EP1653977A2 - Procedes et compositions de traitement de maladie et de troubles associes a des lymphocytes tnk - Google Patents

Procedes et compositions de traitement de maladie et de troubles associes a des lymphocytes tnk

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Publication number
EP1653977A2
EP1653977A2 EP04778424A EP04778424A EP1653977A2 EP 1653977 A2 EP1653977 A2 EP 1653977A2 EP 04778424 A EP04778424 A EP 04778424A EP 04778424 A EP04778424 A EP 04778424A EP 1653977 A2 EP1653977 A2 EP 1653977A2
Authority
EP
European Patent Office
Prior art keywords
cells
beta
galactosyl ceramide
subject
cdld
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04778424A
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German (de)
English (en)
Inventor
John Ortaldo
Robert Wiltrout
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US Department of Health and Human Services
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US Department of Health and Human Services
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Publication date
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Publication of EP1653977A2 publication Critical patent/EP1653977A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the invention relates to methods and compositions useful in the treatment of autoimmune disorders or diseases as well as disorders or diseases having an autoimmune component such as AIDS, to reducing tumor masses of proliferative disorders, and to promoting successful organ and tissue transplants.
  • Preferred methods and compositions of the invention comprise use of a beta Galactosyl Ceramide compound to treat a mammal suffering from or susceptible to such disorders.
  • NKT Natural killer T cells represent a small lymphocyte subpopulation that has been reported to have dramatic and important regulatory functions. NKT cells are characterized by their co-expression of the natural killer (NK) receptors NK1.1 or NKR-PIA (CD161) and a T-cell receptor (TCR). However, these NKT lymphocytes exhibit heterogeneity in both phenotypic characteristics and their functions.
  • NKT cells have recently been categorized into several distinct groups based on their T cell receptor (TcR) repertoire, expression of antigen, ability to recognize antigen presenting co-receptor molecules and their anatomical compartme-italizatiori ⁇ 14008
  • Type I NKT is the T-cell population that has a rearrangement of the V ⁇ l4-J ⁇ l8 variable region of the T cell receptor and is either CD4+ or double negative (CD4-, CD8-).
  • the responsiveness of these NKT cells are CD Id restricted and are primarily located in the thymus, liver, spleen and bone marrow. Reactivity appears restricted to alpha Galactosyl Ceramide ( ⁇ GalCer) and represents a major component of the overall NKT cell population.
  • This NKT type also co-expresses other NK receptors, e.g. Ly49 receptors.
  • a second type of NKT cells is CDld autoreactive and co-expresses a more distinct TcR expression pattern (V ⁇ 3.2-J9/V ⁇ 8, V ⁇ 8).
  • Type 2 NKT cells are CD4+ or double negative and express predominately NK1.1, however, they differ from Type 1 cells in their location in vivo and their antigen reactivity (nonreactive with otGalCer).
  • a third type of NKT cells are not CDld dependent, has a diverse TcR expression and can be either CD4+, CD8+ or double negative. Their antigen reactivity is not known and their in vivo distribution is similar to that of group 1 NKT cells.
  • a fourth type of NKT cells is defined by their expression of cc2-integrin (CD49B), recognized by the NK reactive antibody, DX5. This NKT population has little overlap with Type 1NKT cells and may have a very specialized role in the immune response.
  • NKT cells have centered on the role of the Type 1 and Type 2 - NKT cell as these populations represent a majority of spleen, liver and bone marrow associated NKT cells. These studies have utilized the reactivity of ⁇ GalCer to eliminate these cells and thus evaluate their role in immune regulation. Recently, a number of studies have demonstrated that the administration of ⁇ GalCer, in vivo results in the rapid depletion of NKT cells through a process that largely involves the induction of apoptosis through the TcR stimulation. A bystander effect of this NKT activation is the production of IFN ⁇ and the activation of NK cells that often are found in the spleen and liver, resulting in systemic inflammation.
  • NKT cells have been shown to be involved in autoimmune disease and immune anergy to antigen stimulation. More studies have demonstrated in a number of model systems that NKT cells can regulate autoimmunity and often reverse the adverse effects of immune dysregulation. The application of these models to human disease may provide an avenue for treatment of autoimmune diseases and to increase immuno-reactivity of cancer and AIDS patients to tumors and/or vaccines. To date, the only efficient means to remove NKT cells in vivo has been the use of KRN7000 ( ⁇ GalCer). However, this reagent has been shown to exhibit potent bystander activation of NK cells and strong induction of cytokines in the host, resulting in the side effects of systemic inflammation, particularly in the spleen and liver.
  • KRN7000 ⁇ GalCer
  • ⁇ GalCer is not desirable as a therapeutic agent.
  • Compounds which could act to reduce the numbers of NKT cells involved in such conditions as autoimmunity and AIDS (a viral disease with autoimmune components), while not inducing the production of cytokines and systemic inflammation, would be highly useful as therapeutic agents.
  • NKT Cells Natural Killer T Cells
  • therapies which can deplete a subject's NKT cell population without concomitantly increasing the subject's production of cytokines.
  • Preferred methods and compositions of the invention comprise the use of one or more beta Galactosyl Ceramide compounds to remove or otherwise reduce a population of NKT cells effectively in vivo, particularly in the substantial absence of deleterious side effects caused by the concomitant bystander events (including cellular activation, cytokine production, systemic inflammation, etc) that are found with the use of alpha Galactosyl Ceramides.
  • an administered beta Galactosyl Ceramide compound does not cause a significant increase in production of cytokines.
  • preferred administered beta Galactosyl Ceramide compounds will cause a lower production of cytokines relative to that which would result from the administration of alpha Galactosyl Ceramides.
  • an administered beta Galactosyl Ceramide compound results in no more about 50%, more preferably no more than about 40, 30, 20 or 10 percent or even about a zero percent increase in cytokine production relative to production resulting from administration of alpha Galactosyl Ceramide. Cytokine production can be measured as described herein under Materials and Methods and used in Examples 6 and 7.
  • Suitable and preferred beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention can be readily identified through simple testing.
  • suitable beta Galactosyl Ceramide compounds can be evaluated and identified by an in vitro assay of depletion of NKT cells (obtained from mammalian liver) as exemplified in Example 4 which follows, which in general includes contacting liver tissue with a candidate suitable beta Galactosyl Ceramide compound at room temperature and assessing the NKT cells population of the sample versus a control (same liver cells but not treated with any therapeutic agents) after 24 and 48 after administration.
  • a candidate compound may be considered suitable for use in the methods and compositions of the invention if it provides a discemable decrease in NKT cell population relative to the control sample in either or both the assessment periods, e.g. At least about a 5, 10, 15 or 20 percent decrease in NKT cell amounts relative to the control.
  • References herein to a standard in vitro NKT assay refers to that protocol, as also exemplified in Example 4 which follows.
  • Typical beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention will comprise a beta-configuration galactosyl moiety, suitably optionally substituted at one or more galactosyl ring positions by any of a variety of non-hydrogen substituents such as optionally substituted alkyl; optionally substituted alkyenyl; optionally substituted alkynyl; optionally substituted heteroalkyl; optionally substituted carbocyclic aryl; optionally substituted aralkyl; optionally substituted heteroalicyclic; optionally substituted heteroaryl; and the like.
  • beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention include those of the following Formula I:
  • n is an integer of from 5 to 20, preferably 8 to 15; and m is an integer of from 5 to 30, preferably from 10 to 20; and pharmaceutically acceptable salts or solvates thereof.
  • a preferred compound of Formula I is D-Galactosyl- ⁇ l-l'-N-Dodecanoyl-D- erythroSphingosine .
  • a beta Galactosyl Ceramide compound may be administered or formulated in a composition in conjunction with one or more other agents, including a CDld peptide, a nucleic acid sequence coding for a CDld peptide and or a CD Id-expressing cell, or other therapeutic agent such as agents known for use in autoimmune therapies.
  • agents including a CDld peptide, a nucleic acid sequence coding for a CDld peptide and or a CD Id-expressing cell, or other therapeutic agent such as agents known for use in autoimmune therapies.
  • Preferred therapeutic methods of the invention in general comprise administering an effective amount of one or more beta Galactosyl Ceramide compounds to a subject (e.g., a mammal particularly human) or cells (e.g. Mammalian cells particularly human in need thereof), e.g.
  • a subject or cells that are suffering from or susceptible to a disease or disorder associated with NKT cells and/or autoimmune diseases and disorders such as, systemic lupus erythematosus, multiple sclerosis, diabetes mellitus and rheumatoid arthritis, or disease and disorders with autoimmune components such as Acquired Immunodeficiency Syndrome (AIDS) and treatment of persons suffering from or susceptible to HIV infection as well as treatment of a subject undergoing or having undergone a transplant procedure such as a tissue (e.g. Skin) or organ (e.g. heart, lung, kidney) transplant.
  • AIDS Acquired Immunodeficiency Syndrome
  • Therapeutic methods of the invention also include decreasing a population of mammalian particularly human Natural Killer T cells comprising administering one or more beta Galactosyl Ceramide compounds to cells or a subject.
  • Preferred therapeutic methods also may including identifying and/or selecting a subject or cells (e.g. A mammal or mammalian cells, particularly a human or human cells) that are susceptible to or suffering from a condition disclosed herein, and thereafter administering to the identified and/or selected subject or cells an effective amount of a ⁇ -Galactosyl Ceramide compound.
  • identification and selection can be accomplished by any number of means, e.g. Testing a subject or cells for an autoimmune disease or disorder.
  • the invention also provides use of a beta Galactosyl Ceramide compound for the treatment or prevention (including prophylactic treatment) of a disease or condition as disclosed herein, including autoimmune diseases and disorders such as systemic lupus erythematosus, multiple sclerosis, diabetes mellitus and rheumatoid arthritis, or disease and disorders with autoimmune components such as Acquired Immunodeficiency Syndrome (AIDS) and HIV infections as well as treatment of a subject undergoing or having undergone a transplant procedure.
  • autoimmune diseases and disorders such as systemic lupus erythematosus, multiple sclerosis, diabetes mellitus and rheumatoid arthritis
  • AIDS Acquired Immunodeficiency Syndrome
  • HIV Acquired Immunodeficiency Syndrome
  • the invention provides use of a beta Galactosyl Ceramide compound for the preparation of a medicament for the treatment or prevention (including prophylactic treatment) of a disease or condition as disclosed herein, including autoimmune diseases and disorders such as systemic lupus erythematosus, multiple sclerosis, diabetes mellitus and rheumatoid arthritis, or disease and disorders with autoimmune components such as Acquired Immunodeficiency Syndrome (AIDS) and HIV infections as well as treatment of a subject undergoing or having undergone a transplant procedure.
  • autoimmune diseases and disorders such as systemic lupus erythematosus, multiple sclerosis, diabetes mellitus and rheumatoid arthritis, or disease and disorders with autoimmune components such as Acquired Immunodeficiency Syndrome (AIDS) and HIV infections as well as treatment of a subject undergoing or having undergone a transplant procedure.
  • AIDS Acquired Immunodeficiency Syndrome
  • HIV Acquired Immunodeficiency Syndrome
  • methods to treat proliferative disorders including administering to a subject in need thereof one or more beta Galactosyl Ceramide compounds.
  • one or more beta Galactosyl Ceramide compounds are administered in conjunction with cytokines, preferably, IL2 to treat cancer.
  • Other agents, such as other anti-proliferative compounds may also be administered.
  • the invention also includes pharmaceutical compositions that comprise a beta Galactosyl Ceramide compound optionally admixed with a pharmaceutically acceptable carrier and optionally packaged together with instructions (e.g. written) for use of the composition for a condition as disclosed herein.
  • Figure 1 graphically depicts effects of alpha galactosyl ceramide on various cell functions in vivo on mouse liver lymphocytes, wherein Panel A represents NK activity and is expressed in lytic units; Panel B depicts NK cells based on flow forward scatter; Panel C measures CD69 expression on NK cells (NK1.1+, CD3-);
  • Panel D represents liver NK cell numbers
  • Panel E depicts NKT cells numbers
  • Panel F represents relative percent NK cells in liver
  • Panel G depicts relative percent NKT (NKl .1 +, CD3+) cells in liver
  • Panel H represents total leukocyte number in liver
  • Panel I measures serum IFN ⁇ levels in serum.
  • Figure 2 depicts a graphic representation of the effects of ⁇ GalCer on NK cell size and cell lysis in SCID mice.
  • Figure 3 depicts data graphs illustrating the comparison of ⁇ GalCer and ⁇ GalCer (C12), wherein Panel a represents NK cell percentage ; Panel B represents NK cytolytic activity in lytic units at 20% ; Panel C represents CD69 expression on NK cells; and Panel D represents percentage of NKT cells.
  • Figure 4 depicts various bar graphs illustrating that the administration of ⁇ GalCer but not ⁇ -GalCer (C12) results in a systemic activation of cells that produce cytokines.
  • Figure 5 depicts data graphs illustrating the comparative effects of ⁇ GalCer and ⁇ GalCer on serum cytokines and NKT early events in cell death.
  • Figure 6 depicts data graphs illustrating the binding of CDld with of ⁇ GalCer and ⁇ -GalCer (C12).
  • Figure 7 depicts data graphs illustrating he effects of CD Id-mediated induction of cytokines in NKT and NK cells.
  • Figure 8 depicts data graphs illustrating the effects of ⁇ GalCer and ⁇ -GalCer (C12) on cytokine production from liver lymphocytes.
  • Figure 9 depicts data graphs illustrating the comparative effects of ⁇ GalCer and ⁇ GalCer on in vivo graft rejection of allogeneic bone marrow cells
  • Figure 10 depicts a data graph illustrating the decrease in renal cancer etastases upon administration of IL2 and ⁇ -GalCer.
  • Figure 11 depicts a nucleotide sequences encoding the CDld polypeptide (SEQ ID NO: 1).
  • Figure 12 depicts a CDld polypeptide of SEQ ID NO:2.
  • Beta Galactosyl Ceramide compounds As discussed above, suitable and preferred beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention may be identified by simple testing, including the in vitro NKT cell depletion assay exemplified in Example 4 which follows.
  • beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention will comprise a beta-configuration galactosyl moiety, suitably optionally substituted at one or more galactosyl ring positions by any of a variety of non-hydrogen substituents such as optionally substituted alkyl; optionally substituted alkyenyl; optionally substituted alkynyl; optionally substituted heteroalkyl such as alkoxy, thioalkyl, aminoalkyl, sulfinylalkyb sulfonylalkyl and the like; optionally substituted carbocyclic aryl preferably optionally substituted phenyl and naphthyl; optionally substituted aralkyl; optionally substituted heteroalicyclic; optionally substituted heteroaryl; etc.
  • non-hydrogen substituents such as optionally substituted alkyl; optionally substituted alkyenyl; optionally substituted alkynyl; optionally substituted heteroalky
  • suitable alkyl substituent groups typically have from 1 to about 12 carbon atoms, more preferably 1 to about 8 carbon atoms, still more preferably 1, 2, 3, 4, 5, or 6 carbon atoms.
  • alkyl unless otherwise modified refers to both cyclic and noncyclic as well as branched and straight groups, although of course cyclic groups will comprise at least three carbon ring members.
  • Preferred alkenyl and alkynyl groups of beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention have one or more unsaturated linkages and typically from 2 to about 12 carbon atoms, more preferably 2 to about 8 carbon atoms, still more preferably 2, 3, 4, 5, or 6 carbon atoms.
  • the terms alkenyl and alkynyl as used herein refer to both cyclic and noncyclic groups, although straight or branched noncyclic groups are generally more preferred.
  • Preferred alkoxy groups of beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention include groups having one or more oxygen linkages and from 1 to about 8 carbon atoms, and still more preferably 1, 2, 3, 4, 5 or 6 carbon atoms.
  • Preferred alkylamino groups include those groups having one or more primary, secondary and/or tertiary amine groups, and from 1 to about 8 carbon atoms, still more preferably 1, 2, 3, 4, 5, or 6 carbon atoms.
  • Preferred alkylsulfinyl groups have one or more sulfinyl (SO) groups and from 1 to 12 carbon atoms.
  • Preferred alkylsulfonyl groups have one or more sulfonyl (SO 2 ) and from 1 to 12 carbon atoms.
  • Suitable heteroalicyclic groups of beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention contain one or more N, O or S 14008
  • atoms and include, e.g., tetrahydrofuranyb thienyb piperidinyb morpholino and pyrrolidinyl groups.
  • Suitable heteroaromatic groups of beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention are 5-membered or 6-membered single ring moieties having at least N, O or S rings atoms.
  • Particular examples of heteroaromatic groups include optionally substituted pyridyl, pyrrolyb furyl (furanyl), thienyb imidazolyb oxazolyb isoxazolyb thiazolyb isothiazolyb pyrazolyb 1,2,3- triazolyb 1,2,4-triazolyb 1,2,3-oxadiazolyl, 1,2,4-oxadia-zolyb 1,2,5-oxadiazolyb l,3,4-oxadiazolybl,3,4-triazinyb 1,2,3-triazinyb benzofuryb [2,3-dihydro]benzofuryb isobenzofuryb benzothienyb benzotriazolyb isobenzothieny
  • Suitable carbocyclic aryl groups of beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention include single and multiple ring compounds, including multiple ring compounds that contain separate and/or fused aryl groups.
  • Typical carbocyclic aryl groups of compounds of the invention contain 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms.
  • Specifically preferred carbocyclic aryl groups include phenyl; naphthyl including phenyb 1- naphthyl and 2-naphthyl; biphenyl; phenanthryl; anthracyl; and acenaphthyl.
  • Substituted carbocyclic groups are particularly suitable including substituted phenyb such as 2-substituted phenyb 3-substituted phenyb 4-substituted phenyb 2,3- substituted phenyb 2,4-substituted phenyb and 2,5-substituted phenyl; and substituted naphthyl, including naphthyl substituted at the 5, 6 and/or 7 positions.
  • Suitable aralkyl groups of beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention include single and multiple ring compounds, including multiple ring compounds that contain separate and/or fused carbocyclic aryl groups.
  • Typical aralkyl groups contain 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms.
  • Preferred aralkyl groups include benzyl k i and naphthylmethyl (-CH 2 -naphthyl), and other carbocyclic aralkyl groups, as discussed above.
  • beta Galactosyl Ceramide compounds for use in the methods and compositions of the invention may be optionally substituted.
  • a "substituted"group or other substituent may be substituted by other than hydrogen at one or more available positions, typically 1 to 3 or 4 positions, by one or more suitable groups such as those disclosed herein.
  • suitable groups that may be present on a "substituted" group or other substituent include e.g.
  • halogen such as fluoro, chloro, bromo and iodo; cyano; hydroxyl; nitro; azido; alkanoyl such as a C ⁇ _ 6 alkanoyl group such as acyl and the like; carboxamido; alkyl groups including those groups having 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5, or 6 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 12 carbon, or 2, 3, 4, 5 or 6 carbon atoms; alkoxy groups including those having one or more oxygen linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; aryloxy such as phenoxy; alkylamino groups such as groups having one or more N atoms and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; carbocyclic aryl having 6 or more carbons; aralkyl having 1 to 3
  • beta Galactosyl Ceramide compounds for pharmaceutical compositions and methods of use of the invention include those of the following Formula I: 14008
  • the present invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising D-Galactosyl- ⁇ l-l'-N-Dodecanoyl-D- erythroSphingosine with a pharmaceutically acceptable carrier.
  • This beta Galactosyl Ceramide has the following physical and chemical characteristics: Molecular weight : 643; Molecular Formula: C 36 H 69 N0 8 and Percent Composition: C 67.15%, H 10.80%, N 2.18% O 19.88% (Avanti Product Number: 860544).
  • beta Galactosyl Ceramide compounds for pharmaceutical compositions and methods of use of the invention include D-Galactosyl- ⁇ l-l'-N- Nonanoyl-D-erthroSphingosine, D-Galactosyl- ⁇ 1 -l'-N-Decanoyl-D- erthroSphingosine, D-Galactosyl- ⁇ l-1 '-N-Ondecanoyl-D-erthroSphingosine, D- Galactosyl- ⁇ 1 - 1 '-N-Propdecanoyl-D-erthroSphingosine, D-Galactosyl- ⁇ 1 - 1 '-N- Butdecanoyl-D-erthroSphingosine, and D-Galactosyl- ⁇ l-1 '-N-Pentdecanoyl-D- erthroSphingosine.
  • Beta Galactosyl Ceramide compounds useful in the methods and compositions of the invention are commercially available from a number sources (e.g., Avanti Polar Lipids, Inc., Alabaster, AL, www.avanti.com; Kirin Pharmaceutical Research and Development, Gunma, Japan, www.kirin.co.ip.)
  • Beta Galactosyl Ceramide compounds useful in the methods of the invention also may be readily prepared by known synthetic procedures. For instance, an available compound such as D-Galactosyl- ⁇ 1- l'-N-Dodecanoyl-D- erythroSphingosme may be modified (e.g. Substitution reactions) to include desired substituent groups .
  • CD1 molecules are antigen presenting molecules that are able to bind and present glycolipids. These polypeptides are expressed on dendritic cells, monocytes and some thymocytes and function in antigen presentation to T cells. Without being bound by any theory, it is believed that CDld polypeptides can bind or otherwise associate with a beta Galactosyl Ceramide compound and act to present the thus complexed a beta Galactosyl Ceramide compound as antigens specifically to NKT cells. In doing so, the interaction with NKT cells results in the induction of NKT apoptosis (i.e., cell death) but not in the NKT cell activation of cytokine production and release.
  • NKT apoptosis i.e., cell death
  • the invention provides for the use of one or more beta Galactosyl Ceramide compounds administered in coordination with one or more CDld polypeptide, a nucleic acid sequence coding for a CD1 peptide and/or CDld expressing cells.
  • An isolated CDld polypeptide is characterized as having essentially the amino acid sequence of SEQ ID NO:2.
  • isolated refers to CDld which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify CDld polypeptide using standard techniques for protein purification. The isolated polypeptide will yield a single band on a non-reducing polyacrylamide gel.
  • CDld protein includes functional fragments of the polypeptide, as long as the activity of CDld, such as the ability to bind and present glycolipids remains. Fragments or smaller peptides containing the biological activity of CDld in terms of presenting glycolipids are included in the invention.
  • the invention provides for the use of nucleotide sequences encoding the CDld polypeptide (SEQ ID NO:l) for purposes of producing CDld polypeptides and CDld expressing cells.
  • These nucleotides include DNA, cDNA, and RNA sequences which encode CDld. It is also understood that all nucleotide sequences encoding all or a portion of CDld are also included herein, as long as they encode a polypeptide with CDld activity.
  • Such nucleotide sequences include synthetic, and intentionally manipulated nucleotide sequences. For example, CDld nucleotide sequences may be subjected to site-directed mutagenesis.
  • nucleotide sequences of the invention include sequences that are degenerate as a result of the genetic code. All degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of CDld polypeptide which is encoded by the nucleotide sequence is functionally unchanged in terms of the ability to bind glycolipids.
  • CDld primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the CDld polypeptide described herein.
  • proteins include those as defined by having essentially the amino acid sequence of SEQ ID NO:2.
  • modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the biological activity of CDld still exists.
  • deletions of one or more amino acids can also result in modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule which would have broader utility. For example, one can remove amino or carboxy terminal amino acids which are not required for the CDld biological activity of binding glycolipid.
  • the CDld polypeptide of the invention encoded by the nucleotide sequence of the invention includes the disclosed sequence (SEQ ID NO:2) and conservative variations thereob including the sequences of Accession Nos.: NM_007640, NM_007639, X13170, AK002582, M63697, L38820 or P15813.
  • conservative variation denotes the replacement of an amino acid residue by another, biologically similar residue.
  • conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, or methionine for another, or the substitution of one solar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like.
  • conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
  • the DNA sequences of the invention can be obtained by several methods. For example, the DNA can be isolated using hybridization techniques which are well known in the art.
  • vectors preferably expression vectors, containing a CDld nucleic acid or a portion thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are refe ⁇ ed to herein as "expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • the recombinant expression vectors comprise a nucleic acid encoding CDld or IL2 in a form suitable for expression of the nucleic acid molecule in a host cell.
  • the recombinant expression vectors can include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, operatively linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described in, for example, Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif (1990).
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CDl d proteins, other variant forms of CDld proteins, fusion proteins, and the like, and IL2 proteins, other variant forms of IL2 proteins, fusion proteins, and the like).
  • proteins or peptides including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CDl d proteins, other variant forms of CDld proteins, fusion proteins, and the like, and IL2 proteins, other variant forms of IL2 proteins, fusion proteins, and the like).
  • the recombinant expression vectors can be designed for expression of CDld or IL2 polypeptides in prokaryotic or eukaryotic cells.
  • CDld polypeptides can be expressed in bacterial cells such as E. Coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. And Johnson, K. S.
  • GST glutathione S-transferase
  • One strategy to maximize recombinant protein expression in E. Coli is to express the polypeptide in a bacterial host with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 1 19-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. Coli (Wada et ab, (1992) Nucleic Acids Res. 20:2111- 2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the CDld expression vector can be a yeast expression vector.
  • yeast expression vectors for expression in yeast S. Cerivisae include pYepSecl (Baldari, et ab, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), 4008
  • pJRY88 Schotz et ab, (1987) Gene 54:113-123
  • pYES2 Invitrogen Corporation, San Diego, Calif.
  • picZ InVitrogen Corp, San Diego, Calif
  • CDld polypeptides can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mob Cell Biob 3:2156-21 5) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39
  • a nucleic acid encoding CDld can be expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • viral regulatory elements For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid molecule preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid molecule).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific;
  • pancreas-specific promoters (Edlund et ab (1 985) Science 230:912-916)
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166.
  • Developmentally-regulated promoters are also 14008
  • CDld or IL2 comprising a DNA molecule of CDld or IL2, respectively, cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to CDld mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a CDld polypeptide can be expressed in bacterial cells such as E.
  • Coli insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • Other suitable host cells are known to those skilled in the art
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an CDld protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die.
  • a host cell used in the present invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a CDld polypeptide.
  • the method comprises culturing the host cell (into which a recombinant expression vector encoding an CDld has been introduced) in a suitable medium such that an CDld is produced.
  • the method further comprises isolating a CDld polypeptide from the medium or the host cell.
  • IL2 expression vectors, and cDNA may be constructed, transfected, propagated in host cell lines.
  • IL2 vectors are known in the art and one of skill in the art would be able to readily identify the proper IL2 cDNAs for use in the methods disclosed herein having the benefit of this disclosure. Suitable examples of IL2 sequences and expression vectors may be found, for example, in US Patent Application Publication 20030207832 and 20030129169, which are incorporated herein by reference in their entirety. IL2 protein may be made by standard techniques known to those skilled in the art. The purified IL2 may be used in the methods disclosed herein.
  • purified or “substantially pure” means that the predominant species present (i.e., on a molar basis) is more abundant than any other individual species in the composition, and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • the invention provides therapeutic methods and compositions for the prevention and treatment of autoimmune disease and related conditions such as AIDS and organ/tissue rejection.
  • the invention provides methods and compositions for the prevention and treatment of autoimmune and related diseases in humans as well as other animals through the administration of one or more beta Galactosyl Ceramide compounds.
  • the invention also provides methods of reducing tumor sizes and metastases.
  • the present invention contemplates a method of treatment, comprising: a) providing: i) a mammalian patient particularly human who is either at risk for autoimmune disease or who has symptoms of autoimmune disease, ii) one or more beta Galactosyl Ceramide compounds, and; b) administering the one or more beta Galactosyl Ceramide compounds to the patient.
  • symptoms of autoimmune disease is herein defined as any abnormal symptoms than can be attributed to the generation of autoreactive B and/or T cells.
  • autoantibodies are a common symptom associated with autoimmune disease.
  • at risk for autoimmune disease is herein defined as individuals with familial incidence of autoimmunity.
  • many autoimmune diseases are associated with genetic factors such as certain HLA specificities.
  • autoimmune and related diseases refers to those disease states and conditions wherein the immune response of the patient is directed against the patient's own constituents, resulting in an undesirable and often knowingly debilitating condition.
  • autoimmune disease is intended to further include autoimmune conditions, syndromes and the like.
  • An “autoantigen” is a patient's self-produced constituent, which is perceived to be foreign or undesirable, thus triggering an autoimmune response in the patient, which may in turn lead to a chain of events, including the synthesis of other autoantigens or autoantibodies.
  • An “autoantibody” is an antibody produced by an autoimmune patient to one or more of his own constituents which are perceived to be antigenic.
  • tumors that have spread from cancers elsewhere in the body.
  • Reducing tumor size as used herein refers to reducing the cell numbers of the tumor, the weight, the circumference, or the mass of the tumor.
  • Reducing metastasis refers to reducing the sloughing off of tumor cells from tumors and reducing the ability of tumor cell to adhere to other organs.
  • the present invention is also not limited by the degree of benefit achieved by the administration of beta Galactosyl Ceramide compounds.
  • the present invention is not limited to circumstances where all symptoms are eliminated.
  • administering a beta Galactosyl Ceramide compound reduces the number or severity of symptoms of an autoimmune disease (e.g., the amount of autoantibody is reduced and or the amount of pain is reduced).
  • administering of a beta Galactosyl Ceramide compound may delay the onset of symptoms.
  • beta Galactosyl Ceramide compounds include in particular autoimmune conditions; HIV infection/ AIDS conditions; and conditions associated with or causing transplant rejection, reducing tumor sizes and reducing metastases, e.g., treatment (including amelioration, reduction, elimination or cure of etiology or symptoms) or prevention (including substantial or complete restriction, prophylaxis or avoidance) of the following:
  • an immunological or autoimmune component such as arthritis (for example rheumatoid arthritis, arthritis chronica progrediente and arthritis deformans) and other rheumatic diseases.
  • Specific autoimmune diseases for which the synergistic combination of the invention may be employed include, autoimmune hematological disorders (including e.g.
  • hemolytic anaemia aplastic anaemia, pure red cell anaemia and idiopathic thrombocytopenia
  • systemic lupus erythematosus polychondritis, sclerodoma, Wegener granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven- Johnson syndrome, idiopathic sprue, (autoimmune) inflammatory bowel disease (including e.g.
  • Ulcerative colitis and Crohn's disease Ulcerative colitis and Crohn's disease), endocrine ophthalmopathy, Graves disease, sarcoidosis, multiple sclerosis, primary biliary ci ⁇ hosis, juvenile diabetes (diabetes mellitus type I), uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial lung f ⁇ brosis, psoriatic arthritis, glomerulonephritis (with and without nephrotic syndrome, e.g. Including idiopathic nephrotic syndrome or minimal change nephropathy) and juvenile dermatomyositis.
  • Autoimmune and inflammatory conditions of the skin are also considered to be amenable to treatment and prevention using the synergistic combination of the invention, e.g., psoriasis, contact dermatitis, atopic dermatitis, alopecia areata, erythema multiforma, dermatitis herpetiformis, scleroderma, vitiligo, hypersensitivity angiitis, urticaria, bullous pemphigoid, lupus erythematosus, pemphigus, epidermolysis bullosa acquisita, and other inflammatory or allergic conditions of the skin, as are inflammatory conditions of the lungs and airways including asthma, allergies, and pneumoconiosis; B) HIV infection and all related conditions from initial infection to the full complement of AIDS disease and all other AIDS-related disorders (e.g., ARC);
  • AIDS-related disorders e.g., ARC
  • Acute organ or tissue transplant rejection e.g., treatment of recipients of, e.g., heart, lung, combined heart-lung, liver, kidney, pancreatic, skin, bowel, or corneal transplants, especially prevention and or treatment of T-cell mediated rejection, as well as graft-versus-host disease, such as following bone marrow transplantation; and D) Chronic rejection of a transplanted organ, in particular, prevention of graft vessel disease, e.g., characterized by stenosis of the arteries of the graft as a result of intima thickening due to smooth muscle cell proliferation and associated effects.
  • graft vessel disease e.g., characterized by stenosis of the arteries of the graft as a result of intima thickening due to smooth muscle cell proliferation and associated effects.
  • cytokines are defined as intercellular mediators secreted by the lymphocytes and/or macrophages.
  • cytokines play a role in the generation of an immune response, such as in an immune response to an infection or infectious organism.
  • Cytokines including, for example, interferons (IFN alpha and IFN gamma) and TNFs induce other cytokines which participate in the development of different autoimmune conditions and diseases.
  • Typical subjects for treatment in accordance with the individuals include mammals, such as primates, preferably humans.
  • Cells treated in accordance with the invention also preferably are mammalian, particularly primate, especially human.
  • a subject or cells are suitably identified as in needed of treatment, and the identified cells or subject are then selected for treatment and administered one or more of beta Galactosyl Ceramide compounds.
  • the treatment methods and compositions of the invention also will be useful for treatment of mammals other than humans, including for veterinary applications such as to treat horses and livestock e.g. cattle, sheep, cows, goats, swine and the like, and pets such as dogs and cats.
  • a wide variety of mammals will be suitable subjects including rodents (e.g. mice, rats, hamsters), rabbits, primates and swine such as inbred pigs and the like.
  • rodents e.g. mice, rats, hamsters
  • rabbits e.g. primates and swine
  • primates and swine such as inbred pigs and the like.
  • body fluids e.g., blood, plasma, serum, cellular interstitial fluid, saliva, feces and urine
  • cell and tissue samples of the above subjects will be suitable for use.
  • One or more beta Galactosyl Ceramide compounds may be administered as a "cocktail" formulation with other therapeutics, e.g., coordinated administration of one or more compounds of the invention together with one or more other active therapeutics, such as one or more other agents used to treat immune disorders, and/or one or more agents used to treat HIV infections.
  • a beta Galactosyl Ceramide compound may be administered in coordination with beta interferon or other immune therapy agent, or agents used for treatment of retroviral infections such as a protease inhibitor (e.g., saquinavir, ritonavir, indinavir or AG1343 (Viracept)), AZT, ddl, ddC, d4T, 3TC, FTC, DAPD, acyclovir, and the like.
  • a protease inhibitor e.g., saquinavir, ritonavir, indinavir or AG1343 (Viracept)
  • AZT AZT
  • ddl ddC
  • d4T 3TC
  • FTC FTC
  • DAPD DAPD
  • acyclovir acyclovir
  • beta Galactosyl Ceramide compounds may be useful for treating tumors, for example, one or more beta Galactosyl Ceramide compounds may be administered to a subject with a tumor to reduce the size of the tumor and/or reduce the metastasis of the tumor.
  • One or more beta Galactosyl Ceramide compounds may also be administered with one or more cytokines to reduce the size of the tumor and or reduce the metastasis of the tumor.
  • Administration protocols for administering one or more beta Galactosyl Ceramide compounds for the reduction or tumor size and/or reduction of metastasis is similar to that for preventing autoimmune diseases. 4008
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • lymphokines include lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-.alpha.
  • growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone
  • parathyroid hormone such as thyroxine
  • insulin proinsulin
  • relaxin prorelaxin
  • glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TS
  • TGFs transforming growth factors
  • CSFs colony stimulating factors
  • M-CSF macrophage-CSF
  • GM-CSF granulocyte-macrophage-CSF
  • G-CSF granulocyte-CSF
  • ILs interleukins
  • a tumor necrosis factor such as TNF-.alpha.
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. Preferred is IL-2.
  • tumor types that may be reduced or metastasis reduced or eliminated are, for example, gastrointestinal malignancies, including colorectal, appendiceal, anal, or small bowel cancers; urogenital malignancies, including prostate, bladder, testicular, or penile cancers; gynecologic malignancies, including cervical, endometriab ovarian, vaginal, or vulvar cancers; osteogenic and other sarcomatous malignancies in which pelvic structures are involved.
  • One or more beta Galactosyl Ceramide compounds may be administered prior to, during, and/or after other treatment therapies, for example surgery or radiation therapy.
  • One or more beta Galactosyl Ceramide compounds may be administered, for example, twice a day, three times a day, or four times a day.
  • the one or more beta Galactosyl Ceramide compounds may be administered, for example, in tablet form, powered form, liquid for or in capsules.
  • Cytokines may be administered as cDNA or protein and may be administered with one or more beta Galactosyl Ceramide compounds to treat tumors to reduce their size or to reduce metastases.
  • One or more beta Galactosyl Ceramide compounds administered with, for example, IL2 may also be used in conjunction with other anti- proliferative compounds to treat tumor metastasis,or other compounds, including for example, chemotherapeutic agents, anti-inflammatory agents, anti-pyretic agents radiosensitizing agents, radioprotective agents, urologic agents, anti-emetic agents, and/or anti-dia ⁇ heal agents.
  • cisplatin carboplatin, docetaxel, paclitaxel, flurouracib capecitabine, gemcitabine, irinotecan, topotecan, etoposide, mitomycin, gefitinib, vincristine, vinblastine, doxorubicin, cyclophosphamide, celecoxib, rofecoxib, valdecoxib, ibuprofen, naproxen, ketoprofen, dexamethasone, prednisone, prednisolone, hydrocortisone, acetaminophen, misonidazole, amifostine, tamsulosin, phenazopyridine, ondansetron, granisetron, alosetron, palonosetron, promethazine, prochlorperazine, trimethobenzamide, aprepitant, diphenoxylate with atropine, and or loperamide.
  • Tumor cell types that may respond and benefit from the above described methods include, for example, liver and kidney tumors, hemangiosarcoma, acoustic neuroma, adenoma, astrocytoma, brain stem glioma, chordoma, choroid plexus, craniopharyngioma, ependymoma, ganglioglioma, ganglioglioneurocytoma, glioblastoma multiforme (gbm), glioma, lymphoma, medulloblastoma, meningioma - meningiomas & benign brain tumors, neurofibramatosis, oligodendroglioma, optic nerve glioma, pituitary tumors - pituitary tumors, pineal tumors, pineoblastoma, vascular brain tumors, neuroectodermal, adrenocortical carcinoma, adrenocor
  • Hemangiosarcoma is a tumor of blood vessels. Since blood vessels are essential in tumor metastasis, HSA spreads early and aggressively, even when metastatic lesions are not apparent at initial presentation.
  • the most common sites for HSA are the spleen, liver, right atrium (heart), and skin. The tumor spreads to the lung, most frequently, but can also metastasize to the brain or any other organ.
  • the administration of one or more beta Galactosyl Ceramide compounds may be to a subject at risk for developing a tumor to prevent the tumor, or alternately the one or more beta Galactosyl Ceramide compounds may be administered to a subject that have been diagnosed with a tumor.
  • the cytokines may be admimstered with the one or more beta Galactosyl Ceramide compounds, or they may be administered at a different time than the one or more beta Galactosyl Ceramide compounds.
  • the one or more beta Galactosyl Ceramide compounds may be administered in the morning and the cytokine may be administered in the evening, or the one or more beta Galactosyl Ceramide compounds and the cytokines may be administer at the same time. Either or both may be administered once per day or more than one time per day.
  • Animal species useful for determining the efficacy of the compounds of the invention include, immunodeficient mice and, in particular, nude and scid mice.
  • the observation that the nude mouse with hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this purpose.
  • the autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mice, including, for example, ASW, A/He, AKR, BALB/c, BIO.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS N, NZB, NZC, NZW, P, RIII and SJL.
  • the cells introduced into such animals can be derived from known tumor/cancer cell lines, such as, any of the above-listed tumor cell lines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene); ras-transfected NIH-3T3 cells; Caco-2 (ATCC HTB-37); a moderately well differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-3 8), or from tumors and cancers. Samples of tumor or cancer cells can 4008
  • Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures.
  • the subcutaneous (s.c.) space in mice is very suitable for tumor implantation.
  • Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions.
  • tumor tissue fragments of suitable size are introduced into the s.c. space.
  • Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously. Tumor cells can also be injected as subdermal implants.
  • Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogen was initially isolated), or neutransformed NIH-3T3 cells into nude mice, essentially as described by Drebin et ab, Proc. Natl. Acad. Sci. USA 83:9129-9133 (1986).
  • animal models of colon cancer can be generated by passaging colon cancer cells in animals, e.g., nude mice, leading to the appearance of tumors in these animals.
  • An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et ab, Cancer Research 54:4726-4728 (1994) and Too et ab, Cancer Research, 55:681-684 (1995).
  • This model is based on the so- called "METAMOUSE" sold by AntiCancer, Inc., (San Diego, Calif.).
  • Tumors that arise in animals can be removed and cultured in vitro. Cells from the in vitro cultures can then be passaged to animals. Such tumors can serve as targets for further testing or drug screening.
  • the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest.
  • Such passaging techniques can be performed with any known tumor or cancer cell lines.
  • Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et ab, J. Exp. Med., 146:720 [1977]), which provide a highly controllable model system for studying the anti-tumor activities of various agents (Palladino et al., J. Immunol., 138:4023-4032 [1987]). Briefly, tumor cells are propagated in vitro in cell culture.
  • the cell lines Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about 10.times.106 to lO.times.lO' cells/mb The animals are then infected subcutaneously with 10 to 100 kit of the cell suspension, allowing one to three weeks for a tumor to appear.
  • the Lewis lung (3LL) carcinoma of mice is also useful, which is one of the most thoroughly studied experimental tumors, can be used as an investigational tumor model. Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL).
  • SCCL small cell carcinoma of the lung
  • This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et ab, Br. J. Cancer, 41, suppb 4:309 [1980]), and evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive. For further information about this tumor model see, Zacharski, Haemostasis. 16:300-320 [1986]). Also useful are the cell types used in the examples, which follow.
  • compositions suitable for administration typically comprise the nucleic acid molecule or the protein modulator and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. As discussed above, supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, inhalation, transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermab or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. PH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS).
  • the composition preferably is sterile and should be fluid to the extent that easy syringability exists.
  • the compositions suitably should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerob propylene glycob and liquid polyetheylene glycob and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosab and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., beta Galactosyl Ceramide compound, an CDld nucleic acid molecule, an CDld protein, or anti-CD Id antibody) in a therapeutically effective or beneficial amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. Suitable oral compositions may be e.g., enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogeb or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogeb or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially e.g. from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 0/ED 50 .
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within-this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • compositions can be included in a container, pack, kit or dispenser together with instructions (e.g. written) for administration, particularly such instructions for use of the active agent to treat against a disorder or disease as disclosed herein, including an autoimmune disease or disorder, treatment in connection with an organ or tissue transplant, as well as other diseases or disorders with an autoimmune component such as AIDS.
  • the container, pack, kit or dispenser may also contain CDld polypeptide, a nucleic acid sequence encoding a CDld peptide, or a CD Id-expressing cell.
  • NK cell isolation Liver or spleen NK cells were isolated from C57BIJ6 (B6) mice and grown for 7-10 days in 1000 IU/ml recombinant IL-2 (Chiron Corp.). Liver mononuclear cells were used either fresh or after IL-2 expansion and were 35-70% CD3 ⁇ NK1.1 + .
  • NKl .1 -PE or APC
  • DX-5-PE and CD3_-PcP (Becton Dickinson-Pharmingen, San Jose, CA) as well as CD69-FITC were used for flow cytometric analysis.
  • IFN- ⁇ cytoplasmic detection was performed using kits purchased from Becton Dickinson/Pharmingen. Annexin V was used to evaluate early apoptosis events [Becton Dickinson/Pharmingen].
  • FCA Flow cytometry analysis
  • Sorting Cells were stained and analyzed on a LSR flow cytometer (Becton Dickinson, San Jose, CA). Cells were directly stained using FITC-, phycoerythrin (PE-), Per-CP- and APC-labeled primary abs. Sorting experiments were performed on a MoFlo cell sorter (Dako/Cytomation, Ft. Collins, CO) to isolate NK, and NKT cells using NKl .1 -PE and CD3biotin- SA.Tricolor. Cytokine measurement. Cytokines were measured using IFN- ⁇ , and chemokine ELISA kits (R&D Systems, Minneapolis, MN).
  • RNAse Protection assay The multiprobe RNAse Protection Assay was performed utilizing the mck-1 or mck-5 probe set (Pharmingen, San Diego, CA). Total cellular RNA was extracted utilizing Trisol (Life Technologies, Gaithersburg, MD) and 1-5 ⁇ g of total mRNA was hybridized with a 33 P UTP labeled RNA probe (1 x 10 cpm sample) prepared according to the manufacturers directions (Pharmingen, La Jolla, CA) using the Pharmingen RiboQuant In Vitro Transcription kit. Following hybridization, the samples were treated with RNAse A and Tl according to the procedure provided by Pharmingen.
  • RNAse was inactivated and precipitated utilizing a master cocktail containing 200 ⁇ l Ambion (Austin, TX) RNAse inactivation reagent, 50 ⁇ l ethanol, 5 ⁇ g yeast tRNA and 1 ⁇ l Ambion GycoBlue co- precipitate per RNA sample.
  • the samples were mixed well, incubated at -70EC for 15 min and centrifuged at 14,000 rpm for 15 min at room temperature. The pellets were suspended in 3 ⁇ l of Pharmingen sample buffer and subjected to polyacrylamide gel electrophoresis as recommended by the manufacturer (Pharmingen).
  • NK cytotoxicity was evaluated using Yac-1 prototype NK target. Cytotoxicity was measured in a standard 4-hr 'Cr-release assay.
  • mice used in this study Mice were obtained from the Animal Production Area, NCI-FCRDC and used between 6-12 weeks of age.
  • Ceramide Reagents Various reagents were purchased from AvantiLipids, Alabaster, AL 35007. D-Glucosyl- ⁇ l-1' Ceramide (C8), D-Galactosyl- ⁇ 1-1' Ceramide (C8), D-Glucosyl- ⁇ l-1' Ceramide (C12), and D-Galactosyl- ⁇ 1-1' Ceramide (C12). In addition, alpha-galactosyl-ceramide (KRN7000) was religiously provided by Kirin, Japan. The ceramide reagents were first dissolved in DMSO, then diluted in PBS containing 0.5% tween 20. Control diluent was used for untreated mice.
  • Bone Marrow Transplantation was evaluated using a new label tracking (L. Oberg, Abstract: Soc. Natural Immunity Meeting, San Juan, PR, 2002 ) procedure that utilizes CFSE (Molecular Probes) labeled bone marrow cells that are evaluated after 1-4 days. Briefly, 10xl0 6 CFSE labeled bone marrow cells that are injected into irradiated recipient mice (C57BL/6 - 900R; Balb/c - 800R). The spleens are evaluated for the total number of labeled cells in the autologous versus the allogeneic transfer group with or without treatment. Mice are evaluated individually and the mean and standard deviation for each group is calculated.
  • CFSE Molecular Probes
  • CDld molecules Tetrameric GalCer/CDld complexes. 7etramers of wt CDld molecules were produced as described by Matsuda et al. (19), using sequences of Accession Nos.: NM_007640, NM_007639, X13170, AK002582, M63697, L38820 or P15813.
  • CDld molecules were biotinylated and prepared by the NIH tetramer facility. Biotinylated CDld molecules were then incubated overnight at room temperature with a 3-fold molar excess of ⁇ or ⁇ GalCer (solubilized in 0.5% Tween 20, 0.9% sodium chloride, hereafter called vehicle) or with an equal amount of vehicle alone.
  • CD 1 d monomers were then tetramerized using a 1 :4 molar ratio of APC-conjugated streptavidin (BD PharMingen, San Diego, CA).
  • Tetramer staining All staining and washes were performed in a buffer consisting of 2% FCS, 0.075% sodium bicarbonate, and 0.1 % sodium azide in RPMI medium (Sigma- Aldrich, St. Louis, MO), as previous described (19). Cells were stained for 3h at room temperature, then stained with subsets antibodies, washed two times and fixed in 1% paraformaldehyde (Sigma- Aldrich) in PBS. The intensity of fluorescence on hybridoma cells was determined by flow cytometry analysis using a FACSort flow cytometer (BD Biosciences, Mountain View, CA).
  • this agent (10, 1 and 0.1 ug/mouse) and evaluated an variety of parameters that are depicted in Figure 1.
  • NK cells we could see a rapid and dramatic activation of NK cells as enumerated by increase in NK lysis (panel A), increase in the size of NK cells (panel B), increase of the activation antigen, CD69 (panel C), increase in the total number of NK cells (panel D), increase in the percent of NK cells (panel F), and the expected decrease in NKT cells numbers (panel E) and percent (panel G).
  • NK bystander activation was evaluated at 24 hrs, activation of NK lysis and size (as well as CD69) was observed in all knockout mice (PFP mice lacked NK lysis). However, potent increases in NK frequency and numbers were not observed in FasL knockout mice suggesting a role for the signaling pathway in the bystander effect. It should be noted that 24 hrs is an early time point for evaluation of cellular increases as these increases are generally maximal at 3-5 days after ⁇ GalCer administration.
  • IFN ⁇ knockout mice demonstrate NK bystander activation, indicating that IFN ⁇ was not required for at least some of the bystander effects, and suggesting that the activation of NK cells may not be directly linked to the burst of IFN- ⁇ that occurs coincident with NKT cell depletion.
  • Example 4 Dissociation between NKT depletion and NK augmentation by ⁇ GalCer (C12).
  • ⁇ GalCer C12
  • the results shown above re-emphasize the coordinate induction of two quite different ⁇ GalCer-induced regulatory events in key innate immune parameters, the reduction of NKT cells and the activation of NK cells.
  • This data set demonstrates the potency of ⁇ GalCer for stimulating innate immune functions, but also illustrates the difficulties in using ⁇ GalCer to discriminate the roles of these two cell types in various biological effects in vivo. Studies were performed to evaluate ceramides to deplete NKT cells without causing downstream activation events (e.g. NK activation and IFN- ⁇ production).
  • ⁇ GalCer (C12) potently depletes detectable NKT cells, while other ceramide compounds including ⁇ GalCer (C8), ⁇ -GlucCer (C12), and ⁇ -GlucCer (C8) demonstrated no biological activity when compared to the diluent control.
  • administration of ⁇ GalCer (C12) resulted in a similar reduction in detectable NKT cells as seen for ⁇ GalCer (panel D), but without the broad bystander NK activation obtained after administration of ⁇ GalCer, as illustrated by a lack of increased percentage, lysis and CD69 expression (panels A-C, respectively).
  • ⁇ GalCer (C12) can effectively reduce the number of detectable NKT cells, and this effect can be mediated in the absence of the NKT dependent bystander NK activation that occurs after administration of ⁇ GalCer.
  • Example 5 ⁇ GalCer (C12) reduces the number of detectable NKT cells in the absence
  • Example 6 Cdld development binding of ⁇ GalCer and ⁇ GalCer (C12) to NKT cells. Since the ⁇ GalCer resulted in such a rapid and pronounced increase of NK cell activation and cytokine induction whereas ⁇ GalCer failed to induce this potent stimulation, we evaluated the relative efficiency of binding of both ceramides to NKT cells. The results of a typical binding study are shown in Figure 6. CDld tetramers were either loaded with ⁇ GalCer or ⁇ GalCer (C12) and their binding to NKT- enriched liver lymphocytes was studied.
  • Figure 9a reveals the strong binding of ⁇ GalCer-loaded CDld to NKT cells (bold line), and this was not observed with either NK cells or NK1.1 " T cells.
  • Figure 9b shows binding with ⁇ GalCer (C12)-loaded tetramers were a similar percentage of NKT cells bound the CDld, albeit at lower intensity, while both NK and NKl .1 " T cells failed to demonstrate this binding.
  • both CD4 and CD8 subsets bound ⁇ GalCer with a strong intensity ( Figure 9c and d), while ⁇ GalCer (C12) binding to CD4 + NKT cells was significantly stronger than it was to CD8 + cells (Figure 9d).
  • Binding of unloaded tetramers was used as a control in Figure 9c and d. Similar experiments were performed with flow sorted NKT cells that had been sorted and cultured for 3-4 days in IL-2. These results demonstrated a similar intensity difference in binding of ⁇ GalCer and ⁇ GalCer (C12) to highly purified NKT cells, and a lack of binding of ⁇ GalCer to either NK or T cells.
  • ⁇ GalCer or ⁇ GalCer (C12) In order to more directly investigate the relative abilities of ⁇ GalCer or ⁇ GalCer (C12) to directly interact with the TcR of NKT cells, we employed an in vitro assay system in which control A20 cells or A20 cells transfected with CDld were used to present ⁇ GalCer or ⁇ GalCer (C12) to isolated NKT vs NK cells (negative control). Cultures containing these enriched subsets of NK or NKT cells and A20 cells were stimulated with ⁇ GalCer or ⁇ GalCer (C12) for 24h and supematants evaluated for cytokine release (Figure 7).
  • IFN- ⁇ and IL-4 production was strongly induced from NKT cells by ⁇ GalCer but not ⁇ GalCer (C12) only when CDld transfected A20 cells were used. Only low levels of cytokine production were observed when transfected A20 cells were treated with ⁇ GalCer (C12) or when NK cells were stimulated with either ⁇ GalCer or ⁇ GalCer(C12). These results clearly show that presentation of ⁇ GalCer by CDld is required for cytokine induction, while ⁇ GalCer (CI 2) is either not efficiently presented to NKT cells by CDld or the quality of the signal is insufficient to induce cytokine production.
  • Figure 8 shows a representative experiment where liver lymphocytes (32% NKT) were mixed 1 : 1 with thioglycolate induced peritoneal exudate cells after loading for lh at 37°C with varying doses of ⁇ GalCer and ⁇ GalCer (C12).
  • Example 7 ⁇ GalCer (C12) depletes NKT cells, but fails to induce rejection of allogeneic bone marrow.
  • the accumulated results above demonstrate that ⁇ GalCer (C12) is efficiently recognized by NKT cells, but does not effectively activate NK cells. This conclusion implies that ⁇ GalCer (C12) may not effectively induce cytokines or NK-dependent biological effects in vivo at doses that effectively reduce the number of detectable NKT cells.
  • NK activity in vivo is their ability to reject bone marrow
  • allogeneic transfer of C57BL/6 [H-2 ] marrow into Balb/c [H- 2 ] mice was used as a model to confirm that ⁇ GalCer (C12) was unable to induce NK-mediated functions in vivo.
  • Balb/c mice are known to be relatively weak bone marrow rejecters and thus this model can easily detect even a small amount of NK cell activation.
  • the transfer of C57BL/6 bone marrow cells into allogeneic Balb/c mice was more than 50% rejected by 3 days (Figure 9).
  • Panel D shows two experiments where several doses of ⁇ GalCer and ⁇ GalCer (C12) were evaluated for enhancement of ma ⁇ ow rejection.
  • ⁇ GalCer can potently activate marrow rejection at a dose as low as 0.1 ⁇ g/mouse
  • ⁇ GalCer (C12) at a dose of 10 ⁇ g which is effective at reducing the number of NKT cells, had little enhancing effect on bone marrow graft rejection.
  • ⁇ GalCer (20 ⁇ g) which also had no appreciable NK enhancing effects (Figure 3) did have some ability to enhance rejection of bone marrow cells suggesting that at very high doses ⁇ GalCer (C12) can either induce enough augmentation of NK activity (see Table II) to mediate some effects in vivo or induce some biological effects through a non-NK-mediated mechanism.
  • a dose of 10 ⁇ g ⁇ GalCer can be used in vivo to effectively deplete NKT cells without appreciable NK-activating effects as assessed by NK number, activation stage, cytolytic activity, or very sensitive NK-dependent functions in vitro.
  • NKT cells can complicate interpretation of data involving liver tumors since the NKT cells are highly prominent in the liver.
  • Use of anti-asial-GMl (which does not remove or alter NKT functions) suggested that NKT are not a major effector in eliminating liver metastases.

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Abstract

L'invention concerne des procédés et des compositions utiles dans le traitement de troubles ou de maladies auto-immunes ainsi que de troubles ou de maladies possédant une composante auto-immunitaire, notamment le sida. Des procédés et des compositions préférés de l'invention utilisent un composé de bêta galactosyl céramide afin de traiter un mammifère souffrant ou étant susceptible de souffrir de tels troubles. Ces procédés et ces compositions peuvent réduire les lymphocytes TNK de manière efficace in vivo en l'absence substantielle d'événements tiers non désirés (activation cellulaire, production de cytokine, inflammation systémique, etc.). Ces procédés et ces compositions peuvent également réduire la taille de tumeurs ou la capacité à métastaser.
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WO2006060117A2 (fr) 2004-11-02 2006-06-08 The Board Of Trustees Of The Leland Stanford Junior University Methodes d'inhibition des cellules nkt
WO2006122296A1 (fr) * 2005-05-11 2006-11-16 Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University Isolement et structure des turbostatines 1 a 4
IL172175A0 (en) * 2005-11-24 2011-08-01 Hadasit Med Res Service Beta glycolipids as immuno-modulators
US10045953B2 (en) 2006-07-06 2018-08-14 Case Western Reserve University Ceramide composition and method of use
WO2011112889A2 (fr) * 2010-03-12 2011-09-15 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Β-mannosylcéramide et stimulation de l'immunité anticancéreuse de cellules nkt
GB201517859D0 (en) 2015-10-08 2015-11-25 Altermune Ltd Novel compounds and therapeutic uses thereof
WO2019184991A1 (fr) * 2018-03-29 2019-10-03 中国医学科学院基础医学研究所 Utilisation d'un composé ou d'un extrait de médecine chinoise traditionnelle dans la préparation d'un agent d'administration d'acide nucléique, et produits associés
EP3820994A1 (fr) * 2018-07-10 2021-05-19 Nantkwest, Inc. Génération de cellules cik nkt à partir de sang de cordon
CN110776544B (zh) * 2019-11-11 2023-03-10 三峡大学 一类2,3-不饱和半乳糖硫苷类化合物及其合成方法
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