539, Jones et al (1986) Nature 321 552-25, Verhoeyan et al (1988) Science 239 1534, Beidler et al (1988) J Immunol 141 4053-60, Winter, U S Patent No 5,225,539, the contents of all of which are hereby incorporated by reference Winter describes a CDR-grafting method that may be used to prepare the humamzed antibodies of the present invention (UK Patent Application GB 2188638A, Winter, U S Patent No 5,225,539), the contents of which are hereby incorporated by reference All of the CDRs of a particular human antibody may be replaced with at least a portion of a nonhuman CDR, or only some of the CDRs may be replaced with nonhuman CDRs It is only necessary to replace the number of CDRs required for bmdmg of the humamzed antibody to a predetermined antigen
[0115] Monoclonal, chimeric and humanized antibodies that have been modified by, e g, deleting, adding, or substituting other portions of the antibody, e g, the constant region, are also within the scope of the mvention For example, an antibody can be modified as follows (l) by deleting the constant region, (n) by replacmg the constant region with another constant region, e g, a constant region meant to increase half-life, stability, or affinity of the antibody, or a constant region from another species or antibody class, or (in) by modifying one or more ammo acids in the constant region to alter, for example, the number of glycosylation sites, effector cell function, Fc receptor (FcR) bmdmg, complement fixation, etc
[0116] Methods for altering an antibody constant region are known in the art Antibodies with altered function, e g altered affinity for an effector ligand, such as FcR on a cell, or the CI component of complement, can be produced by replacmg at least one ammo acid residue in the constant portion of the antibody with a different residue (see, e g, EP 388,151 Al, U S 5,624,821 and U S 5,648,260, the contents of all of which are hereby incorporated by reference) Similar types of alterations to the murine (or other species') immunoglobulin may be applied to reduce or eliminate these functions Such alterations are known in the art
[0117] For example, it is possible to alter the affinity of an Fc region of an antibody (e g, an IgG, such as a human IgG) for an FcR (e g, Fc gamma Rl), or for Clq binding by replacmg the specified residue(s) with a residue(s) having an appropriate functionality on its side chain, or by introducing a charged functional group, such as glutamate or aspartate, or an aromatic nonpolar residue such as phenylalanine, tyrosine, tryptophan or alanine (see, e g, U S 5,624,821)
[0118] Anti-GITRL antibodies of the invention may be useful for isolating, purifying, and/or detecting GITRL polypeptides in supernatant, cellular lysate, or on the cell surface Antibodies disclosed in this mvention can be also used diagnosucally to monitor GITRL protein levels as part of a clinical testing procedure, or clinically to target a therapeutic modulator to a cell or tissue compnsmg the antigen of the GITRL antibody For example, a therapeutic such as a small molecule, or other therapeutic of the mvention can be linked to the GITRL antibody in order to target the therapeutic to the cell or tissue expressmg GITRL Neutralizing or nonneutrahzrng antibodies (preferably monoclonal antibodies) binding to GITRL protein may also be useful in the treatment of conditions involving disregulation of the immune system, e g, autoimmune diseases These neutralizing monoclonal antibodies maybe capable of blocking GITRL binding to GITR The present mvention further provides compositions compnsmg an antibody that specifically reacts with GITRL Similarly, anti-GITR antibodies may be useful in isolating, punfying and/or detecting GITR, diagnosucally monitoring GITR levels, or chmcally targeting a therapeutic modulator to a cell or tissue compnsmg GITR Agomstic antibodies to GITR (preferably monoclonal antibodies) may also be useful in the treatment of conditions involving disregulation of the immune system e g, cancer or infectious diseases These agomstic antibodies may be capable of inducing GITR activity Thus the present mvention further provides compositions compnsmg an antibody to GITR
GITRL Screening Assays
[0119] The polynucleotides and polypeptides of the present mvention may be used in screening assays to identify pharmacological agents or lead compounds for agents that are capable of modulating the activity of GITRL, and thereby GITR, in a cell or organism, and are thereby potential regulators of immune
responses For example, samples containing GITRL (either natural or recombinant) can be contacted with one of a plurality of test compounds (either biological agents or small orgamc molecules), and the activity of GITRL in each of the treated samples can be compared with the activity of GITRL in untreated samples or in samples contacted with different test compounds Such comparisons will determine whether any of the test compounds results in 1) a substantially decreased level of expression or activity of GITRL, thereby indicating an inhibitor of GITRL (e g, a compound that restores or enhances immune suppression), or 2) a substantially mcreased level of expression or activity of GITRL, thereby indicating an activator of GITRL (e g, a compound that reverses immune suppression) In one embodiment, the identification of test compounds capable of modulating GITRL activity is performed using high-throughput screening assays, such as BIACORE (Biacore International AB, Uppsala, Sweden), BRET (bioluminescence resonance energy transfer), and FRET (fluorescence resonance energy transfer) assays, as well as ELISA and cell-based assays
Small Molecules
[0120J Decreased GITR activity in an orgamsm (or subject) afflicted with (or at nsk for) autoimmune disorders, inflammatory diseases, or transplant rejection, or in a cell from such an orgamsm (or subject) mvolved in such disorders, may also be achieved through the use of small molecules (usually orgamc small molecules) that antagonize, l e, inhibit the activity of, GITR Novel antagonistic small molecules may be identified by the screening methods described above and may be used in the treatment methods of the present invention described below Conversely, mcreased GITR activity in an orgamsm (or subject) afflicted with (or at nsk for) cancer or infectious disease, or in a cell from such an orgamsm (or subject) involved in such disorders, may also be achieved through the use of small molecules (usually orgamc small molecules) that agonize, l e, enhance the activity of, GITR. Novel agonistic small molecules may be identified by the screening methods descnbed above and may be used in the methods of treating cancer and/or infectious disease as descnbed below
Methods for Diagnosing, Prognosing, and Mointonng the Progress of Autoimmune Disorders and Cancers
[0121] It is well known in the art that immunological mechanisms studied in
animal models, particularly murine models, may be and often are, translatable to
the human immune system As such, although the Examples disclosed herein
demonstrate the role of GITR in immune suppression by CD4+CD25+ regulatory
cells in a murine model, the disclosed methods for diagnosing, prognosing, and
mointonng disorders related to disregulation of the immune system, e g,
autoimmune disorders, inflammatory disorders and transplant rejection, and
cancer and infectious disease, will be particularly useful for diagnosing,
prognosing and mointonng such disorders in humans In practicing the disclosed
methods, a skilled artisan will recognize that the human homologs of GITR and
GITRL, as well as human GITR agomsts and antagonists, may be used in the
claimed methods of diagnosing, prognosing, and mointonng such disorders m
humans
[0122] The present invention provides methods for diagnosing, prognosing, and mointonng the progress of autoimmune disorders in a subject (e g, that directly or indirectly involve mcreases in the levels of GITRL) by detecting an upregulation of GITR activity, e g, by detecting the upregulation of GITRL, mcludmg but not limited to the use of such methods in human subjects One of skill in the art will recognize that these methods can apply to mflammatory diseases and transplant rejection as well These methods may be performed by utilizing prepackaged diagnostic kits compnsing at least one of the group compnsmg GITRL polynucleotide or fragments thereof, GITRL polypeptide or portions thereof (mcludmg fusion proteins thereof), or antibodies to GITRL polypeptides or denvatives thereof, or modulators of GITRL polynucleotides and/or polypeptides as descnbed herein, which may be convemently used, for example, in a clinical setting In addition, one of skill in the art would recognize that the upregulation of GITRL could also be detected by indirect methods, such as counting the number of immune cells
[0123] "Diagnostic" or "diagnosing" means identifying the presence or absence of a pathologic condition Diagnostic methods include detecting upregulation of GITRL by determining a test amount of GITRL gene product (e g, mRNA,
cDNA, or polypeptide, including fragments thereof) in a biological sample from a subject (human or nonhuman mammal), and companng the test amount with a normal amount or range (1 e, an amount or range from an individuals) known not to suffer from autoimmune disorders) for the GITRL gene product Although a particular diagnostic method may not provide a definitive diagnosis of autoimmune disorders, it suffices if the method provides a positive mdication that aids in diagnosis
[0124] The present invention also provides methods for prognosing such autoimmune disorders by detecting the upregulation of GITR activity, e g, by detecting upregulation of GITRL "Prognostic" or "prognosing" means predicting the probable development and/or seventy of a pathologic condition Prognostic methods include determining the test amount of a GITRL gene product in a biological sample from a subject, and companng the test amount to a prognostic amount or range (1 e, an amount or range from individuals with varying seventies of autoimmune disorders) for the GITRL gene product Vanous amounts of the GITRL gene product in a test sample are consistent with certain prognoses for autoimmune disorders The detection of an amount of GITRL gene product at a particular prognostic level provides a prognosis for the subject
[0125] The present invention also provides methods for monitoring the progress or course of such autoimmune disorders by detecting the upregulation of GITR activity, e g, by detecting upregulation of GITRL Monitoring methods include determining the test amounts of a GITRL gene product in biological samples taken from a subject at a first and second time, and companng the amounts A change in amount of GITRL gene product between the first and second times indicates a change in the course of autoimmune disorders, with a decrease in amount indicating remission of autoimmune disorders, and an increase in amount indicating progression of autoimmune disorders Such monitoring assays are also useful for evaluating the efficacy of a particular therapeutic intervention in patients being treated for autoimmune disorders
[0126] Increased expression of GITRL in methods outlined above can be detected in a vanety of biological samples, including bodily fluids (e g, whole blood, plasma, and urine), cells (e g, whole cells, cell fractions, and cell
extracts), and tissues Biological samples also include sections of tissue, such as biopsies and frozen sections taken for histological purposes Preferred biological samples include blood, plasma, lymph, tissue biopsies, urine, CSF (cerebrospinal fluid), synovial fluid, and BAL (bronchoalveolar lavage) It will be appreciated that analysis of a biological sample need not necessarily require removal of cells or tissue from the subject For example, appropriately labeled agents that bind GITRL gene products (e g, antibodies, nucleic acids) can be administered to a subject and visualized (when bound to the target) using standard imaging technology (e g, CAT, NMR (MRI), and PET)
[0127] In the diagnostic and prognostic assays of the present invention, the GITRL gene product is detected and quantified to yield a test amount The test amount is then compared with a normal amount or range An amount significantly above the normal amount or range is a positive sign in the diagnosis of autoimmune disorders Particular methods of detection and quantitation of GITRL gene products are described below
[0128] Normal amounts or baselme levels of GITRL gene products can be detennmed for any particular sample type and population Generally, baseline (normal) levels of GITRL protein or mRNA are detennmed by measuring the amount of GITRL protein or mRNA in a biological sample type from normal (l e, healthy) subjects Alternatively, normal values of GITRL gene product can be detennmed by measuring the amount in healthy cells or tissues taken from the same subject from which the diseased (or possibly diseased) test cells or tissues were taken The amount of GITRL gene product (either the normal amount or the test amount) can be detennmed or expressed on a per cell, per total protein, or per volume basis To determine the cell amount of a sample, one can measure the level of a constitutively expressed gene product or other gene product expressed at known levels in cells of the type from which the biological sample was taken
[0129] It will be appreciated that the assay methods of the present invention do not necessanly require measurement of absolute values of GITRL gene product because relative values are sufficient for many applications of these methods It will also be appreciated that in addition to the quantity or abundance of GITRL gene products, vanant or abnormal GITRL gene products or their expression
patterns (e g, mutated transcripts, truncated polypeptides) may be identified by comparison to normal gene products and expression patterns
[0130] The diagnostic, prognostic, and mointonng assays of the present invention involve detecting and quantifying GITRL gene products in biological samples GITRL gene products include GITRL mRNA and GITRL polypeptide, and both can be measured using methods well known to those skilled in the art
[0131] For example, GITRL mRNA can be directly detected and quantified using hybndization-based assays, such as Northern hybndization, in situ hybndization, dot and slot blots, and oligonucleotide arrays Hybndization-based assays refer to assays in which a probe nucleic acid is hybndized to a target nucleic acid In some formats, the target, the probe, or both are immobilized The immobilized nucleic acid may be DNA, RNA, or another oligonucleotide or polynucleotide, and may compnse naturally or nonnaturally occurnng nucleotides, nucleotide analogs, or backbones Methods of selecting nucleic acid probe sequences for use in the present invention are based on the nucleic acid sequence of GITRL and are well known in the art
[0132] Alternatively, GITRL mRNA can be amplified before detection and quantitation Such amplification-based assays are well known in the art and include polymerase chain reaction (PCR), reverse-transcnption-PCR (RT-PCR), PCR-enzyme-hnked immunosorbent assay (PCR-ELISA), and hgase chain reaction (LCR) Primers and probes for producmg and detecting amplified GITRL gene products (e g, mRNA or cDNA) may be readily designed and produced without undue experimentation by those of skill in the art based on the nucleic acid sequence of GITRL Amplified GITRL gene products may be directly analyzed, for example, by gel electrophoresis, by hybndization to a probe nucleic acid, by sequencing, by detection of a fluorescent, phosphorescent, or radioactive signal, or by any of a vanety of well-known methods In addition, methods are known to those of skill in the art for increasing the signal produced by amplification of target nucleic acid sequences One of skill in the art will recognize that whichever amplification method is used, a vanety of quantitative methods known in the art (e g, quantitative PCR) may be used if quantitation of GITRL gene products is desired
[0133] GITRL polypeptide (or fragments thereof) can be detected using various well-known immunological assays employing the anti-GITRL antibodies described above Immunological assays refer to assays that utilize an antibody (e g, polyclonal, monoclonal, chimeric, humanized, scFv, and fragments thereof) that specifically binds to GITRL polypeptide (or a fragment thereof) Such well-known immunological assays suitable for the practice of the present invention include ELIS A, radioimmunoassay (RIA), lmmunoprecipitation, immunofluorescence, fluorescence-activated cell sorting (FACS), and Western blotting GITRL polypeptide can also be detected using labeled GITR
[01341 One of skill in the art will understand that the aforementioned methods can be applied to autoimmune disorders and other disorders (such as inflammatory diseases), including, but not limited to, rheumatoid arthritis, osteoarthritis, multiple sclerosis, autoimmune gastritis, systemic lupus erythematosus, psoriasis and other inflammatory dermatoses, type I diabetes, asthma, allergy, and inflammatory bowel diseases, including Crohn's disease and ulcerative colitis
[0135} One of skill in the art will also recognize that the aforementioned methods or variations thereupon can also be used for diagnosing, prognosing, and monitoring the progress of various cancers and infectious diseases in a subject (e g, that directly or indirectly involve decreases in the levels of GITRL) by detecting a downregulation of GITR activity, e g, by detecting the downregulation of GITRL, including but not limited to the use of such methods in human subjects
Uses of GITRL and Related Molecules in Therapy
[0136} Applicants believe they are the first to recognize that binding of GITR on effector T cells by GITRL, or other GITR agonists, provides a costrmulatory signal to effector T cells, wherein such signal renders the effector T cells less susceptible to suppression by CD4+CD25+ regulatory T cells and increases the ability of effector T cells to proliferate in response to anti-CD3 or other activating signals Although the murine model was used to uncover the mechanism, it is well known in the art that immunological mechanisms studied in murine models, may be and often are, translatable to the human immune system As such, the
disclosed methods for using GITRL and related molecules, 1 e , GITR agonists or GITR antagonists, to treat disorders related to the disregulation of the of the immune system, e g, autoimmune disorders, inflammatory disorders and transplant rejection, and cancer and infectious disease, will be particularly useful for treating such disorders in humans In practicmg the disclosed methods, a skilled artisan will recognize that the human homologs of GITR and GITRL, as well as human GITR agonists and antagonists, may be used in the claimed methods of using GITRL and GITRL-related proteins, 1 e, GITR agonists and antagonists, in treating autoimmune disorders, inflammatory disorders and transplant rejection, and cancer and infectious diseases in humans
[01371 The GITRL-related molecules disclosed herein, 1 e, GITR agonists and antagonists, mcludmg modulators of GITRL polynucleotide and/or polypeptide activity identified using the methods described above, can be used in vitro, ex vivo, or incorporated into pharmaceutical compositions and administered to individuals in vivo to treat, for example, autoimmune disorders by administration of a GITR antagonist (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, neutralizing anti-GITR antibodies, and/or neutralizing anti-GITRL antibodies), or, e g, cancers by administration of a GITR agonist (e g, GITRL polynucleotides, GITRL polypeptides, or fusion proteins thereof, agonistic small molecules and/or agomstic anti-GITR antibodies) Such GITRL and/or related molecules (mcludmg modulators) include, but are not limited to, mouse GITRL and its homologs (and antibodies to such molecules), and such homologs include, but are not limited to, human GITRL Several pharmacogenomic approaches to be considered in determining whether to administer GITRL and/or GITRL related molecules are well known to one of skill in the art and include genome-wide association, candidate gene approach, and gene expression profiling A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration (e g, oral compositions generally include an inert diluent or an edible earner) Other nonlimiting examples of routes of administration include parenteral (e g, intravenous), intradermal, subcutaneous, oral (e g, inhalation), transdermal (topical), transmucosal, and rectal administration The pharmaceutical compositions compatible with each intended route are well known in the art
[0138] GITR agomsts or antagonists may be used as pharmaceutical compositions when combined with a pharmaceutically acceptable earner Such a composition may contain, in addition to the GITR agonists or antagonists and earner, vanous diluents, fillers, salts, buffers, stabilizers, solubilizers, and other matenals well known in the art The term "pharmaceutically acceptable" means a nontoxic material that does not interfere with the effectiveness of the biological activity of the active mgredient(s) The charactenstics of the earner will depend on the route of administration
[0139] The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, EL-6, IL-7, IL-8, IL-9, IL-10, DL-11, EL-12, DL-14, IL-15, G-CSF, stem cell factor, and erythropoietin The pharmaceutical composition may also include anticytokine antibodies as desenbed in more detail below The pharmaceutical composition may contain thrombolytic or antithrombotic factors such as plasminogen activator and Factor Vm The pharmaceutical composition may further contain other anti-inflammatory agents as desenbed in more detail below Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with GITR agomsts or antagonists, or to minimize side effects caused by the GITR agomsts or antagonists Conversely GITR agomsts or antagonists may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or antithrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or antithrombotic factor, or anti-inflammatory agent
[0140] The pharmaceutical composition of the invention may be in the form of a liposome in which GITR agomsts or antagonists are combmed, in addition to other pharmaceutically acceptable earners, with ampmpathic agents such as lipids that exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution Suitable lipids for liposomal formulation include, without limitation, monoglycendes, diglycendes, sulfatides, lysolecithin, phospholipids, sapomn, bile acids, etc Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, mUS Pat No 4,235,871, US Pat No 4,501,728, US Pat No
4,837,028, and U S Pat No 4,737,323, all of which are hereby incorporated by reference
[0141] As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, e g, amelioration of symptoms of, healing of, or increase in rate of healing of such conditions When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether admmistered in combmation, serially or simultaneously
[0142] In practicing the method of treatment or use of the present invention, a therapeutically effective amount of a GITR agomst (e g, a GITRL polynucleotide or GITRL polypeptide expressed therefrom) or a GITR antagonist (eg,a neutralizing anti-GITRL antibody or a neutralizing anti-GITR antibody) is administered to a subject, e g, a mammal (e g, a human) A GITR agomst or antagonist may be administered in accordance with the method of the mvention either alone or in combmation with other therapies, such as treatments employing cytokines, lymphokmes or other hematopoietic factors, or anti-inflammatory agents When coadministered with one or more agents, GITR agomsts or antagonists may be administered either simultaneously with the second agent, or sequentially If admmistered sequentially, the attending physician will decide on the appropriate sequence of administering, e g, a GITRL polypeptide (or fusion protein thereof) or neutralizing anti-GITRL antibody in combmation with other agents
[0143] When a therapeutically effective amount of a GITR agomst or antagonist is admmistered orally, the binding agent will be in the form of a tablet, capsule, powder, solution or elixir When admmistered in tablet form, the pharmaceutical composition of the mvention may additionally contain a sohd earner such as a gelatm or an adjuvant The tablet, capsule, and powder contain from about 5 to 95% binding agent, and preferably from about 25 to 90% bmdmg agent When administered in liquid form, a liquid earner such as water, petroleum, oils of animal or plant ongm such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added The liquid form of the pharmaceutical
composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol When administered in liquid form, the pharmaceutical composition contains from about 0 5 to 90% by weight of the binding agent, and preferably from about 1 to 50% by weight of the binding agent
[0144] When a therapeutically effective amount of a GITR agonist or antagonist is administered by intravenous, cutaneous or subcutaneous injection, the GITR agonist or antagonist will be in the form of a pyrogen-free, parenterally acceptable aqueous solution The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill of those in the art A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contam, in addition to the GITR agomst or antagonist, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art The pharmaceutical composition of the present invention may also contam stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art
[0145] The amount of a GITR agomst or antagonist in the pharmaceutical composition of the present mvention will depend upon the nature and seventy of the condition bemg treated, and on the nature of pnor treatments that the patient has undergone Ultimately, the attending physician will decide the amount of GITR agomst or antagonist with which to treat each individual patient Initially, the attending physician will administer low doses of GITR agomst or antagonist and observe the patient's response Larger doses of GITR agomst or antagonist may be administered until the optimal therapeutic effect is obtamed for the patient, and at that pomt the dosage is not generally mcreased further It is contemplated that the various pharmaceutical compositions used to practice the method of the present mvention should-contam about 0 1 fig to about 100 mg of e g, GITRL polypeptide or neutralizing anti-GITRL antibody per kg body weight
[0146] The duration of intravenous (l v ) therapy using a pharmaceutical composition of the present mvention will vary, depending on the seventy of the
disease being treated and the condition and potential idiosyncratic response of each individual patient It is contemplated that the duration of each application of the GITR agomst or antagonist may be in the range of 12 to 24 hours of continuous 1 v admmistration Also contemplated is subcutaneous (s c) therapy using a pharmaceutical composition of the present mvention These therapies can be administered daily, weekly, or, more preferably, biweekly, or monthly It is also contemplated that where the GITR agomst or antagonist is a small molecule, the therapies may be administered daily, twice a day, three times a day, etc Ultimately the attending physician will decide on the appropriate duration of 1 v or s c therapy, or therapy with a small molecule, and the tuning of admmistration of the therapy, using the pharmaceutical composition of the present invention
[0147] The polynucleotides and proteins of the present mvention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below Uses or activities described for proteins of the present mvention may be provided by administration or use of such proteins or by admmistration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA)
Uses of GITRL and Other GITR Agomsts to Enhance an Immune Response [0148] In one aspect, the present mvention provides methods for mcreasmg immune cell, e g, T cell (e g, an effector T cell) proliferation by contacting an immune cell or a population of immune cells with a GITR agomst, e g, a GITRL polynucleotide or polypeptide of the mvention (e g, a fusion protein thereof) and/or an agonistic anti-GITR antibody, which potentiates or enhances the activity of GITR. These methods are based, at least in part, on the finding that an agonistic anti-GITR antibody reversed CD4+CD25+ T cell-mediated suppression of CD4+CD25" T cell proliferation (Example 5) The methods are also based, in part, on the finding that GITR binding by, e g, GITRL or an agonistic anti-GITR antibody, induces proliferation of effector T cells (e g, CD4+CD25" and CD8+ T cells) (Example 9 and Example 13) Applicants also showed that GITR binding by GITRL provides a costimulatory signal to effector T cells (e g, CD4+ and CD8+ T cells), thereby mcreasmg the abilities of effector T cells to overcome

suppression mediated by CD4+CD25+ regulatory T cells and proliferate in response to anti-CD3 (Examples 11 and 13), 1 e, binding of GITR expressed on effector T cells by GITR agonists (e g, GITRL polypeptide, active fragments thereof, and/or agonistic anti-GITR antibody) renders effector T cells less susceptible to suppression by CD4+CD25+ regulatory T cells Accordingly, GITR agonists that stimulate the GITR activity in effector T cells can be used by themselves or in combmation with an antigen, e g, as an adjuvant (e g, a vaccine adjuvant), to upregulate an immune response in vivo, e g, for use in treating cancer and infectious disorders
[0149] In one embodiment, GITR agonists (e g, GITRL polynucleotides, polypeptides, active fragments and/or fusion proteins thereof, agomstic small molecules and/or agomstic anti-GITR antibodies) may be useful in the treatment of various immune deficiencies and disorders (including severe combmed immunodeficiency (SOD)), e g, in upregulating growth and proliferation of T cells These immune deficiencies may be genetic or be caused by viral (e g, HTV) as well as bacterial or fungal infections, or may result from autoimmune disorders More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HTV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp, malaria spp and various fungal infections such as candidiasis Of course, in this regard, a protein of the present mvention may also be useful where a boost to the immune system generally may be desirable, l e, in the treatment of cancer
[0150] Upregulation of antigen presenting cell (APC) antigens (e g, upregulation of B7 1, B7 2, and B7 3), as a means of upregulating immune responses, may also be useful in therapy Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response For example, enhancing an immune response through stimulating dendritic cell antigen presenting functions may be useful in cases of viral infection In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms antigen presenting molecules, e g, dendntic cell antigens, systemically
[0151] Alternatively, antiviral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells ex vivo with viral antigen-pulsed professional APCs (e g, B cells, macrophages and/or dendritic cells) and GITR agonists (e g, GITRL polynucleotides, polypeptides, active fragments and/or fusion proteins thereof, agonistic small molecules and/or agomstic anti-GITR antibodies) GITR agonists (e g, GITRL polynucleotides, polypeptides, active fragments and/or fusion proteins thereof, and/or agomstic anti-GITR antibodies) may be supplied either as soluble protein or as expressed by the APCs Another method of enhancing antiviral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a GITRL protein of the present mvention as described herein, such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient The infected cells would now be capable of delivering a cosumulatory signal to effector T cells in vivo, 1 e, expression of GITRL protein, or an active fragment thereof, by the mfected cell, and bmdmg of such GITRL protein to GITR on effector T cells could render the effector T cells less susceptible to suppression by CD4+CD25+ regulatory T cells
[0152] In another application, upregulation or enhancement of an APC antigen function may be useful in the induction of tumor immunity Tumor cells (e g, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, and carcinoma) transfected with a nucleic acid encoding at least one peptide of the present mvention can be administered to a subject to overcome tumor-specific tolerance in the subject If desired, the tumor cell can be transfected to express a combination of peptides For example, tumor cells obtamed from a patient can be transfected ex vivo with an expression vector directing the expression of a GITR agomst (e g, GITR polypeptides, active fragments and/or fusion proteins thereof, and/or agomstic anti-GITR antibodies), alone or in combination with a peptide having B7 2-like activity alone, or in conjunction with a peptide having B7 1-hke activity, etc The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo
[0153] The presence of a GITR agomst (e g, a GITRL polypeptide, active fragments and/or fusion proteins thereof, an agomstic small molecule and/or an
agomstic anti-GITR antibody), in combination with a peptide having the activity of an APC antigen (e g, B7 1, B7 2, etc) on the surface of the tumor cell provides the necessary costimulatory signals to T cells to induce a T cell-mediated immune response against the transfected tumor cells In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acids encodmg all or a portion (e g, a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and 02 microglobulin protein or an MHC class II a chain protein and an MHC class II (5 chain protein (or corresponding human HLA nucleic acids) to thereby express MHC class I or MHC class II proteins (or corresponding HLA molecules) on the cell surface Expression of the appropriate class I or class II MHC in conjunction with a GITR agomst (e g, a GITRL polypeptide, active fragments and/or fusion proteins thereof, and/or an agonistic anti-GITR antibody), and/or a peptide havmg the activity of an APC antigen (e g, B7 1, B7 2, etc ) induces a T cell-mediated immune response against the transfected tumor cell Optionally, a gene encodmg an antisense construct which blocks expression of an MHC class H-associated protein, such as the invariant cham, can also be cotransfected with a DNA encoding a GITR agomst (e g, a GITRL polypeptide, active fragment thereof, a fusion protein thereof, and/or an agomstic anti-GITR antibody) and/or a peptide having the activity of an APC antigen to promote presentation of tumor associated antigens and induce tumor specific immunity Thus, the induction of a T cell-mediated immune response in a human subject maybe sufficient to overcome tumor-specific tolerance in the subject
[0154] In other embodiments, GITR agomsts (e g, GITRL polypeptides, active fragments and/or fusion proteins thereof, fusion proteins thereof, agomstic small molecules, and/or agomstic anti-GrTR antibodies) of the mvention may be used as vaccme adjuvants Adjuvants are immune modulating compounds that have the ability to enhance and/or steer the development and profile of immune responses against various antigens that are themselves poorly immunogemc Cytokines and/or lymphokmes can be used as adjuvants The appropriate selection of adjuvants can induce good humoral and cellular immune responses that would not develop in the absence of adjuvant In particular, adjuvants have
significant effects in enhancing the immune response to subunit and peptide antigens in vaccmes Their stimulatory activity is also beneficial to the development of antigen-specific immune responses directed agamst protein antigens For a variety of antigens that require strong mucosal responses, high serum titers, induction of CTL (cytotoxic T lymphocytes) and vigorous cellular responses, adjuvant and cytokine/lymphokme combinations provide stimuli that are not provided by most antigen preparations
[0155] As used herein, the phrase "vaccme adjuvant" or "vaccme therapy" is intended to mean the use of a GITR agomst (e g, a GITRL polynucleotide, GITRL polypeptide, an active fragment thereof, a fusion protein thereof, and/or an agomstic anti-GITR antibody), in combination with an antigen (e g, viral, parasitic and bacterial polypeptides, proteins or peptides), or other antigens (e g, tumor or cancer cell polypeptides, proteins or peptides) or polynucleotides encodmg the antigen to enhance, suppress or otherwise modulate an immune response to the antigen For the purpose of this definition, "combination" shall mean use in conjunction with, simultaneous with (combined or uncombrned) or sequentially with an antigen
[0156] The term "vaccme adjuvant composition" refers to a vaccme adjuvant that additionally includes rmmunologically acceptable diluents or carriers in a conventional manner to prepare injectable liquid solutions or suspensions The vaccme adjuvant composition may additionally include agents that further enhance an immune response ehcited by a GITR agomst For example, the vaccme adjuvant composition may additionally include 3-O-deacylated monophosphoryl lipid A (MPL®, Conxa Corporation, Seattle, WA) or monophosphoryl lipid A and denvatives and analogs thereof MPL® can be used in a range of 1-100 ng/dose
[0157] The antigens used for vaccme therapy include proteins, peptides or polypeptides derived from immunogenic and nommmunogenic proteins, as well as any of the following saccharides, proteins, polynucleotides or oligonucleotides, or other macromolecular components, or fragments thereof As used in this section, a "peptide" comprises a series of at least six ammo acids and contains at least one antigenic determinant, while a "polypeptide" is a longer molecule than a peptide, but does not constitute a full-length protein As used
herein, a "fragment" comprises a portion, but less than all of a saccharide, protein, polynucleotides or oligonucleotide, or other macromolecular components
[0158] As used herein, the term "effective adjuvanting amount" means a dose of the combination of adjuvants described herein, which is suitable to elicit an increased immune response in a vertebrate host The particular dosage will depend in part upon the age, weight and medical condition of the host, as well as on the method of administration and the antigen
[0159] The vaccme adjuvant composition of the invention can be admimstered to a human or nonhuman vertebrate by a vanety of routes, including, but not limited to, intranasal, oral, vaginal, rectal, parenteral, intradermal, transdermal (see, e g, International application WO 98/20734, which is hereby incorporated by reference), intramuscular, intraperitoneal, subcutaneous, intravenous and intraarterial The amount of the antigen component or components of the antigemc composition will vary depending in part upon the identity of the antigen, as well as upon the age, weight and medical condition of the subject, as well as on the method of administration Again, suitable doses are readily determined by persons skilled in the art It is preferable, although not required, that the antigen and the combination of adjuvants be administered at the same fame The number of doses and the dosage regunen for the antigemc composition are also readily determined by persons skilled in the art In some instances, the adjuvant properties of the combination of adjuvants may reduce the number of doses needed or the time course of the dosage regimen
[0160] The combmations of adjuvants of this mvention are suitable for use in combmation with wide vanety of antigens from a wide vanety of pathogenic microorganisms, mcludmg but not limited to those from viruses, bactena, fungi or parasitic microorganisms that infect humans and nonhuman vertebrates, or from a cancer cell or tumor cell (e g, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, and carcinoma) The antigen may compnse peptides or polypeptides derived from proteins, as well as fragments of any of the following sacchandes, proteins, polynucleotides or oligonucleotides, cancer or tumor cells, or other macromolecular components In some instances, more than one antigen is included in the antigemc composition
[0161] Desirable viral vaccines containing the adjuvant combinations of this invention include those directed to the prevention and/or treatment of disease caused by, without limitation, Human lrnrnunodeficiency virus, Simian immunodeficiency virus, Respiratory syncytial virus, Parainfluenza virus types 1-3, Influenza virus, Herpes simplex virus, Human cytomegalovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human papillomavirus, poliovirus, rotavirus, cahciviruses, Measles virus, Mumps virus, Rubella virus, adenovirus, rabies virus, canine distemper virus, rinderpest virus, coronavrrus, parvovirus, infectious rhinotracheitis viruses, feline leukemia virus, feline infectious peritonitis virus, avian infectious bursal disease virus, Newcastle disease virus, Marek's disease virus, porcine respiratory and reproductive syndrome virus, equine arteritis virus and various Encephalitis viruses
[0162] Desirable bactenal vaccmes contaimng the adjuvant combmations of this invention include those directed to the prevention and/or treatment of disease caused by, without limitation, Haemophilus influenzae (both typable and nontypable), Haemophilus somnus, Moraxella catarrhahs, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis, Salmonella typhi, Salmonella typhimunum, Salmonella choleraesuis, Escherichia coh, Shigella, Vibrio cholerae, Corynebactenum diphthenae, Mycobacterium tuberculosis, Mycobacterium avium- Mycobacterium intracellular complex, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Clostridium tetani, Leptospira interrogans, Borreha burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae and Mycoplasma gallisepttcum
[0163] Desirable vaccmes against fungal pathogens containing the adjuvant combinations of this mvention include those directed to the prevention and/or treatment of disease caused by, without limitation, Aspergilhs, Blastomyces, Candida, Coccidiodes, Cryptococcus and Histoplasma
[0164] Desirable vaccmes against parasites containing the adjuvant combmations of this mvention include those directed to the prevention and/or treatment of disease caused by, without limitation, Leishmania major, Ascans, Tnchuns,
Giardia, Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii and Pneumocystis carinu
[0165] Desirable vaccines for eliciting a therapeutic or prophylactic anticancer effect in a vertebrate host, which contain the adjuvant combinations of this invention, include those utilizing a cancer antigen or tumor-associated antigen including, without limitation, prostate specific antigen (PSA), prostate-specific membrane antigen (PSMA), carcino-embryonic antigen (CEA), MUC-1, Her2, CA-125, MAGE-3, EGFR, HELP, GCC, CD66-C, prostasin, TMPRSS3, TADG 12andTADG15
[0166] In the case of HTV and SIV, the antigenic compositions comprise at least one protein, polypeptide, peptide or fragment derived from said virus In some instances, multiple HIV or SIV proteins, polypeptides, peptides and/or fragments are included in the antigenic composition
[0167] The adjuvant combmation formulations of this mvention are also suitable for inclusion as an adjuvant in polynucleotide vaccines (also known as DNA vaccines) Such vaccmes may further include facilitating agents such as bupivicarne (see U S Patent No 5,593,972, which is hereby incorporated by reference)
[0168] Methods of 1) stimulating antigen presenting cell function, e g, dendritic cell functions, 2) removing T cells from the patient, costunulating them ex vivo, and reintroducing them into the subject, 3) transfecting tumor cells to induce tumor immunity, and 4) using vaccme adjuvants are well known in the art (see, e g, Cerundolo et al (2004) Dendritic cells a journey from laboratory to clinic Nat Immunol 5(1) 7-10, Ko et al (2003) Immunotherapy of malignant diseases Int Arch Allergy Immunol 132 294-309, Valmon et al (1999) An antigen-targeted approach to adoptive transfer therapy of cancer Cancer Res 59 2167-73)
Uses of GITR Antagonists to Decrease Immune Cell Activity [0169] In yet another aspect, the invention features a method for mamtaimng the susceptibility of effector T cells, e g, CD4+ and CD8+ T cells, or a population thereof, to suppression by CD4+CD25+ regulatory T cells The method may
compnse contacting a population of T cells with a GITR antagomst (e g, GITRL inhibitory polynucleotides, an antagonistic small molecule, a neutralizing anti-GITR antibody, and/or a neutralizing anti-GITRL antibody) in an amount sufficient to inhibit the activity of the unmune cell or population Antagonists of GITR may also be administered to subjects for whom suppression of an immune response is desired These conditions include, e g, autoimmune disorders (e g, arthritic disorders), inflammatory diseases, or organ transplantation
[0170] These methods are based, at least in part, on the finding that reduction of GITR activity, e g, by using a neutralizing anti-GITRL antibody, restores CD4+CD25+-mediated suppression (Example 13), l e, neutralizing anti-GITRL antibody maintains the susceptibility of effector T cells, e g, CD4+ and CD8+ T cells, to suppression by CD4+CD25+ regulatory T cell Additionally, applicants have demonstrated that incubation of effector T cells with neutralizing anti-GiTRL antibody ameliorates disease in murine experimental autoimmune encephalitis (EAE) (Example 14) Accordingly, GITR antagonists, l e, molecules that inhibit GITR activity (e g, anti-GITRL antibodies) may be used to maintain the susceptibility of effector T cells to suppression by CD4+CD25+ T cells in vivo, e g, for treating or preventing unmune cell-associated pathologies, including transplant rejection, inflammatory diseases, and autoimmune disorders
[0171] The methods of using GITR antagonists may also be used inhibit the activity (e g, proliferation, differentiation, survival) of an effector T cell, and thus, can be used to treat or prevent a variety of immune disorders Nonhmiting examples of the disorders that can be treated or prevented include, but are not limited to, transplant rejection, autoimmune diseases (including, for example, diabetes melhtus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer, mtis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, spondyoarthropathy, ankylosing spondylitis, intrinsic 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, chrome active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, and allergy such as, atopic allergy Preferred disorders that can be treated using methods which comprise the administration of GITR antagonists, e g, a neutralizing GITRL antibody, include arthritic disorders (e g, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and ankylosing spondylitis (preferably, rheumatoid arthritis)), multiple sclerosis, type I diabetes, lupus (SLE), IBD, Crohn's disease, asthma, vasculitis, allergy, scleroderma and psoriasis
[0172] In another embodiment, GITR antagonists, alone or in combination with, other therapeutic agents as described herein (e g, TNF antagonists) can be used to treat multiple myeloma and related B lymphocytic mahgnancies (Brenne, A et al (2002) Blood99(10) 31'56-62)
[0173] Usmg GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL), it is possible to modulate immune responses in a number of ways Downregulation may be in the form of inhibiting or blocking an immune response already in progress, or may mvolve preventing the induction of an immune response The functions of activated T cells may be inhibited by enhancing the suppression of T cell responses, or by inducmg specific tolerance in T cells, or both Immunosuppression of T cell responses is generally an active, non-antigen-specific, process that requires continuous exposure of the T cells to the suppressive agent Tolerance, which involves inducmg nonresponsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolenzing agent has ceased Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolenzing agent
[0174] Downregulating or preventing one or more functions of an antigen presenting cell antigen (e g, B7 1), and thus preventing high level lymphokme
synthesis by activated T cells will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD) For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant The adrninistration of a GITR antagonist (e g, GITRL inhibitory polynucleotides, an antagonistic small molecule, a neutralizing anti-GITR antibody, and/or a neutralizing anti-GITRL antibody), in combination with a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomelic form of a peptide having B7 2 activity alone or in conjunction with a monomenc form of a peptide having an activity of another B lymphocyte antigen (e g, B7 1) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal Blocking B7 lymphocyte antigen function in this manner prevents cytokine synthesis by immune cells, such as effector T cells, and thus acts as an immunosuppressant Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject Induction of long-term tolerance by B7 lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combmation of B lymphocyte antigens
[0175] The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans Examples of appropriate systems which can be used include allogenic cardiac grafts in rats and xenogemc 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 (1992) Science 257 789-92 and Turka et al (1992) Proc Natl Acad Sci USA 89 11102-05 In addition, murine models of GVHD (see, e g, Paul ,ed, Fundamental Immunology, Raven Press, New York, 1989, pp 846-47) can be used to
determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease
[0176] Blocking the function of an APC antigen 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 and which promote the production of cytokines and autoantibodies mvolved in the pathology of the diseases Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms Administration of GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) in combination with reagents that block costunulation of T cells by disrupting receptor ligand interactions of APC antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines that may be mvolved in the disease process Additionally, GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) in combination with blocking reagents may induce antigen-specific tolerance of autoreactive T cells, which could lead to long-term relief from the disease The efficacy of these agents in preventing or alleviating autoimmune disorders can be determined using a number of well-charactenzed animal models of human autoimmune diseases Examples include murine experimental autoimmune encephalitis (EAE), systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes melhtus in NOD mice and BB rats, and murme experimental myasthema gravis (see, e g, Paul, ed, Fundamental Immunology, Raven Press, New York, 1989, pp 840-56)
[0177] In one embodiment, GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL), including pharmaceutical compositions thereof, are administered in combination therapy, I e, combmed with other agents, e g, therapeutic agents, that are useful for treating pathological conditions or disorders, such as immune disorders and inflammatory diseases The term "in combination" in this context means that the agents are given substantially contemporaneously, either simultaneously or sequentially If given sequentially,
at the onset of administration of the second compound, the first of the two compounds is preferably still detectable at effective concentrations at the site of treatment
[01781 For example, the combmation therapy can include one or more GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizmg antibodies to GITR and/or GITRL), coformulated with, and/or coadministered with, one or more additional therapeutic agents, e g, one or more cytokine and growth factor inhibitors, immunosuppressants, antiinflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below Furthermore, one or more GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizmg antibodies to GITR and/or GITRL) described herein may be used in combmation with two or more of the therapeutic agents described herein Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoidmg possible toxicities or complications associated with the various monotherapies Moreover, the therapeutic agents disclosed herein act on pathways that differ from the GITRL receptor pathway, and thus are expected to enhance and/or synergize with the effects of the GITR antagonists, 1 e wherein effector T cells maintain their susceptibility to suppression by CD4+CD25+ regulatory T cells
[0179] Preferred therapeutic agents used in combmation with a GITRL antagonist are those agents that interfere at different stages in the autoimmune and subsequent inflammatory response In one embodiment, one or more GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizmg antibodies to GITR and/or GITRL) described herein may be coformulated with, and/or coadministered with, one or more additional agents such as other cytokine or growth factor antagonists (e g, soluble receptors, peptide inhibitors, small molecules, ligand fusions), or antibodies or antigen binding fragments thereof that bmd to other targets (e g, antibodies that bmd to other cytokmes or growth factors, their receptors, or other cell surface molecules), and anti-inflammatory cytokmes or agonists thereof Nonlimiting examples of the agents that can be used in combmation with the GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small
molecules, and/or neutralizing antibodies to GITR and/or GITRL) descnbed herein, include, but are not limited to, antagomsts of one or more mterleukins (ILs) or their receptors, e g, antagonists of IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, and IL-22, antagonists of cytokines or growth factors or their receptors, such as tumor necrosis factor (TNF), LT, EMAP-II, GM-CSF, FGF and PDGF GITR antagomsts (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) can also be combined with inhibitors of, e g, antibodies to, cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7 1), CD86 (B7 2), CD90, or their ligands, including CD154 (gp39 or CD40L), or LFA-l/ICAM-1 and VLA-4/VCAM-1 (Yusuf-Makagiansar et al (2002) Med Res Rev 22 146-67) Preferred antagomsts that can be used in combination with GITR antagomsts (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) descnbedherein include antagomsts of IL-1, IL-12, TNFa, IL-15, IL-17, IL-18, and IL-22
[0180] Examples of those agents include IL-12 antagomsts, such as chimeric, humanized, human or in vtfro-generated antibodies (or antigen bmdmg fragments thereof) mat bmd to IL-12 (preferably human IL-12), e g, the antibody disclosed in WO 00/56772, IL-12 receptor inhibitors, e g, antibodies to human IL-12 receptor, and soluble fragments of the IL-12 receptor, e g, human IL-12 receptor Examples of IL-15 antagonists include antibodies (or antigen bmdmg fragments thereof) against EL-15 or its receptor, e g, chimeric, humanized, human or in vtfro-generated antibodies to human IL-15 or its receptor, soluble fragments of the IL-15 receptor, and IL-15-binding proteins Examples of IL-18 antagomsts include antibodies, e g, chimeric, humanized, human or in vifro-generated antibodies (or antigen binding fragments thereof), to human IL-18, soluble fragments of the IL-18 receptor, and IL-18 bmdmg proteins (IL-18BP, Mallet et al (2001) Ore Res 28) Examples of EL-1 antagomsts include Interleukin-1-converting enzyme (ICE) inhibitors, such as Vx740, EL-1 antagomsts, e g, EL-1RA (Amkinra, Amgen), sILlRII (Immunex), and anti-DL-1 receptor antibodies (or antigen bmdmg fragments thereof)
[0181] Examples of TNF antagonists include chimeric, humanized, human or in vzfro-generated antibodies (or antigen bmdmg fragments thereof) to TNF (e g, human TNFa), such as D2E7, (human TNFa antibody, U S 6,258,562), CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNFa antibody, Celltech/Pharmacia), cA2 (chimeric anti-TNFa antibody, RemicadeTM, Centocor), anti-TNF antibody fragments (e g, CPD870), soluble fragments of the TNF receptors, e g, p55 or p75 human TNF receptors or derivatives thereof, e g, 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, Enbrel™, Immunex, see, e g, Arthritis & Rheumatism (1994) 37 S295, J Invest Med (1996) 44 235A), p55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein (Lenercept)), enzyme antagonists, e g, TNFa converting enzyme (TACE) inhibitors (e g, an alpha-sulfonyl hydroxamic acid derivative, WO 01/55112, and N-hydroxyformamide TACE inhibitor GW 3333, -005, or -022), and TNF-bp/s-TNFR (soluble TNF bmding protein, see e g, Arthritis & Rheumatism (1996) 39(9)(supplement) S284, Amer J Physiol -Heart and Circulatory Physiology (1995) 268 37-42) Preferred TNF antagonists are soluble fragments of the TNF receptors, e g, p55 or p75 human TNF receptors or derivatives thereof, e g, 75 kdTNFR-IgG, and TNFa converting enzyme (TACE) inhibitors
[0182] In other embodiments, the GITR antagonists described herein can be administered in combination with one or more of the following EL-13 antagonists, e g, soluble EL-13 receptors (sEL-13) and/or antibodies agamst EL-13, IL-2 antagonists, e g, DAB 486-IL-2 and/or DAB 389-EL-2 (IL-2 fusion proteins, Seragen, see, e g, Arthritis & Rheumatism (1993) 36 1223), and/or antibodies to EL-2R, e g, anti-Tac (humanized anti-EL-2R, Protein Design Labs, Cancer Res (1990) 50(5) 1495-502) Yet another combination includes GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) in combination with nondepleting anti-CD4 inhibitors (DOEC-CE9 1/SB 210396, nondepleting pnmatized anti-CD4 antibody, EOEC/SmithKhne) Yet other preferred combinations include antagonists of the costimulatory pathway CD80 (B7 1) or CD86 (B7 2), including antibodies, soluble receptors or antagonistic ligands, as well as p-selectm glycoprotein ligand (PSGL), anti-inflammatory cytokines, e g, IL-4 (DNAX/Schenng), EL-10 (SCH 52000, recombinant EL-10
DNAX/Schermg), IL-13 and TGF-p, and agomsts thereof (e g, agomst antibodies)
[01831 In other embodiments, one or more GITR antagonists can be coformulated with, and/or coadministered with, one or more anti-mflammatory drugs, immunosuppressants, or metabolic or enzymatic inhibitors Nonlimiting examples of the drugs or inhibitors that can be used in combmation with the GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) described herein, include, but are not limited to, one or more of nonsteroidal antiinflammatory drug(s) (NS AIDs), e g, lbuprofen, temdap (see, e g, Arthritis & Rheumatism (1996) 39(9)(supplement) S280)), naproxen (see, e g, Neuro Report (1996) 7 1209-13), meloxicam, piroxicam, diclofenac, and indomethacin, sulfasalazine (see, e g, Arthritis & Rheumatism (1996) 39(9)(supplement) S281), corticosteroids such as prednisolone, cytokine suppressive anti-inflammatory drag(s) (CSAIDs), inhibitors of nucleotide biosynthesis, e g, inhibitors of purine biosynthesis, folate antagonists (e g, methotrexate (N-[4-[[(2,4-diamino-6-ptendmyl)methyl]methylammo]benzoyl]-L-glutamic acid), and inhibitors of pynmidme biosynthesis, e g, dihydroorotate dehydrogenase (DHODH) inhibitors (e g, leflunomide (see, e g, Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), S131, Inflammation Research (1996) 45 103-07) Preferred therapeutic agents for use in combmation with GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) include NSAIDs, CSAIDs, (DHODH) inhibitors (e g, leflunomide), and folate antagonists (e g, methotrexate)
[0184] Examples of additional inhibitors include one or more of corticosteroids (oral, inhaled and local injection), lmmunosuppresants, e g, cyclosporin, tacrolimus (FK-506), and mTOR inhibitors, e g, sirohmus (rapamycin) or rapamycin derivatives, e g, soluble rapamycin derivatives (e g, ester rapamycin derivatives, e g, CCI-779 (Eht (2002) Current Opinion Investig Drugs 3(8) 1249-53, Huang et al (2002) Current Opinion Investig Drugs 3(2) 295-304), agents which interfere with signaling by proinflammatory cytokmes such as TNFa or BL-1 (e g IRAK, NIK, IKK, p38 or MAP kinase inhibitors), COX2 inhibitors, e g, celecoxib, rofecoxib, and variants thereof, see, e g, Arthritis &
Rheumatism (1996) Vol 39, No 9 (supplement), S81), phosphodiesterase inhibitors, e g, R973401 (phosphodiesterase Type IV inhibitor, see, e g, Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), S282)), phosphohpase inhibitors, e g, inhibitors of cytosolic phosphohpase 2 (cPLA2) (e g, tnfluoromethyl ketone analogs (U S 6,350,892)), inhibitors of vascular endothelial cell growth factor or growth factor receptor, e g, VEGF inhibitor and/or VEGF-R inhibitor, and inhibitors of angiogenesis Preferred therapeutic agents for use in combination with GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) are lmmunosuppresants, e g, cyclosporin, tacrolimus (FK-506), mTOR inhibitors, e g, sirolimus (rapamycin) or rapamycin derivatives, e g, soluble rapamycin derivatives (e g, ester rapamycin derivatives, e g, CCI-779), COX2 inhibitors, e g, celecoxib and variants thereof, and phosphohpase inhibitors, e g, inhibitors of cytosolic phosphohpase 2 (cPLA2), e g, tnfluoromethyl ketone analogs
[0185] Additional examples of therapeutic agents that can be combmed with a GrTRL antagonist include one or more of 6-mercaptopurmes (6-MP), azathioprme sulphasalazine, mesalazme, olsalazine
chloroquimne/hydroxychloroqume, pencillamine, aurothiornalate (intramuscular and oral), azathioprme, cocmcine, beta-2 adrenoreceptor agomsts (salbutamol, terbutahne, salmeteral), xanthines (theophylline, arninophylhne), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, mycophenolate mofetil, adenosme agomsts, antithrombotic agents, complement inhibitors, and adrenergic agents
[0186] The use of the GITR antagonists disclosed herein in combmation with other therapeutic agents to treat or prevent specific immune disorders is discussed in further detail below
[0187] Nonhmiting examples of agents for treating or preventing arthritic disorders (e g, rheumatoid arthritis, inflammatory arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis), with which a GITR antagonists can be combined include one or more of the following IL-12 antagonists as described herein, NSAIDs, CSAIDs, TNFs, e g, TNFa, antagonists as described herein, nondepleting anti-CD4 antibodies as described
herein, IL-2 antagonists as descnbed herein, anti-inflammatory cytokines, e g, IL-4, IL-10, IL- 13 and TGFa, or agomsts thereof, EL-1 or IL-1 receptor antagomsts as descnbed herein, phosphodiesterase inhibitors as descnbed herein, COX-2 inhibitors as descnbed herein, lloprost (see, e g, Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), S82), methotrexate, thalidomide (see, e g, Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), S282) and thalidomide-related drugs (e g, Celgen), leflunomide, inhibitor of plasminogen activation, e g, tranexamic acid, see, e g, Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), S284), cytokine inhibitor, e g, T-614, see, e g, Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), S282), prostaglandin El (see, e g, Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), S282), azathiopnne (see, e g , Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), S281), an inhibitor of mterleukm-1 converting enzyme (ICE), zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase zap-70 or lck), an inhibitor of vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor as descnbed herein, an inhibitor of angiogenesis as descnbed herein, corticosteroid anti-inflammatory drugs (e g, SB203580), TNF-convertase inhibitors, IL-11 (see, e g, Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), S296), IL-13 (see, e g , Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), S308), IL-17 inhibitors (see, e g, Arthritis & Rheumatism (1996) Vol 39, No 9 (supplement), SI20), gold, penicillamine, chloroquine, hydroxychloroquine, chlorambucil, cyclophosphamide, cyclosponne, total lymphoid uradiation, antithymocyte globulin, CD5-toxins, orally administered peptides and collagen, lobenzant disodium, cytokine regulating agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc), ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302, Isis Pharmaceuticals, Inc), soluble complement receptor 1 (TP10, T Cell Sciences, Inc ), prednisone, orgotem, glycosaminoglycan polysulphate, minocycline, anti-IL2R antibodies, manne and botanical lipids (fish and plant seed fatty acids, see, eg,DeLucaetal (1995) Rheum Dis Clin North Am 21 759-77), auranofin, phenylbutazone, meclofenamic acid, flufenamic acid, intravenous immune globulin, zileuton, mycophenohc acid (RS-61443), tacrolimus (FK-506), sirolimus (rapamycm), amipnlose (therafectin), cladnbme (2-chlorodeoxyadenosine), and azanbine Preferred combinations include one or
more GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) in combmation with methotrexate or leflunomide, and in moderate or severe rheumatoid arthritis cases, cyclosponne
[0188] Preferred examples of inhibitors to use in combmation with GITR antagonists to treat arthritic disorders include TNF antagonists (e g, chimeric, humanized, human or in vtfro-generated antibodies, or antigen bmdmg fragments thereof, that bind to TNF, soluble fragments of a TNF receptor, e g, p55 or p75 human TNF receptor or derivatives thereof, e g, 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, Enbrel™), p55 kD TNF receptor-IgG fusion protein, TNF enzyme antagonists, e g, TNFa converting enzyme (TACE) inhibitors), antagonists of IL-12, IL-15, IL-17, IL-18, TL-22, T cell and B cell-depleting agents (e g, anti-CD4 or anti-CD22 antibodies), small molecule inhibitors, e g, methotrexate and leflunomide, sirohmus (rapamycin) and analogs thereof, e g, CCI-779, cox-2 and cPLA2 mhibitors, NSAIDs, pS8 mhibitors, TPL-2, Mk-2 and NFkb inhibitors, RAGE or soluble RAGE, P-selechn or PSGL-1 mhibitors (e g, small molecule inhibitors, antibodies thereto, e g, antibodies to P-selectin), estrogen receptor beta (ERB) agonists or ERB-NFkb antagonists Most preferred additional therapeutic agents that can be coadministered and/or coformulated with one or more GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) include one or more of a soluble fragment of a TNF receptor, e g, p55 or p75 human TNF receptor or derivatives thereof, e g, 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, Enbrel™), methotrexate, leflunomide, or a sirohmus (rapamycin) or an analog thereof, e g, CCI-779
[0189] Nonlimiting examples of agents for treating or preventing multiple sclerosis with which a GITR antagonist can be combmed include the following interferons, e g, interferon-alphala (e g, Avonex™, Biogen) and interferon-lb (Betaseron™, Chiron/Berlex), Copolymer 1 (Cop-1, Copaxone™, Teva Pharmaceutical Industries, Inc), hyperbaric oxygen, intravenous immunoglobulin, cladribine, TNF antagonists as described herein, corticosteroids, prednisolone, methylprednisolone, azathiopnne, cyclophosphamide, cyclosponne, methotrexate, 4-aminopyndine, and tizanidine
Additional antagomsts that can be used in combination with GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) include antibodies to or antagomsts of human cytokines or growth factors, for example, TNF, LT, EL-1, DL-2, IL-6, IL-7, IL-8, IL-12 IL-15, IL-16, IL-18, EMAP-11, GM-CSF, FGF, and PDGF GITR antagomsts (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) as described herein can be combmed with antibodies to other cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands The GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) may also be combined with agents, such as methotrexate, cyclosponne, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, lbuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signaling by proinflammatory cytokines as described herein, IL-Ib converting enzyme inhibitors (e g, Vx740), anti-P7s, PSGL, TACE inhibitors, T cell signaling inhibitors such as kinase inhibitors, metal loproteinase inhibitors, sulfasalazine, azathlopnne, 6-mercaptopunnes, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof, as described herein, and anti-mflammatory cytokines (e g IL-4, IL-10, LL-13 and TGF)
[0190J Preferred examples of therapeutic agents for multiple sclerosis with which the GITR antagomsts (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) can be combmed include interferon-b, for example, IFNb-la and IFNb-lb, Copaxone, corticosteroids, IL-1 inhibitors, TNF inhibitors, antibodies to CD40 ligand and CD80, and IL-12 antagonists
[0191] Nonhmiting examples of agents for treating or preventing inflammatory bowel disease (e g, Crohn's disease, ulcerative colitis) with which a GITR antagonist (e g, GITRL inhibitory polynucleotides, an antagonistic small molecule, a neutralizing anti-GITR antibody, and/or neutralizing anti-GITRL antibody) can be combmed include the following budenoside, epidermal growth
factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, 6-mercaptopunne, azathiopnne, metromdazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL-1 monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, pyndmyl-imidazole compounds, TNF antagomsts as described herein, IL-4, IL-10, EL-13 and/or TGFb cytokmes or agomsts thereof (e g, agomst antibodies), IL-11, glucuronide- or dextran-conjugated prodrugs of prednisolone, dexamethasone or budesomde, ICAM-1 antisense phosphorothioate ohgodeoxynucleotides (ISIS 2302, Isis Pharmaceuticals, Inc), soluble complement receptor 1 (TP10, T Cell Sciences, Inc ), slow-release mesalazine, methotrexate, antagomsts of platelet activating factor (PAF), ciprofloxacm, and hgnocaine
[0192] In one embodiment, a GITR antagomst (e g, GITRL inhibitory polynucleotides, an antagonistic small molecule, a neutralizing anti-GITR antibody, and/or neutralizing anti-GITRL antibody) can be used in combmation with one or more antibodies directed at other targets involved in regulating immune responses, e g, transplant rejection or graft-v-host disease Nonlimiting examples of agents for treating or preventing immune responses with which a GITR antagomst (e g, GITRL inhibitory polynucleotides, an antagonistic small molecule, a neutralizing anti-GITR antibody, and/or neutralizing anti-GITRL antibody) of the invention can be combmed include the following antibodies against other cell surface molecules, including but not limited to CD25 (mterleukm-2 receptor-a), CDlla (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7 1) and/or CD86 (B7 2) In yet another embodiment, a GITR antagomst (e g, GITRL inhibitory polynucleotides, an antagonistic small molecule, a neutralizing anti-GITR antibody, and/or neutralizing anti-GITRL antibody) is used in combmation with one or more general immunosuppressive agents, such as cyclosporin A or FK506
[0193] In other embodiments, GITR antagomsts (e g, GITRL inhibitory polynucleotides, an antagonistic small molecule and/or neutralizing anti-GITRL antibody) are used as vaccine adjuvants against autoimmune disorders, inflammatory diseases or transplant rejection The combmation of adjuvants for treatment of these types of disorders are suitable for use in combmation with a
wide variety of antigens from targeted self-antigens, 1 e, autoantigens, involved in autoimmunity, e g, myelin basic protein, inflammatory self-antigens, e g, amyloid peptide protein, or transplant antigens, e g, alloantigens The antigen may comprise peptides or polypeptides derived from proteins, as well as fragments of any of the following saccharides, proteins, polynucleotides or oligonucleotides, autoantigens, amyloid peptide protein, transplant antigens, allergens, or other macromolecular components In some instances, more than one antigen is included in the antigenic composition
[0194] For example, desirable vaccines for moderating responses to allergens in a vertebrate host, which contam the adjuvant combmations of this invention, include those containing an allergen or fragment thereof Examples of such allergens are described mUS Patent No 5,830,877 and published International Patent Application No WO 99/51259, which are hereby incorporated by reference in their entireties, and include pollen, insect venoms, animal dander, fungal spores and drugs (such as penicillin) The vaccmes interfere with the production of IgE antibodies, a known cause of allergic reactions In another example, desirable vaccmes for preventing or treating disease characterized by amyloid deposition in a vertebrate host, which contain the adjuvant combmations of this mvention, include those containing portions of amyloid peptide protein (APP) This disease is referred to variously as Alzheimer's disease, amyloidosis or amyloidogenic disease Thus, the vaccmes of this mvention include the adjuvant combmations of this mvention plus A|3 peptide, as well as fragments of Ap peptide and antibodies to Ap peptide or fragments thereof
[0195] Methods of 1) downregulating antigen presenting cell function, and 2) combination therapy for managing immunosuppression are well known in the art (see, e g, Xiao et al (2003) Dendntic cell vaccine design strategies for eliciting peripheral tolerance therapy of autoimmune diseases BioDrugs 17 103-11, Kuwana (2002) Induction of anergic and regulatory T cells by plasmacytoid dendntic cells and other dendntic cell subsets Hum Immunol 63 1156-63, Lu et al (2002) Manipulation of dendntic cells for tolerance induction in transplantation and autoimmune disease Transplantation 73 S19-S22, Rifle et al (2002) Dendntic cells and second signal blockade a step toward allograft
tolerance Transplantation 73 S1-S2, Mancim et al (2004) The management of immunosuppression the art and the science Cnt Care Nurs Q 27 61-64)
[0196] Another aspect of the present mvention accordingly relates to kits for carrying out the combmed administration of GITR antagonists (e g, GITRL inhibitory polynucleotides, antagonistic small molecules, and/or neutralizing antibodies to GITR and/or GITRL) with other therapeutic compounds In one embodiment, the kit compnses one or more binding agents formulated in a pharmaceutical earner, and at least one agent, e g, therapeutic agent, formulated as appropriate, in one or more separate pharmaceutical preparations
[0197J The present mvention is illustrated by the following Examples related to a novel mouse cDNA, designated mouse GITRL cDNA, encoding a novel ligand polypeptide designated mouse GITRL, as well as novel antibodies to GITRL One of skill in the art would understand the teachings of the Examples to be applicable to all homologs of mouse GITRL
EXAMPLES [0198] The Examples which follow are set forth to aid in the understanding of the mvention but are not intended to, and should not be construed to, limit its scope in any way The Examples do not include detailed descriptions of conventional methods, such as flow cytometry (e g, FACS), PCR, Northern and in situ hybridization, or those methods employed in the construction of vectors, the insertion of genes encoding the polypeptides into such vectors and plasmids, the introduction of such vectors and plasmids into host cells, and the expression of polypeptides from such vectors and plasmids in host cells Such methods are well known to those of ordinary skill in the art
EXAMPLE 1
Identification of GITRL DNA sequences
Example 1 1 Identification of the Mouse GITRL cDNA and Genomic Sequences [0199] Two approaches were taken to identify the murine GITRL homolog In one approach, the ammo acid sequence of human GITRL (from GenBank Ace
No AX077015) was used in a Tblastn search against divl, div2, div3, div4, gbdivcu, Celera mouse (cm), and draftmouse-dna databases Genomic sequence ga_69772862 cm_4 was identified as one of the possible hits to investigate Missing ammo acid sequences were identified in a Tblastn search with the ammo acid sequence of human GITRL (GenBank Ace No AX077015) against the unmasked Celera mouse genomic assembly (cm) using expectation value (E) =10 (default), 100 and 1000 Genomic sequence ga_x5j8b7w7wj5_041 cm_aa_2 was identified as the genomic sequence containing the missing amino acid sequences
[0200] In another approach, the ammo acid sequence of human GITRL (from GenBank Ace No NM_005092) was used in a Tblastn search against the unmasked Celera mouse genomic assembly (cm) using default expectation value (E) =10 and 1000 Genomic sequence ga_x5j8b7w7wj5_041 cm_aa_2 was identified as the genomic sequence containing three high scoring pair (HSP) regions
[0201] A putative mouse cDNA sequence was constructed based on the three HSP regions obtained in the above-described Tblastn search This cDNA sequence was edited based on the comparison between the alignments of the three human exon sequences with the corresponding human genomic sequence from Celera (ga_x2htbl3vud5_66 ch_r25h_l) and the three derived mouse putative exon sequences with the correspondmg mouse genomic sequence from Celera (ga_x5j8b7w7wj5_041 cm_aa_2) This editing took into account the sphce junctions for the human sequence The edited mouse GITRL cDNA sequence contained an open reading frame of 519 bp (coding sequence of 522 bp), correspondmg to a protein of 173 ammo acids
[0202] Primers were designed based on the putative exons of mouse GITRL genomic sequence and used to isolate the corresponding physical cDNA clone from a murine thymus cDNA library by PCR The sequences of the forward (SEQ ID NO 4) and reverse (SEQ ID NO 5) PCR primers were 5' ATGGAGGAAATGCCTTTGAGAG 3' (forward pnmer), and 5' GAATGGTAGATCAGGCATTAAGATG 3' (reverse pnmer)
[0203] The resulting fragment was subcloned and the DNA sequence was
determined using standard methods The resulting fragment confirmed the
existence of a mouse GITRL cDNA comprising all three predicted exons (see
below) This fragment was extended to include the final coding segment (two
amino acids) of the cDNA by PCR amplification of this resulting cDNA clone
The sequences of the forward (SEQ ID NO 6) and reverse (SEQ ID NO 7) PCR
primers for this step were
5' TTTAAAGTCGACCCACCATGGAGGAAATGCCTTTGAGAG 3' (forward
primer), and
5' TTTAAAGAATTCTCATTAAGAGATGAATGGTAGATCAGGCAT 3'
(reverse pnmer)
[0204] The forward PCR primer contained a Sail site, a Kozak sequence for translation initiation, and the ATG encoding the initiating methionine The reverse pnmer contained an EcoRI site The Sail and EcoRI sites were used for directional subcloning, and sequence determination of the final cDNA clone was performed
[0205] The full-length mouse GITRL cDNA sequence and its deduced ammo acid sequence are set forth in SEQ ID NO 1 and SEQ ID NO 2, respectively Alignment of the human GITRL cDNA (SEQ ID NO 8) and mouse GITRL cDNA sequences revealed 69 6% identity Alignment of the human GITRL ammo acid (SEQ ID NO 9) and mouse GITRL ammo acid sequences (Figure 1) revealed 54 1% identity and 60 0% similanty This degree of ammo acid identity is similar to that which exists in general between human and mouse homologs of other TNFR ligands (Oshima et al (1998) Int Immunol 10 517-26)
[0206] Companson of the cloned mouse GITRL cDNA sequence (SEQ ID NO 1) with publicly available murine databases revealed a single nucleotide polymorphism (SNP) in the coding region of mouse GITRL (an A/C transversion at nucleotide position 470 of SEQ K) NO 1 in exon 3, which results in an asparagine to threonine change at amino acid position 157 of SEQ ID NO 2)
[0207] Companson of the mouse GITRL cDNA sequence with the mouse genomic sequence from Celera (ga_x5j8b7w7wj5_041 cm_aa_2) descnbed above revealed that the mouse GITRL locus contains three exons and two introns
(see Table 2 below), with exonic size and position well conserved between mouse and human GITRL The mouse GITRL genomic DNA sequence is set forth in SEQ ID NO 3
(Table Removed)
[0208] A comparison of the genomic structure of mouse GTRL (Table 2) with the genomic structure of human GITRL (see Table 3 below) shows that exomc size and rntronic position are well conserved between the human and mouse GITRL genomic DNA sequences The human GITRL genomic DNA sequence is set forth in SEQ ID NO 10
(Table Removed)
Example 1 2 Hydrophobicity Profile of Mouse GITRL
[0209] The hydrophobicity profile of mouse GITRL was determmed by TopPred (Claros and von Heijne (1994) Comput Appl Biosci 10 685-6) A plot of the hydrophobicity score against the ammo acid residues of mouse GITRL (SEQ ID NO 2) revealed a single putative hydrophobic region located approximately between amino acids 25-50, similar to human GITRL This hydrophobic segment corresponds to the predicted transmembrane region for type II transmembrane proteins
EXAMPLE 2
Tissue Expression of Mouse GITRL
[0210] Oligonucleotide probes based on the mouse GITRL cDNA sequence (SEQ ID NO 1) were used to test several murine tissue samples for GrTRL expression by Northern hybndization, in situ hybndization, and real-time PCR (eg,Heid et al (1996) Genome Res 6 986-94, Mullah et al (1998) Nucleic Acids Res 26 1026-31, Giuhetti et al (200l}Methods 25 386-401)
[0211] Although Northern hybndization revealed barely detectable transcnpts in heart, spleen, lung, lymph node, kidney, and hver, subsequent in situ hybndization revealed GITRL expression in the heart, spleen, lymph node and thymus GITRL expression in these tissues was generally limited to the pericardial and endocardial cells of the heart, the white pulp of the spleen, the cortical, paracortical and medullary zones of lymph nodes, and the cortical and medullary zones of the thymus
[0212] GITRL expression in thymus, spleen and lymph node was further confirmed by real-time PCR analysis GITRL was expressed at the highest levels in spleen and hpopolysacchande (LPS)-stimulated spleen cells, which are pnmanly B lymphocytes Vanishingly low levels of GITRL expression were detected in stomach, brain, and kidney Real-time PCR analysis also revealed GITRL transcnpts in liver, activated CD25" cells, activated CD25+ cells, and concanavalin A-activated lymph node cells, although to a lesser degree than the spleen and LPS blasts No GITRL expression was detected in resting CD25" or
CD25+ cells Real-time PCR analysis of immature and LPS-stimulated bone marrow-derived dendritic cells (DC) also demonstrated baselme GITRL expression by immature DC that increased upon stimulation with LPS for 24 hours, but decreased below baselme after 48 hours of LPS stimulation GITRL expression was also detected to varying degrees in all endothelial cell lines tested (bEND3, C166, EOMA, MSI and SVEC4-10), and was demonstrated to remain relatively unchanged when the cell lines were stimulated with LPS In contrast, GITRL cDNA was not detected by PCR in the following unstimulated murine cell lines of specified origin E10 T cell line, T2 fetal thymus line, T10 plasmacytoma, EL4 thymoma, BAF3 and PREB pre-B cell lmes, B9 B cell hybndoma, DA1G monocytic, Ml monocytic, FBMD-1 fetal bone marrow, P19 embryonic carcinoma, MDF liver, and E14 embryonic stem cell lme
EXAMPLE 3
Functional Expression of Recombinant Mouse GITRL Example 3 1 Binding of GITRL to Cell-Surface GITR
[0213] To determine whether the mouse cDNA isolated in Example 1 encoded a functional GITR ligand (GITRL), Cos cells expressmg mouse GITRL fused to the FLAG epitope (GITRL-Flag-Cos) or control mouse IL-21 receptor fused to the FLAG epitope (IL-21R-Flag-Cos) were incubated for various lengths of tune with 293T cells expressmg mouse GITR (GITR-293T) Cell-cell mteracUon was detected by flow cytometry using phycoerythnn-labeled anti-Flag antibody (PE-FLAG) and fluorescein lsothiocyanate (FITC)-labeled anti-GITR Even 1 mm post cocentnfugation of the GITR-293T and GITRL-Flag-Cos cells, -90% of GITR-293T cells (as detected by FITC fluorescence) costained for FLAG (as detected by PE fluorescence), indicating that the GITR-293T cells were bound to the GrrRL-Flag-Cos cells, and this interaction persisted throughout the entire 60 mm of the experiment In contrast, GITR-293T cells mcubated with IL-21R-Flag-Cos cells did not significantly costain for FLAG, even at 60 mm post cocentnfugation These data demonstrate that the mouse cDNA isolated in Example 1 encodes for a functional GITRL capable of bmdmg cell-surface GITR
Example 3 2 Bindmg of GITRL to Soluble GITR
[0214] The ability of mouse GITRL to bind GITR was confirmed by incubating Cos cells expressing mouse GITRL (GITRL-Cos) or mock-transfected Cos cells with recombinant GITR fused to the Fc portion of human IgG (GLTR-Fc) or control human IgG (HIgG) Bmding of GITR-Fc to GLTRL was determined by flow cytometry using donkey antihuman antibody conjugated to FITC (FITC-Ab) Incubation of GITRL-Cos cells with GITR-Fc resulted in a 3 6-fold mcrease in FITC-Ab bmdmg (28 8%) compared to incubation of GITRL-Cos cells with control HIgG (7 9%) Unstained GITRL-Cos cells, GITRL-Cos cells mcubated with CTLA-4 Fc fusion protein and FITC-Ab, and GITRL-Cos cells incubated with FITC-Ab alone exhibited no fluorescence Neither treated nor untreated mock-transfected Cos cells exhibited any appreciable fluorescence These data demonstrate that the mouse cDNA isolated in Example 1 encodes for a functional GITRL capable of bmdmg soluble GITR
EXAMPLE 4
Bmdmg of Mouse GITRL to GITR Results in Proliferation of CD4+CD25+ Cells [0215] The effect of GITRL GITR bmdmg on cellular proliferation was determined by stimulating -50,000 murine T cells with ~50,000 irradiated T cell-depleted splenocytes, and 100 IU/ml BL-2 for 65-72 hrs in the absence or presence of varying concentrations of a GITR-bmdmg protein Two GITR-binding proteins were used in these assays either an agonistic anti-GITR antibody (see, e g, McHugh et al (2002) Immunity 16 311-23, see also U S Patent Apphcation 10/194,754) or murine GITRL expressed on the surface of modified rat YB2/0 cells (GITRL-YB2/0) Cellular proliferation was assayed by pulsing cells with l|iCi 3H-thymidme for the last 6-12 hr of culture and then measuring 3H-thymidine incorporation via scintillation counting
[0216] As shown in Figure 2A, CD4+CD25" T cells did not respond to any concentraton of anti-GITR antibody In contrast, anti-GITR antibody stimulated the proliferation of CD4+CD25+ T cells at all concentrations tested For example, culture of CD4+CD25+ T cells in the presence of the lowest titre of anti-GITR antibody tested (~0 02p.g/ml) resulted in a ~3-fold mcrease in 3H-thymidine
mcorporation (-15,000 cpm) over cells cultured in the absence of anti-GITR antibody The ability of anti-GITR antibody to stimulate CD4+CD25+ T cell proliferation reached a plateau of-45,000 cpm at an antibody concentration of -0 3|ig/ml, corresponding to a -9-fold increase in H-thymidine incorporation over cells cultured in the absence of anti-GITR antibody
[0217] Similar to the results obtained with the anti-GITR antibody, GITRL-YB2/0 cells did not stimulate proliferation of CD4+CD25" T cells (Figure 2B) In contrast, GITRL-YB2/0 cells markedly stimulated the proliferation of CD4+CD25+ T cells For example, culture of CD4+CD25+ T cells in the presence of-10,000 GITRL-YB2/0 cells resulted in a ~4-5-fold mcrease in 3H-thymidine incorporation over cells cultured in the presence of an equal number of unmodified YB2/0 cells Increasing the number of YB2/0 cells to -50,000 resulted in a -15-fold mcrease in 3H-thymidine incorporation over cells cultured in the presence of an equal number of unmodified YB2/0 cells (Figure 2B)
EXAMPLE 5
Binding of Mouse GITRL to GITR Reverses CD4+CD25+ T Cell-Mediated Suppression of CD4+CD25" T Cells
[0218] The T cell suppressor assay used in these Examples has been previously described (see, e g, Thornton and Shevach (2000) / Immunol 164 183-90, McHugh et al (2002) Immunity 16 311-23, both hereby incorporated by reference) Briefly, -50,000 CD4+CD25" responder T cells were cultured in the presence of-50,000 irradiated T cell-depleted splenocytes, 0 5 |ig/ml anti-CD3 antibody, and vanous numbers of freshly isolated suppressor CD4+CD25+ T cells The ability of-50,000 irradiated GITRL-YB2/0 cells or 2 fig/ml agonistic anti-GITR antibody to reverse suppression of CD4+CD25" proliferation was then assessed by measuring 3H-thymidine incorporation via scintillation counting
[0219] As shown in Figure 3A, CD4+CD25+ cells reduced proliferation of CD4+CD25" cells in a dose-dependent manner Both anti-GITR antibody and GITRL-YB2/0 cells were able to completely reverse the suppression of CD4+CD25" proliferation over the entire range of number of CD4+CD25+
suppressor cells tested Thus binding of GITRL to its receptor GITR, like binding of agonistic anti-GITR antibody to GITR, blocked the suppressive function of CD4+CD25+ cells The ability of GITRL-YB2/0 cells to reverse suppression occurred in a dose-dependent manner, with as few as -3,000 GITRL-YB2/0 cells at least partially reversing or decreasing suppression in this assay (Figure 3B) Neither unmodified YB2/0 nor YB2/0 cells expressing GITR had an appreciable effect on CD4+CD25+ T cell-mediated suppression (Figure 3 A)
[0220] In contrast to the results obtamed with freshly isolated CD4+CD25+ T cells, GITRL GITR binding had little-to-no effect on suppression mediated by CD4+CD25+ T cells activated with anti-CD3 antibody, T cell-depleted splenocytes, and EL-2 (Figure 4) Neither the addition of anti-GITR antibody nor -50,000 GITRL-YB2/0 cells was able to reverse suppression mediated by -25,000 activated CD4+CD25+ T cells When fewer activated CD4+CD25+ T cells were added to the assay (e g, -1,500-12,500 cells), however, anti-GITR antibody and GITRL-YB2/0 cells were able to partially decrease, but not completely abrogate, suppression
EXAMPLE 6
Anti-Mouse GITRL Antibody Restores Suppression Mediated by CD4+CD25+ T cells
Example 6 1 Isolation of Anti-Mouse GITRL Antibodies [0221] Antibodies specific for mouse GITRL were produced by immunizing rats with rat YB2/0 cells expressmg the mouse GITRL cDNA (GITRL-YB2/0) Usmg methods well known in the art, antibody hybndomas were created and screened against Phoenix cells expressmg mouse GITRL using flow cytometry Two antibodies, 5F1 and 10F12, were identified that bound specifically to GiTRL-Phoenix cells and not mock-transfected Phoenix control cells These antibody hybndomas were deposited with the American Type Culture Collection (ATCC) on July 22,2003, ATCC assigned number PTA-5336 to hybndoma 5F1, and number PTA-5337 to hybndoma 10F12
Example 6 2 Anti-Mouse GITRL Antibodies Block the Effects of GITRL on Suppressor Activity of CD4+CD25+ T Cells
[0222] As descnbed in Example 5 above, YB2/0 cells expressing GITRL on then-cell surface were able to reverse the suppression of CD4+CD25" T cell proliferation mediated by freshly isolated CD4+CD25+ T cells To determine whether anti-GITRL antibodies could restore CD4+CD25+ T cell-mediated suppression, the T cell suppressor assay as descnbed in Example 5 was performed in the presence or absence of either 5F1 or 10F12 anti-GITRL antibody or control antibodies
[0223] As seen in Example 5, culture of CD4+CD25" responder T cells and freshly isolated CD4+CD25+ suppressor T cells in the presence of GITRL-YB2/0 cells resulted in complete reversal of suppression of CD4+CD25" cell prohferation (Figure 5A and 5B) The addition of 10% hybndoma culture supernatants containing 5F1 anti-GITRL antibody to the assay resulted in partial (Figure 5B) to almost complete (Figure 5A) restoration of CD4+CD25+-mediated suppression Addition of 1 OF 12 anti-GITRL antibody gave similar results As expected, the presence of control antibodies ("control Ig") had no appreciable effect on the ability of GITRL-YB2/0 cells to reverse suppression (Figure 5B) These data demonstrate that anti-GITRL antibodies block the ability of GITRL to turn off the suppressor activity of CD4+CD25+ cells
Example 6 3 Anti-Mouse GITRL Antibodies Suppress T Cell Responses Only in the Presence of CD4+CD25+ T Cells
[0224] Lymph node cell cultures were stimulated in the presence of varying concentrations of agonistic anti-CD3 antibody pnor to (Figure 6A) or after the depletion of (Figure 6B) CD4+CD25+ T cells, and prohferation was measured by determining H-thymidine incorporation via scintillation counting To determine the effects of anti-GITRL antibody on prohferation, 10% hybndoma culture supernatants containing 5F1 anti-GITRL antibody were added to parallel cultures
[0225] As shown in Figure 6 A, addition of anti-GITRL antibody suppressed proliferation of lymph node cells containing CD4+CD25+ T cells when the cells
were stimulated with ~0 075 |ig/ml to ~0 75 |ig/ml anti-CD3 antibody Suppression was not seen in the presence of the anti-GITRL antibody when the lymph node cells contammg CD4+CD25+ T cells were stunulated with 1 0 |xg/ml anti-CD3 antibody In contrast to the results obtained with lymph node cell cultures contammg CD4+CD25+ T cells, the addition of anti-GITRL antibody generally had no suppressive effects on lymph node cell cultures that had been depleted of CD4+CD25+ T cells (Figure 6B) Taken together, these data suggest that anti-GITRL antibody blocks the interaction between GITR expressed on CD4+CD25+ T cells and GITRL expressed on other cells, and that blockade of this GrrR/GITRL mteraction enhances the regulatory function of CD4+CD25+ T cells to restore immunosuppression
EXAMPLE 7
Distribution of GITRL-expressing cells in lymphoid tissues [0226] The agomstic anti-GITRL antibody was used to examine the expression of GITRL in mouse tissues by flow cytometry Freshly isolated GDI 1 c+ splenic dendritic cell (DC) subsets expressing CD4 only, CD8 only, or both CD4 and CD8, conshtutively expressed low levels of GITRL (Figure 7A) However, surface expression of GITRL was noticeably higher among CD1 lclowB220+ plasmacytoid dendritic cells (Nakano et al, 2001) In Figure 7B, staining with anti-GITRL mAb or an isotype control was done on the indicated subsets of freshly isolated CD1 lc+ splemc DCs from BALB/c mice
[0227] Similarly, freshly isolated B220+ splemc B cells constitutively expressed GITRL, as did pentoneal B-l B cells (perC CD1 lb4B2204), although at higher levels (Fig 7C, top) Resting pentoneal macrophages (perC CD1 lb+B220") were also found to express this ligand (Fig 7C, bottom)
[0228] Thymocyte subsets undergoing selection did not express measurable amounts of GITRL (Fig 7D) In contrast, as shown in Figure 7E, expression of GITRL was detectable on all subsets of CD4CD8" thymic precursors, with CD44+CD25+ (R2) and CD44"CD25+ (R3) subsets expressing the highest levels
[0229] GITRL was undetectable on unstimulated lymph node cells (Fig 7F) GITRL was also undetectable on unstimulated splemc T cells (data not shown) These data demonstrate the expression of GITRL by professional antigen presenting cells (DCs, B cells and macrophages, Fig 7A-7C) and thymic CD4" CD8" precursor cells (Fig 7E), but not T cells undergoing selection (Fig 7D) or resting T cells in the periphery (unstimulated lymph node and splemc cells, Fig 7F, and data not shown) These data correlated with data obtained by Northern hybndization, in situ hybndization, and real-tune PCR as descnbed in Example 2 (above)
EXAMPLE 8
APCs downregulate GITRL followmg TLR stimulation [0230] The effects of B cell activation on GITRL expression were examined following treatment with Toll-like receptor (TLR) ligands, or anti-CD40 and DL-4, or anti-IgM Stimulation of either splemc B cells (B220+ splenocytes) or pentoneal B-l B cells (B220+CD1 lb+ PerC) resulted in a rapid but transient upregulation of GITRL, which was apparent after 4 hours with most of the treatments (Fig 8A) Followmg 48-60 hours of stimulation, expression declmed to below prestimulation levels where it stabilized An exception was polyl C-treated B-l B cells from the pentoneal cavity, which did not display this downregulation during the time pomts examined As expected, levels of CD86 mcreased over the course of the experiment in all groups, indicating that the observed downregulation of GITRL was not secondary to cell death (data not shown)
[0231] The reduction of GITRL expressed by B cells after treatment with anti-CD40 and IL-4 suggested that expression could be modulated followmg the provision of T cell help GITRL expression by B cells among total splenocytes was assessed after culture with anti-CD3 antibody Under these culture conditions, expression of GITRL on B220+ splenocytes was also downregulated after 48 hours (Fig 8B) Thus, physiological levels of T cell activity also led to a reduction in the expression of GITRL by splemc B cells
[0232] Splenic CD1 lc+ DCs were enriched with magnetic beads and examined for expression of GITRL after 12 and 36 hours of culture in the presence of LPS DCs cultured in either medium or LPS expressed GITRL during the initial 12 hours, with modest upregulahon induced by LPS However, by 36 hours, expression of GITRL was undetectable on both LPS-treated DCs as well as those cultured only in medium Figure 8C shows expression of GITRL (top histogram panels) and CD86 (B7 2) (bottom histogram panels) by purified CD1 lc+ DCs following culture with or without LPS (0 5jj.g/ml) at the indicated time points As shown, CD86 (B7 2) expression was upregulated as expected (Banchereau and Steinman, 1998) The reduction in GITRL expression by splemc DCs cultured in medium suggests that the "spontaneous" DC maturation that occurs during in vitro culture (Vremec and Shortman, 1997) is sufficient to downregulate expression of GITRL For this reason, only results following LPS stimulation are shown, although DCs were subjected to the same treatments shown for B cells Similar to the results of another published report (Tone et al, 2003), it was found that bone marrow-derived DCs express GITRL constitutively, and that such expression was only marginally reduced after treatment with various TLR ligands (data not shown)
[0233] Both CD4 and CD8 T cells expressed measurable levels of GITRL after a 48-hour culture of splenocytes in the absence ("+Med ") or presence ("+aCD3") of soluble anti-CD3 antibody (Fig 8D), confirming previous real time PCR (see also Example 2)
EXAMPLE 9
Blockade of GITR/GITRL interaction inhibits lymphocyte proliferation [0234] Because GITRL was constitutively expressed by APCs, and because GITR/GITRL interactions were proposed to abrogate the suppressive functions of CD4 CD25+ T cells, the ability of anti-GITRL antibody to enhance suppression mediated by the endogenous population of CD4+CD25+ T cells resident in secondary lymphoid organs was tested A comparison was made of the proliferative responses of total lymph node cells (LN), total splenocytes (Sp), and LN or Sp depleted of CD25+ cells (A25), each of which were cultured with the
anti-GITRL antibody (filled circles) or a control antibody (Rat IgG, open cu-cles The addition of anti-GITRL antibody reduced the proliferative response of total lymph node cells (Fig 9 A, top left) and to a lesser degree, total splenocytes (Fig 9A, bottom left) However, the inhibitory effect was also apparent in cultures depleted of CD25+ cells (Fig 9 A, top right (LN), bottom right (Sp)), raising the possibility that GITR/GITRL interactions provide costimulatory signals for CD25' T cells
[0235] To directly test mis possibility, the proliferative responses of purified CD4+CD25" and CD8+ T cells were examined in the presence of APCs and YB2/0 cells expressing GITRL The proliferation of both CD4+CD25" and CD8+ T cells was substantially enhanced in the presence of GITRL-expressing cells (Fig 9B), which was particularly evident at low concentrations of anti-CD3 for CD4+CD25" T cells
[0236] It may appear discrepant that the anti-GITR antbody mediates its effects by acting on CD4+CD25* T cells, as resting T cells express only low levels of GITR However, the discrepancy is resolved by the demonstration that GITR expression is rapidly upregulated following T cell activation (Fig 9C), reaching maximal levels between 48 and 72 hours after activation These results support the possibility GITR/GITRL interactions can influence CD4+CD25" T cell activation independently of regulatory CD4+CD25+ T cells
EXAMPLE 10
Reversal of suppression requires GITR expression by CD25" T cells [0237] Previous studies suggested that ligation of GITR on CD4+CD25+ T cells inhibited then* suppressive capabilities (McHugh et al, 2002, Shimizu et al, 2002) However, because activated T cells also express GITR, we sought to determine the relevant cellular target(s) of GITR engagement resulting in the abrogation of suppression Proliferation was measured either in the presence or absence of anti-GITR mAb (DTA-1) in cocultures using combinations of CD4+CD25+ and CD4+CD25" T cells from GITR+/+ and GTTR^ mice (Fig 10) As previously reported (Shimizu et al, 2002), when both the CD4+CD25+ and
CD4+CD25" T cells expressed GITR, the addition of anti-GITR mAb to cocultures resulted in an increase in the proliferative response compared to cocultures receiving isotype antibody (Fig 10A, panel a) When CD4+CD25", but not CD4+CD25+, T cells expressed GITR in cocultures, the addition of the anti-GITR antibody led to an enhancement in T cell proliferation similar to that seen when CD4+CD25+T cells expressed GITR (Fig 10A,panelb) However, in cocultures of CD4+CD25 'GTTEC1' and CD4+CD25+G/77?V+ T cells, addition of the anti-GITR antibody had no effect on proliferation (Fig 10A, panel c) As expected, the addition of anti-GITR antibody to cocultures of CD4+CD25"G/77?'A and CD4+CD25+G77K~/" T cells also had no effect on T cell proliferation (Fig 10A, panel d) Results similar to those described above were obtained with a polyclonal anti-mGITR antibody preparation obtained commercially (data not shown)
[0238] Strong evidence in support of the hypothesis that the abrogation of suppression was a consequence of ligation of GITR expressed by CD4+CD25+ T cells was obtained in a previous study, which used combmations of rat responders and mouse CD4+CD25+ regulatory T cells (Shimizu et al, 2002) The anti-GITR mAb (DTA-1) used in these studies was generated in a rat, and consequently did not bind to rat cells (id) Experiments analogous to those described above were performed using cocultures of rat CD4+CD25" responders, mouse CD4+CD25+ suppressors, and irradiated rat APCs (Fig 10B, panel b) Cocultured mouse CD4 CD25" responders, mouse CD4+CD25+ suppressors, and irradiated rat APCs were included as a control (Fig 10B, panel a) Similar to the data obtained from GTTK1' mice, no abrogation of CD4+CD25+-mediated suppression occurred (Fig 10B, panel b), unless GITR could be cross-linked on the responding CD25" population (Fig 10B, panel a)
[0239] A further analysis of the rat/mouse system was accomplished by examining the dilution of CFSE by cocultured rat CD4+CD25" and mouse CD4 CD25 T cells by flow cytometry In the presence of the isotype control antibody, the rat CD4+CD25" T cells were only partially suppressed by mouse CD4 CD25+ T cells when cultured at a 1 8 suppressor to responder ratio (Fig 10C, left histogram panels) However, the addition of the anti-GITR antibody led to an additional expansion of the mouse CD4+CD25+ T cells, and a consequent
mcrease in the suppression of the rat T cells (Fig IOC, nght histogram panels) The increased CFSE dilution of mouse CD4+CD25+ T cells following GITR ligation could be partially inhibited by the addition of blocking ann-CD25 antibodies, suggesting that IL-2 initially made by the responder T cells was also required for this expansion (data not shown) Together, these results unequivocally demonstrate that engagement of GITR on responder CD4+CD25" T cells is required to overcome CD4+CD25+ T cell-mediated suppression
EXAMPLE11
Expression of GITR signals is required to regulate and overcome suppression mediated by endogenous regulatory T cells
[0240] Since both CD28 and GITR appeared to provide costimulatory signals during the activation of T cells, we sought to determine if they played distinct roles during the primary response We compared the capacity of GITR and CD28 to promote T cell prohferation in the presence or absence of endogenous lymph node CD4+CD25+ T cells, and in the presence or absence of exogenous IL-2 (Fig 11) The same samples used for prohferation studies were simultaneously assessed for CFSE dilution following the 72-hour culture period In the absence of exogenous IL-2, CD4+ and CD8+ T cells from both GITR''' and CD28'A animals failed to proliferate (Fig HA,panela) LN cells from GITR+A animals displayed a phenotype intermediate between those from wild type and GITR''' animals (Fig 11 A, panel a) The response ofT cells from wild type mice was significantly enhanced following depletion of CD25+ T cells, mdicating that suppression under these culture conditions was mediated by the CD25+ T cells resident in the normal lymph node (Fig 11 A, compare panels a and b) Most importantly, in the absence of CD4+CD25+ T cells, the responses of CD4+ and CD8+ lymph node T cells from GITR''' mice were comparable to those of wild type mice as assessed by 3H-thyrrndine incorporation (Fig 11 A, panel b) and CSFE dilution (Fig 11B, top set of panels) However, after 72 hours, CD4+ and CD8+ T cells from CD28^' animals were not prohferating even in the absence of CD4+CD25+T cells (Fig HA,panelb)
[0241] A very different pattern of response was observed when exogenous IL-2 was added to the cultures of intact lymph node cells CD4+ and CD8+ T cell proliferation was completely inhibited in the absence of GITR as assayed by 3H-thymidine uptake (Fig 11 A, panel c) or by the lack of CFSE dilution (Fig 11B, middle set of panels) In contrast, measurable proliferation of T cells from
/ 1
CD2&' animals was detected by H-thymidine incorporation (Fig 11 A, panel c), although the CFSE profile demonstrated that CD8+ T cells were largely responsible for the proliferation measured (Fig 1 IB) In the presence of IL-2 (50U/ml), lymph nodes depleted of CD4+CD25+ T cells from all animals
displayed similar levels of proliferation as assessed by H-thymidine incorporation (Fig 11 A, panel d) and CFSE dilution (Fig 11B, bottom set of panels) Taken together, these results indicate that the defects in T cell activation in GITR'A and CD28TA mice are distinct
[0242] The inability of total T cells present in lymph nodes of GITK1' mice to proliferate in the presence of exogenous IL-2 suggested that the expression of the high affinity IL-2 receptor might be affected in these animals Since the expression of the IL-2Ra chain is primarily induced by its kgand (Depper et al, 1985, Malek and Ashwell, 1985), the ability of anti-CD3-activated CD4+CD25" T cells from GITR''' mice to express this chain was examined in the presence or absence of CD4+CD25+ T cells, and in the presence or absence of IL-2 (Fig 11C) In the presence of regulatory T cells, the addition of IL-2 to cocultures resulted in enhanced expression of CD25 by GITR+/+, but not by GITK1', CD4+CD25" T cells after 24 hours (Fig 11C, bottom right histogram panel) However, the ability of GITR'1' CD4+CD25" T cells to undergo IL-2-induced CD25 expression could be readily restored by removing CD4+CD25+ T cells (Fig 11C, bottom left histogram panel) Thus, the impairment in IL-2 responsiveness by GITR''' T cells in the presence of CD4+CD25+ T cells was due, at least in part, to their inability to express the high affinity IL-2 receptor in the presence of concentrations of exogenous IL-2 sufficient to induce CD25 expression on wild type cells
EXAMPLE 12
CD28-dependent costrmulation enhances GITR expression and responsiveness [0243] Although the data presented above suggested that engagement of either GITR or CD28 on CD25" T cells provided a signal that allowed the responder T cells to escape suppression, the nature of the signals involved in this process remained unclear The partial rescue of the proliferative responses of CD28;' CD8+ T cells by IL-2 in the presence of CD4+CD25+ T cells suggested that CD28/B7 signaling might regulate T cell sensitivity to GrrR/GrrRL interactions Furthermore, previous studies have demonstrated that CD28-CD80/CD86 interactions can enhance expression of some TNFR-family members (Gilfillan et
al, 1998, Rogers et al, 2001) Thus, we examined this possibility with respect to GITR Purified CD4+CD25" and CD8+ T cells from CD28*' or wild type mice either remained unstimulated or were activated with low concentrations of plate-bound anti-CD3 antibody in the presence or absence of plate-bound anti-CD28 antibody (Fig 12A) Although wild type T cells exposed to anti-CD3 alone only slightly upregulated GITR, the expression of GITR by both CD4+CD25" and CD8+ T cells was greatly enhanced by the inclusion of anti-CD28
[0244] Similarly, the upregulation of GITR expression on CD4+CD25' T cells was markedly inhibited by the addition of anti-CD80/CD86 (anti-B7 1/7 2) (Fig 12B, left histogram panel) The inhibition of GITR expression in cultures containing anti-CD80/CD86 was not secondary to reduced production of IL-2, as GITR upregulation by CD4+CD25" T cells was not prevented in cultures containing anti-IL-2/IL-2R mAbs (Fig 12B (right histogram panel)
[0245] The enhanced expression of GITR induced by CD28-denved coshmulatory signals was paralleled by an enhanced responsiveness to GITR signaling (Fig 12C) The addition of the anti-GITR antibody substantially enhanced the proliferation of both CD4+ and CD8+ effector T cells from wild type mice (Fig 12C, left panels) However, when anti-CD80/CD86 was added to these cultures, the presence of the anti-GITR antibody only marginally mcreased the proliferation of GITR+/+ CD4+CD25' T cells (Fig 12C, GITR+/+, top left, GITR-A, top right) and GITR+/+ CD8+ T cells (Fig 12C, GITR+/+, bottom left, GITR'', bottom right) over the range of anti-CD3 concentrations tested Treatment with anti-GITR did not affect the responses of purified CD4+CD25" and CD8+ T cells from GITR''' mice (Fig 12C, right panels) These data suggest that CD28-mediated signals, distinct from costimulation of IL-2 production, enhance GITR expression and facilitate GITR-mediated signaling
EXAMPLE 13
GITRL binding to GITR provides a costimulatory signal to effector T cells [0246] Forty-thousand effector HT-2 T helper cells (GITRYrCR*) were cultured in the absence or presence of the following reagents 1) anti-CD3 coated beads at
a 1 1 or 1 2 ratio, 2) ten-thousand YB/2 cells that were either not modified to express GITRL (YB2/0 parental) or modified to express GITRL (YB2/0 muGITRL), and 3) increasing concentrations of an isotype control antibody or four different anti-GITRL antibodies (5F1, MGTL-10, MGTL-15, or a polyclonal anti-GITRL antibody) Proliferation was measured by 3H-thymidine incorporation for the last 5 hours of a 44-hour culture period Fig 13 A demonstrates that GITRL enhances the proliferation of T cells stimulated with anti-CD3 Additionally, GITRL-mediated enhancement of T cell prohferation may be blocked with 5F1 antibody, but not the isotype control antibody (Fig 13B) and commercially available MGTL-10, MGTL-15 and polyclonal anh-GITRL antibodies (Fig 13C) These data provide further evidence that GITRL provides a costunulatory signal, and also, that 5F1 is a neutralizing antibody to GITRL
EXAMPLE 14
Blocking GITR-GITRL binding with an anti-GITRL antibody prevents adoptive transfer of PLP induced experimental autoimmune encephalomyelitis [0247] Nme-week-old female SJL mice were immunized with 150 fig PLP peptide (ammo acids 139-151) [HSLGKWLGHPDKF (SEQ ID NO 12)] in complete Freund's adjuvant Ten days after immunization, splenocytes were harvested and restrmulated ex vivo for 3 days with 10 fig/ml PLP (ammo acids 139-151) in the absence (no treatment) or presence of 10 ug/ml antibody (anti-GITRL antibody or control antibody) After restimulation, 5x106 splenocytes were adoptively transferred into 10-week-old naive SJL mice, and the mice were monitored for 52 days for the experimental autoimmune encephalomyelitis (EAE), as measured on a scale from 0 to 5 EAE developed in 40% and 80% of mice that received splenocytes restimulated in the absence of any antibody (no treatment) and mice that received splenocytes restimulated in the presence of control antibody (CK01), respectively (Fig 14) Additionally, there was no significance difference in disease scale between animals that received splenocytes with no treatment and animals that received splenocytes treated with control antibody Significantly, none of the mice that received splenocytes that were
restimulated in the presence of anti-GITRL antibody (5F1) developed EAE (p = 0 0023 vs control antibody treatment) (Fig 14) These data suggest that blockade of the GITR/GITRL pathway will limit the ability of CD25' T cells to overcome suppression, thereby downmodulating their ability to effect autoimmune responses
EXAMPLE 15
Experimental Procedures Example 15 1 Antibodies and reagents
[0248] All antibodies used for flow cytometry or functional studies were from BD-Pharmingen, except tn-color labeled aCD4 (clone CT-CD4) and aB220 (clone RA3-6B2) which were purchased from Caltag (Burhngame, CA), and MGTL-10, MGTL-15, and polyclonal anti-GITRL antibodies, which were purchased from Alexis Biochemicals (San Diego, CA) Purified F(ab')2 fragment of goat-anti-IgM p.-chain was purchased from Jackson Immunoresearch (West Grove, PA) Anti-IL-2 (clone S4B6) was used as ascites fluid Human recombinant IL-2 was obtained from the National Cancer Institute (Frederick, MD) IL-4, IFN-y, IL-12, and T cell enrichment columns were purchased from R&D Systems (Minneapolis, MN) Poly IC and LPS were purchased from Sigma CpGs were purchased from InvivoGen (San Diego, CA) Anu-GITRL (clone 5F1, also clone 10F12) and anti-GITR (clone DTA-1) and the PLP peptide were produced "ln-house " Anu-B220, -CD1 lc, -CD1 lb -CD8, -CD4 and -PE magnetic beads were purchased from Miltenyi (Auburn, CA)
Example 15 2 Mice
[0249] BALB/c and C57B1/6 mice (6-8 week old females) were purchased from the NCI Frederick animal facility (Frederick, MD) CD28~/" mice were provided by Dr Alfred Smger (NIH/NCI) GrTR+/" embryos (Svl29 x B6) were provided by C Ricarrdi (Perugia Umversity Medical School, Italy) (Ronchetti et al, 2002) The redenved GITR+/' mice were backcrossed once with C57BL/6 mice, and the resulting progeny were screened for the mutant allele by PCR The identified
GITR+/" progeny were then intercrossed to generate GITK' mice All mice were bred and housed at NIH/NIAID facilities under SPF (specific pathogen-free) conditions
Example 15 3 cDNA cloning and expression
[0250] The ammo acid sequence for human GITRL (GenBank Ace No NM005092) was used to search the Celera database for the mGITRL Genomic sequence ga_x5j8b7w7wj5_041 cm_aa_2 contained three high scoring pair (HSP) regions Based on the assumption that these regions correspond to exons for mouse GITRL, primers were designed for PCR amplification The forward primer (5'-ATGGAGGAAATGCCTTTGAGAG-3') (SEQ ID NO 4) and reverse primer (5'-GAATGGTAGATCAGGCATTAAGATG-3') (SEQ ID NO 5) amplified a cDNA clone from a mouse thymus library The resulting fragment was subcloned and the DNA sequence was determined A subsequent full-length clone was amplified by PCR from the previous construct using 5'-TTTAAAGTCGACCCACCATGGAGGAAATGCCTTTGAGAG-3'(forward) (SEQ ID NO 6) and 5'-
TTTAAAGAATTCTCATTAAGAGATGAATGGTAGATCAGGCAT-3' (reverse) (SEQ ID NO 7) primers The resulting PCR fragment was subcloned into the GFP-RV retroviral vector (Ouyang et al, 1998), and sequence determination of the final cDNA clone was performed This vector was then transfected into the Phoenix cell lme Supernatants from the transfected Phoenix cells were used to transduce the YB2/0 cell lme GFP-expressing YB2/0 cells were then FACS sorted and maintained in culture The predicted mGITRL ammo acid sequence is identical to that of another group (Kim et al, 2003), except for the substitution of an alanine for a valme at ammo acid position 48 in their sequence
Example 15 4 Production and purification of monoclonal antibodies [0251] Lewis rats were immunized once s q with lOOxlO6 YB2/0-GITRL cells in CFA Two weeks later, these rats were immunized s q with lOOxlO6 YB2/0-GITRL cells in IFA Two weeks later, rats were boosted with 50xl06 YB2/0-GITRL cells in PBS Four days later, the spleen was harvested and cell fusion
was performed as previously described (Cohgan et al, 2003) The supernatants from the resulting hybndomas were screened by flow cytometry using Phoenix-GITRL and Phoenix cells Antibodies were purified from cell culture supernatants using protein G-loaded columns, and eluted antibodies were dialyzed against PBS
Example 15 5 Cell purification
[0252] T cells were purified from peripheral lymph nodes of mice CD25+ T cells were labeled with magnetic beads and purified on an autoMACS (Miltenyi Biotech, Auburn, CA) according to the manufacturer's protocol Purity of the CD25+ cells was typically between 97 to 99 percent Cells remaining in the negative fraction were subsequently labeled with either anti-CD4 or anti-CD8 microbeads and purified using the positive selection procedure on the autoMACS Purity was routinely 90-95 percent T cell-depleted spleen suspensions were prepared from erythrocyte-lysed suspensions by depleting Thyl 2+ cells using the autoMACS B220+ cells were purified from splenocytes in a similar fashion using anti-B220 microbeads (Miltenyi Biotech, Auburn, CA), and purity of the resulting preparations was routmely greater than 90% Peritoneal cells were prepared by flushing the peritoneal cavity (PerC) with 10ml of cold HBSS For splenic DCs, splenocyte suspensions were prepared in a manner similar to that described (Vremec et al, 2000) Splenic DCs were then purified from the suspensions using anti-CDl lc microbeads (Miltenyi Biotech, Auburn, CA) The resulting DC suspensions were routmely 85 to 90 percent pure Rat CD4+CD25" cells were generated by depleting rat splenocytes of CD25+ cells using PE-anti-rat CD25 (OX-39) antibodies followed by anti-PE microbeads After depletion, CD4+ cells were then selected from the depleted fraction using anti-rat-CD4 microbeads
Example 15 6 In vitro proliferation assays
[0253] Suppression assays were performed as described (Thornton and Shevach, 1998) Briefly, (5xl04) cells were cocultured in FBS-supplemented RPMI1640 (Atlanta Biologicals, Atlanta, GA) with irradiated (3000R), T cell-depleted splenocytes (5xl04) in the presence of 0 5ug/ml anti-CD3 mAb (2C11) in 96-well
flat-bottom plates To some cultures, antibodies specific for GITR or a Rat Ig isotype were added at a final concentration of 2µg/ml Titrated numbers of CD4+CD25+ cells were added at final suppressor to responder ratios of 0 1,1 2, 1 4, or 1 8 Cultures were pulsed with 1µC1 of 3H-thymidine for the final 5-8 hours of a 72-hour culture, and were performed in triplicate unless otherwise indicated Cocultures of rat and mouse T cell subsets were setup in a similar manner except irradiated (3000R) CD4-depleted rat splenocytes were used as APCs, and rat and mouse T cells were stimulated using a cocktail of anti-rat-CD3 (0 25µg/ml) and anti-mouse-CD3 (0 25µg/ml) mAbs
Example 15 7 In vitro culture of lymph node cells and CFSE labeling [0254] CD25+-depleted lymph node cell suspensions were prepared on the autoMACS as descnbed above Cells were labeled with CFSE at a concentration of 2µM for 8 minutes in a 37°C water bath Cells were then washed in complete RPMI1640 Cells (5xl04/well) were then cultured in 96-well plates in the presence or absence of rhIL-2 (50U/ml) Duplicate wells were either pulsed with 1µC1 of 3H-thymidine for the final 5-8 hours of a 72-hour culture or used for analysis of CFSE dilution
EXAMPLE 16
Discussion
[0255] The role of GITR in the function of CD4+CD25+ T cells was inferred from the demonstration that both polyclonal and monoclonal antibodies to GITR reversed the suppressive effects of CD4+CD25+ T cells when added to cocultures of CD25+ and CD25" T cells (McHugh et al, 2002, Shimizu et al, 2002) The CD4+CD25+ T cells appeared to be the likely target for the anti-GITR reagents because freshly explanted CD25+ T cells expressed GITR at higher levels than resting CD25" T cells and because anti-GITR antibody together with IL-2 triggered the proliferation of CD25+, but not CD25", T cells in the absence of a TCR signal Furthermore, when Shimizu et al added a rat anti-mGITR mAb, which was nonreactive with rat cells, to cocultures of mouse CD25+ suppressors
and rat responder T cells, a reversal of suppression was observed These studies led to the hypothesis that engagement of GITR by agomstic anti-GITR antibodies and, presumably, by its physiological ligand, generated a signal that both inhibited the suppressor activity of CD4+CD25+ T cells and reversed the nonresponsiveness of the CD25+ T cells to exogenous IL-2
[0256] Expenments were designed to attempt to extend and confirm these studies by cloning the mouse GITRL, analyzing its tissue distribution, and definitively determining the target for the agomstic anti-GITR antibodies by using mixtures of CD25+ and CD25" T cells from wild type and GITR-/- mice Collectively, the studies demonstrate that the anti-GITR antibodies and GITRL abrogate the suppressive effects of CD4+CD25+ T cells by providing CD25- T cells a unique signal that raises their threshold for suppression by CD4+CD25+ T cells The studies indicate that GITRL is selectively expressed on the cell surface of APC, with the highest level of expression on B-l B lymphocytes, intermediate levels on conventional B-2 B lymphocytes, macrophages, and B220+ DCs, and lower levels on B220- DC subsets GITRL is unique among members of the TNF superfarmly in that it is expressed on resting APC, and its expression is downregulated by triggering the B cell receptor, CD40, or different Toll-like receptors Other members of the TNF superfarmly (4-1BB-L, OX40-L, LIGHT, CD70, CD30-L) are not detectable on resting APC, and their expression is upregulated by activation of the APC via Toll-like receptor stimulation (Croft, 2003) The present studies do not address whether the downregulation of GITRL expression from the cell surface is accompanied by secretion of soluble GITRL, but Tone et al (2003) have reported that LPS stimulation of DC, macrophages and B cells results in downregulation of GITRL mRNA The expression of GITRL on resting APC strongly suggests that it functions early in the process of T cell activation Other cell types, including endothelial cells (Gurney et al, 1999, Kwon et al, 1999), activated T cells, and certam subsets of DN thymocytes were also shown to express GITRL, and the function of this molecule on these latter cell types remains to be determined
[0257] The ability of both the anti-GITR antibodies and the GITRL expressing cells to enhance the activation of CD25" T cells alone, as well as the ability of anti-GITRL to partially inhibit the activation of CD25" T cells in the absence of
CD25+ T cells, prompted a careful reexammation of the cellular target(s) of these reagents The addition of anti-GITR to cocultures of CD25+ and CD25" T cells from wild type and GITR.'' mice demonstrated that the target for the antibody-mediated reversal of suppression was the CD25" T cell This was further supported by experiments using rat CD25" responder and mouse CD25+ suppressor T cells in cocultures, which conclusively demonstrated GITR ligation on the CD25+ T cell subset does not abrogate their suppressive function In fact, an examination of CFSE dilution demonstrated that GITR ligation promoted the expansion of CD25+ T cells in cocultures, which ultimately enhanced the suppression of the rat CD25" responders Therefore, it is possible that the enhancement in proliferation in anti-GITR-treated rat/mouse cocultures reported by Shimizu et al (2002), which was presumed to be due to rat T cells, actually reflected proliferation of the mouse CD25+ suppressor cells This would not have been apparent by measuring 3H-thymidrne incorporation alone
[0258] One previous report suggested that GITR-deficient T cells are hyperreactive to TCR stimulation (Ronchetti et al, 2002) However the extrapolation of those observations to the present observations are difficult as that report did not analyze the responses of purified CD4+, CD8+ T lymphocytes, nor did it evaluate the role of CD4+CD25+ T cells In the present studies, the responses of highly purified CD4+CD25" and CD8+ T cells from GITR-/- mice were comparable to those of controls However, in the presence of physiological numbers of regulatory T cells, both CD4+ and CD8+ T cells from GITR-/- animals were completely unresponsive to CD3 cross-linking This result clearly demonstrates that in the absence of GITR/GITRL interactions suppression is dominant Indeed, the suppression of activation of CD4+CD25" T cells from GITR''' mice was so strong that it could not be overcome by the addition of a high concentration of exogenous IL-2, which normally abrogates the suppressive effects of much higher numbers of CD25+ T cells on the responses of wild type CD4+CD25" T cells (Takahashi et al, 1998, Thornton and Shevach, 1998) The suppressive effects of the CD4+CD25+ were mediated by inhibition of both IL-2 production and expression of CD25 by the CD4+CD25" GITR-/- responder population, which resembles what has been previously described for the effects
of CD4+CD25+ T cells on normal CD8+ T cell responses (Piccmllo and Shevach, 2001)
[0259] The costimulatory signals delivered by GITR and CD28 appeared to be distinct, but interrelated In the absence of regulatory T cells, the responses of both CD4+ and CD8+ T cells from GITR-/- mice were identical to those of cells from wild type mice In contrast, under the culture conditions used in this study, CD4+ and CD8+ T cells from CD28-/- mice were nonresponsive Conversely, when IL-2 was added to cultures containing regulatory T cells, the responses of CD4+ and CD8+ T cells from the GITR-/- mice were not restored, while the responses of CD8+ cells from the CD28-/- mice were partially restored On the other hand, a potential cooperative relationship and shared signaling hierarchy between CD28 and GITR was supported by the demonstration that (1) CD4+CD25" and CD8 T cells failed to upregulate GITR in the absence of CD28 cross-linking and (2) anti-CD80/CD86 antibodies markedly inhibited both the upregulation of GITR expression and responsiveness to anti-GITR antibodies Thus, the results suggest that an additional important function of the CD28-CD80/CD86 signaling pathway during T cell activation is to license T cell resistance to CD25+-mediated suppression by enhancing the expression and function of GITR
[0260] Similar to the results suggested by other published studies (Shimizu et al, 2002, Tone et al, 2003), these studies indicate that ligation of GITR on CD25" T cells, in the absence of regulatory T cells, did provide some degree of costimulauon However, GITR ligation is not required for costimulating CD25" T cells in the same manner as CD28, as CD4+CD25" and CD8 T cells from GITR-/- mice respond similar to wild type T cells We favor the view that engagement of GITR on both CD4+CD25" and CD8+ effector T cells by GITRL early during the course of an immune response serves primarily to render the effector population resistant, e g, less susceptible, to the suppressive effects of the CD4+CD25+T cells Durmg the course of the response, inflammatory signals ultimately result in the downregulation of GITRL expression, thereby mcreasing the susceptibility of the effector cells to CD25+-mediated suppression Although only limited data is available as to when CD25+-mediated suppression of immune responses to infectious agents occurs in vivo, some studies have suggested that it
operates at the contraction, rather than the initiation, phase of the response to prevent tissue damage secondary to exuberant inflammation (Suvas et al, 2003) It should be pointed out that engagement of GITR on CD25+ T cells in the presence of EL-2 induces their expansion (McHugh et al, 2002) It remains possible that in vivo engagement of GITR on CD25+ T cells by GITRL on resting APC results in the nonspecific expansion of regulatory T cells in the presence of EL-2 secreted by effector T cells early in the course of the immune response This nonspecific expansion may be cntical to the subsequent generation of a pool of antigen-specific suppressor cells that function to inhibit effector cell activity later in the response
[0261] We have previously proposed that manipulation of GITR/GITRL interactions may prove to be an effective way of manipulating regulatory T cell function in vivo (McHugh and Shevach, 2002) Although this concept was based on data suggesting that the CD4+CD25+ T cell was the target of the anti-GITR antibody, it is still the case that the GITR/GITRL interaction represents an important therapeutic target Thus, treatment with an agonistic anti-GITR antibody or an agonistic GITRL-Fc should render effector cells resistant, e g, less susceptible, to the suppressive effects of CD4+CD25+ T cells and should prove to be effective for treating cancers and infectious diseases, for enhancement of immune responses to cancers (used alone or in combmation with other tumor therapies, such as tumor vaccmes), and for enhancement of immune responses to infectious pathogens, including viruses, bacteria, etc (used alone or in combmation with other therapies for infectious pathogens, such as a vaccine adjuvant to weak vaccmes for infectious agents) Similarly, inhibition of GITR/GITRL interactions with a GITR agonist, e g, through the use of a neutralizing anti-GITRL antibody or GITR-Fc, should lower the threshold of effector T cells to suppression and thus be useful for the prevention and/or treatment of autoimmune disorders, inflammatory diseases, transplant (or graft) rejection, and graft-versus-host disease
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WHATTS CLAIMED:
1. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3.
2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule is operably linked to at least one expression control sequence.
3. A host cell transformed or transfected with the nucleic acid molecule of claim 2.
4. An isolated nucleic acid molecule that specifically hybridizes under highly stringent conditions to the nucleotide sequence set forth in SEQ ID NO:l or SEQ ID NO:3, or the complement thereto.
5. An isolated nucleic acid molecule that encodes a protein comprising the amino acid sequence of SEQ ID NO:2 or an active fragment of the amino acid sequence of SEQ ID NO:2.
6. The isolated nucleic acid molecule of claim 5, wherein the nucleic acid molecule is operably linked to at least one expression control sequence.
7. A host cell transformed or transfected with the isolated nucleic acid molecule of claim 6.
8. A nonhuman transgenic animal in which the somatic and germ cells contain DNA comprising the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3.
9. An isolated protein comprising the amino acid sequence encoded for by the isolated nucleic acid of claim 4.
10. An isolated protein comprising the amino acid sequence of SEQ ID NO:2, or an active fragment thereof.
11. An isolated nucleic acid molecule comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3, or a fragment thereof, wherein expression of the nucleic acid molecule in a cell results in decreased production of GITRL.
12. The isolated nucleic acid molecule of claim 11, wherein the nucleic acid molecule is operably linked to at least one expression control sequence.
13. An antisense oligonucleotide complementary to a mRNA corresponding to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:3, or a fragment thereof, wherein the oligonucleotide inhibits expression of GITRL.
14. A siRNA molecule corresponding to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3, or a fragment thereof, wherein the siRNA molecule inhibits expression of GITRL.
15. An isolated antibody capable of specifically binding to the protein of claim 10.
16. The antibody of claim 15, wherein the antibody neutralizes GITRL activity.
17. The antibody of claim 16, wherein the antibody is 5F1, having ATCC number PTA-5336, or 10F12, having ATCC number PTA-5337.
18. A method of screening for test compounds capable of inhibiting the interaction of GiTRL with GITR comprising the steps of:
contacting a sample containing GITRL and GITR with the compound; and determining whether the interaction of GITRL with GITR in the sample is
decreased relative to the interaction of GITRL with GITR in a sample not
contacted with the compound,
whereby a decrease in the interaction of GITRL with GITR in the sample contacted
with the compound identifies the compound as one that inhibits the interaction of
GITRL with GITR.
19. A method of screening for test compounds capable of enhancing or
mimicking the interaction of GITRL with GITR comprising the steps of:
contacting a sample containing GITRL and GITR with the compound; and detennining whether the interaction of GITRL with GITR in the sample is
increased relative to the interaction of GITRL with GITR in a sample not contacted
with the compound,
whereby an increase in the interaction of GITRL with GITR in the sample
contacted with the compound identifies the compound as one that enhances or
mimics the interaction of GITRL with GITR.
20. A method for diagnosing an autoimmune disorder, an inflammatory
disease, or transplant rejection in a subject comprising the steps of:
detecting a test amount of a GITRL gene product in a sample from the subject; and
comparing the test amount with a normal amount of the GITRL gene product in a control sample,
whereby a test amount significantly above the normal amount provides a positive indication in the diagnosis of an autoimmune disorder, an inflammatory disease, or transplant rejection.
21. A method for diagnosing cancer or an infectious disease in a subject
comprising the steps of:
detecting a test amount of a GITRL gene product in a sample from the subject; and
comparing the test amount with a normal amount of the GITRL gene product in a control sample,
whereby a test amount significantly below the normal amount provides a positive indication in the diagnosis of cancer or an infectious disease.
22. A method of treating a subject at risk for, or diagnosed with, an
autoimmune disorder, inflammatory disease, or transplant rejection comprising
administering to the subject a GITR antagonist.
23. The method of claim 22, wherein the method comprises administering the GITR antagonist such that the susceptibiUty of the effector T cells in the subject to suppression by CD4+CD25+ regulatory T cells is maintained.
24. The method of claim 22, wherein the GITR antagonist is selected from the group consisting of a neutralizing anti-GITRL antibody, a neutralizing anti-GITR antibody, a fusion protein containing GITR, a fusion protein containing an active fragment of GITR, an antagonistic small molecule, an antisense GITRL nucleic acid molecule, and a siRNA GITRL nucleic acid molecule.
25. The method of claim 22, wherein the autoimmune disorder or inflammatory disease is selected from the group consisting of rheumatoid arthritis, encephalomyelitis, osteoarthritis, multiple sclerosis, autoimmune gastritis, systemic lupus erythematosus, psoriasis and other inflammatory dermatoses, type I diabetes, asthma, allergy, and inflammatory bowel diseases, including Crohn's disease and ulcerative colitis.
26. The method of claim 22, wherein the autoimmune disorder is multiple sclerosis.
27. A method of treating a subject at risk for, or diagnosed with, cancer or an infectious disease comprising administering to the subject a GITR agonist.
28. The method of claim 27, wherein the method comprises administering the GITR agonist such that the GITR agonist provides a costimulatory signal to effector T cells in the subject and renders them less susceptible to suppression by CD4+CD25+ regulatory T cells in the subject.
29. The method of claim 27, wherein the GITR agonist is selected from the group consisting of GITRL, an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containing an active fragment of GITRL, and an agonistic GITR antibody.
30. A method of inducing proliferation of a cell population containing effector T cells comprising administering a GITR agonist to the cell population.
31. The method of claim 30, wherein the GITR agonist is selected from the group consisting of GITRL, an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containing an active fragment of GITRL, and an agonistic GITR antibody.
32. The method of claim 30, wherein the effector T cells are CD4+ T cells or CD8+T cells.
33. A method of inhibiting proliferation of a cell population containing effector T cells comprising administering a GITR antagonist to the cell population.
34. The method of claim 33, wherein the GITR antagonist is selected from the group consisting of a neutralizing anti-GrrRL antibody, a neutralizing anti-GITR antibody, a fusion protein containing GITR, a fusion protein containing an active fragment of GITR, an antagonistic small molecule, an antisense GITRL nucleic acid molecule, and a siRNA GITRL nucleic acid molecule.
35. The method of claim 33, wherein the effector T cells are CD4+ T cells or CD8+T cells.
36. A method of blocking suppression of a cell population including effector T cells in the presence of CD4+CD25+ regulatory T cells comprising administering a GITR agonist to the cell population.
37. The method of claim 36, wherein the method comprises administering the GITR agonist such that the GITR agonist provides a costimulatory signal to the effector T cells and renders them less susceptible to suppression by the CD4+CD25+ regulatory T cells.
38. The method of claim 36, wherein the GITR agonist is selected from the group consisting of GITRL, an active fragment of GITRL, a fusion protein
containing GITRL, a fusion protein containing an active fragment of GITRL, and an agonistic GITR antibody.
39. The method of claim 36, wherein the effector T cells are CD4+ T cells or CD8+T cells.
40. A method of suppressing of a cell population including effector T cells in the presence of CD4+CD25+ regulatory T cells comprising administering a GITR antagonist to the cell population.
41. The method of claim 40, wherein the method comprises administering the GITR antagonist such that the susceptibility of the effector T cells to suppression by the CD4+CD25+ regulatory T cells is maintained.
42. The method of claim 40, wherein the GITR antagonist is selected from the group consisting of a neutralizing anti-GITRL antibody, a neutralizing anti-GITR antibody, a fusion protein containing GITR, a fusion protein containing an active fragment of GITR, an antagonistic small molecule, an antisense GITRL nucleic acid molecule, and a siRNA GITRL nucleic acid molecule.
43. The method of claim 40, wherein the effector T cells are CD4+T cells or CD8+T cells.
44. A method of treating cancer or an infectious disease in a subject, the method comprising the steps of obtaining a population of effector T cells, treating the population with a GITR agonist, and administering the treated population to the subject afflicted with cancer or an infectious disease.
45. The method of claim 44, wherein the GITR agonist is selected from the group consisting of GITRL, an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containing an active fragment of GITRL, an agonistic small molecule, and an agonistic anti-GITR antibody.
46. The method of claim 44, wherein the subject is afflicted with cancer, and wherein the treated population is used as a tumor vaccine.
47. A pharmaceutical composition comprising a GITR agonist and a pharmaceutically acceptable carrier.
48. The pharmaceutical composition of claim 47, wherein the GITR agonist is selected from the group consisting of GITRL, an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containing an active fragment of GITRL, an agonistic small molecule, and an agonistic anti-GITR antibody.
49. A pharmaceutical composition comprising a GITR antagonist and a pharmaceutically acceptable carrier.
50. The pharmaceutical composition of claim 49, wherein the GITR antagonist is selected from the group consisting of a neutralizing anti-GITRL antibody, a neutralizing anti-GITR antibody, a fusion protein containing GITR, a fusion protein containing an active fragment of GITR, an antagonistic small molecule, an antisense GITRL nucleic acid molecule, and a siRNA GITRL nucleic acid molecule.
51. A vaccine adjuvant comprising a GITR agonist and an antigen selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a cancer antigen, a tumor-associated antigen, and fragments thereof.
52. The vaccine adjuvant of claim 51, wherein the GITR agonist is selected from the group consisting of GITRL, or an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containing an active fragment of GITRL, an agonistic small molecule, and an agonistic anti-GITR antibody.
53. A vaccine adjuvant comprising a GITR antagonist and an antigen selected from the group consisting of an autoantigen, amyloid peptide protein, an alloantigen, a transplant antigen, an allergen, and fragments thereof.
54. The vaccine adjuvant of claim 53, wherein the GITR antagonist is selected from the group consisting of a neutralizing anti-GITRL antibody, a neutralizing anti-GITR antibody, a fusion protein containing GITR, a fusion protein containing an active fragment of GITR, an antagonistic small molecule, an antisense GITRL nucleic acid molecule, and a siRNA GITRL nucleic acid molecule.