WO2013070563A1 - Traitement de troubles auto-immuns et inflammatoires par inhibition de blimp-1 - Google Patents

Traitement de troubles auto-immuns et inflammatoires par inhibition de blimp-1 Download PDF

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WO2013070563A1
WO2013070563A1 PCT/US2012/063635 US2012063635W WO2013070563A1 WO 2013070563 A1 WO2013070563 A1 WO 2013070563A1 US 2012063635 W US2012063635 W US 2012063635W WO 2013070563 A1 WO2013070563 A1 WO 2013070563A1
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blimp
cells
expression
antagonist
prdml
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PCT/US2012/063635
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Renu Jain
Daniel J. Cua
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Merck Sharp & Dohme Corp.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors

Definitions

  • the present invention relates generally to methods of treating autoimmune and inflammatory disorders by inhibiting the transcription regulator Blimp- 1 (PRDM1), methods of enhancing immune response by enhancing the activity of Blimp- 1, and methods of screening for compounds that inhibit or activate Blimp- 1.
  • PRDM1 transcription regulator Blimp- 1
  • Interleukin-23 is an important pro-inflammatory cytokine that has been shown to be an essential factor in the maturation and maintenance of pathogenic Thl7 cells, which are involved in a variety of human autoimmune diseases, including psoriasis, multiple sclerosis, and rheumatoid arthritis.
  • CD4 + T cells that produce interleukin-17 are recognized as a unique population of cells that have critical roles in both host defense against extracellular pathogens and the pathogenesis of human inflammatory diseases including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, psoriasis, and asthma.
  • Thl7 cells are recognized as a unique population of cells that have critical roles in both host defense against extracellular pathogens and the pathogenesis of human inflammatory diseases including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, psoriasis, and asthma.
  • the early development of Thl7 cells is initiated by pro-inflammatory
  • Thl7 cells Signaling through IL-23R is essential for Thl7 cells to differentiate into a "pathogenic" cell population. McGeachy et al. (2009) Nat. Immunol. 10:314; McGeachy et al. (2007) Nat. Immunol. 8: 1390; Langrish et al. (2005) J. Exp. Med. 201 :233; Ghoreschi et al. (2010) Nature 467:967; Hirota et al. (2011) Nat. Immunol. 12:255. In the absence of IL-23 signal, Thl7 cell development is stalled at the early activation stage (McGeachy et al. (2009) Nat. Immunol.
  • GWAS genome-wide association studies
  • Thl7-mediated inflammatory response in subjects where this response is inhibiting an alternative, beneficial immune response. Examples include inhibition of Thl7-mediated inflammation to promote elimination of tumors (Langowski et al. (2006) Nature 442:461; WO 2004/081990) and to promote clearance of chronic infections, such as chronic fungal infections (Zelante et al. (2007) Eur. J. Immunol. 37:2695; WO 2008/153610).
  • a reciprocal need also exists for improved methods of enhancing Thl7 immune responses in subject for whom such response would be expected to be beneficial, for example to enhance the memory response caused by vaccination.
  • Thl7-mediated inflammatory disorders such as autoimmune diseases
  • the subject has multiple sclerosis, psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, and anklyosing spondyitis.
  • Blimp- 1 activity is inhibited by administration of a Blimp- 1 antagonist, including but not limited to a small molecule drug, and antisense nucleic acid molecule, a small interfering nucleic acid molecule ⁇ e.g. siRNA), antibodies or fragments thereof, or other antagonists.
  • a Blimp- 1 antagonist including but not limited to a small molecule drug, and antisense nucleic acid molecule, a small interfering nucleic acid molecule ⁇ e.g. siRNA), antibodies or fragments thereof, or other antagonists.
  • the invention also provides methods of treating cancers and chronic infections, such as chronic fungal infections, by inhibition of the activity of the transcriptional regulator Blimp- 1 (PRDM1).
  • Blimp- 1 activity is inhibited by administration of a Blimp- 1 antagonist, including but not limited to a small molecule drug, an antisense nucleic acid molecule, a small interfering nucleic acid molecule ⁇ e.g. siRNA), antibodies or fragments thereof, or other antagonists.
  • Blimp-1 activity is selectively inhibited in Thl7 cells.
  • a Blimp-1 antagonist is delivered selectively to Thl7 cells by
  • a Blimp-1 antagonist is a nucleic acid sequence, such as an antisense nucleic acid or siRNA, in an expression vector and under the control of a RORyT or IL-23R transcriptional promoters, such that its expression is enhanced in Thl7 cells.
  • the invention further provides methods of enhancing Thl7 response in subjects in which such Thl7 responses would be expected to be beneficial, by enhancing Blimp-1 (PRDM1) activity.
  • the subject is receiving a vaccination and the Blimp-1 agonist enhances generation of a memory response to the immunogen.
  • Blimp-1 activity is enhanced by administration of a Blimp-1 agonist, including but not limited to a small molecule drug, and nucleic acid encoding Blimp-1 or an active fragment thereof, Blimp-1 protein or an active fragment thereof, agonist antibodies or fragments thereof, or other agonists.
  • Blimp-1 activity is selectively enhanced in Thl7 cells.
  • a Blimp-1 agonist is delivered selectively to Thl7 cells by conjugation to an antibody, or antigen binding fragment thereof, that binds to one or more surface markers on Thl7 cells selected from the group consisting of CCR6, CD161 and IL-23R.
  • a Blimp-1 agonist is a nucleic acid sequence encoding Blimp-1 cloned into an expression vector in which it is under the control of a RORyT or IL-23R transcriptional promoters, such that its expression is enhanced in Thl7 cells.
  • FIG. 1 A shows Prdml and Bcl6 gene expression (in arbitrary units) in Thl7
  • FIG. IB shows Prdml and Bcl6 gene expression (in arbitrary units) in wt
  • CD4 + / IL-17 + and CD4 + / IL-17 " cells in IL-23R “7" (IL-23R KO) CD4 + / IL-17 + and CD4 + / IL-17 " cells, as described in Example 2.
  • IL-17 + cells as shown as shaded bars, and IL-17 " cells are shown as open bars. Wt cells are shown as non-hatched bars, and IL-23R " " cells are shown as hatched bars.
  • FIG. 1C shows Prdml and 112 gene expression (in arbitrary units) in CD4 + /
  • FIG. 2A shows the percentage IL-17 + cells in a population of CD4 YFP + T cells from draining lymph nodes of MOG 35 _55 immunized PrdmV 1' after ex vivo stimulation with PMA and ionomycin at day seven post immunization (left plot), and the percentage of GM-CSF + and IFNy + cells within these IL-17 + / CD4 + T cells (right plots) See Example 3.
  • percentages were determined by flow cytometry with intracellular staining for the respective cytokines, and data are representative of three independent experiments with four to five mice per group. Error bars in all graphs are SEM.
  • FIG. 2B shows the percentage of T-bet + cells in a population of CD4 YFP + T cells and IL-17 CD4 YFP + T cells at days seven and 12 post immunization, respectively. See Example 3.
  • FIG. 2C shows the percentages of IL-17 + IL-2 + , IL-17 + IL-2 " , IL-17TL-2 + cells in a population of CD4 YFP + T cells . See Example 3.
  • FIG. 3 A shows EAE clinical score for various mice (wt-Prdml +/+ , Prdml +/ ⁇ ,
  • Prdml 7 was a function of the number of days post immunization with MOG 35 _55 in CFA. See Example 4 for details of the conditional knockout mice used in the experiments.
  • FIG. 3B presents summaries of the maximal clinical scores for three experiments of the type described for FIG. 3A, and at Example 4.
  • FIG. 3C shows percentage of CD4 + T cells and total CD4 + T cells in the CNS of the heterozygous and Prdml KO mice described for FIG. 3A, and at Example 4, 12 days after EAE induction.
  • FIGS. 3D, 3E and 3F show the percentages and absolute numbers of IL-17 + .
  • FIG. 4A shows the percentage of CCR6 + and CXCR3 + cells in a population of
  • CD4 + YFP + T cells in Prdml heterozygotes and knockouts as described in greater detail at Example 5.
  • FIGS. 4B and 4C show percentages of Ki-67 + and Bcl-2 + cells in a population of CD4 + YFP + and IL-17 + CD4 + YFP + T cells, respectively, as indicated. See Example 5.
  • Blimp-1 antagonist refers to any agent that inhibits Blimp-1 activity.
  • Blimp-1 agonist refers to any agent that enhances or mimics Blimp-1 activity.
  • administering refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell.
  • administering also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell.
  • Treatment refers to therapeutic treatment, prophylactic or preventative measures, to research and diagnostic applications.
  • Treatment as it applies to a human, veterinary, or research subject, or cell, tissue, or organ, encompasses contact of an agent with animal subject, a cell, tissue, physiological compartment, or physiological fluid.
  • a binding compound that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, that do not materially affect the properties of the binding compound.
  • Effective amount encompasses an amount sufficient to ameliorate or prevent a symptom or sign of the medical condition.
  • Effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • An effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side affects. See, e.g., U.S. Pat. No. 5,888,530.
  • An effective amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects.
  • the effect will result in an improvement of a diagnostic measure or parameter by at least 5%, usually by at least 10%, more usually at least 20%>, most usually at least 30%>, preferably at least 40%>, more preferably at least 50%, most preferably at least 60%, ideally at least 70%, more ideally at least 80%>, and most ideally at least 90%>, where 100% is defined as the diagnostic parameter shown by a normal subject. See, e.g., Maynard et al. (1996) A Handbook of SOPs or Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK.
  • the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples without the agent.
  • Control samples i.e., not treated with agent, are assigned a relative activity value of 100%.
  • Inhibition is achieved when the activity value relative to the control is about 90%> or less, typically 85%> or less, more typically 80%> or less, most typically 75%> or less, generally 70%> or less, more generally 65%> or less, most generally 60%> or less, typically 55%> or less, usually 50%> or less, more usually 45%> or less, most usually 40%> or less, preferably 35%> or less, more preferably 30%) or less, still more preferably 25%> or less, and most preferably less than 25%>.
  • Activation is achieved when the activity value relative to the control is about 110%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
  • Endpoints in activation or inhibition can be monitored as follows. Activation, inhibition, and response to treatment, e.g., of a cell, physiological fluid, tissue, organ, and animal or human subject, can be monitored by an endpoint.
  • the endpoint may comprise a predetermined quantity or percentage of, e.g., an indicia of inflammation, oncogenicity, or cell degranulation or secretion, such as the release of a cytokine, toxic oxygen, or a protease.
  • the endpoint may comprise, e.g., a predetermined quantity of ion flux or transport; cell migration; cell adhesion; cell proliferation; potential for metastasis; cell differentiation; and change in phenotype, e.g., change in expression of gene relating to inflammation, apoptosis, transformation, cell cycle, or metastasis
  • a predetermined quantity of ion flux or transport cell migration; cell adhesion; cell proliferation; potential for metastasis; cell differentiation; and change in phenotype, e.g., change in expression of gene relating to inflammation, apoptosis, transformation, cell cycle, or metastasis
  • An endpoint of inhibition is generally 75% of the control or less, preferably
  • an endpoint of activation is at least 150% the control, preferably at least two times the control, more preferably at least four times the control, and most preferably at least 10 times the control.
  • the present invention relates to the discovery that zinc finger domain containing molecule Blimp- 1 is a key regulator of the IL-23 -mediated Thl7 effector differentiation pathway.
  • Blimp- 1 zinc finger domain containing molecule Blimp- 1 is a key regulator of the IL-23 -mediated Thl7 effector differentiation pathway.
  • Excessive Thl7 responses are believed to be responsible for the pathogenesis of a number of autoimmune and chronic inflammatory disorders, including but not limited to multiple sclerosis, psoriasis, rheumatoid arthritis, Crohn's disease, and ankylosing spondylitis.
  • Thl7 responses in subjects for which such an inflammatory response is detrimental such as those with cancers or chronic infections, e.g. chronic fungal infections.
  • enhancement of Blimp- 1 expression or activity will find use in enhancing Thl7 responses in subjects for which such responses are beneficial, such as those receiving vaccinations.
  • Blimp- 1 (B lymphocyte-induced maturation protein- 1), encoded by PRDM1, is a zinc finger domain-containing transcription factor that is induced by cytokine signaling during early T cell activation. It directly represses il-2 transcription to ensure properly controlled immune response. Martins et al. (2008) J. Exp. Med. 205:1959. It was first discovered 16 years ago as a transcriptional repressor of human interferon-beta (IFN- ⁇ ) promoter that was induced upon viral infection. Ectopic expression of Blimp- 1 is sufficient to drive B cells into mature Ig-secreting plasma cells. Blimp- 1 is also a key regulator of terminal differentiation in various cell types.
  • Blimp- 1 has been reported to control effector cytokine production in human NK cells. Smith et al. (2010) J. Immunol. 185:6058. Blimp-1 promotes differentiation of B cells to antibody secreting plasma cells (Turner et al. (1994) Cell 77:297; Shapiro-Shelef et al. (2003) Immunity 19:607), CD8 + T cells to cytotoxic T lymphocytes (CTLs) (Rallies et al. (2009) Immunity 31 :283; Shin et al. (2009) Immunity 31 :309; Rutishauser et al.
  • CTLs cytotoxic T lymphocytes
  • Blimp-1 (PRDM1) is also known as PR domain zinc finger protein 1; positive regulatory domain 1 -binding factor 1; as well as BLIMP 1; PRDI-BF1; PRDIBF1;
  • Nucleic acid sequences are provided at RefSeqs NM 001198.3 (longer isoform 1, PRDMla) and NM 182907.2 (shorter isoform 2, PRDMip), and corresponding polypeptide sequences are provided at RefSeqs NP 001189.2 and NP 878911.1, the sequences of which are hereby incorporated by reference in their entireties, and also provided at SEQ ID NOs: 1 and 3 (nucleic acid sequences) and SEQ ID NOs: 2 and 4 (polypeptide sequences). See Table 1.
  • Mouse Blimp- 1 may find use in screening for antagonists and agonists of
  • Blimp- 1 for use as drugs in humans.
  • Mouse Blimp- 1 (PRDM1) is described further at Gene ID No. 12142.
  • Nucleic acid and polypeptide sequences are provided at RefSeqs
  • NM_007548.3 and NP_031574.2 the sequences of which are hereby incorporated by reference in their entireties, and also provided at SEQ ID NOs: 5 and 6. See Table 1.
  • Blimp-1 is a key transcription factor that regulates IL-23- mediated differentiation of pathogenic Thl7 cells.
  • Blimp-1 represses IL-2 expression, allowing Thl7 cells to transition to a mature phenotype.
  • Blimp-1 facilitates co-expression of effector cytokines including GM-CSF and IFNy in activated IL-17-producing T cells and endows the Thl7 cells with inflammatory functions, and enhances the expression of survival and homing markers associated with pathogenic function.
  • T cell-specific Blimp-1 deficiency renders mice resistant to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, consistent with its essential role in the disease.
  • EAE experimental autoimmune encephalomyelitis
  • Example 2 Results are provided at FIG. 1A. Prdml expression was specific to Thl 7 cells as compared to Thl cells, which had reduced Prdml but increased Bcl6 expression. Bcl6 is an antagonistic regulator of Blimp-1. These results indicate that Blimp-1 expression is specifically elevated in Thl 7 cells.
  • IL-23 signaling induces Blimp-1 expression
  • gene expression was measured in sorted wt and IL-23R-deficient Thl 7 cells, as described in Example 2.
  • IL-23 signaling induces Prdml expression in Thl 7 cells, and reduced Bcl6 expression, as show in FIG. IB.
  • IL-2 is involved in Blimp- 1 -mediated effects described herein. It has been previously observed that IL-23R " " fail to downregulate IL-2 expression, and the resulting continued IL-2 expression inhibits maturation of Thl7 to their fully differentiated
  • Blimp-1 exhibit the hallmark properties of pathogenic Thl7 cells, including elevated IL-17, INF- ⁇ , GM-CSF, and T-bet expression. Details are provided at Example 3. To test whether Blimp-1 deletion affects Thl 7 differentiation we assessed draining lymph nodes at day seven post immunization with MOG 3 5_55 peptide in CFA. As shown in FIG. 2A (left plot), the proportion of YFP + CD4 + T cells producing only IL-17 was reduced by Prdml deficiency. However, there was a significant defect in the ability of Prdml "7" IL-17 producers to co- express GM-CSF and IFNy.
  • IL-23 promotes GM-CSF expression by T cells and that GM-CSF is essential for the ability of Thl 7 cells to drive inflammation in the central nervous system (CNS).
  • CNS central nervous system
  • Thl7 cells had transitioned towards a mature IL-17 single producer phenotype, and were no longer IL-2 co-producers. See FIG. 2C. But in the absence of IL-23 signal (due to Prdml ablation), nearly all the Thl7 cells were trapped at the early activation (IL-2 co-producer) stage and were unable to downregulate their IL-2 expression. All together these results confirm that IL-23 drives full effector differentiation of Thl7 cells in lymph nodes by inducing Blimp-1.
  • IFNy producing Thl7 cells are known to be present in the lesional tissue in EAE and preferentially accumulate in the CNS of patients with multiple sclerosis (Kebir et al. (2009) Ann. Neurol. 66:390), highlighting their importance in promoting inflammation.
  • the invention provides a method of treating autoimmune diseases or chronic inflammatory diseases, comprising administering an antagonist of Blimp- 1.
  • the inflammatory or autoimmune disease is selected from the group consisting of diabetes, multiple sclerosis, uveitis, rheumatoid arthritis, psoriasis, asthma, chronic obstructive pulmonary disease, atherosclerosis, and inflammatory bowel diseases. See, e.g., Duvallet et al. (2011) Annals Med. 43:503.
  • the inflammatory bowel disease is selected from the group consisting of Crohn's disease, ulcerative colitis, sprue and food allergies.
  • the methods of the present invention may be used in the treatment of inflammatory bowel disease (IBD). Ulcerative colitis (UC) and Crohn's disease are the two major forms of idiopathic Inflammatory Bowel Disease (IBD) in humans. Kirsner et al. eds. (1988) Inflammatory Bowel Disease: 3rd ed. Goldner et al. (1990) Idiopathic Inflammatory Bowel Disease, in Stein,, ed., Internal Medicine, Little Brown & Co., Boston, pp. 369-380; Cello et al. (1989) Ulcerative Colitis, in Sleisenger et al. eds. (1989) Gastrointestinal Disease: Pathophysiology Diagnosis Management, W. B. Saunders Co., Philadelphia, p. 1435.
  • MS multiple sclerosis
  • MS is a multi-factorial inflammatory disease of the human central nervous system resulting in the slowing of electrical conduction along the nerve.
  • the disease is characterized by an increase in the infiltration of inflammatory cells, loss of oligodendrocytes, and increased gliosis (astrocyte hypertrophy and proliferation).
  • Myelin is the target of this cellular autoimmune inflammatory process, leading to impaired nerve conduction. See Thompson (1996) Clin. Immunother. 5:1.
  • Clinical manifestations are variable, but are usually characterized by an initial relapsing-remitting course, with acute exacerbation followed by periods of clinical stability.
  • EAE Experimental Autoimmune Encephalomyelitis
  • CNS central nervous system
  • SJL mice susceptible strains of mice
  • CD4 + T cells antigen stimulated CD4 + T cells.
  • EAE is widely recognized as an acceptable animal model for multiple sclerosis in primates. Alvord et al. eds.
  • RA rheumatoid arthritis
  • RA is a chronic, systemic and articular inflammatory disorder characterized by an imbalance in the immune system that causes overproduction of proinflammatory cytokines, e.g. tumor necrosis factor alpha (TNFa) and interleukin 1 (IL-I), and a lack of anti-inflammatory cytokines, e.g. IL-10.
  • cytokines e.g. tumor necrosis factor alpha (TNFa) and interleukin 1 (IL-I)
  • IL-10 interleukin 1
  • RA is characterized by synovial inflammation, which progresses to cartilage destruction, bone erosion and subsequent joint deformity.
  • the primary symptoms of RA are joint inflammation, stiffness, swelling, fatigue, difficulty moving, and pain.
  • Activated T-lymphocytes produce cytotoxins and pro-inflammatory cytokines, while macrophages stimulate the release of prostaglandins and cytotoxins.
  • Vasoactive substances histamine, kinins, and prostaglandins are released at the site of inflammation and cause edema, warmth, erythema, and pain associated with inflamed joints.
  • One of the models used to test for new therapies for arthritis includes the collagen-induced arthritis (CIA) model. Myers et al. ⁇ 997) Life Sci. 61 : 1861. In this model, immunization of genetically susceptible rodents or primates with Type II collagen leads to the development of a severe polyarticular arthritis that is mediated by an autoimmune response. It mimics the synovitis and erosions of cartilage and bone that are characteristic of RA.
  • CIA collagen-induced arthritis
  • the methods of the present invention may be used in the treatment of diabetes
  • IDDM The main clinical feature of IDDM is elevated blood glucose levels
  • hypoglycemia hyperglycemia
  • the elevated blood glucose level is caused by autoimmune destruction of insulin producing cells in the islets of Langerhans of the pancreas. This is accompanied by a massive cellular infiltration surrounding and penetrating the islets (insulitis) composed of a heterogeneous mixture of CD4 + and CD8 + T-lymphocytes, B-lymphocytes, macrophages and dendritic cells.
  • IDDM is the NOD mouse.
  • the NOD mouse represents a model in which autoimmunity against beta-cells is the primary event in the development of IDDM. Diabetogenesis is mediated through a multi-factorial interaction between a unique MHC class II gene and multiple, unlinked, genetic loci, as in the human disease. Moreover, the NOD mouse demonstrates the critical interaction between heredity and environment, and between primary and secondary auto-immunity. Its clinical manifestation, for example, depends on various external conditions, most importantly on the micro-organism load of the environment in which the NOD mouse is housed.
  • STZ streptozotocin
  • Hartner et al. (2005) BMC Nephrol. 6:6 This model has been used extensively as an animal model to study the mechanisms involved in the destruction of pancreatic beta cells in IDDM.
  • diabetes is induced in rodents by the beta-cell toxin streptozotocin (STZ).
  • STZ is taken up by the pancreatic beta cell through the glucose transporter GLUT-2, decomposes intracellularly, and causes damage to DNA either by alkylation or by the generation of NO.
  • the methods of the present invention may also be useful in treatment of other disorders with which a Thl7 inflammatory response is undesirable.
  • Thl7-mediated inflammation may promote, rather than inhibit, the growth of tumors. Langowski et al. (2006) Nature 442:461; WO 2004/081990.
  • the invention provides a method of promoting immunosurveillance and treatment of cancer comprising administering an antagonist of Blimp- 1.
  • Exemplary tumors that may be treated using the methods of treatment of the present invention include tumors of the gastrointestinal tract, such as, but not limited to, tumors of the stomach, bowel and intestine, and also tumors of the lung, liver, pancreas, breast, bone and any other solid tumor or blood borne tumor.
  • the treatment may result in inhibition of tumor cell growth or proliferation, or may result in preventing the further spread (metastasis) of tumor cells to other tissues or organs.
  • Thl7-mediated inflammation may prevent the mounting of a productive immune response to chronic infections, such a chronic fungal infections.
  • the invention also provides a method of promoting clearance of chronic infections, such as chronic fungal infections, comprising administering an antagonist of Blimp- 1.
  • the present invention involves, in one aspect, antagonism of Blimp- 1 activity as a means of treating autoimmune and inflammatory disorders, or in treating cancers or chronic infections, such as chronic fungal infections.
  • Many of the experiments herein make use of Prdml knockout mutants, and demonstrate that cells lacking Blimp- 1 have different properties than wt cells. These different properties can be used to screen compounds for Blimp- 1 antagonist activities, since compounds that induce effects mimicking the properties Prdml knockout mutants in wt cells will be Blimp- 1 antagonists.
  • Compounds identified on the basis of their ability to inhibit mouse Blimp- 1 in mouse models would be expected to also inhibit human Blimp- 1 in most cases, and thus be potential human therapeutics.
  • Small molecule drugs can be selected from natural or synthetic libraries of compounds, isolated from plant or animal extracts, or synthesized based on rational drug design considerations.
  • Blimp- 1 antagonists can be obtained using any of the numerous suitable approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds.
  • Lam (1997) Anticancer Drug Des . 12: 145; U.S. Pat. Nos.
  • Another approach uses recombinant bacteriophage to produce large peptide libraries. Using the "phage method" (Scott and Smith (1990) Science 249:386; Cwirla et al. (1990) Proc. Nat'l Acad. Sci. 87:6378; Devlin et a/.(1990) Science 249:404), very large libraries can be constructed (10 6 -10 8 peptides).
  • a second approach uses primarily chemical methods, of which the Geysen method (Geysen et al. (1986) Molecular Immunology 23:709; Geysen et al. (1987) J. Immunologic Method 102:259) and the method of Fodor et al.
  • selector molecules immobilized on a solid support can be used to select peptides that bind to them. This procedure reveals a number of peptides that bind to the selector and that often display a common consensus amino acid sequence.
  • Bio amplification of selected library members and sequencing allows the determination of the primary structure of the peptide(s).
  • Peptides are expressed on the tip of the filamentous phage Ml 3, as a fusion protein with the phage surface protein pilus (at the N-terminus).
  • a filamentous phage carries on its surface three to five copies of pili and therefore of the peptide.
  • Synthetic libraries can also be used to screen for novel peptides, or mimetics or fragments thereof, according to the present invention. Needels et al . (1993) Proc. Nat'l Acad. Sci. USA 90:10700; Ohlmeyer et al . (1993) Proc. Nat'l Acad. Sci. USA 90:10922; Lam et al . Infl Pat. Appl. Pub. WO 92/00252; Kocis et al . Infl Pat. Appl. Pub. WO 94/28028, each of which is incorporated herein by reference in its entirety.
  • Additional compounds that can be used to selectively inhibit Blimp- 1 include siRNA (see, e.g., Stevenson (2004) New. England. J. Med. 351 : 1772), antisense nucleic acids, and ribozymes, as disclosed at U.S. Pat. Nos. 6,211,164, 6,677,445 and 6,846,921; U.S. Pat. App. Publication Nos. 2004/0097446 and 2005/01533925; and PCT publications
  • nucleic acid-based antagonists can be designed in light of the known nucleic acid sequence for human Blimp- 1, as provided at SEQ ID NOs: 1 and 3 herein.
  • exemplary methods of using siRNA in gene silencing and therapeutic treatment are disclosed at PCT publications WO 02/096927 (VEGF and VEGF receptor); WO 03/70742 (telomerase); WO 03/70886 (protein tyrosine phosphatase type IVA (Prl3)); WO 03/70888 (Chkl); WO 03/70895 and WO 05/03350 (Alzheimer's disease);
  • WO 03/70983 protein kinase C alpha
  • WO 03/72590 Map kinases
  • WO 03/72705 cyclin D
  • WO 05/45034 Parkinson's disease.
  • Exemplary experiments relating to therapeutic uses of siRNA have also been disclosed at Zender et al. (2003) Proc. Nat'l. Acad. Sci. (USA) 100:7797; Paddison et al. (2002) Proc. Nat'l Acad. Sci. (USA) 99: 1443; and Sah (2006) Life Sci. 79: 1773.
  • siRNA molecules have also been used in clinical trials, e.g., of chronic myeloid leukemia (CML) (ClinicalTrials.gov Identifier: NCT00257647) and age-related macular degeneration (AMD) (ClinicalTrials.gov Identifier: NCT00363714).
  • CML chronic myeloid leukemia
  • AMD age-related macular degeneration
  • siRNA is used herein to refer to molecules used to induce gene silencing via the RNA interference pathway (Fire et al. (1998) Nature 391 :806)
  • siRNA molecules need not be strictly polyribonucleotides, and may instead contain one or more modifications to the nucleic acid to improve its properties as a therapeutic agent.
  • agents are occasionally referred to as "siNA” for short interfering nucleic acids.
  • siNA short interfering nucleic acids.
  • siRNA duplexes comprise two 19 - 25 nt (e.g.
  • shRNA short hairpin RNAs
  • Such shRNA molecules may be delivered, e.g., using a retroviral vector, as disclosed at U.S. Pat. App. Pub. 2007/0154487 (see Figures 42 and 43, and Example 9, therein), the disclosure of which is hereby
  • siRNA duplexes are simply the reverse complement of the sense strand, with the caveat that both strands have
  • 2-nucleotide 3' overhangs That is, for a sense strand "n" nucleotides long, the opposite strand is the reverse complement of residues 1 to (n-2), with 2 additional nucleotides added at the 3' end to provide an overhang.
  • the opposite strand also includes two U residues at the 3 ' end.
  • the opposite strand also includes two dT residues at the 3 ' end.
  • Double stranded RNA or a small interfering RNA, including shRNA can be delivered to the target cell in a patient in a number of different ways. For example, it may be conveniently administered by microinjection; by bombardment by particles covered by the dsRNA; by soaking the cell or organism in a solution of the dsRNA; by electroporation of cell membranes in the presence of the dsRNA; by liposome-mediated delivery of dsRNA and transfection mediated by chemicals such as calcium phosphate; by viral infection; by transformation; and the like.
  • the dsRNA may be introduced along with components that enhance RNA uptake by the cell, stabilize the annealed strands, or otherwise increase inhibition of Prdml gene expression.
  • dsRNA or shRNA can be administered via an implantable extended release device.
  • the siRNA or shRNA is conjugated to an antibody, or antigen-binding fragment thereof, that specifically binds to the surface of Thl7 cells, such as antibodies specific for CCR6, CD161 and/or IL-23R, including bispecific constructs binding to two or more of these surface markers. See, e.g., WO 08/106131.
  • Antisense is an antibody, or antigen-binding fragment thereof, that specifically binds to the surface of Thl7 cells, such as antibodies specific for CCR6, CD161 and/or IL-23R, including bispecific constructs binding to two or more of these surface markers. See, e.g., WO 08/106131.
  • Antisense nucleic acids can also be used as Blimp- 1 antagonists in the methods of the present invention.
  • a Blimp- 1 /Prdml antisense nucleic acid sequence can comprise the complement of any contiguous segment within the sequence of the Prdml genes of the invention (SEQ ID NOs: 1 and 3).
  • "Antisense" nucleic acids are DNA or R A molecules that are complementary to at least a portion of a specific mRNA molecule. See Weintraub (1990) Sci. Amer. 262:4046; Marcus-Sekura (1987) Nucl. Acid Res. 15:5749; Marcus-Sekura (1988) Anal. Biochem. 172:289; Brysch et al.
  • 5' untranslated sequence up to and including the AUG initiation codon are considered preferred for antisense applications because, in general, they efficiently inhibit translation.
  • sequences complementary to the 3' untranslated sequences of mRNAs have also been shown to be effective at inhibiting translation of mRNAs. See generally Wagner (1994) Nature 372:333.
  • oligonucleotides complementary to the 5 '-non-translated region, the 3 '-non-translated region, or any other suitable region of the transcript ⁇ e.g., part of a coding region) could be used in an antisense approach to inhibit translation of endogenous Prdml gene mRNA.
  • Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
  • Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5'-, 3'-, or coding region of a gene mRNA, antisense nucleic acids should be at least six nucleotides long, and are preferably oligonucleotides ranging from six to about 50 nucleotides long.
  • the oligonucleotide is at least ten nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (Letsinger et al. (1989) Proc. Nat'l Acad. Sci. USA 86:6553; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648; PCT Publication No. WO 88/09810) or the blood-brain barrier (PCT Publication No. WO 89/10134), hybridization- triggered cleavage agents (Krol et al. (1988) BioTechniques 6:958) or intercalating agents (Zon (1988) Pharm. Res.
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane Letsinger et al. (1989) Proc. Nat'l Acad. Sci. USA 86:6553; Lemai
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl -2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methyIguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2- fluoroarabinose, xylulose, and hexose.
  • the antisense may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2- fluoroarabinose, xylulose, and hexose.
  • oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res. 16:3209, 1988), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Nat'l Acad. Sci. U.S.A. 85:7448), etc.
  • R A to cells e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • a preferred approach employs a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong transcriptional promoter. The use of such a construct to transfect target cells in the patient results in the transcription of sufficient amounts of single stranded RNAs that form complementary base pairs with the endogenous Pdrml transcripts and thereby prevent translation.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be effected by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bemoist and Chambon (1981) Nature 290:304), the promoter contained in the 3D long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980) Cell 22:787), the herpes thymidine kinase promoter (Wagner et al. (1981) Proc. Nat'l Acad. Sci. USA 78:1441), the regulatory sequences of the SV40 early promoter region (Bemoist and Chambon (1981) Nature 290:304), the promoter contained in the 3D long terminal repeat of Rous sarcoma virus
  • viral vectors can be used that selectively infect the desired tissue or cell type (e.g. Thl7 cells).
  • An effective dose of an antisense oligonucleotide ranges from about 0.01 to
  • the antisense oligonucleotide may be delivered subcutaneously, intravenously, or by any other suitable delivery method.
  • Nucleic acid-based antagonists and agonists of Blimp- 1, such as antisense nucleic acids, siRNAs, and Blimp- 1 encoding constructs, may be delivered to target cells as follows.
  • a gene encoding a Blimp- 1 antagonist or agonist can be introduced either in vivo, ex vivo, or in vitro in a viral vector.
  • Such vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred.
  • Defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • the striatal subventricular zone SVZ
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al. (1991) Molec. Cell. Neurosci. 2:320), an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest. 90:626 (1992)), and a defective adeno-associated virus vector (Samulski et al.
  • HSV1 herpes virus 1
  • the inhibitor of Prdml can be introduced in a retroviral vector, e.g., as described in U.S. Pat. No. 5,399,346; Mann et al. (1983) Cell 33:153; U.S. Pat. No. 4,650,764; U.S. Pat. No. 4,980,289; Markowitz et al. (1988) J. Virol, 62: 1120; U.S. Pat. No. 5,124,263; Infl Pat. App. Pub. WO 95/07358; and Kuo et al. (1993) Blood 82:845.
  • Targeted gene delivery is described in Int'l Pat. App. Pub. WO 95/28494.
  • the vector can be introduced by lipofection.
  • Liposomes may be used for encapsulation and trans fection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated
  • transfection can be used to prepare liposomes for in vivo transfection of an expression vector encoding Blimp-1 or an antagonist thereof. Feigner et. al. (1987) Proc. Nat'l Acad. Sci.
  • DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, e.g., Wu et al. (1992) J. Bioi. Chem., 267:963-967; Wu and Wu (1988) J. Biol. Chem. 263: 14621; Can. Pat. App. Pub.No. 2,012,311.
  • Antibodies including intrabodies ⁇ e.g. Alvarez et al. (2000) Clinical Cancer
  • Blimp-1 protein, or antigenic peptide fragments thereof may be used to generate these antibodies, based on the known amino acid sequence of Blimp-1, as provided at SEQ ID NOs: 2 and 4 herein. Such antibodies or fragments may be chimeric, humanized, human or single chain antibodies or fragments.
  • a nucleic acid molecule encoding the antibody such as a recombinant expression vector encoding the antibody, is introduced into the cell.
  • the antibody used to modulate protein expression or function is a single chain Fv (scFv) fragment, although whole antibodies, or antigen binding fragments thereof (e.g., Fab fragments) may also be useful.
  • an antibody is expressed intracellularly in a Thl7 cell, or precursor thereof, to inhibit the activity of Blimp-1.
  • a nucleic acid molecule encoding the antibody may optionally be introduced using a recombinant viral vector, such a retroviral vectors ⁇ e.g. lentiviral vectors), adenoviral vectors and adeno-associated viral (AAV) vectors, or any other vector system suitable for delivery of genes to cells in vivo.
  • Blimp-1 has been reported to play a role in promoting differentiation of regulatory T cells (Tregs) to IL-10-expressing effector Tregs (Cretney et al. (2011) Nat. Immunol. 12:304) and in inhibiting Bcl-6-driven differentiation of CD4 + T cells to T follicular helper cells (T FH ) (Johnston et al. (2009) Science 325:1006; Nurieva et al. (2009) Science 325 : 1001 ; Yu et al. (2009) Immunity 31 :457). Given these disparate effects on different T cell subsets, the methods of the present invention are best performed by antagonizing Blimp-1 activity specifically in Thl 7 cells. Such targeted inhibition would avoid effects of Blimp-1 antagonists on non-Thl7 cells, which might otherwise lead to undesired side effects caused by reduction of Treg function and enhanced T FH function.
  • Thl 7 cells are characterized, inter alia, by expression of the transcription factor RORyt, IL-23 receptor (IL-23R), and CCR6.
  • RORyt and IL-23R promoters may be used to drive expression of Blimp- 1 antagonistic nucleic acid constructs in gene therapy embodiments of the present invention.
  • Such vectors would limit expression of Blimp- 1 antagonistic nucleic acid constructs to those cells in which RORyt or IL-23R genes are being expressed, and thus target their effects to the desired Thl7 cell population.
  • Blimp- 1 antagonistic nucleic acid constructs may comprise, for example, antisense nucleic acids or siRNA constructs designed to block the activity of Blimp- 1.
  • IL-23R and RORyt promoters are discussed at Gocke et al. (2007) J. Immunol. 178:1341 and Eberl et al. (2003) Immuno. Rev. 195:81, respectively, the disclosures of which are hereby incorporated by reference in their entireties.
  • Antibodies exhibiting specific for Thl7 cells may be used to target Blimp- 1 antagonist compounds to Thl7 cells.
  • Anti-CCR6 antibodies have been proposed for therapeutic use. See, e.g., U.S. Pat. No. 7,465,448, and U.S. Pat. App. Pub. Nos. 2002/0138860 and 2009/0041787.
  • Anti-CCR6 monoclonal antibodies include, e.g., clone 11 A9 (Pharmingen, San Diego, Calif, USA), clone 4C6 (Acris
  • Blimp- 1 antagonists may be bound to the anti-CCR6 antibodies to form antibody-drug conjugates.
  • Antibodies that bind to the cell surface receptor CCR6 would be expected to be internalized, along with any conjugated drug ⁇ e.g. an siRNA molecule), based on the internalization of CCR6 upon binding of its natural ligands (CCL20/MIP3a) and ⁇ -defensin.
  • Antibodies that bind to other surface markers on Thl7 cells may also be used to deliver Blimp- 1 antagonists to Thl7 cells.
  • Bispecific and multispecific binding compounds such as antibodies, antigen binding fragments, or antibody-like constructs that bind to two or more of CCR6, CD161 and IL-23R may also be used in the methods of treatment of the present invention to deliver Blimp- 1 antagonists to Thl7 cells.
  • Exemplary humanized antibodies to human IL-23R are disclosed at WO 2008/106134.
  • Exemplary anti-human CD161 are available as clone B199.2 (Pierce Antibodies, Rockford, III, USA) and clone DX12 (BD Pharmingen, San Diego, Calif, USA). Evaluation of Antagonist Activity
  • Blimp- 1 antagonists Compounds that mimic the effects of the Pdrml "7" mutants used in the experiments herein will be considered Blimp- 1 antagonists.
  • Potential Blimp- 1 antagonists can be tested for their ability to produce the same biological effects as the knockout mice used in many of the experiments disclosed herein.
  • Agents that alter the phenotype of wild-type mice to mimic that of homozygous Prdml knockout mice are likely to be Blimp- 1 antagonists.
  • agents that promote IL-2 expression, or inhibit GM-CSF, IFN- and/or IL-17 expression, in CD4 + T cells will be likely Blimp-1 antagonists.
  • Preferred antagonists include those that act specifically in Thl7 (and precursor) cells, e.g.
  • agents that are preferentially targeted to Thl7 cells or agents that are active preferentially in Thl7 cells such as nucleic acid constructs that are selectively expressed in those cells.
  • the present invention involves enhancement of Blimp-1 activity as a means of enhancing Thl7 response where such enhancement may be beneficial, such as during vaccinations.
  • Any agent capable of enhancing Blimp-1 activity, or enhancing its expression or protein levels, can act as a Blimp-1 agonist in the methods of the present invention.
  • Small molecule drugs can be selected from natural or synthetic libraries of compounds, or synthesized based on rational drug design considerations. Compounds that exhibit effects opposite to the effects of the Pdrml "7" mutants used in the experiments herein will be considered potential Blimp-1 agonists.
  • Blimp-1 protein itself or a biologically active fragment or sequence variant thereof, would be a Blimp-1 agonist.
  • Thl7 cells are characterized, inter alia, by expression of the transcription factor RORyt and IL-23R, their respective promoters may be used to drive Blimp- 1 expression in gene therapy embodiments of the present invention.
  • Such vectors would limit Blimp- 1 expression to those cells in which RORyt or IL-23R genes are being expressed, and thus target their effects to the desired Thl7 cell population.
  • IL-23R and RORyt promoters are discussed at Gocke et al. (2007) J. Immunol. 178: 1341 and Eberl et al. (2003) Immuno. Rev. 195:81, respectively, the disclosures of which are hereby incorporated by reference in their entireties.
  • compositions for use in the methods of the present invention, the agent or agents are admixed with a pharmaceutically acceptable carrier or excipient, see, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).
  • Formulations of therapeutic agents or combinations thereof may be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions. See, e.g., Hardman et al. (2001) Goodman and Gilman 's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications , Marcel Dekker, NY; Lieberman et al. (eds.) (1990)
  • inert, pharmaceutically acceptable carriers can be either solid or liquid.
  • Solid form preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories.
  • the powders and tablets may be comprised of from about 5 to about 95 percent active ingredient.
  • Suitable solid carriers are known in the art, e.g., magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration. Examples of
  • Liquid form preparations include solutions, suspensions and emulsions.
  • Liquid form preparations may also include solutions for intranasal administration.
  • Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, e.g. nitrogen.
  • a pharmaceutically acceptable carrier such as an inert compressed gas, e.g. nitrogen.
  • solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration.
  • liquid forms include solutions, suspensions and emulsions.
  • the compounds of the invention may also be deliverable transdermally.
  • the transdermal compositions can take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
  • the pharmaceutical preparation is in a unit dosage form.
  • the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose.
  • the quantity of active compound in a unit dose of preparation may be varied or adjusted from about 1 mg to about 100 mg, preferably from about 1 mg to about 50 mg, more preferably from about 1 mg to about 25 mg, according to the particular application and the properties of the specific active compound in question ⁇ e.g. the affinity, toxicity or pharmacokinetic profile).
  • the actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage regimen for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day as required.
  • a typical recommended daily dosage regimen for oral administration can range from about 1 mg/day to about 500 mg/day, preferably 1 mg/day to 200 mg/day, in two to four divided doses.
  • Toxicity and therapeutic efficacy of the therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD 5 o and ED 5 o.
  • Therapeutic combinations exhibiting high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • an administration regimen for a therapeutic agent depends on several factors, including the serum or tissue turnover rate of the agent, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix.
  • an administration regimen maximizes the amount of therapeutic agent delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of agent delivered depends in part on the particular agent and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub.
  • Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.
  • Important diagnostic measures include those of symptoms of, e.g., reduction in the rate of growth of tumor tissue, or alteration of biomarkers associated with therapeutic efficacy.
  • Methods for flow cytometry including fluorescence activated cell sorting detection systems (FACS ® ), are available. See, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2 nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available. Molecular Probes (2003) Catalogue,
  • mice were obtained from Jackson Research Labs. OT-II and
  • CD45.1 + mice were obtained from Jackson Research Labs and interbred at Merck. The generation of U23ra ⁇ ' ⁇ mice has been described. Chan et al. (2006) J. Exp. Med. 203:2577. Il23ra ⁇ ' ⁇ OT-II CD45.1 + mice were generated at Merck. IL-17-GFP Knockin reporter mice were generated and provided by Biocytogen. Blimp- 1 conditional knockout mice ⁇ Prdmlfl/fl) were purchased from Jackson Research Labs and Rosa-YFP ⁇ DlcJf mice were provided by Pamela J. Fink (originally from Nigel Killeen) and interbred at Merck.
  • mice C57BL/6 mice (including IL-17-GFP Knockin reporter mice and Prdml ⁇ Rosa-YFF ⁇ Dlck cre mice) were immunized in four sites on the back with 100 ⁇ g MOG35-55 in 200 ml CFA containing 100 ⁇ g tuberculosis strain H37Ra. All mice also received 100 ng pertussis toxin (List Biological Laboratories) intraperitoneally on days 0 and 2.
  • mice were assigned clinical scores for EAE according to the following scale: 1, flaccid tail; 2, impaired righting reflex and hindlimb weakness; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, hindlimb paralysis with partial forelimb paralysis; 6, moribund.
  • CD4 (RM4-5), CD45.1 (A20), CD44 (IM7), IFN- ⁇ (XMG1.2), IL-17 (TC11- 18H10), IL-2 (JES6-5H4), CCR6 (140706), Ki-67 (B56), Bcl-2 (Bcl-2/100) and pSTAT3 (pS727).
  • GM-CSF MP1-22E9
  • T-bet (4B10) T-bet (4B10)
  • CXCR3 (173) antibodies were purchased from eBioscience.
  • Intracellular cytokine staining was performed with Cytofix-cytoperm kit from BD according to the manufacturer's instructions.
  • pSTAT3 staining was performed with BD Phosflow kit according to the manufacturer's instructions.
  • OVA(323-339) was obtained from Biosynthesis Inc (Lewisville, TX).
  • recipient mice received 1 10 5 CD45.1 + 23ra ' ⁇ or Il23ra +I+ OT-II CD4 + T cells intravenously 1 day before being immunized subcutaneously in the flank with 100 mg OVA (323-339) in CFA.
  • the phenotype of OT-II + cells was assessed by flow cytometry with gating on live CD4 + CD45.1 + cells.
  • CD4 T cells For stimulation of CD4 T cells ex vivo for cytokine analysis, draining lymph nodes were collected at various time points after immunization and single-cell suspensions were obtained. Cells were stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin (500 ng/ml; both from Sigma- Aldrich) in the presence of GolgiPlug (BD Biosciences) in complete medium (RPMI medium containing 10% (vol/vol) FCS, supplemented with penicillin and streptomycin, L-glutamine, HEPES, pH 7.2, sodium pyruvate and 2-mercaptoethanol), followed by flow cytometry staining and analysis.
  • PMA phorbol 12-myristate 13-acetate
  • ionomycin 500 ng/ml
  • GolgiPlug BD Biosciences
  • complete medium RPMI medium containing 10% (vol/vol) FCS, supplemented with penicillin
  • ribonuclease vanadyl complex RNase inhibitor (Sigma-Aldrich) was added during the fixation, intracellular staining and cell sorting steps to keep the RNA intact.
  • Fixed cells were sorted based on IL-17 and IFNy protein expression and gene expression analysis was performed.
  • For sorting ex vivo cytokine stimulated IL-17 + cells draining lymph node cells were isolated from MOG immunized IL-17.GFP Knockin mice and rested for 1.5 hours, followed by stimulation with IL-23 or IL-6 for 2 hours. Stimulated cells were sorted based on IL-17.GFP and CD4 expression.
  • TAQMAN ® real-time quantitative PCR gene expression analysis was performed as follows. Total RNA was isolated from cells using Arcturus PicoPure RNA Isolation method, according to manufacturer's protocol (Applied Biosystems, Life).
  • OT-II ovalbumin peptide323-339-TCR-tg cells from wildtype (wt) and IL-23 receptor knockout (IL-23R ⁇ ⁇ ) were transferred to wild-type C57B1/6 recipient mice.
  • OVA ovalbumin
  • CFA complete Freund's adjuvant
  • Prdml and Bcl-6 expression in Thl7 and Thl cells was determined as follows. Cells from draining lymph nodes of C57BL/6J mice immunized with MOG 35 _55 in CFA were obtained at day seven post immunization. These cells were stimulated ex vivo with phorbol 12-myristate 13 -acetate (PMA) and ionomycin, surface stained, and fixed along with an RNase inhibitor to keep the RNA intact. Fixed cells were stained intracellularly and sorted based on IL-17 and IFNy protein expression.
  • PMA phorbol 12-myristate 13 -acetate
  • TAQMAN ® gene expression analysis was performed on the resulting IL-17 + / IFNy " and IFNy + / IL-17 " cell populations (representing 2.0% and 1.94% of total sorted cells, respectively). Data are representative of three independent experiments. Error bars in all graphs are SEM. Data are presented at FIG. IB for Prdml (left plot) and Bcl6 (right plot).
  • Thl7 cells draining lymph node cells were collected from IL-17-GFP Knockin reporter mice at day seven post immunization with MOG 35 _55 peptide in CFA and allowed to rest briefly before stimulating with cytokine (IL-6 or IL-23). About 55-60% of the IL-17. GFP + cells responded to IL-23 and IL-6 stimulation as measured by phosphorylation of STAT-3 (data not shown).
  • CD4 + IL-17.eGFP + cells from untreated, IL-23 treated, and IL-6 treated were sorted two hours after cytokine stimulation and TAQMAN ® gene expression analysis was performed. Results are presented at FIG. 1C for Prdml (left plot) and 112 (right plot).
  • Prdml expression was significantly upregulated in IL-17 + cells by IL-23 but not IL-6 stimulation indicating that Prdml gene transcription is specifically induced by IL-23 and is a proximal event following IL-23 stimulation.
  • 112 expression was greatly diminished in IL-17 + cells as compared to IL-17 " cells.
  • YFP + cells from Prdml "7” were compared to YFP + cells from either Prdml ⁇ /wt Rosa-YF ⁇ Dlck cre (Prdml "7+ ) mice or Prdml wt/wt Rosa-YF ⁇ Dlck cre (Prdml +/+ ) mice.
  • Intracellular GM-CSF and IFNy expression was determined in IL-17 + cells from flow cytometry of intracellular IL-17-producing CD4 + T cells from draining lymph nodes of MOG 35 _ 5 5 immunized Prdml '1' after ex vivo stimulation with PMA and ionomycin at day seven post immunization. Results are provided at FIG. 2A.
  • Intracellular T-bet expression was determined in CD4 + T cells and IL-17 +
  • Blimp- 1 that promotes co-expression of GM-CSF, IFNy and T-bet and inhibits IL-2 expression by IL-17-producing cells.
  • Intracellular staining was used to sort for cells expressing IL-17, GM-CSF and
  • Results are provided at FIGS. 3D, 3E and 3F. Data are representative of three independent experiments. Error bars in all graphs are SEM.
  • Thl7 cells as follows. Flow cytometry of CCR6 and CXCR3 expressing CD4 + T cells was performed on cells from draining lymph nodes of MOG 35 _55 immunized Prdml conditional knockout mice at day seven post immunization. Results are shown in FIG. 4A. Intracellular Ki-67 expression was measured in CD4 + T cells (left panel) and IL-17 + CD4 + T cells (right panel) from draining lymph nodes at day seven post immunization. Results are shown in FIG. 4B. Intracellular Bcl-2 expression was measured in CD4 + T cells (top panel) and IL-17 + CD4 + T cells (bottom panel) from draining lymph nodes at day seven post immunization. Data are representative of three independent experiments. Error bars in all graphs are SEM.

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Abstract

La présente invention concerne des méthodes de traitement de troubles auto-immuns et inflammatoires par l'inhibition de l'activité Blimp-1, et des procédés de criblage pour des antagonistes de Blimp-1 à utiliser dans de telles méthodes de traitement. L'invention concerne également des procédés d'amélioration de la réponse immunitaire par Th17, et des procédés de criblage pour la recherche d'agonistes de Blimp-1 à utiliser dans de tels procédés d'amélioration des réponses immunitaires par Th17.
PCT/US2012/063635 2011-11-07 2012-11-06 Traitement de troubles auto-immuns et inflammatoires par inhibition de blimp-1 WO2013070563A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014195496A1 (fr) * 2013-06-07 2014-12-11 Institut National De La Sante Et De La Recherche Medicale (Inserm) Modulateur de blimp-1 a employer dans le traitement de l'infection par le vih
US20200384094A1 (en) * 2014-04-11 2020-12-10 Cellectis Method for generating immune cells resistant to arginine and/or tryptophan depleted microenvironment

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US20080060087A1 (en) * 2004-02-12 2008-03-06 Davies Collison Cave Modified Cells and Methods of Using Same
US20100323383A1 (en) * 2008-04-15 2010-12-23 Nicolas Manel Methods for in vitro differentiation of Th-17+cells
US20110293629A1 (en) * 2010-05-14 2011-12-01 Bastid Jeremy Methods of Treating and/or Preventing Cell Proliferation Disorders with IL-17 Antagonists
US20120177597A1 (en) * 2011-01-12 2012-07-12 Baylor Research Institute Compositions and methods of alteration of autoimmune diseases

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US20100323383A1 (en) * 2008-04-15 2010-12-23 Nicolas Manel Methods for in vitro differentiation of Th-17+cells
US20110293629A1 (en) * 2010-05-14 2011-12-01 Bastid Jeremy Methods of Treating and/or Preventing Cell Proliferation Disorders with IL-17 Antagonists
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014195496A1 (fr) * 2013-06-07 2014-12-11 Institut National De La Sante Et De La Recherche Medicale (Inserm) Modulateur de blimp-1 a employer dans le traitement de l'infection par le vih
US20200384094A1 (en) * 2014-04-11 2020-12-10 Cellectis Method for generating immune cells resistant to arginine and/or tryptophan depleted microenvironment

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