WO2009053481A1 - Compositions et procédés destinés à moduler la circulation des lymphocytes nk et t - Google Patents

Compositions et procédés destinés à moduler la circulation des lymphocytes nk et t Download PDF

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WO2009053481A1
WO2009053481A1 PCT/EP2008/064487 EP2008064487W WO2009053481A1 WO 2009053481 A1 WO2009053481 A1 WO 2009053481A1 EP 2008064487 W EP2008064487 W EP 2008064487W WO 2009053481 A1 WO2009053481 A1 WO 2009053481A1
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cells
cell
antibody
patient
agent
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PCT/EP2008/064487
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Thierry Walzer
Eric Vivier
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons

Definitions

  • the present invention relates to methods of inhibiting the trafficking of NK cells and activated T cells, and for the prevention and/or treatment of inflammatory and autoimmune conditions.
  • the present methods involve the use of agents that bind and/or inhibit S1P5, including but not limited to antagonists and agonists having the ability to act as functional antagonists, in order to modulate the in vivo circulation of NK cells and activated T cells.
  • Natural killer (NK) cells are lymphocytes of the innate immune system that are involved in anti-tumor immune surveillance ⁇ and in the early control of several viral, bacterial and parasitic infections 2 ' 3 .
  • NK cells possess the capacity to kill cells recognized as targets and to produce cytokines such as interferon (IFN)- ⁇ .
  • IFN interferon
  • NK cell development occurs in the bone marrow 4 .
  • lymph nodes, and to some extent the thymus also support NK cell development 4 ' 5 .
  • NK cells undergo a maturation process and relocate throughout the body in lymphoid and non-lymphoid organs 6 .
  • Chemokine receptors such as CCR2, CCR5, CXCR3, and CX3CR1 are involved in the rapid NK cell mobilization that occurs in inflammatory conditions 7"17 .
  • the mechanisms governing NK cell trafficking during steady-state situations are unclear.
  • No gross abnormality in NK cell distribution was detected in mouse strains lacking chemokine receptors such as CXCR3, CXCR6, CCRl, CCR5 or CX3CR1 10 ' 15 ' 16 ' 18 (and data not shown), suggesting that individual chemokine receptors do not play a major role in NK cell trafficking during steady-state conditions.
  • SlP sphingosine 1 -phosphate
  • Circulating T and B cells are absent in mice lacking SlPi, and SlPi-def ⁇ cient thymocytes accumulate in the thymus 24 .
  • Intravenously transferred S IPi -deficient thymocytes can enter secondary lymphoid organs but they are unable to exit 24 .
  • S IPi -deficient B cells cannot exit secondary lymphoid organs 24 . Therefore, it has been proposed that a common S IPi -dependent mechanism controls lymphocyte egress from thymus and secondary lymphoid organs 19 .
  • FTY720 a pharmacological agonist of SlP receptors, FTY720 (fingolimod), inhibits the egress of T cells and B cells from lymphoid organs 22 .
  • FTY720 which is structurally similar to sphingosine, is phosphorylated in vivo; this phosphorylated metabolite acts as an agonist for four of the five known SlP receptors (SlPi, SlP 3 _s).
  • SlPi SlP 3 _s
  • FTY720 is used as an immunosuppressant, and a recent phase II clinical trial suggested that FTY720 may provide an effective treatment for multiple sclerosis 22 .
  • FTY720 also regulates vascular integrity by binding to SlPi expressed on endothelial cells; in this way FTY720 indirectly influences lymphocyte trafficking 21 ' 25 .
  • SlP 5 also referred as to GPCR NRG-I or edg-8
  • GPCR NRG-I or edg-8 GPCR NRG-I or edg-8
  • SlP 5 -deficient immature oligodendrocytes display reduced responses to SlP in vitro; however, no deficits in myelination are observed in SIP 5 - deficient mice 28 . Therefore, the precise role of SIP 5 in oligodendrocyte function remains to be determined.
  • lymphocyte trafficking While influencing lymphocyte trafficking would be extremely useful for the development of therapeutic and prophylactic strategies for treating and preventing diseases such as inflammatory and autoimmune diseases, currently strategies for modulating trafficking that act on SlP receptors appear to have only limited efficacy. There is therefore a need for means to improve control of lymphocyte trafficking.
  • the present invention addresses this and other needs.
  • the present application discloses that SIP5 is expressed in the immune system, specifically in mature NK cells and in activated cytolytic CD8 T cells, and that SIP5 regulates trafficking of cells expressing SIP 5 in vivo via a FTY720-resistant mechanism.
  • the inventors show that S IPs-deficient mice exhibit aberrant NK cell homing during steady-state conditions.
  • SIP5 is required for NK cell mobilization to inflamed organs.
  • the present invention provides methods for modulating (e.g. inhibiting) the activity, particularly the trafficking of cells of the immune system expressing SIP 5 , particularly activated NK cells and T cells, e.g. mature NK cells and activated cytolytic CD 8 T cells.
  • trafficking when referring to a cellular activity comprises any individual or combination of cellular activities generally understood to be related to the movement (e.g.
  • NK and T cells e.g. activated cytolytic CD8 T cells
  • cytolytic CD8 T cells e.g. activated cytolytic CD8 T cells
  • NK cells and/or activated T cells provide the possibility to treat an established inflammatory or autoimmune disorder (e.g. a disorder characterized by chronic and/or ongoing inflammation).
  • an established inflammatory or autoimmune disorder e.g. a disorder characterized by chronic and/or ongoing inflammation.
  • This has advantages over therapies such as SlPl inhibition (without inhibition of S1P5) which might prevent trafficking of na ⁇ ve cells but lack effect on trafficking of NK cells and/or activated cytolytic CD8 T cells which would be present in an established inflammatory or autoimmune disorder.
  • the present methods and compositions can use any of a variety of different means to specifically bind to and/or inhibit S1P5 polypeptides, such as the use of antibodies or other compounds that specifically recognize S1P5 polypeptides in order to specifically bind to S1P5 and thereby inhibit S 1P5 -expressing cells, and the use of compounds that alter the amount of such S1P5 polypeptides on the surface of immune cells.
  • agents that specifically recognize S1P5 polypeptides include (a) antibodies or other polypeptide-based agents that bind S1P5, (b) sphingosine analogues or derivatives, including but not limited to (i) compositions that act as S1P5 antagonists and (ii) compositions that have S1P5 agonist properties so long as they can be used in a way that results in the functional inhibition of S1P5.
  • the present invention provides methods for producing agents useful in the treatment of autoimmune and inflammatory disorders.
  • the agents produced using the present methods are capable of specifically targeting and/or inhibiting S1P5 on immune cells such as activated T cells, e.g., activated cytolytic CD8 + T cells, in patients.
  • the agents can limit the pathological effects of cells expressing S1P5, e.g., by interfering with the trafficking of the cells, or, alternatively, by killing them directly by contacting them with a cytotoxic agent such as a radioisotope, toxin, or drug.
  • kits comprising the herein-described antibodies and/or compounds as well as instructions for their use.
  • the present invention provides a method for inhibiting NK and/or T cell trafficking, said method comprising contacting said NK and/or T cell with an effective amount of an agent that binds and/or inhibits S1P5.
  • a method for detecting or isolating a mature NK cell or an activated cytolytic CD8 T cell said method comprising contacting said cell with an agent that binds to S1P5.
  • the methods can be carried out in vitro or in vivo.
  • a method of treating or preventing an inflammatory or autoimmune disorder in a patient comprising administering to said patient a pharmaceutical composition comprising an agent that binds and/or inhibits S1P5, and a pharmaceutically acceptable carrier.
  • the invention provides a method of reducing the number or proliferation of mature NK cell or activated cytolytic CD8 T cells in a patient, said method comprising administering to said patient a compound that specifically binds to a S1P5 polypeptide.
  • Another embodiment encompasses a method for diagnosing or assessing susceptibility to an inflammatory or autoimmune disease in a patient, comprising detecting the prevalence of S1P5 expressing cells in said patient, wherein a detection of elevated levels of said cells in said patient indicates that the patient has or is susceptible to an inflammatory or autoimmune disease.
  • Yet another embodiment encompasses a method of modulating an NK or T cells -mediated response in a patient, said method comprising administering to a patient a compound that alters the amount of S1P5 on the surface of NK or T cells.
  • the agent that binds and/or inhibits S1P5 and the test compounds can be of a variety of suitable compositions.
  • the agent or test compound selectively binds and/or inhibits S1P5 compared to SlPl, S1P2, S1P3 and/or S1P4.
  • the agent or test compound is substantially free of binding and/or activity, including agonist and antagonist activity, at any or all of human SlPl, S1P2, S1P3 and/or S1P4 polypeptides.
  • S1P5 is expressed only on oligodendrocytes and current SlPl antagonists can lead to capillary leakage and pulmonary edema (Sanna et al. (2006) Nature Chemical Biology 2:434-441)
  • such agents that are antagonists and have selectivity for S1P5 over other SlP receptors are expected to have improved side effect profiles.
  • agent or test compound is an S1P5 antagonist.
  • the agent or test compound is a S1P5 agonist and is provided under conditions or in an effective amount in which the S1P5 agonist acts as a functional inhibitor of S1P5.
  • the agent or test compound prevents the interaction of a S1P5 polypeptide with a ligand of S1P5, optionally wherein said ligand of S 1P5 is SlP or a sphingosine analogue or derivative.
  • the agent or test compound is an antibody or an antibody derivative or fragment; optionally wherein said antibody or an antibody derivative or fragment is an antagonist of S1P5; optionally, wherein said antibody is a monoclonal antibody; optionally wherein said antibody is human, humanized, or chimeric; optionally wherein said antibody comprises a constant region capable of binding to a human Fcgamma3A receptor; optionally wherein said antibody specifically binds to one or more NK or T cells in the patient and leads to their destruction by the immune system.
  • the agent or test compound comprises a sphingosine analogue or derivative.
  • the agent or test compound is FTY720 or an analogue or derivative.
  • the agent or test compound is conjugated with a toxic moiety.
  • the agent or test compound may modulate, preferably inhibit, in a mature NK or activated cytolytic CD8 T cells, one or more of the activities selected from the group consisting of: circulation, homing, migration, migration towards a SlP gradient, extravasion, chemotaxis, accumulation in organs, accumulation in spleen, lung, liver, epithelial tissues and blood, and exit from lymph nodes and/or bone marrow.
  • the agent is a monoclonal antibody, or a fragment or derivative thereof.
  • the antibody is humanized, chimeric, or human.
  • the agent is a sphingosine analogue or derivative.
  • the inhibitor interferes with the binding of an Sl P5 ligand to S1P5.
  • the agent is an aptamer, an siRNA or an antisense agent.
  • the antibody is a cytotoxic antibody.
  • the cytotoxic antibody comprises an element selected from the group consisting of radioactive isotope, toxic peptide, and toxic small molecule.
  • the method further comprises a diagnostic step in which, prior to the administration of the inhibitor, the prevalence of Sl P5 -expressing immune cells, optionally also detecting. NK cells or activated cytolytic CD8 T cells and/or expression of S1P5 on such cells, in the patient is assessed, wherein a detection of elevated levels of such cells in the patient indicates that the patient is suitable for the administration of the inhibitor.
  • the method further comprises a diagnostic step in which, prior to the administration of the inhibitor, the prevalence of NK cells or T cells (e.g. activated cytolytic CD8 + T cells) in the patient is assessed, wherein a detection of elevated levels of such cells in the patient indicates that the patient is suitable for the administration of the inhibitor.
  • the diagnostic step may comprise for example an immunoassay to detect the presence of CD8, an NK cell marker and/or S1P5 on NK and/or T cells obtained from the patient.
  • Methods for identifying, screening or producing agents specifically bind to and/or inhibit S1P5 polypeptides can be carried out using standard methods.
  • the assay steps will generally be carried out in vitro, but certain steps can also be carried out in vitro in mammals (e.g. assessing cell trafficking, for example in mice).
  • the method provides a method of screening for a compound, said method comprising: contacting a S1P5 polypeptide with a test compound and determining whether the test compound binds and/or inhibits S1P5; selecting a test compound binds and/or inhibits S1P5, and determining whether the test compound selected in (b) modulates cell trafficking.
  • the method further comprises determining whether the test compound selectively binds and/or inhibits S1P5 compared to SlPl, S1P2, S1P3 and/or S1P4.
  • the method comprises a step in which the activity of said polypeptide is assessed in the presence and absence of said compound.
  • the method comprises selecting the candidate compound identified in for use as a medicament.
  • the method comprises making the candidate compound suitable for administration to a human.
  • the method comprises preparing the quantity of the candidate compound for administration to a human.
  • Sl P5 is a G-protein coupled receptor, well-known screening systems can be used to provide agents.
  • the present invention provides a method of producing an antibody or other compound suitable for use in the treatment of an autoimmune or inflammatory disorder, said method comprising the following steps: a) providing a S1P5 receptor-expressing CD8 + T cell and a plurality of monoclonal antibodies or other compounds; b) testing the ability of each of the antibodies or compounds to bind to the S1P5 receptor on said cells, or to interfere with the binding of a ligand to the S1P5 receptor on said cells; c) selecting an antibody or compound from the plurality that bind to the S1P5 receptor and/or reduces the binding of the ligand to the receptor; and d) if an antibody is selected in step c), rendering the antibody suitable for human administration.
  • the antibody is made suitable for human administration by humanizing or chimerizing it.
  • the method further comprises a step in which a cytotoxic agent is linked to the antibody.
  • the cytotoxic agent is a radioactive isotope, a toxic polypeptide, or a toxic small molecule.
  • the antibody or compound reduces the binding of the ligand (e.g. SlP or an analogue thereof) to S1P5 by at least a percentage selected from the group consisting of 50%, 60%, 70%, 80%, 90%, 95%, and 99%.
  • the ligand e.g. SlP or an analogue thereof
  • the present invention provides antibodies or compounds produced using any of the herein-described methods.
  • the invention also encompasses fragments and derivatives of the antibodies having substantially the same antigen specificity and activity (e.g., which can bind to the same antigens as the parent antibody).
  • fragments include, without limitation, Fab fragments, Fab '2 fragments, CDR and ScFv.
  • kits comprising any one or more of the herein-described antibodies or compounds.
  • the kit also comprises instructions for using the antibodies according to the present methods.
  • the invention also comprises pharmaceutical compositions comprising one or more of the present antibodies, or a fragment or derivative thereof, or any of the present compounds, and a pharmaceutically acceptable carrier or excipient.
  • the invention comprises cells obtained using the methods of the invention.
  • the invention provides a population of isolated NK or activated T cells, preferably human cells, expressing S1P5.
  • the NK cells are characterized as also expressing CD 16, CD56, CD57 and/or NKp46.
  • the NK cells are characterized as also expressing CD8, and/or CD3.
  • Figure 1 Expression of genes encoding SlP receptors in mouse lymphocytes. Quantitative RT-PCR analysis of transcripts encoding SlP receptors, normalized to Hprtl mRNA, in various sorted lymphocyte populations. Results are the mean ⁇ s.d. of three independent experiments.
  • FIG. 3 Tissue distribution of NK cells in wild-type and S IPs-deficient mice treated with IL-15.
  • FIG. 5 SlP 5 deficiency most severely affects CD27 du11 NK cell tissue distribution
  • (b) Tissue distribution of NK cell subsets in wild-type (WT) and S IPs- deficient (SIP5-KO) mice as determined by flow cytometry. Bars depict percentages of each NK cell subset in various organs of S IPs-deficient and wild-type littermates (n 5- 10, depending on the organ).
  • FIG. Defective in vivo and in vitro migration of S IPs-deficient NK cells.
  • (a,b) Chemotactic responses of splenic T cells (a) and NK cells (b) from wild-type (WT) and SlP5-deficient (SIP5-KO) mice to SlP, as indicated.
  • WT wild-type
  • SIP5-KO SlP5-deficient mice
  • NK cells are wild-type.
  • the migration index was determined by the ratio of the number of migrated cells under control and test conditions. Results are representative of three independent experiments.
  • FIG. 7 Pharmacological modulation of NK cell trafficking
  • (b) Wild-type C57BL/6 mice were treated for seven days with drinking water containing FTY720 or DOP. S IP-induced chemotaxis of splenic T and NK cells from untreated and treated wild-type mice was assessed. The migration index was determined by the ratio of migrated cells under control and test conditions. Results are expressed as the mean ⁇ s.d. percent of inhibition of chemotaxis by FTY720 or DOP for each cell population (n 4).
  • FIG. 8 Expression of genes encoding SlP receptors in mouse lymphocytes. Microarray analysis of transcripts encoding SlP receptors is shown, in various sorted lymphocyte populations, demonstrating specific expression of S1P5 in CD8 effector T cells in addition to NK cells.
  • FIG. 9 Expression of genes encoding SlP receptors in human NK cells.
  • Microarray data of sorted human NK cell subsets were downloaded from Pubmed Geo (GDS571). Results show the expression of mRNA encoding each of the five SlP receptors, relative to HPRT.
  • FIG. 10 Effector functions and tissue distribution of NK cell subsets, (a) Flow cytometric analysis of wild-type (WT) spleen cells stained for NKl.1, CD3, CD27 and CDl Ib. Results show the expression of CD27 and CDl Ib in gated NK cells in a representative experiment of three independent experiments. Gates used to define the three NK cell subsets are shown, (b-c) NK cell subsets (as indicated in a) from various organs of wild-type C57BL/6 mice were cultured in the absence or presence of IL- 12 and IL- 18, or YAC-I tumor cells. IFN- ⁇ secretion was assessed by intracellular staining.
  • NK cells were assessed by flow cytometry, (c) Flow cytometric analysis of the percentage of splenic CD27dull NK cells expressing cell surface proteins indicated on x-axis in wild-type (WT, black bars) and S 1P5 -deficient (S1P5-KO, white bars) mice, (d) Viability of NK cells freshly isolated from the indicated organs from wild-type (WT, black bars) and S 1P5 -deficient (S1P5- KO, white bars) mice, (e) Splenocytes from wild-type (WT, black bars) and S1P5- deficient (S1P5-KO, white bars) mice were cultured in vitro in complete medium in the presence or absence of IL-2 or IL- 15.
  • NK cell viability was measured 24 h later
  • Figure 12, 13 and 14 S1P5 microarray data from disease biopsies.
  • Figure 12 shows data from accession number GDS2382, where AD patients, both lesional and non- lesional, appear to have higher S1P5 expression than healthy control patients.
  • Figure 13 shows data from accession number GDS1330, where it appears that S1P5 expression is generally somewhat higher in Crohn's disease and ulcerative colitis patients than in normal control patients.
  • Figure 14 shows data from accession number GDS2014, where it appears that S1P5 expression is higher in ulcerative colitis patients than in irritable bowel syndrome patients.
  • the present disclosure provides novel methods for modulating the activity and effects of NK and activated T cells, particularly activated cytolytic CD8+ cells, and for identifying compounds capable of doing the same.
  • the present methods and compounds are useful for treating and preventing conditions related to of NK and activated T cells, particularly CD8+ T effectors cells activity, particularly inflammatory and autoimmune diseases.
  • NK cells were not completely absent from these organs, suggesting the existence of other mechanisms that compensate, at least in part, for the lack of SIP 5 . This phenomenon is reminiscent of previous observations indicating that NK cell recruitment to inflamed sites in mice lacking various chemokine receptors is diminished but not completely abrogated 7 ' 8 ' 10 ' 16 . Thus, alternative NK cell recruitment mechanisms co-exist with SIP5, and NK cell trafficking relies on several pathways.
  • the present disclosure also demonstrates that SIP5 expression increases with NK cell maturation and with differentiation and/or activation of T cells.
  • Immature CDl lb du11 NK cells were less affected by SlP 5 deficiency than were DP and CD27 dull NK cells. Consequently, S IPs-deficient CDl lb du11 NK cells may seed peripheral organs where they can complete their maturation. This phenomenon could also explain why the NK cell defect in S IPs-deficient mice was less severe than the T cell defect in S IPi -deficient mice 24 . Indeed, unlike NK cells that complete maturation in various organs throughout the body, T cells develop and mature in the thymus.
  • SIP5 operates to facilitate egress of NK cells from the BM and LN in response to SlP gradients. Indeed, NK cells accumulated in the BM and LN and withdrew from the blood, spleen and lung in S IPs-deficient mice. Moreover, SIP 5 functioned as a chemotactic receptor for SlP in vitro. However, our data clearly show that FTY720 and DOP had no effect on NK cell trafficking in vivo, and thus suggest that SlPi and SIP5 operate through distinct molecular mechanisms. The inventors provide three different yet not mutually exclusive possible explanations for these observations.
  • NK cells and T cells might be due to different localizations of NK cells and T cells within lymphoid organs. Indeed, spleen NK cells are mostly found within red pulp sinuses whereas T cells are located in the white pulp. Similarly, LN NK cells are located mainly in lymphatic sinuses of the medulla, whereas T cells are found in the cortex 6 ' 29 ' 42 . The precise concentration of SlP in these different compartments is not known and is difficult to assess, but could be differentially affected by DOP. Similarly, FTY720 may differentially access the various compartments of lymphoid organs.
  • SlPi and SIP 5 may trigger activation of different intracellular signaling pathways. Indeed, SlPi primarily couples with GCV 0 heterotrimeric G proteins, whereas SIP5 can couple with both GCV 0 and Gcci 2/13 proteins 43 . GCC12/13 proteins are different from the GCV 0 variety as they specifically interact with and regulate RGS-Rho guanine nucleotide exchange factors (GEFs) 44 . Thus, RGS-RhoGEFs relay signals from heterotrimeric GCC12/13 protein-coupled receptors to Rho GTPases.
  • GEFs RGS-Rho guanine nucleotide exchange factors
  • SIP5 receptors engage this Gcci2/i3-coupled Rho-ROCK signaling pathway to impede oligodendrocyte precursor cell migration 43 .
  • SIP5 coupling to GCC12/13 and Rho activation might be less susceptible than SlPi to FTY270 and DOP.
  • S IP5 might not control the egress of cells from BM and LN, but instead prevent the exit of cells from blood vessels. Such a possibility would imply that SlP gradients are not required for the normal function of SIP5 and would explain the absence of an effect of FTY720 and DOP treatment on cell migration.
  • novel pharmacological tools such as SlPs-specific agonists 45 and antagonists should facilitate delineation of the SIP 5 mechanism of action.
  • SIP5 the role of SIP5 in NK cell migration is likely to be similar in mice and humans. Consistent with the mice data, SIP 5 was preferentially expressed by the most mature CD56 dim human NK cells. This differential SIP5 expression correlates with the tissue distribution of human NK cell subsets, as CD56 dim NK cells prevail in the blood and spleen and CD56 b ⁇ ght NK cells are predominant in LN 47 . In addition, the absence of effect of FTY720 on NK cell distribution in mouse correlates with the absence of an effect of FTY720 on human NK cells during clinical trials 48 .
  • NK cell trafficking is dependent on a dedicated SlP receptor with an apparently unique pharmacology that could potentially be used to specifically manipulate the activity of these cells for therapeutic purposes 49 .
  • the present invention is therefore based on the discovery that SIP5 is expressed in the immune system, specifically in NK cells and in cytolytic CD8 T cells, and that SIP5 furthrmore regulates trafficking of such cells in vivo via a FTY720-resistant mechanism.
  • the inventors show that S IPs-deficient mice exhibit aberrant NK cell and cytolytic CD8 T cell homing during steady-state conditions.
  • SIP5 is required for NK cell and activated cytolytic CD8 T cell mobilization to inflamed organs.
  • SlP receptor agonists as functional antagonists (e.g. SlPl agonist, including those that also act as agonists at S1P5) will not substantially inhibit NK cell and cytolytic CD8 T cell trafficking. While not wishing to be bound by theory, the inventors provide that the amounts of SlP receptor agonist used to generate a functional inhibition of SlPl is below the amount of SlP receptor agonist required to lead to functional inhibition of S1P5. Thus, the present invention provides means of inhibiting NK cell and cytolytic CD8 T cell trafficking by inhibiting S1P5.
  • agents that bind and/or inhibit S1P5 can be used to specifically modulate the activity and effects of NK cells and cytolytic CD8 T cells, e.g., to modulate their activity such as trafficking, to selectively destroy immune cells (e.g. NK cells or cytolytic CD8 T cells), or to identify immune cells, particularly activated immune cells (e.g. NK cells or cytolytic CD8 T cells), for example in diagnostic methods to detect an inflammatory or autoimmune condition.
  • Agents that bind and/or inhibit S1P5 can be used as lead compounds or reference compounds to screen for or develop new S1P5 modulating compounds.
  • Inhibiting S1P5 may comprise inhibiting any biological activity of an S1P5 polypeptide, including but not limited to inhibiting ligand binding (e.g. binding of an endogenous or exogenous S1P5 ligand), inhibiting S1P5 expression, reducing S1P5 polypeptide numbers on the cell surface, and/or inhibiting the ability of S1P5 to transduce a signal (e.g. to transduce an extracellular signal across the plasma membrane).
  • ligand binding e.g. binding of an endogenous or exogenous S1P5 ligand
  • NK cell refers to a cell from a sub-population of lymphocytes that is involved in non-conventional immunity.
  • NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD16, CD56, CD57 and/or NKp46, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express "self MHC/HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art.
  • T cells refers to a sub-population of lymphocytes that mature in the thymus, and which display, among other molecules, T cell receptors on their surface.
  • T cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including the TCR, CD4 or CD8, the ability of certain T cells to kill tumor or infected cells, the ability of certain T cells to activate other cells of the immune system, and the ability to release cytokines or other soluble mediators that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify T cells using methods well known in the art.
  • active or “activated” T cells designate biologically active T cells, more particularly T cells having the capacity of cyto lysis or of stimulating an immune response by, e.g., secreting cytokines.
  • an "active" CD8 + T cell will have cytolytic activity. Active cells can be detected in any of a number of well known methods, including functional assays and expression-based assays such as the expression of cytokines such as TNF-alpha.
  • SlP refers to sphingosine 1-phosphate
  • a "SlP receptor”, used interchangeable with the terms “Edg” or “Edg receptor” refers to a member of a family of proteins composed of eight G-protein-coupled receptors that bind to specific lysophospholipids. Edg receptors are divided into two subgroups according to ligand specificity and sequence. Members of the first subgroup bind SlP and include Edgl (also called SlPi), Edg3 (also called SlP 3 ), Edg5 (also called SlP 2 ), Edg6 (also called SlP 4 ) and Edg8 (SlP 5 ). Members of the second subgroup bind lysophosphatidic acid 31 . SIP receptors are described, for example in WO 03/061567 the disclosure of which is herein incorporated by reference.
  • S1P5 refers to a mammalian, preferably human, sphingosine 1-phosphate receptor expressed primarily on NK cells and T cells capable of mediating cell trafficking. It is also referred to as Edg8.
  • Human S1P5 is described at Genbank accession number NP-110387, WO 02/05731 land WO 03/061567, the disclosures of which are herein incorporated by reference.
  • Rat S1P5 is described at Genbank accession number NP 068543; murine S1P5 is described at Genbank accession number NP 444420, the disclosures of which are herein incorporated by reference.
  • Autoimmune disorders include any disorder, condition, or disease in which the immune system mounts a reaction against self cells or tissues, due to a breakdown in the ability to distinguish self from non-self or otherwise.
  • autoimmune disorders include Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, polymyositis, Guillain Barre, Wegener's granulomatosus, polyarteritis nodosa, polymyalgia rheumatica, temporal arteritis, Bechet's disease, Churg-Strauss syndrome, Takayasu's arteritis, and others. Autoimmune disorders
  • Inflammatory diseases refer to any disorder, condition, or disease characterized or caused by excessive or uncontrolled inflammation, or any aspect of inflammation such as redness, swelling, heat, pain, etc.
  • Inflammatory diseases include allergies, including allergic rhinitis/sinusitis, skin allergies such as urticaria/hives, angioedema, atopic dermatitis, food allergies, drug allergies, insect allergies, and rare allergic disorders such as mastocytosisasthma, asthma, arthritis, including osteoarthritis, rheumatoid arthritis, and spondyloarthropathies, gastrointestinal inflammation, neuro inflammatory disorders, and autoimmune disorders.
  • the terms “inhibiting,” “reducing,” “blocking,” “downmodulating,” and “downregulating,” with respect to the activity of a polypeptide refer to any process, method, or compound that can slow down, reduce, reverse, or in any way negatively affect the stimulation or expression of the polypeptides (e.g. S1P5) on cells, preferably NK or cytolytic CD8 T cells. These terms can refer to compounds that inhibit the stimulation of the polypeptide (e.g.
  • the inhibiting compound or method prevents the binding of the receptor by a ligand, e.g. SlP or a sphingosine analogue or derivative.
  • the number of S1P5 molecules or any of the herein-described activities can be measured in any standard way, e.g. as disclosed elsewhere in the present application.
  • antibody refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as IgGl, IgG2, IgG3, IgG4, and the like.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
  • each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.
  • the heavy- chain constant domains that correspond to the different classes of immunoglobulins are termed "alpha,” “delta,” “epsilon,” “gamma” and “mu,” respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • IgG and/or IgM are the preferred classes of antibodies employed in this invention, with IgG being particularly preferred, because they are the most common antibodies in the physiological situation, and because they are most easily made in a laboratory setting.
  • the antibody of this invention is a monoclonal antibody. Particularly preferred are humanized, chimeric, human, or otherwise-human-suitable antibodies.
  • the term "specifically binds to” means that an antibody or compound can bind preferably in a competitive binding assay to the binding partner, e.g. S1P5.
  • a competitive binding assay to the binding partner
  • competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.
  • immunoglobulin any polypeptidic or peptidic fragment or other molecular entity which is capable of eliciting an immune response such as (i) the generation of antibodies binding said fragment and/or binding any form of the molecule comprising said fragment, including the membrane-bound receptor and mutants derived therefrom, (ii) the stimulation of a T-cell response involving T-cells reacting to the bi-molecular complex comprising any MHC molecule and a peptide derived from said fragment, (iii) the binding of transfected vehicles such as bacteriophages or bacteria expressing genes encoding mammalian immunoglobulins.
  • an immune response such as (i) the generation of antibodies binding said fragment and/or binding any form of the molecule comprising said fragment, including the membrane-bound receptor and mutants derived therefrom, (ii) the stimulation of a T-cell response involving T-cells reacting to the bi-molecular complex comprising any MHC molecule and a peptide derived from said fragment, (iii
  • an immunogenic fragment also refers to any construction capable of eliciting an immune response as defined above, such as a peptidic fragment conjugated to a carrier protein by covalent coupling, a chimeric recombinant polypeptide construct comprising said peptidic fragment in its amino acid sequence, and specifically includes cells transfected with a cDNA of which sequence comprises a portion encoding said fragment.
  • a "human-suitable" agent or antibody refers to any agent or antibody, derivatized antibody, or antibody fragment that can be safely used in humans for, e.g. the therapeutic methods described herein.
  • Human-suitable antibodies include all types of humanized, chimeric, or fully human antibodies, or any antibodies in which at least a portion of the antibodies is derived from humans or otherwise modified so as to avoid the immune response that is provoked when native non-human antibodies are used.
  • a “humanized” antibody refers to an antibody in which the constant and variable framework region of one or more human immunoglobulins is fused with the binding region, e.g. the CDR, of an animal immunoglobulin.
  • Such humanized antibodies are designed to maintain the binding specificity of the non- human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody.
  • a "chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • isolated refers to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.
  • biological sample includes but is not limited to a biological fluid (for example serum, lymph, blood), cell sample or tissue sample (for example bone marrow).
  • a biological fluid for example serum, lymph, blood
  • cell sample for example bone marrow
  • a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.
  • the present invention is based on the discovery that SIP5 is expressed in the immune system, specifically in NK cells and in cytolytic CD8 T cells, and that SIP 5 regulates trafficking of such cells in vivo via a FTY720-resistant mechanism, and in particular that eliminating SIP 5 activity results in aberrant NK cell and cytolytic CD8 T cell homing during steady- state conditions, including cell mobilization to inflamed organs.
  • agents that bind and/or inhibit S1P5, and particularly anti-SlP5 antibodies and sphingosine analogues and derivatives, e.g., with enhanced specificity, avidity or affinity for S1P5, as described elsewhere herein) are useful in a number of ways.
  • the ability of the agents to specifically bind S1P5 makes them useful for the purification of S1P5 polypeptides or of cells expressing them, e.g., NK cells and cytolytic CD8 T cells, activated NK cells and activated CD8+ T cells.
  • the ability to purify NK cells and cytolytic CD8 T cells, activated NK cells and activated CD8+ T cells is useful for a number of applications, e.g., to characterize the cells for research purposes, for diagnostic purposes in a patient (e.g. to detect whether a patient has an inflammation or autoimmune disorder characterized by the presence of such cell), or for ex vivo procedures.
  • Purification of cells and proteins using ligands can be carried out using any of a variety of well known methods, e.g., affinity purification using ligands bound to a solid support, FACS sorting, immunoprecipitation, etc.
  • Labeled agents that bind and/or inhibit S1P5, e.g., fluorescently labeled sphingosine or analogues, or anti-SlP5 antibodies, can also be used to visualize S1P5- expressing cells in vitro or in vivo.
  • labeled anti-SlP5 antibodies (or derivatives or modified forms thereof) can be used to specifically mark S1P5 expressing cells, e.g., to monitor the number or activity of such cells in a patient who is suspected of having an inflammatory or autoimmune disease, or who is undergoing or who is a candidate for a therapy aimed to alter the activity or number of NK to T cells.
  • Methods of labeling ligands and using them to visualize receptor-expressing cells in vivo or in vitro are also well known in the art.
  • the agents that bind and/or inhibit S1P5 can be used to modulate NK and cytolytic CD8 T cell activity in various ways.
  • certain agents that bind and/or inhibit S1P5 can be used in vivo in a "dominant negative" fashion to prevent the interaction of S1P5 with endogenous ligands (e.g. sphingosine-1 phosphate), or to block or prevent S1P5 signaling induced by an endogenous ligand.
  • S15P antagonists that bind S1P5, including but not limited to antibodies and fragments or derivatives thereof, will generally have such activity.
  • the agent is an agonist of S1P5 under certain conditions (e.g. certain concentrations) but acts as a functional antagonist of S1P5 under other conditions (e.g. certain concentrations).
  • S1P5 agonists may be used under conditions in which the extracellular concentration of S1P5 agonist is increased beyond the range where the agent has agonist properties, resulting in inhibition of S1P5 signaling, as has been demonstrated for various receptors of the GPCR type.
  • the agents will preferably compete with endogenous S1P5 ligands for interaction with S1P5. Agents may also have other effects such as leading to the downregulation of S1P5 expression on a cell surface.
  • Such agents would be useful for treating or preventing conditions caused by excessive NK and/or cytolytic CD8 T cell activity, for example those involving or characterized by acticated NK and/or cytolytic CD8 T cells, and/or which can be treated or prevented by reducing NK and/or cytolytic CD8 T cell activity, such as certain autoimmune and inflammatory disorders.
  • S1P5 binding and/or inhibiting agents including S1P5 antagonists, S1P5 agonists and conditions under which they act as functional inhibitors of S1P5, and antibodies and fragments and derivatives that bind S1P5, using the herein-described methods and assays.
  • any of a wide variety of agents that bind and/or inhibit S1P5 can be used in the practice of the present invention.
  • the agents can be used alone or linked to another compound such as an amino acid residue, peptide or polypeptide, other polymer or small molecule.
  • any agent that leads to the inhibition of S1P5 activity and to the resulting inhibition of cell (e.g. NK and/or CD8+ T cell) trafficking can be used.
  • Agents may inhibit the trafficking, pharmacology, and/or signaling capabilities of an S1P5 polypeptide.
  • the agent may act directly on S1P5 or may act on an S1P5 binding partner, (e.g.
  • an extracellular ligand preferably a ligand other than SlP; a receptor activity-modifying proteins (RAMPs) - a small family of 3 single-transmembrane spanning proteins that can form complexes with G-protein coupled receptors; and/or another S1P5 or other GPCR receptor which binds S1P5 to form an oligomer or homo- oligomer).
  • RAMPs receptor activity-modifying proteins
  • agents that leads to inhibition of S1P5 activity include but not limited to agents that:
  • S1P5 inhibit interaction of S1P5 with a ligand thereof (e.g. by competing with an endogenous S1P5 agonist such as SlP for binding to S1P5);
  • S1P5 signaling by causing S1P5 to be unable or have decreased ability to transduce an extracellular signal across the plasma membrane, e.g. by any mechanism, including but not limited to affecting the conformation of the S1P5 polypeptide (e.g. allosteric regulators);
  • S1P5 polypeptides at the surface of a cell (e.g. inducing receptor-mediated endocytosis);
  • a downstream signaling element causes a downstream signaling element to be unable or have decreased ability to transmit a signal (e.g. inhibiting signaling activity mediated by GCV 0 and/or GCC12/13 proteins.
  • agents that inhibit expression of the S1P5 protein including for example aptamers, siRNA and antisense agents.
  • an antagonist is an agent that does not provoke a biological response itself upon binding to its receptor, but blocks or attenuates agonist-mediated responses.
  • the antagonist may be competitive (or surmountable), i.e. it binds reversibly to a region of the receptor in common with an agonist, but occupies the site without activating the effector mechanism.
  • the effects of a competitive antagonist may be overcome by increasing the concentration of agonist, thereby shifting the equilibrium and increasing the proportion of receptors which the agonist occupies.
  • antagonists may be unsurmountable or irreversible, where no amount of agonist can completely overcome the inhibition once it has been established.
  • Unsurmountable antagonists may bind covalently to the agonist binding site (competitive irreversible antagonists), in which case there is a period before the covalent bond forms during which competing ligands can prevent the inhibition.
  • Other types of unsurmountable antagonists act allosterically at a different site on the receptor or an associated ion channel.
  • the potency of an antagonist is usually defined by its IC50 value. This can be calculated for a given antagonist by determining the concentration of antagonist needed to elicit half inhibition of the maximum biological response of an agonist. Elucidating an IC50 value is useful for comparing the potency of drugs with similar efficacies. The lower the IC 50 , the greater the potency of the antagonist the lower the concentration of drug that is required to inhibit the maximum biological response.
  • Antagonists of S1P5 may be agents that inhibit the interaction of S1P5 with a ligand thereof, preferably by competing with an endogenous S1P5 agonist such as SlP for binding to S1P5.
  • an antagonist of S1P5 may be an agent that causes S1P5 to be unable or have decreased ability to transduce an extracellular signal across the plasma membrane, by any mechanism including but not limited to affecting the conformation of the S1P5 polypeptide. Examples of antagonists include sphigosine derivatives, phosphonates, and antibodies that bind S1P5.
  • antagonists of S1P5 will have at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold or 1000-fold selectivity for binding to S1P5 or inhibition of S1P5 activity over other members of the S1P5 protein family, including but not limited to any one or combination of SlPl, S1P2, S1P3 or S1P4.
  • Selectivity is preferably measured by the ratio of EC50 for the S1P5 to the EC50 for another SlP receptor, in the particular assay used (e.g. GTP ⁇ s binding, calcium mobilization).
  • An agonist is an agent that binds to a receptor and triggers a response in the cell.
  • the agonist may be an endogenous ligand or may mimics the action of an endogenous ligand (e.g. SlP) that binds to the same receptor.
  • Agonists have both affinity and maximum efficacy for their cognate receptors.
  • Agonists may be full agonists, partial agonist or co-agonists.
  • a full agonist binds (has affinity) and activates a receptor, displaying full efficacy for the receptor.
  • a partial agonist also binds and activates a receptor, but only has partial efficacy for the receptor compared to a full agonist.
  • a partial agonist may also be considered as a ligand which displays both agonistic and antagonistic effects (e.g. in the presence of a full agonist partial agonist it may act as competitive antagonists).
  • a co-agonist works with other co-agonists to produce the cellular response.
  • a S1P5 agonist may, for example, have the ability to act as an agent that inhibits S1P5 by attenuating S1P5 signalling by receptor desensitization, in which S1P5 receptors no longer transduce a signal even if the stimulus (e.g. endogenous ligand) is still present, and/or by attenuating receptor signalling by removing S1P5 from the cell surface by receptor-mediated endocytosis resulting from S1P5 activation.
  • a S1P5 agonist could also, for example, have the ability to act as an agent that inhibits S1P5 by causing the elimination of S 1P5 -expressing cells (e.g. a S1P5 agonist linked to a toxic moiety).
  • agonists of S1P5 will have at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold or 1000-fold selectivity for binding to S1P5 or inhibition of S1P5 activity over other members of the S1P5 protein family, including but not limited to any one or combination of SlPl, S1P2, S1P3 or S1P4.
  • Selectivity can be measured for example by the ratio of EC50 for the S1P5 to the EC50 for another SlP receptor, in the particular assay used (e.g. GTP ⁇ S binding, calcium mobilization).
  • an agent that inhibits S1P5 is an analogue or derivative a sphingosine (2-amino-4-octadecene-l,3- diol), preferably a compound which is capable of retaining at least part of the conformation of SlP so as to be capable of positioning in the active site of Sl P5.
  • the agent that inhibits S1P5 is a phospholipid analogue or derivative, a sphingo lipid analogue or derivative.
  • the agent that inhibits S1P5 is a phosphonate, for example an alpha-aminophosphonate, a beta- aminophosphonate or a gamma-aminophosphonate, an aryl-amine or aryl-ether containing phosphonate, or an aryl-amide containing phosphate.
  • the alkyl chain is in a meta conformation with the amino residue (e.g. [3-amino-3-(3- octylphenylcarbamoyl)propyl]-phosphonic acid compounds, such as VPC44116).
  • the S1P5 inhibitors may or may not be phosphorylated for administration to an individual.
  • the agent that inhibits S1P5 is an azacylic compound, preferably a azacyclic analogue of sphingosine (as described in Hanessian et al 2006 Bioog. Med. Chem. Lett) or of a sphingosine analogue (e.g. FTY720).
  • the agent that inhibits S1P5 comprises a saturated hydrocarbon chain of at least 4, 5, 6, 7, 8, 10, 12, 14, 16 or 18 carbons, for example a CgHj ⁇ chain.
  • Other examples of agents include those described in PCT publication nos. WO 01/098301 (e.g. JTE-013), wherein the disclosure of each of the aforementioned references are incorporated herein by reference in their entireties.
  • these agents are agonists of SlPl that are able to act as functional inhibitors of SlPl and/or other SlP family members, although are believed to have been used only under conditions which did not lead to inhibition of S1P5.
  • These agents are typically sphingosine analogues, such as 2- substituted 2-amino- propane- 1,3-diol or 2- amino-propanol derivatives.
  • SlPl and S1P5 agonist is FTY720 or FTY720-P (described for example in Hanessian et al 2006 Bioog. Med. Chem. Lett. Doi:10.1016/j.bmcl.2006.10.014).
  • KRP-203 (Shimizu et al. I l l (2): 222. (2005) http://circ.ahajournals.Org/cgi/content/full/l 11/2/222).
  • An agent that inhibits S1P5 is a phospholipid analogue or derivative, a sphingo lipid analogue or derivative, for example an azacylic compound (see Hanessian et al 2006 Bioog. Med. Chem. Lett.).
  • the agent that inhibits S1P5 is a phosphonate, for example an alpha- aminophosphonate, a beta-aminophosphonate or a gamma-aminophosphonate, an aryl- amine or aryl-ether containing phosphonate, or an aryl-amide containing phosphate.
  • the alkyl chain is in a para conformation with the amino residue (e.g. [3-amino-4-(4-octylphenoxy)-butyl ⁇ -phosphonic acid compounds as set out in F. W. Foss et al, Bioorg Med Chem 15 (2007) 663-677).
  • a range of agents are described in PCT publication no.
  • WO 2006058316 including particularly the compounds of formula X at page 3, formula I, formula II, formula III, formulas IVa and IVb, formula V, formula VI, formula VII, formula VIII, formula IX, formula X at page 9, formula XI, formulas XIIa and XIIb, formula XIII and formula XIV described in PCT publication no. WO2006058316; each of these formulas and individual compounds disclosed in the reference are incorporated herein by reference.
  • Other examples of agents include those described in European patent application nos. EP 627406A1 and EP 1002792A1, PCT publication nos.
  • S1P5 inhibitors can be designed based on known chemical structure that interact, act as agonists or antagonists of SlP receptors.
  • azacyclic analogues of FTy720 act as agonists of S1P4 and S1P5 but are inactive on SlPl and S1P3 (Hanessian et al 2006 Bioog. Med. Chem. Lett., the disclosure of which is incorporated herein by reference). It may therefore be possible to prepare S1P5 antagonists based on the azacyclic compounds that selectively access the binding pocket of S1P5.
  • SlP receptor antagonists include gamma-aminophosphonate, an aryl-amine or aryl- ether containing phosphonate, or an aryl-amide containing phosphate, as described in Foss et al. ((2007) Bioorg. Med. Chem. 15: 663-677) the disclosure of which is incorporated herein by reference.
  • Sphingosine analogues can therefore be adapted to obtain S1P5 antagonists or agonist capable of acting as function inhibitors; determining S1P5 activity can be readily tested using assays well know in the art.
  • An agent that binds and/or inhibits S1P5 can be used alone or, preferably, covalently linked to one or more additional molecular entities.
  • they may be linked to a polymer, peptide or polypeptide.
  • they may be linked to a cytotoxic compound in order to specifically destroy S 1P5 -expressing cells, e.g. NK cells or cytolytic CD8 T cells.
  • the agent is conjugated to a label allowing the visualization and monitoring of Sl P5 -expressing cells, in vivo or in vitro.
  • they may be linked to a solid support, e.g., to allow the purification of S1P5 polypeptides or SlP5-expressing cells.
  • any of the agents that bind and/or inhibit S1P5 for use in the present methods can be assessed in various ways.
  • the ability of the agent to bind to the S1P5 polypeptide, or to a cell (e.g. an NK cell, a cytolytic CD8 T cell) expressing a S1P5 polypeptide can be assessed.
  • Any of a large number of standard methods can be used to assess the binding of the agent to the S1P5 polypeptide, including affinity chromatography, titration calorimetry, surface plasmon resonance, ELISA-type assays, IC50-hapten inhibition, fluorescence binding assays, precipitation, and others.
  • the ability of the agent to act as an agonist or antagonist of the S1P5 polypeptide is assessed using one of various standard assays applicable to GPCR proteins.
  • One exemplary method for determining binding to S1P5 is a GTP ⁇ S binding assay, for example as described in Foss et al. ((2007) Bioorg. Med. Chem. 15: 663-677) or in PCT patent publication no. WO05070886A1, the disclosures of which are incorporated herein by reference.
  • GPCR occupation by agonists leads to guanine nucleotide exchange. GDP bound to Ga, of the Ga ⁇ complex, dissociates and is replaced by GTP. G ⁇ -GTP then detaches from the complex.
  • Dissociation of the complex leads to ⁇ and G ⁇ -GTP subunits that are capable of downstream signaling.
  • Activity of the subunits is controlled by GTPase activity that hydro lyzes the GTP to form G ⁇ -GDP, which then associates with the ⁇ subunits to reform the G ⁇ complex.
  • GTPase activity that hydro lyzes the GTP to form G ⁇ -GDP, which then associates with the ⁇ subunits to reform the G ⁇ complex.
  • This assay can be carried out by expressing a target protein (e.g. preferably an individual SlP receptor, in this case S1P5) in a host cell (e.g. CHO, HEK293 cells) and preparing membrane-bound proteins therefrom. When contacted with a test compound, binding of GTP ⁇ S to membrane bound G protein ⁇ subunits can be detected.
  • a target protein e.g. preferably an individual SlP receptor, in this case S1P5
  • a host cell e.
  • the assay involves generating CHO clones expressing large amounts of human S1P5 can be used and adapted.
  • Membrane proteins are prepared as described above.
  • the basic protocol of I GTP ⁇ s - binding assay used is according to Brinkmann et al. ((2002), J. Biol. Chem, 277, 21453) with the modifications as follows.
  • WGA coated PVT beads SPA-bead, Amersham Bio sciences
  • the plates are centrifuged to minimize non- specific effects caused by the non bound GTP ⁇ - ⁇ S.
  • the assay is performed in 96-well Optiplates (Packard) in final volume of 225, ul/well.
  • the membrane proteins are resuspended at different concentrations (between 25 to 150,ug/ml) in 50 mM HEPES, 100 mM NaCI, 10 mM MgCI2, 20 ug/ml saponin (Riedel-de-Haen: Cat. N° 16109), 0.1% fat free BSA (Sigma) pH 7.4.
  • the membrane proteins are -1 1 mixed with 1 mg/weil SPA-bead, 10 VIM GDP, different concentrations of agonists and incubated for 10-15 min at RT.
  • the GTPy35S binding reaction is started by the addition of pM GTPy35S (Amersham; lOOOCi/mmol).
  • Optiplates are sealed and incubated at RT for 110-120 minutes with constant shaking. The plates are then centrifuged for 10 minutes at 2000 rpm and counted with a TopCount instrument (Packard). Calculations of the EC50 are performed with a non-linear regression fit program as available in the Origin 7 RS2 software package (OriginLab Corporation, MA, USA).
  • a calcium mobilization assay is used to assess the ability of the agent to act as an agonist or antagonist of the S1P5 polypeptide.
  • a candidate agonist or antagonist is added to cells over-expressing S1P5, the cells are loaded with a
  • Ca ⁇ + specific fluorescent probe and activation of the S1P5 signal promotes a concentration-dependent increase of intracellular Ca ⁇ + , resulting in a increase of fluorescence.
  • a suitable calcium mobilization assay is described in Osada et al. ((2002) Biochem. Biophys. Res. Comm. 299: 483-487, the disclosure of which is incorporated herein by reference.
  • Another example is reproduced from WO05070886A1 and Hanessian et al 2006 (Bioog. Med. Chem. Lett.), the disclosures of which are incorporated herein by reference, and is carried out as follows, optionally adapted as needed.
  • CHO cells are plated in black Costar plate (96 or 384 well, 50'0OO cells or 12.500 cells, respectively) in a MEM with FCS and cultured for 20-24 h at 37°C in a CO2 incubator. After the removal of the culture medium cells are incubated in HBSS medium containing 2, ⁇ M Fluo4AM (Molecular Probes; 1 mg/ml stock in DMSO), 5 mM probenicid for 1 h at 37°C, rinsed with HBSS buffer, 2.5 mM probenicid and overlaid with the same medium (75 ul for 96- well plates, 50,ul for 384 plates). Plates are transferred to FLIPR (fluorometric imaging plate reader).
  • FLIPR fluorometric imaging plate reader
  • the agonist in HBSS is added and the fluorescence is measured at intervals of 2 sec for 3 to 5 min.
  • the cells are pre-treated for 5 h with 50 ng/ml pertussis toxin (Sigma).
  • 2-aminoethoxydiphenyl borate (2- APB, Calbiochem, Juro Supply, Lucerne, Switzerland) a blocker of the release of calcium from the endoplasmic reticulum (Ascher- Landsberg et al., 1999, Biochem. Biophys. Res. Commun. 264, 979) at 50 ⁇ M and/or 150 M is added directly to the cell medium 20- 40 min prior to the measurements.
  • Calculations of the EC50 are performed using a non linear regression fit program provided in the Origin 7 RS2 software package (Origin LabCorporation) .
  • agents are identified that bind to S1P5 and/or have agonist or antagonist activity are identified, their ability to inhibit S1P5 can be assessed in a functional assay designed to assess agents' activity on cells, for example for effects on cell trafficking, homing, migration, proliferation, adherence and/or morphogenesis.
  • a functional assay designed to assess agents' activity on cells, for example for effects on cell trafficking, homing, migration, proliferation, adherence and/or morphogenesis.
  • any of the assays in the Examples section can be used.
  • Assays for detecting the ability of an agent that binds and/or inhibits S1P5 to affect cell trafficking can be carried out in any suitable way.
  • a cell migration assay is used (see for example Osada et al. ((2002) Biochem. Biophys. Res. Comm. 299: 483-487).
  • Chemotactic migration of cells can be measured in a modified Boyden chamber (for example Transwell culture chambers, Costar, Cambridge MA) using polycarbonate filters (25 X 80 mm; 12 ⁇ m pore size) coated with collagen type I (50 ⁇ g/ml in 5% acetic acid), which promotes uniform attachment to and migration across the filter without formation of a barrier.
  • Cells that migrate to the lower side of the filter are fixed, stained and counted.
  • the cells are preferably NK cells or cytolytic CD8 T cells, but can also be any cell made to express functional S1P5 polypeptides.
  • the agents decrease the trafficking, homing, migration, proliferation, adherence and/or morphogenesisactivity of Sl P5- expressing cells, NK cells or T cells (e.g. activated cytolytic CD8 T cells), using any relevant assay or test by at least 20%, preferably at least 30%, 40% or 50%.
  • binding to S1P5 polypeptides binding to cells expressing S1P5 polypeptoides (e.g. host cells made to express S1P5, cytolytic CD8 T cells, NK cells), or functional assays, e.g., the above-described binding and cell activation or migration assays can be used as a readout in order to assay standard or lead compounds in, in order to assess the ability of test compounds to bind S1P5 or affect its activity.
  • compounds of any type e.g.
  • sphingosine analogues antibodies that bind S1P5 can be screened in the assays in order to identify other compounds that have equal or greater binding affinity or ability to modulate S1P5 activity than the sphingosine analogues (e.g. FTY720).
  • sphingosine analogues can be modified in various ways in order to identify variants with enhanced binding or modulatory activities or with, e.g., improved specificity, reduced side effects, or enhanced biochemical, pharmacokinetic, or pharmacodynamic properties.
  • high throughput screening methods are used to identify small molecules or other compounds that are capable of binding specifically to S1P5 and/or modulating its activity. Such methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or binding compounds). Such "combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al, Nature 354:84-88 (1991)).
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No.
  • WO 93/20242 random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc.
  • High throughput screening assays for G-protein coupled receptors are well known in the art and can be purchased from commercial suppliers.
  • GTP ⁇ 3f>S assays that are homogeneous, eliminates the need for filtration and washing, can be purchased from Perkin-Elmer Life Sciences (Boston, MA).
  • WGA Wheat Germ Agglutinin
  • FlashPlates 96- or 384-well plates
  • WGA Wheat Germ Agglutinin
  • the FlashPlate is then coated with membrane S1P5 proteins followed by WGA which captures receptors.
  • Calcium mobilization assays can also be readily adapted to high throughput screening.
  • a variety of other screening assays that have been developed for G- protein coupled receptors is described in Eglen et al. ((2007) Assay and Drug Dev. Tech. 5(3):524-451), the disclosure of which is incorporated herein by reference in its entirety.
  • a method screening for a compound can comprise: (a) providing a SlP receptor-modulator test compound, (b) contacting a cell expressing a S1P5 polypeptide with said test compound; and (c) determining whether the test compound modulates cell trafficking.
  • a method screening for a compound can comprise: (a) contacting a S1P5 polypeptide with a test compound and determining whether the test compound binds and/or inhibits S1P5; (b) selecting a test compound binds and/or inhibits S1P5; and (c) determining whether the test compound selected in (b) modulates cell trafficking.
  • Another example is a method of screening for a compound that modulates NK or T cell activity (e.g. activation, proliferation, trafficking) and/or for screening for a compound for the treatment or prevention of a disorder mediated by activated NK or T cells (e.g. an inflammatory or autoimmune disorder), said method comprising: (a) contacting an S1P5 polypeptide with a test compound; and (b) determining whether the test compound binds and/or inhibits S1P5, wherein a detection that said test compound binds and/or inhibits S1P5 indicates that said test compound is a candidate compound for modulating NK or T cell activity and/or for a compound for the treatment or prevention of a disorder mediated by activated NK or T cells.
  • a disorder mediated by activated NK or T cells e.g. an inflammatory or autoimmune disorder
  • a method of screening for a compound that modulates NK or T cell activity e.g. activation, proliferation, trafficking
  • a compound for the treatment or prevention of a disorder mediated by activated NK or T cells e.g.
  • an inflammatory or autoimmune disorder said method comprising: (a) contacting an S1P5 polypeptide with a S1P5 ligand in the presence of a test compound; (b) contacting an S1P5 polypeptide with a S1P5 ligand in the absence of said test compound; and (c) comparing the binding of S1P5 ligand to said polypeptide in steps a) and b); wherein a detection that said binding differs between steps a) and b) indicates that said test compound is a candidate compound for modulating NK or T cell activity and/or for a compound for the treatment or prevention of a disorder mediated by activated NK or T cells.
  • the binding of said S1P5 ligand e.g. SlP or sphingosine analogue or a derivative
  • the binding of said S1P5 ligand e.g. SlP or sphingosine analogue or a derivative
  • a method of producing an agent, suitable for use in the treatment of an autoimmune or inflammatory disorder comprising: i) providing a S 1P5 -expressing cell and a plurality of test agents; ii) testing the ability of each of said test agents to bind and/or inhibit S1P5 in said cells, optionally to interfere with the binding of a S1P5 ligand to S1P5 on said cells; iii) selecting an agent from said plurality that bind and/or inhibits S1P5, optionally that reduces the binding of said S1P5 ligand to S1P5; iv) optionally, rendering said agent suitable for human administration; and v) optionally, producing a quantity of said agent.
  • S 1P5 -expressing cell can be a host cell made to express S1P5 (e.g. a mammalian, human host cell), or an NK or T cells that naturally expresses S1P5.
  • S1P5 ligands include SlP, FTY720, or a composition comprising a sphingosine analogue or derivative.
  • the assays may comprise determining whether the test compound selectively binds and/or inhibits S1P5 compared to SlPl, S1P2, S1P3 and/or S1P4, and in one embodiment the compound is selective to have selectivity for S1P5 over SlPl, S1P2, S1P3 and/or S1P4; optionally, the test compound binds and/or inhibits SlPl, S1P2, S1P3 and/or S1P4 in addition to S1P5.
  • the test compound is or comprises a sphingosine analogue or derivative.
  • the assays can also comprise a step in which the activity of said polypeptide is assessed in the presence and absence of said compound.
  • the method can further comprise selecting the candidate compound identified in one of the screening steps for use as a medicament (e.g. if a test compound bind and/or inhibits S1P5, or if it inhibits cell trafficking, etc.).
  • any of the methods can further comprise further comprising making the candidate compound suitable for administration to a human (e.g. formulating the compound for use as a medicament, humanizing an antibody, etc.) and/or preparing the quantity of the candidate compound for administration to a human.
  • the S1P5 inhibitor may be an aptamer having affinity with an S 1P5 -encoding nucleic acid or a S1P5 polypeptide.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., (1990) Science 249(4968):505-10.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA.
  • each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S. D., (1999) Clin Chem. 45(9): 1628-50.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, (1996) Nature 380(6574):548-50).
  • Inhibition of S1P5 can be performed with an inhibitor of S1P5 gene expression gene expression.
  • Inhibitors of S1P5 gene expression for use in the present invention may be also based on anti-sense oligonucleotide constructs.
  • Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of S1P5 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of S1P5, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding S1P5 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
  • Small inhibitory RNAs can also function as inhibitors of S1P5 gene expression for use in the present invention.
  • S1P5 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that S1P5 gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl T et al. (1999) Genes Dev.;13(24):3191-7; Elbashir SM, et al.
  • shRNAs can also function as inhibitors of S1P5 or S1P5 receptor expression for use in the present invention.
  • Ribozymes can also function as inhibitors of S1P5 gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of S1P5 mRNA sequences are thereby useful within the scope of the present invention.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable.
  • Both antisense oligonucleotides and ribozymes useful as inhibitors of S1P5 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and preferably cells expressing S1P5.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • adenovirus adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • vaccinia virus
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high- efficiency transduction of genes in vivo.
  • viruses for certain applications are the adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • AAV adeno-associated virus
  • 12 different AAV serotypes AAVl to 12
  • Recombinant AAV are derived from the dependent parvovirus AA V2 (Choi, VW J Virol 2005; 79:6801-07).
  • the adeno-associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species (Wu, Z MoI Ther 2006; 14:316-27).
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the present invention provides methods of specifically targeting S1P5 and cells expressing S1P5, using antibodies or antigen binding compound such as a protein or peptide that binds S1P5. Such methods are useful for a number of purposes. Specifically, the discovery that S1P5 is expressed on immune cells, particularly NK cells, mature NK cells, T cells, cytolytic CD8 T cells, provides that antibodies and antigen binding compounds that bind S1P5 can be used as antagonists to inhibit S1P5 activity (e.g.
  • S1P5 polypeptide by "masking" the S1P5 polypeptide so as to interfere with binding to a ligand, preferably an endogenous ligand), or to bind S1P5 expressing cells in order to eliminate them (e.g. by linking the antibody to a cytotoxic moiety, or by mediating antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity).
  • a ligand preferably an endogenous ligand
  • S1P5 expressing cells in order to eliminate them (e.g. by linking the antibody to a cytotoxic moiety, or by mediating antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity).
  • using antibodies or other compounds to specifically bind to S1P5 and thereby inhibit or destroy cells expressing them can be used to selectively eliminate NK or T cells, for example as might be involved in an inflammatory or autoimmune condition in a patient.
  • any antibody or antigen binding compound that specifically binds to S1P5 can be used, e.g., as assessed using the herein-described binding assays, and can be optionally conjugated with a functional compound such as a label or cytotoxic moiety.
  • Labeled antibodies can be used for example to monitor, in vitro or in vivo, the number, activity, or location of immune cells (e.g. NK cells, mature NK cells, T cells, cytolytic CD8 T cells) in a patient or in a biological sample.
  • antibodies linked to a cytotoxic moiety can be used to selectively kill S1P5 presenting cells, e.g., NK or cytolytic CD8 T cells involved in an inflammatory or autoimmune disorder. It will be appreciated that such antibodies, can be generated de novo, or derived from any known SlP5-binding compound, e.g., antibodies generated specifically against S1P5.
  • antibodies When antibodies are used to bind to S1P5, they may be produced by any of a variety of techniques known in the art. Typically, they are produced by immunization of a non-human animal, preferably a mouse, with an immunogen comprising a S1P5 polypeptide, peptide, or cells expressing S1P5.
  • the S1P5 protein may be isolated, a fragment or derivative of full protein (e.g., an immunogenic fragment or derivative), and may be present in any suitable form.
  • the S1P5 protein used to generate antibodies is is present on the extracellular surface of an NK or T cell.
  • Preferred isolated antibodies of the invention are directed S1P5, and do not bind substantially to non-cancer or uninfected cells, or at least bind with substantially less affinity to SlPl, S1P2, S1P3 and/or S1P4 polypeptides.
  • Antibodies can exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab) T 2, a dimer of Fab which itself is a light chain joined to V H -C HI by a disulfide bond.
  • the F(ab) T 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab) T 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term "antibody,” as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).
  • GPCR proteins have been developed, including those by M-Phasys (Tubingen, Germany), based on methods for preparing GPCR proteins as immunogens (see U.S. patent no. 6,939,951, the disclosure of which is incorporated herein by reference).
  • transgenic mice, or other organisms such as other mammals may be used to express humanized, chimeric, or similarly-modified antibodies.
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)).
  • the method comprises selecting, from a library or repertoire, a monoclonal antibody or a fragment or derivative thereof that cross reacts with S1P5.
  • the repertoire may be any (recombinant) repertoire of antibodies or fragments thereof, optionally displayed by any suitable structure (e.g., phage, bacteria, synthetic complex, etc.).
  • the step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is herein incorporated by reference).
  • the immunogen is suspended or dissolved in a buffer, optionally with an adjuvant, such as complete Freund's adjuvant.
  • an adjuvant such as complete Freund's adjuvant.
  • the location and frequency of immunization sufficient to stimulate the production of antibodies is also well known in the art.
  • the non- human animals are injected intraperitoneally with antigen on day 1 and again about a week later. This is followed by recall injections of the antigen around day 20, optionally with adjuvant such as incomplete Freund's adjuvant.
  • the recall injections are performed intravenously and may be repeated for several consecutive days. This is followed by a booster injection at day 40, either intravenously or intraperitoneally, typically without adjuvant.
  • This protocol results in the production of antigen-specific antibody-producing B cells after about 40 days.
  • lymphocytes from an unimmunized non-human mammal are isolated, grown in vitro, and then exposed to the immunogen in cell culture. The lymphocytes are then harvested and the fusion step described below is carried out.
  • the next step is the isolation of cells, e.g., lymphocytes, splenocytes, or B cells, from the immunized non- human mammal and the subsequent fusion of those splenocytes, or B cells, or lymphocytes, with an immortalized cell in order to form an antibody-producing hybridoma.
  • cells e.g., lymphocytes, splenocytes, or B cells
  • preparing antibodies from an immunized animal includes obtaining B-cells/splenocytes/lymphocytes from an immunized animal and using those cells to produce a hybridoma that expresses antibodies, as well as obtaining antibodies directly from the serum of an immunized animal.
  • splenocytes e.g., from a non-human mammal
  • isolation of splenocytes involves removing the spleen from an anesthetized non-human mammal, cutting it into small pieces and squeezing the splenocytes from the splenic capsule and through a nylon mesh of a cell strainer into an appropriate buffer so as to produce a single cell suspension.
  • the cells are washed, centrifuged and resuspended in a buffer that lyses any red blood cells.
  • the solution is again centrifuged and remaining lymphocytes in the pellet are finally resuspended in fresh buffer.
  • the antibody-producing cells are fused to an immortal cell line.
  • This is typically a mouse myeloma cell line, although many other immortal cell lines useful for creating hybridomas are known in the art.
  • Preferred murine myeloma lines include, but are not limited to, those derived from MOPC-21 and MPC-11 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, Calif. U.S.A., X63 Ag8653 and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland U.S.A.
  • the fusion is effected using polyethylene glycol or the like.
  • the resulting hybridomas are then grown in selective media that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT- deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • HAT medium thymidine
  • the hybridomas can be grown on a feeder layer of macrophages.
  • the macrophages are preferably from littermates of the non-human mammal used to isolate splenocytes and are typically primed with incomplete Freund's adjuvant or the like several days before plating the hybridomas. Fusion methods are described, e.g., in (Goding, "Monoclonal Antibodies: Principles and Practice,” pp. 59-103 (Academic Press, 1986)), the entire disclosure of which is herein incorporated by reference.
  • the cells are allowed to grow in the selection media for sufficient time for colony formation and antibody production. This is usually between 7 and 14 days.
  • the hybridoma colonies are then assayed for the production of antibodies that specifically recognize the desired substrate, e.g. the S1P5 polypeptide.
  • the assay is typically a colorimetric ELISA-type assay, although any assay may be employed that can be adapted to the wells that the hybridomas are grown in. Other assays include immunoprecipitation and radioimmunoassay.
  • the wells positive for the desired antibody production are examined to determine if one or more distinct colonies are present.
  • the cells may be re-cloned and grown to ensure that only a single cell has given rise to the colony producing the desired antibody. Positive wells with a single apparent colony are typically recloned and re-assayed to insure only one monoclonal antibody is being detected and produced.
  • Hybridomas that are confirmed to be producing a monoclonal antibody of this invention are then grown up in larger amounts in an appropriate medium, such as DMEM or RPMI- 1640.
  • an appropriate medium such as DMEM or RPMI- 1640.
  • the hybridoma cells can be grown in vivo as ascites in an animal.
  • the growth media containing the monoclonal antibody (or the ascites fluid) is separated away from the cells and the monoclonal antibody present therein is purified. Purification is typically achieved by gel electrophoresis, dialysis, chromatography using protein A or protein G- Sepharose, or an anti-mouse Ig linked to a solid support such as agarose or Sepharose beads (all described, for example, in the Antibody Purification Handbook, Amersham Biosciences, publication No. 18-1037-46, Edition AC, the disclosure of which is hereby incorporated by reference).
  • the bound antibody is typically eluted from protein A/protein G columns by using low pH buffers (glycine or acetate buffers of pH 3.0 or less) with immediate neutralization of antibody-containing fractions. These fractions are pooled, dialyzed, and concentrated as needed.
  • low pH buffers glycine or acetate buffers of pH 3.0 or less
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • Recombinant expression in bacteria of DNA encoding the antibody is well known in the art (see, for example, Skerra et al. (1993) Curr. Op. Immunol. 5:256; and Pluckthun (1992) Immunol. Revs. 130:151.
  • Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in Ward et al. (1989) Nature 341 :544.
  • the binding of various antibodies or other compounds to S1P5 can be assessed using various assays, e.g., to identify binding compounds with higher affinity or greater specificity, where the test antibodies to be examined are obtained from different source animals, or are even of a different Ig isotype.
  • a simple competition assay may be employed in which control and test antibodies or compounds are admixed (or pre-adsorbed) and applied to a sample containing the epitope-containing compound, e.g. S1P5. Protocols based upon ELISAs, radioimmunoassays, Western blotting and the use of BIACORE are suitable for use in such simple competition studies and are well known in the art.
  • control antibodies or compounds with varying amounts of the test antibodies or compounds (e.g., 1 : 10 or 1 :100) for a period of time prior to applying to the antigen (e.g. S1P5) containing sample.
  • the control and varying amounts of test antibodies or compounds can simply be admixed during exposure to the antigen sample.
  • test antibodies or compounds As long as one can distinguish bound from free antibodies or compound (e.g., by using separation or washing techniques to eliminate unbound antibodies or compound) and the control antibody or compound from the test antibodies or compound (e.g., by using species- or isotype-specific secondary antibodies or by specifically labeling the control antibody or compound with a detectable label) one will be able to determine if the test antibodies or compounds reduce the binding of the control antibody or compound to the antigen, indicating that the test antibody or compound recognizes substantially the same epitope as the control. The binding of the (labeled) control antibodies or compounds in the absence of a completely irrelevant antibody or compound would be the control high value.
  • the control low value would be obtained by incubating the labeled control antibodies or compounds with unlabeled antibodies or compounds of exactly the same type, where competition would occur and reduce binding of the labeled antibodies or compounds.
  • a significant reduction in labeled antibody or compound reactivity in the presence of a test antibody or compound is indicative of a test antibody or compound that recognizes the same epitope, i.e., one that "cross-reacts with” or “specifically recognizes” or “specifically binds to" the labeled control antibody or compound.
  • test antibody or compound that reduces the binding of the labeled control to the antigen by at least 50% or more preferably 70%, at any ratio of control :test antibody or compound between about 1 :10 and about 1 : 100 is considered to be an antibody or compound that binds to substantially the same epitope or determinant as the control.
  • test antibody or compound will reduce the binding of the control to the antigen by at least 90%.
  • the test antibody or compound is pre-adsorbed and applied at saturating concentrations to a surface onto which is immobilized the substrate for the antibody or compound binding, e.g. the S1P5 polypeptide.
  • the surface is preferably a BIACORE chip.
  • competition can be assessed by a flow cytometry test.
  • Cells bearing a S1P5 polypeptide e.g., T or NK cells or any suitable host cell made to express S1P5
  • a control antibody or compound that is known to specifically bind to the S1P5 polypeptide are incubated first with a control antibody or compound that is known to specifically bind to the S1P5 polypeptide, and then with the test antibody or compound that has been labeled with, e.g., a fluorochrome or biotin.
  • the test antibody or compound is said to compete with the control if the binding obtained with preincubation with saturating amounts of control antibody or compound is 80%, preferably, 50, 40 or less the binding (mean of fluorescence) obtained by the antibody or compound without preincubation with the control.
  • a test antibody or compound is said to compete with the control if the binding obtained with a labeled control (by a fluorochrome or biotin) on cells preincubated with saturating amount of antibody or compound to test is 80%, preferably 50%, 40%, or less the binding obtained without preincubation with the antibody or compound.
  • monoclonal antibodies are produced, generally in non-human animals that can specifically bind to S1P5, the antibodies are modified so as to make them suitable for therapeutic use in humans.
  • they may be humanized, chimerized, or selected from a library of human antibodies using methods well known in the art.
  • Humanized forms of antibodies according to this invention are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from the murine or other non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of the original antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody.
  • CDR complementary-determining region
  • a humanized antibody according to the present invention has one or more amino acid residues introduced into it from the original antibody. These murine or other non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. (1986) Nature 321 :522; Riechmann et al. (1988) Nature 332:323; Verhoeyen et al. (1988) Science 239:1534 (1988)).
  • humanized antibodies are “chimeric” antibodies (Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al. (1984) Proc. Natl. Acad. Sci. 81 :6851; the entire disclosures of which are herein incorporated by reference), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from the original antibody.
  • humanized antibodies according to this invention are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in the original antibody. The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity.
  • the sequence of the variable domain of an antibody of this invention is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the mouse is then accepted as the human framework (FR) for the humanized antibody (Sims et al. (1993) J. Immunol, 151 :2296; Chothia and Lesk (1987) J. MoI. Biol. 196:901).
  • Another method uses a particular framework from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. 89:4285; Presta et al. (1993) J. Immunol. 51 :1993)).
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • Another method of making fully “humanized” or “human” monoclonal antibodies is to use a XenoMouse® (Abgenix, Fremont, CA) as the mouse used for immunization.
  • a XenoMouse is a murine host that has had its immunoglobulin genes replaced by functional human immunoglobulin genes.
  • antibodies produced by this mouse or in hybridomas made from the B cells of this mouse are already humanized.
  • the XenoMouse is described in United States Patent No. 6,162,963, which is herein incorporated in its entirety by reference.
  • An analogous method can be achieved using a HuMAb-MouseTM (Medarex).
  • Human antibodies may also be produced according to various other techniques, such as by using, for immunization, other transgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et al, Nature 362 (1993) 255), or by selection of antibody repertoires using phage display methods. Such techniques are known to the skilled person and can be implemented starting from monoclonal antibodies as disclosed in the present application.
  • Other derivatives within the scope of this invention include functionalized antibodies, i.e., antibodies that are conjugated or covalently bound to a toxin, such as ricin, diphtheria toxin, abrin and Pseudomonas exotoxin; to a detectable moiety, such as a fluorescent moiety, a radioisotope or an imaging agent; or to a solid support, such as agarose beads or the like.
  • a toxin such as ricin, diphtheria toxin, abrin and Pseudomonas exotoxin
  • a detectable moiety such as a fluorescent moiety, a radioisotope or an imaging agent
  • solid support such as agarose beads or the like.
  • antibodies or other compounds are obtained that are known to specifically bind to S1P5, they will generally be assessed for their ability to bind to or interact with S 1P5 -expressing cells, modulate S1P5 activity (e.g. cell trafficking, calcium mobilization, etc.) Any suitable assay can be used, including but not limited to those described above.
  • S1P5 activity e.g. cell trafficking, calcium mobilization, etc.
  • Any suitable assay can be used, including but not limited to those described above.
  • the ability of the antibodies to interact with S1P5 and S 1P5 -expressing cells can be assessed using any of a large number of well known methods.
  • the assays described herein can be equally applied to detect the interaction of humanized, chimeric, or other human-suitable SlP5-specific antibodies or other compounds with S1P5 and S1P5- expressing cells.
  • the cells are human cells.
  • Antibodies can also be selected to be selective for binding and/or inhibition of activity (e.g. interfering with ligand binding) of S1P5 over other SlP receptors, including but not limited to SlPl, S1P2, S1P3 and/or S1P4.
  • the anti-SlP5 antibody will not substantially bind to and/or inhibit the activity of to SlPl, S1P2, S1P3 and/or S1P4 polypeptides.
  • the ability of the humanized or human-suitable antibody, or fragment thereof, or other compound, to bind to S1P5 or SlP5-expressing cells will be compared with the ability of a control protein or other compound, e.g. a non-human antibody or a non-Ig peptide or protein, to bind to the same target.
  • Antibodies or compounds that bind to the S1P5 and S 1P5 -expressing cells using any suitable assay with 25%, 50%, 100%, 200%, 1000%, or higher increased affinity relative to the control protein, are said to "specifically bind to," “specifically recognize,” or “specifically interact with” the target, and are preferred for use in the methods described elsewhere herein.
  • any antibodies, fragments thereof, or other compounds that bind to the S1P5 polypeptide or S 1P5 -expressing cells with 25%, 50%, 100%, 200%, 1000%, or higher increased affinity relative to their binding to control compounds are said to "specifically bind to,” “specifically interact with,” or “specifically recognize” S1P5.
  • the present invention provides a method for producing an antibody suitable for use in the treatment of a disorder such as an inflammatory or autoimmune disorder, the method comprising the following steps: a) providing a monoclonal antibody that binds specifically to S1P5; b) humanizing the antibody or otherwise rendering it suitable for use in humans; and c) assessing the ability of the humanized or human-suitable antibody to specifically interact with S1P5 presenting target cells, wherein a determination that the humanized or human-suitable antibody binds specifically to the S1P5 presenting target cell indicates that the antibody is suitable for use in the treatment of the disorder.
  • antibodies or compounds that specifically bind to S1P5 present on the surface of a cell (e.g. S 1P5 -expressing host cell, NK cell, cytolytic CD8 T cell), their ability to inhibit the trafficking, homing, growth, proliferation, or activity of the cells, or to kill the cells, will generally be assessed, in vivo or in vitro.
  • a cell e.g. S 1P5 -expressing host cell, NK cell, cytolytic CD8 T cell
  • Any of a number of standard methods can be used to monitor, e.g., the number, proliferation, location, trafficking, homing, survival, or activity of cells in vitro or in vivo, where any compound or antibody that reduces the trafficking, homing, presence in circulation or at a site of inflammation, extravasation, proliferation or number of such cells, or increases the killing of such cells, relative to in the presence of a control compound (e.g. one that does not bind specifically to S1P5) or in the absence of any additional compounds by 10%, 20%, 30%, 40%, 50%, 100%, 200%, 500%, 1000%, or more, is considered suitable for use in the present methods.
  • a control compound e.g. one that does not bind specifically to S1P5
  • any additional compounds e.g. one that does not bind specifically to S1P5
  • 10%, 20%, 30%, 40%, 50%, 100%, 200%, 500%, 1000%, or more is considered suitable for use in the present methods.
  • a compound or antibody can be selected for its ability (or inability) to interfere with binding of Sl P5 to a ligand, for example SlP or a sphingosine analogue.
  • a ligand for example SlP or a sphingosine analogue.
  • Methods for determining S1P5 binding to a ligand are described herein.
  • an antibody is selected that specifically binds to S1P5 and which interferes with or decreases binding of the example SlP or a sphingosine analogue to Sl P5.
  • an antibody is selected that specifically binds to S1P5 and which decreases S1P5 signalling induced by an agonist, for example which decreases calcium mobilization induced by SlP or a sphingosine analogue.
  • Such antibody compositions and their uses are encompassed by the present invention.
  • Antibodies or other compounds designed to specifically bind to S1P5 and thereby inhibit or destroy cells expressing, such as for use to eliminate NK cells or cytolytic CD8 T cells in a patient having an inflammatory or autoimmune disease can advantageously comprise an Fc region capable of mediating a cytoxic (e.g. ADCC or CDC) toward the cells.
  • a cytoxic e.g. ADCC or CDC
  • human IgGl or IgG3 type Fc regions are generally suitable.
  • the cytotoxic antibody advantageously comprises an element selected from the group consisting of radioactive isotope, toxic peptide, and toxic small molecule.
  • Such antibody compositions and their uses are encompassed by the present invention.
  • antibodies designed to specifically bind to S1P5 and inhibit S1P5 on NK cells or cytolytic CD8 T cells in a patient having an inflammatory or autoimmune disease, but which are not intended to destroy the S 1P5 -expressing cell may advantageously contain Fc regions of the human IgG4 or IgG2 subtype, may lack effective Fc regions (e.g. F(ab')2 antibodies), or may comprise Fc regions modified so as to reduce their ability to induce cell desctruction (e.g. IgG4 type Fc regions containing one of more amino acid substitutions to reduce binding to Fcgamma receptors).
  • S1P5 bind and/or inhibits S1P5 are expected to be particularly effective at treating autoimmune and inflammatory disorders.
  • S1P5 has been found to be expressed selectively by mature NK cells and cytolytic CD8 T cells, it is expected that the present methods can be used advantageously to treat inflammatory and autoimmune conditions that are established and characterized by chronic and ongoing, inflammation.
  • the present methods can be used to treat any disorder caused at least in part by the presence or activity of Sl P5 -expressing cells, e.g., NK cells, T cells, activated T cells, cytotoxic CD8 + T cells expressing S1P5, and which can therefore be effectively treated by selectively killing or inhibiting the activation of Sl P5 -expressing cells, e.g., by inhibiting the activation of or by downregulating the expression of the receptor.
  • Sl P5 -expressing cells e.g., NK cells, T cells, activated T cells, cytotoxic CD8 + T cells expressing S1P5, and which can therefore be effectively treated by selectively killing or inhibiting the activation of Sl P5 -expressing cells, e.g., by inhibiting the activation of or by downregulating the expression of the receptor.
  • Other suitable diseases include other autoimmune disorders and inflammatory disorders, particularly those involving CD8 + cells, particularly cytotoxic CD8 + T cells. In any case, however, any disorder whose pathology involves at least in
  • the expression of S1P5 on cells underlying the particular disorder will be assessed, and/or the presence or involvement of NK cells, T cells, activated T cells, cytotoxic CD8 + T cells in the disorder is assessed.
  • This can be accomplished by obtaining a sample of PBLs or cells from the site of the disorder (e.g., from the synovium in RA patients), and testing e.g., using immunoassays, to determine the relative prominence of markers such as CD3, CD8, NKp46, etc., as well as S1P5 on the cells.
  • Other methods can also be used to detect expression of S1P5 and other genes, such as RNA-based methods, e.g., RT-PCR or Northern blotting.
  • the treatment may involve multiple rounds of administration of an agent that binds and/or inhibits S1P5.
  • an agent that binds and/or inhibits S1P5. For example, following an initial round of administration, the presence, trafficking, level and/or activity of Sl P5 -expressing T cells, e.g., NK cells, cytotoxic CD8 + T cells in the patient will generally be re-measured, and, if still elevated, an additional round of administration can be performed. In this way, multiple rounds of cell and/or receptor detection and agent administration can be performed, e.g., until the disorder is brought under control.
  • compositions that comprise any of the present compounds, e.g. agents that bind and/or inhibit S1P5, including but not limited to S1P5 agonists, antagonists, sphingosine analogues and derivatives, and antibodies and other compounds that specifically recognize S1P5, in any suitable vehicle in an amount effective to inhibit S1P5 and/or an SlP5-expressing cell (preferably NK cell and/or cytolytic CD8 T cells) activity, to specifically bind to and/or modulate or preferably inhibit the trafficking, circulation, homing, migration, extra vasion, proliferation, survival, or activity of S 1P5 -expressing cells in a patient or in a biological sample, or to eliminate or kill S 1P5 -expressing cells in a patient or in a biological sample.
  • the composition generally further comprises a pharmaceutically acceptable carrier. Kits comprising the present compounds, e.g., for use in treatment methods, are also provided, typically also including instructions for their use
  • the agents that bind and/or inhibit S1P5 may be employed in a method of modulating the activity of or binding to SlP5-expressing cells (e.g. immune cells, particularly NK cell and/or cytolytic CD8 T cells), or of altering the amount of S1P5 on the surface of cells, in a patient or a biological sample.
  • SlP5-expressing cells e.g. immune cells, particularly NK cell and/or cytolytic CD8 T cells
  • the composition must be formulated for administration to the patient.
  • the agents that bind and/or inhibit S1P5 are particularly effective at treating autoimmune or inflammatory disorders, and can generally be used to treat any disorder caused at least in part by an excess of NK cells or T cells, or which could be treated by decreasing the number or activity of NK or T cells, preferably mature NK cells or acticated cytolytic CD8 T cells.
  • the autoimmune disease is not associated with large granular lymphocyte leukaemia (LGL or T-LGL), which is a disorder characterized by a clonal expansion of NK-like or cytotoxic T cells and thus an increased count of large granular lymphocytes.
  • LGL or T-LGL large granular lymphocyte leukaemia
  • Exemplary autoimmune disorders treatable using the present methods include, inter alia, hemolytic anemia, pernicious anemia, polyarteritis nodosa, systemic lupus erythematosus, Wegener's granulomatosis, autoimmune hepatitis, Behcet's disease, Crohn's disease, inflammatory bowel disease, primary bilary cirrhosis, scleroderma, ulcerative colitis, Sjogren's syndrome, Type 1 diabetes mellitus, uveitis, Graves' disease, Alzheimer's disease, thyroiditis, myocarditis, rheumatic fever, scleroderma, ankylosing spondylitis, rheumatoid arthritis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, Guillain-Barre syndrome, multiple sclerosis, alopecia areata, pemphi
  • NK cells and/or T cells have been reported to be involved in many inflammatory disorders. Because inflammation is a fundamental defense mechanism, inflammatory disorders can affect virtually any tissue of the body. Accordingly, agents that bind and/or inhibit S1P5 have uses in the treatment of tissue-specific inflammatory disorders, including, but not limited to, allergies, including allergic rhinitis/sinusitis, skin allergies such as urticaria/hives, angioedema, atopic dermatitis, food allergies, drug allergies, insect allergies, and allergic disorders such as mastocytosisasthma, asthma, arthritis, including osteoarthritis, rheumatoid arthritis, and spondyloarthropathies, gastrointestinal inflammation, in particular adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis,
  • the present methods of administering agents to patients can also be used to treat animals, or to test the efficacy of any of the herein- described methods or compositions in animal models for human diseases.
  • the antibodies or compounds will be ensured to be capable of specifically binding to S1P5 present on NK cells and/or cytolytic CD8 T cells from the relevant animal.
  • the antibody or compound will be modified to be suitable for administration into the animal.
  • the agents that bind and/or inhibit S1P5 will be evaluated in vivo in suitable animal models.
  • mouse models containing human NK cells and/or cytolytic CD8 T cells are administered compounds of the invention, and the activity of the human NK cells in the mouse is addressed (in terms of trafficking, circulation, homing, migration, extravasion, proliferation, survival, or activity of Sl P5-expressing cells, etc.).
  • mouse models containing human NK cells and/or cytolytic CD8 T cells are administered antibodies or other compounds that specifically recognize S1P5, and the biological effect of the antibodies or compounds on human cells is assessed.
  • the animal model contains NK cells and/or cytolytic CD8 T cells, and the ability of the compounds to lyse, kill, reduce the number of, inhibit the growth of, or otherwise inhibit the S1P5- exprsesing cells, NK cells and/or cytolytic CD8 T cells is determined.
  • compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial gly
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • the compositions are administered orally, intraperitoneally or intravenously.
  • Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3- butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • Other commonly used surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • compositions of this invention may be administered in the form of suppositories for rectal administration.
  • suppositories for rectal administration.
  • suppositories can be prepared by mixing the agent with a suitable non- irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable non- irritating excipient include cocoa butter, beeswax and polyethylene glycols.
  • the antibodies or other compounds of this invention may be incorporated into liposomes, alone or together with another substance for targeted delivery to a patient or an animal.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used. In one embodiment, the compositions are applied directly to a site of inflammation or autoimmunity.
  • compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride.
  • the compositions may be formulated in an ointment such as petrolatum.
  • compositions of this invention may also be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • Several monoclonal antibodies have been shown to be efficient in clinical situations, such as Rituxan (Rituximab), Herceptin (Trastuzumab) or Xolair (Omalizumab), and similar administration regimens (i.e., formulations and/or doses and/or administration protocols) may be used with the antibodies of this invention. Schedules and dosages for administration can be determined in accordance with known methods for these products, for example using the manufacturers' instructions.
  • a monoclonal antibody can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials.
  • the product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection.
  • the pH is adjusted to 6.5.
  • An exemplary suitable dosage range for an anti-SlP5 antibody of the invention may between about 10 mg/m2 and 500 mg/m2.
  • these schedules are exemplary and that optimal schedule and regimen can be adapted taking into account the affinity of the antibody and the tolerability of the antibody which must be determined in clinical trials.
  • Quantities and schedule of injection of the antibodies of the invention can be, e.g., 24 hours, 48 hours 72 hours or a week or a month and will be determined considering the affinity of the antibody and its pharmacokinetic parameters.
  • compositions of the invention are formulated in a unit dosage form, each dosage typically containing from about 0.001 to about 1 g, more usually about 1 to about 30 mg, of the active ingredient.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. It will be appreciated that the compounds of the invention are effective over a wide dosage range and are generally administered in a pharmaceutically effective amount.
  • the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • the skilled artisan is directed to "Remington's Pharmaceutical Sciences” 15th Edition, the entire disclosure of which is herein incorporated by reference; chapter 33, pages 624-652, are particularly relevant to dosage determination.
  • an antibody, sphingosine analogue or derivative or other compound may further comprise another therapeutic agent, including agents normally utilized for the particular therapeutic purpose for which the compound is being administered.
  • the additional therapeutic agent will normally be present in the composition in amounts typically used for that agent in a monotherapy for the particular disease or condition being treated.
  • Such therapeutic agents include, but are not limited to, therapeutic agents used in the treatment inflammatory and autoimmune disorders.
  • the additional therapeutic agent will generally be administered at a dose typically used for that agent in a monotherapy for the particular disease or condition being treated.
  • an agent that binds and/or inhibits S1P5 is used in combination with another anti-inflammatory agent.
  • non-limited examples of combinations include the use of an agent that binds and/or inhibits S1P5 with an anti- TNF-alpha or an anti-IL-15 agent such as anti-TNF-alpha or anti-IL-15 antibodies, or IL-IO.
  • the present invention also provides methods for detecting mature NK cells and/or activated cytolytic CD8 T cells in a sample, comprising using an agent that binds S1P5, or a nucleic acid that hybridizes to a nucleic acid encoding a S1P5 polypetide.
  • said method comprises: (a) contacting a biological sample (e.g. a sample from a patient suffering or suspected of suffering from an inflammatory or autoimmune disorder) with an antibody or fragment thereof that binds S1P5, optionally attached to a detectable label or to a solid support, under conditions appropriate for immune complex formation, then (b) detecting S1P5 polypeptide positive cells.
  • said method comprises: (a) contacting said sample with a compound selected from a polynucleotide or an oligonucleotide of the gene coding for a S1P5 polypeptide as above defined, or a sequence complementary thereto, under conditions appropriate for the formation of hybridation products, and (b) detecting, and quantifying if desired, the hybridation products when formed.
  • the invention also relates to a method for the removal of mature NK cells and/or activated cytolytic CD8 T cells from a biological sample, comprising: (a) contacting said sample with a antibody or fragment thereof that binds S1P5, and (b) eliminating the immune complexes when formed.
  • a method for enrichment of NK cells and/or activated cytolytic CD8 T cells from a sample is also part of the invention. Such a method comprises: (a) contacting said sample with antibody or fragment thereof that binds S1P5, and (b) recovering the immune complexes when formed.
  • the sample can also be contacted with an anti-CD3 antibody with a distinct label to detect and distinguish T cells, and/or with an anti-CD 16, CD56, CD57 and/or NKp46 antibody with a distinct label to detect and distinguish NK cells
  • mice adoptive transfer and treatments. All inbred mice (C57BL/6 CD45.1 + and CD45.2 + ) were purchased from Charles River Laboratories. The generation of S IPs- deficient mice (5 generations backcrossed to C57BL/6 mice) was described previously 28 . In all experiments, mice were 6-8 weeks old and control mice were littermates. Mixed BM chimeras were generated as described previously 50 . For adoptive transfer experiments, 20 ⁇ 10 6 splenocytes from S IPs-deficient and wild-type mice were labeled with 0.2 ⁇ M or 2 ⁇ M CFSE (Invitrogen), mixed at a 1 :1 ratio and injected intravenously into littermate recipients.
  • S IPs- deficient mice 5 generations backcrossed to C57BL/6 mice was described previously 28 . In all experiments, mice were 6-8 weeks old and control mice were littermates. Mixed BM chimeras were generated as described previously 50 .
  • FTY720 (kindly provided by V. Brinkmann, Novartis) and DOP (Sigma) were provided in the drinking water (5 ⁇ g/ml and 30 ⁇ g/ml, respectively) supplemented with glucose.
  • RLI fusion protein was given intravenously (5 ⁇ g per injection).
  • PoIy(LC) (Invivogen) was injected intraperitoneally (150 ⁇ g/mouse). Experiments were conducted in accordance with institutional guidelines for animal care and use.
  • Flow cytometry and lymphocyte preparation Lung and liver lymphocytes were prepared as described previously 29 . Flow cytometry was carried out on a FACS Canto flow cytometer (Becton-Dickinson). NK cells were identified as CD3 NKp46 + or CD3 NKLl + . NKp46 expression was detected using the rat 29Al.4 monoclonal antibody 29 . All other antibodies were from BD Biosciences (CD3: 2Cl 1, NK1.1 :PK136).
  • NK cell stimulation assay Splenic NK cells were enriched by a negative selection procedure. Briefly, 1-A + , 1-E + , CD5 + , and Ly76 + cells were labeled with rat antibodies (home-made 53-7.3 culture supernatants or antibodies from BD Biosciences: IA/IE: 2G9, Ly76: Terl 19) and removed with goat anti-rat IgG magnetic beads (Qiagen). Antibodies to NKl.1 (PK136) NKp46 (29Al.4) and Ly49D (4E5) were allowed to bind to plastic (96-well plates, Thermo Scientific) overnight in carbonate buffer.
  • NK cells were stimulated for 4 h in the presence of FITC-coupled anti-CD 107a and Golgi-stop (BD-Biosciences).
  • Various samples were incubated with IL-2 (3,000 U/ml, Peprotech), IL- 12 (R&D Systems, 20 ng/ml), IL- 18 (R&D Systems, 5 ng/ml), phorbol myristate acetate (PMA, Sigma, 50 ng/ml) and ionomycin (Sigma, 500 ng/ml) as indicated.
  • IL-2 3,000 U/ml, Peprotech
  • IL- 12 R&D Systems, 20 ng/ml
  • IL- 18 R&D Systems, 5 ng/ml
  • PMA phorbol myristate acetate
  • PMA phorbol myristate acetate
  • ionomycin Sigma, 500 ng/ml
  • NK cell subsets were analyzed by 6-color staining after stimulation (anti-CD3-Alexa700, anti-NKl.l -APC, anti-IFN- ⁇ -PE-Cy7, anti-CD l lb-Percp, anti-CD27-PE, anti-CD 107a- FITC).
  • NK cells can be identified across mammalian species as CD3 NKp46 lymphocytes 29 ' 30 .
  • human and mouse microarray databases were screened for genes with an expression pattern similar to the gene encoding NKp46 (Ncrl).
  • Endothelial differentiation gene 8 (Edg8) which encodes SIP5, met this criterion (http://symatlas.gnf.org/SymAtlas/).
  • the Edg family is composed of eight G-protein-coupled receptors that bind to specific lysophospho lipids. Edg receptors are divided into two subgroups according to ligand specificity and sequence.
  • SIP5 is preferentially expressed on CD56 dim human NK cells (Fig. 9), which constitute the most mature subset of human NK cells. SIP5 was also specifically expressed in mouse NK cells (Fig. 1). No transcripts encoding SlPi_3 were detected in mouse NK cells, but the gene encoding Sl P 4 was expressed in similar amounts in all mouse lymphocytes.
  • microarray data were downloaded from the NCBI repository (reference GSE8678, corresponding to CD8 T cell subsets (either IL7Rhi or IL7Rlo CD8 T cells, respectively) coming from Lymphocytoid choriomeningitis virus-infected C57BL/6 mice). All data analyses were performed by using Bioconductor version 1.5 for the statistical software R. Expression values were background corrected, normalized, and summarized by using the default settings of the gcrma package. . SIP5 was found specifically expressed in mouse activated effector CD8 T cells (Fig. 8).
  • SIP 5 The function of SIP 5 by comparing NK cells from S IPs-deficient and wild-type was studied in mice.
  • S IPs-deficient mice were born at Mendelian frequencies, developed normally and were fertile 28 .
  • NK cells were present in all organs tested in S IPs-deficient mice, but the tissue distribution of these cells was very different from that in wild-type mice. Percentages of NK cells in the blood, the spleen and the lung were much lower in SlPs-deficient than in wild-type mice (2-3 fold reduction) (Fig. 2a, b). Conversely, percentages of NK cells in the bone marrow (BM) and in lymph nodes (LN) were twice as high in S IPs-deficient as in wild-type mice.
  • BM bone marrow
  • LN lymph nodes
  • IL- 15 Inter leukin 15
  • IL- 15 Inter leukin 15
  • IL- 15 is trans- presented by IL- 15Rcc to lymphocytes displaying surface IL-2R ⁇ - ⁇ complexes.
  • Soluble IL- 15PvCC bound or fused to IL- 15 can also efficiently activate and trigger proliferation of NK cells 33 .
  • Wild-type and S IPs-deficient mice were treated with the hyperagonist PvLI fusion protein composed of IL- 15 attached via a flexible linker to an IL- 15R sushi domain 33 .
  • PvLI treatment induced a 10-20 fold expansion of NK cell populations in blood, spleen, LN, BM and liver in both wild-type and S IPs-deficient mice (Fig. 3).
  • spleen and lung NK cell numbers were reduced by half in PvLI -treated S IPs- deficient compared to wild-type RLI-treated mice (Fig. 3).
  • the number of NK cells in the BM and LN was twice as high in PvLI -treated S IPs-deficient versus wild- type mice.
  • NK cells differing in expression of CDl Ib and CD27 have been described in the mouse (Fig. 10a) 34 .
  • Adoptive transfer experiments indicate that these three subsets represent various stages of NK cell maturation; CDl lb du11 NK cells are the most immature, 'double positive' (DP) CD27 hlgh CDl lb hlgh cells constitute an intermediate population, and CD27 du11 NK cells represent the most mature subset.
  • DP and CD27 du11 NK cells exhibit comparable capacities to kill target cells and to secrete IFN- ⁇ in a broad range of in vitro stimulation conditions (Fig. 10b, c). However, these three NK cell subsets differ widely in tissue distribution 34 .
  • CDl lb du11 NK cells were predominantly found in BM and LN, whereas CD27 du11 NK cells were more abundant in blood, spleen, lung and liver than in BM and LN and DP NK cells were more equally distributed throughout all organs examined (Fig. 1Od).
  • SIP5 expression correlated with the pattern of NK cell distribution as it gradually increased with NK cell maturation and was expressed in highest amounts in CD27 du11 NK cells (Fig. 5a). Nevertheless, the expression of SlP 5 in CDl l du11 NK cells, although low compared to that in CD27 du11 NK cells, was greater than that observed in T cells (Fig. 5a).
  • CD27 du11 NK cells (which in wild-type mice express high quantities of Sl P 5 ) constituted lower percentages of the lymphocyte populations in blood, spleen and lung, and higher percentages of the lymphocyte populations in BM and LN, in S IPs-deficient compared to wild-type mice (Fig. 5b and data not shown).
  • the percentages of CDl lb du11 and DP NK cells within the lymphocyte populations in each organ were less affected by Sl P 5 deficiency, except in BM and LN.
  • SIP5 might act as a chemotactic receptor for SlP and thereby regulate NK cell recirculation 24 .
  • This hypothesis was first tested by comparing migration of wild-type and S IPs-deficient splenocyes in response to SlP gradients in vitro. Wild-type T and NK cells migrated in response to SlP gradients in a dose-dependent manner, as previously reported 24 ' 37 ' 38 . Whereas wild-type and S IPs- deficient T cells migrated similarly towards to SlP (Fig. 6a), S IPs-deficient NK cells were unresponsive to SlP (Fig. 6b).
  • SIP5 acts a non- redundant chemotactic receptor for SlP in NK cells.
  • SIP5 acts a non- redundant chemotactic receptor for SlP in NK cells.
  • SIP5 acts a non- redundant chemotactic receptor for SlP in NK cells.
  • the S IP-induced chemotactic responsiveness of wild-type NK cells increased with maturation, consistent with parallel increases in SIP5 expression (Fig. 6b).
  • splenocytes from wild-type and S IPs-deficient mice were labeled with distinct concentrations of 5-(and 6-)-carboxyfluorescein diacetate succinimidyl ester (CFSE), and adoptively transferred these differentially labeled cells at a 1 :1 ratio into littermate recipient mice (Fig. 6c).
  • CFSE-labeled NK cells were present in all organs analyzed; thus transferred NK cells can rapidly traffic through lymphoid and non- lymphoid organs (data not shown).
  • CDl lb du11 NK cell and T cell populations in recipient mice contained approximately equal proportions of cells derived from wild-type and S IPs-deficient donors (Fig. 6d).
  • S IPs-deficient donor cells were enriched in CD27 du11 populations in the LN and BM but were depleted in the spleen, blood and lung (Fig. 6d).
  • polyinosine polycytidylic acid, poly(LC), a compound established as stimulating NK cells and inducing NK cell recruitment from spleen to liver 39 .
  • NK cells accumulated in the liver (4-5 fold increase) and decreased in the spleen after poly(LC) treatment (Fig. 6e).
  • poly(LC) treatment failed to elicit significant alterations in NK cell abundance in the spleen or liver of S IPs-deficient mice (Fig. 6e).
  • FTY720 is an agonist for SlPi as well as SlP3_s 31 .
  • mice were treated with FTY720 and examined the distribution of various lymphocyte subsets. Substantially fewer T cells were observed in the blood of mice treated with FTY720 compared to control mice, as previously described 22 ' 40 .
  • FTY720 treatment did not affect trafficking of NK cell subsets (Fig. 7a).
  • S IP-dependent function can also be assessed using 4'deoxypyridoxine (DOP), a vitamin-B6 antagonist that irreversibly inhibits SlP lyase 23 .
  • DOP 4'deoxypyridoxine
  • DOP abrogates the endogenous SlP gradient between the blood and lymphoid organs.
  • DOP treatment inhibits the function of SlP chemotactic receptors, such as SlPi, and results in the disappearance of T cells from the blood 23 .
  • DOP treatment induced substantial decreases in the numbers of T cells in the blood, but left NK cell numbers unchanged (Fig. 7a).
  • the in vitro migration of NK cells and T cells to SlP gradients following in vivo FTY720 and DOP treatment was also measured. As previously reported, FTY720 and DOP induced a drastic inhibition of S IPi -dependent chemotaxis of T cells in vitro.
  • SlPs regulates the trafficking and the tissue distribution of NK cells in vivo via a mechanism different from that of SlPi in T and B cells and resistant to effects of FTY720 and DOP.
  • Example 6 [00220] S1P5 microarray data from disease biopsies
  • S1P5 expression is the disease tissues is attributable to lymphocytes present in the biological sample.
  • the methods described herein notably binding and/or inhibiting S1P5, and diagnostic methods to detect S1P5 or S 1P5 -expressing cells, may be useful in the treatment, prevention or diagnosis or Crohns' disease, ulcerative colitis and atopic dermatitis.
  • accession number GDS2382 in the GEOpro files provides an analysis of lesional (6 patients) and non-lesional (6 patients) skin biopsy specimens from adult patients with atopic dermatitis (AD) (Fig. 12). It appears that AD patients, both lesional and non-lesional, have higher S1P5 expression than healthy control patients. GDS2382 is issued from a study using high-density oligonucleotide Affymetrix Human Ul 33 A GeneChip arrays to identify cutaneous gene transcription changes associated with early AD inflammation as potential disease control targets. Skin biopsy specimens analyzed included normal skin from five healthy nonatopic adults and both minimally lesional skin and nearby or contralateral nonlesional skin from six adult AD patients (Plager et al.
  • AD has been reported to involve CD8 T cells; based on the role of S1P5 it is believed that S1P5 expression observed in GDS2382 may arise from the presence of CD8 T cells and/or NK cells in the biopsy.
  • accession number GDS 1330 provides an analysis of mucosal biopsy samples from sigmoid colons of 10 patients with Crohn's disease and 10 patients with ulcerative colitis, compared to 11 normal control patients. (Fig. 13). It appears that S1P5 expression is generally somewhat higher in Crohn's disease and ulcerative colitis patients than in normal control patients. GDS 1330 is described as a study to identify new genes involved in the pathophysiology of inflammatory bowel disease (Crohn's disease and ulcerative colitis). A group of 31 samples was represented, subdivided into 3 groups: 1) Normal controls: 11 samples; 2) patients with Crohn's diseases: 10 samples; 3) patients with ulcerative colitis: 10 samples.
  • accession number GDS2014 provides an analysis of expression profiling from colonic mucosa from 3 patients with ulcerative colitis and 5 patients with irritable bowel syndrome.
  • Fig. 14 GDS2014 describes the patients in IBS as patients who have undergone a diagnostic program for gastrointestinal symptoms and where the diagnosis irritable bowel syndrome was reached, and for UC as Patients with well-diagnosed ulcerative colitis. It appears that S1P5 expression is higher in ulcerative colitis patients than in irritable bowel syndrome patients.
  • Table 1 List of oligonucleotides. The oligonucleotide primers (Sigma) used for the quantitative RTPCR experiments are listed herein. F: forward; R: reverse.

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Abstract

La présente invention a pour objet des procédés d'inhibition de la circulation des lymphocytes NK et des lymphocytes T activés, destinés à la prévention et/ou au traitement d'affections inflammatoires et auto-immunes. De manière générale, les présents procédés comprennent l'utilisation d'agents qui inhibent S1P5 afin de moduler la circulation in vivo des lymphocytes NK et des lymphocytes T activés.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8791100B2 (en) 2010-02-02 2014-07-29 Novartis Ag Aryl benzylamine compounds
WO2017053990A1 (fr) * 2015-09-24 2017-03-30 The Regents Of The University Of California Molécules de type sphingolipide synthétiques, médicaments, procédés pour leur synthèse et procédés de traitement
US10077236B2 (en) 2013-07-15 2018-09-18 The Regents Of The University Of California Azacyclic constrained analogs of FTY720

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1481680A1 (fr) * 2003-05-30 2004-12-01 Aventis Pharma Deutschland GmbH Utilisation de S1P
WO2005014525A2 (fr) * 2003-08-12 2005-02-17 Mitsubishi Pharma Corporation Compose bi-aryle presentant une activite immunosuppressive
WO2007085451A2 (fr) * 2006-01-27 2007-08-02 Novartis Ag Isoxasoles inverses
EP1826197A1 (fr) * 2004-12-13 2007-08-29 Ono Pharmaceutical Co., Ltd. Dérivé d'acide aminocarboxylique et applications thérapeutiques dudit dérivé

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1481680A1 (fr) * 2003-05-30 2004-12-01 Aventis Pharma Deutschland GmbH Utilisation de S1P
WO2005014525A2 (fr) * 2003-08-12 2005-02-17 Mitsubishi Pharma Corporation Compose bi-aryle presentant une activite immunosuppressive
EP1826197A1 (fr) * 2004-12-13 2007-08-29 Ono Pharmaceutical Co., Ltd. Dérivé d'acide aminocarboxylique et applications thérapeutiques dudit dérivé
WO2007085451A2 (fr) * 2006-01-27 2007-08-02 Novartis Ag Isoxasoles inverses

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BRINKMANN ET AL: "Sphingosine 1-phosphate receptors in health and disease: Mechanistic insights from gene deletion studies and reverse pharmacology", PHARMACOLOGY AND THERAPEUTICS, ELSEVIER, GB, vol. 115, no. 1, 1 July 2007 (2007-07-01), pages 84 - 105, XP022134156, ISSN: 0163-7258 *
GREGOIRE CLAUDE ET AL: "The trafficking of natural killer cells", IMMUNOLOGICAL REVIEWS, vol. 220, December 2007 (2007-12-01), pages 169 - 182, XP002511849, ISSN: 0105-2896 *
JAILLARD C ET AL: "Edg8/S1P5: An oligodendroglial receptor with dual function on process retraction and cell survival", JOURNAL OF NEUROSCIENCE, vol. 25, no. 6, 9 February 2005 (2005-02-09), pages 1459 - 1469, XP002512297, ISSN: 0270-6474 *
KVEBERG LISE ET AL: "Sphingosine 1 phosphate induces the chemotaxis of human natural killer cells. Role for heterotrimeric G proteins and phosphoinositide 3 kinases", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 32, no. 7, July 2002 (2002-07-01), pages 1856 - 1864, XP002511847, ISSN: 0014-2980 *
MANDALA S ET AL: "ALTERATION OF LYMPHOCYTE TRAFFICKING BY SPHINGOSINE-1-PHOSPHATE RECEPTOR AGONISTS", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, WASHINGTON, DC, vol. 296, no. 5566, 12 April 2002 (2002-04-12), pages 346 - 349, XP001128118, ISSN: 0036-8075 *
VAESSEN ET AL: "FTY720 treatment of kidney transplant patients: A differential effect on B cells, naive T cells, memory T cells and NK cells", TRANSPLANT IMMUNOLOGY, ELSEVIER, vol. 15, no. 4, 1 April 2006 (2006-04-01), pages 281 - 288, XP005401554, ISSN: 0966-3274 *
WALZER THIERRY ET AL: "Natural killer cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor", NATURE IMMUNOLOGY, vol. 8, no. 12, December 2007 (2007-12-01), pages 1337 - 1344, XP002511850, ISSN: 1529-2908 *
WEI SINDY H ET AL: "Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses", NATURE IMMUNOLOGY, vol. 6, no. 12, December 2005 (2005-12-01), pages 1228 - 1235, XP002511848, ISSN: 1529-2908 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8791100B2 (en) 2010-02-02 2014-07-29 Novartis Ag Aryl benzylamine compounds
US10077236B2 (en) 2013-07-15 2018-09-18 The Regents Of The University Of California Azacyclic constrained analogs of FTY720
WO2017053990A1 (fr) * 2015-09-24 2017-03-30 The Regents Of The University Of California Molécules de type sphingolipide synthétiques, médicaments, procédés pour leur synthèse et procédés de traitement
US10995068B2 (en) 2015-09-24 2021-05-04 The Regents Of The University Of California Synthetic sphingolipid-like molecules, drugs, methods of their synthesis and methods of treatment
CN108366990B (zh) * 2015-09-24 2021-09-03 加利福尼亚大学董事会 合成的鞘脂类分子、药物、它们的合成方法及治疗方法
US11479530B2 (en) 2015-09-24 2022-10-25 The Regents Of The University Of California Synthetic sphingolipid-like molecules, drugs, methods of their synthesis and methods of treatment
US11999693B2 (en) 2015-09-24 2024-06-04 The Regents Of The University Of California Synthetic sphingolipid-like molecules, drugs, methods of their synthesis and methods of treatment

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