WO2012112079A1 - Amélioration de l'efficacité de vaccins immunothérapeutiques cellulaires par une répression génique dans les cellules dendritiques et les lymphocytes t à l'aide de sirna - Google Patents

Amélioration de l'efficacité de vaccins immunothérapeutiques cellulaires par une répression génique dans les cellules dendritiques et les lymphocytes t à l'aide de sirna Download PDF

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WO2012112079A1
WO2012112079A1 PCT/RU2012/000101 RU2012000101W WO2012112079A1 WO 2012112079 A1 WO2012112079 A1 WO 2012112079A1 RU 2012000101 W RU2012000101 W RU 2012000101W WO 2012112079 A1 WO2012112079 A1 WO 2012112079A1
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tim
fas
gene
rna
lymphocytes
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PCT/RU2012/000101
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Mikhail Valentinovich Filatov
Anna Il'inichna GUTKINA
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Vitaspero, Inc.
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
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    • A61K39/12—Viral antigens
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to medicine and immunology and can be used for methods of improving the efficacy of cellular immunotherapeutic vaccines through manipulation of gene expression in the vaccine cells.
  • immune system cells comprising tumor antigens, such cells including dendritic cells and/or activated lymphocytes (Janikashvili et al., Immunotherapy (2010) 2(1):57; Eshhar, Curr Opin Mol Ther. (2010) 12(l):55-63).
  • virus infected cells are recognized by the immune system as foreign, though such a natural immune response is often not strong enough to block the development of viruses.
  • One purpose of immunotherapy is to enhance the ability of the immune system to recognize cells infected with viruses, for example, and to establish effective mechanisms to reduce viral load.
  • One important issue for any immunotherapy is the selection of antigens for directional effects and opt imal presentation of antigens to the immune system.
  • Dendritic cells that contain antigenic material.
  • Dendritic cells' use as immuno-stimulatmg agents is of growing interest for both in vivo and in vitro applications.
  • a method used in oncology for the treatment of tumors includes the administration of dendritic cells induced in vitro for the beginning of maturation and characterized by their ability to capture and process an antigen in vivo.
  • Pharmaceutical compositions used in these methods include the dendritic cells in combination with a pharmaceutically acceptable carrier for their administration.
  • This method and pharmacological composition provide induction of antitumor immune response by efficiently capturing and processing, by dendritic cells, of tumor antigens at the site of a tumor, secretion of the cytokines and contact with T-cells in the lymph node.
  • An antigen can be a tumor-specific antigen, tumor- associated antigen, viral antigen, bacterial antigen, a tumor cell, a nucleic acid encoding the antigen isolated from tumor cells, a bacterial cell, recombinant cell expressing the antigen, a cell lysate, a membrane. preparation, a recombinant antigen, a peptide antigen or an isolated antigen.
  • an autologous vaccine for treating oncological diseases which includes lymphocytes that have been activated with intei eukin-2 and dendritic cells that have been prepared by incubating the immature dendritic cells with a tumor lysate.
  • the vaccine has T- lymphocytes that were specifically activated by mature dendritic cells and is characterized by the manner in which it was produced.
  • the method of preparation includes the isolation of mononuclear lymphocytes (MNK) from the peripheral blood of a subject, cultivation of MNKs in the DMLM medium, separation of cells into monocytes that adhere to the substrate and lymphocytes that do not adhere to the substrate, placing of the MNKs into a culture medium, isolation of non-adhering lymphocytes and subsequent addition of IL-2 to obtain lymphokin-activatcd killer cells (LAK-cells), adding to a remanding adhering monocytes of a growth factor, stimulating DC cells' maturation by an autologous tumor lysate in vitro and the subsequent addition of maturation factors for the duration of 1 day.
  • MNK mononuclear lymphocytes
  • MNKs are cultivated in the medium augmented with 10% FSC for 1 hour, after which immature dendritic cells are obtained.
  • the adhering monocytes are cultivated in a medium with neupogen at a 50 ng/inl concentration during 48 hours, and after obtaining mature dendritic cells, the immature DCs are cultivated in a medium with 2000 MU/ml of Reofcron and 50 ng/ml beta-leukin.
  • LAK cells from the non- adhering fraction of MNK cells are cultivated in a medium containing 100 U/ml Roncoleukin for 72 hours.
  • the mDCs and LAKs are washed by centrifugation in saline solution for 10 min at 1500 rpm, after which mDCs and LAKs are cultivated together in a medium with 100 U/ml of roncoleukin for 24 hours, after which the adhering fraction and non-adhering fraction are washed separately by centrifugation in saline solution for 10 min at 1,500 rpm.
  • the resultant population comprises the vaccine.
  • a prototypical method of treatment of malignant brain tumors comprising the isolation of a patient's blood monocytes, cultivation with growth factors, and addition of the monocytes to ihose dendritic cells obtained from monocytes of the antigenic material from patients' tumor.
  • the resulting dendritic cells are injected back into the patient subcutaneously, in conjunction with additional immune modulation by activated lymphocytes.
  • This method purportedly allows activation of specific antitumor immunity and is characterized by the fact that the patients' peripheral blood mononuclear cells are obtained and cultivated with growth factors, then added to a medium with dendritic cells obtained from patient monocytes.
  • the antigenic material prepared from a patient's tumor is added by electroporation, then the dendritic cells are injected intradermally, followed by immunomodulation using activation by phytohemagglutinin lymphocytes of a patient.
  • autologous activated lymphocytes are injected, providing production of cytokines that can catalyze the immunological antitumor response.
  • a fragment of a patient's tumor that has been. taken during the surgery is dissociated into protein extract, to be used as the antigen in later work.
  • the patient's peripheral blood monocytes are cultivated with growth factors.
  • the antigenic material is added first to monocytes, and then to the dendritic cells.
  • dendritic cells are injected intradermally into a subject.
  • immunomodulation of the patient is performed with activated lymphocytes, wherein the lymphocytes were derived from mononuclear leukocytes' of the subject, and activated by phytohemagglutinin; Activated lymphocytes are then also injected intradermally.
  • the method provides treatment of malignant brain tumors, reducing side effects and complications, increases the life expectancy of patients, and adds to the- quality of life of the patient by offering T-lymphocytes of tumor antigen "professional" antigen-presenting cells of the body, i.e., dendritic cells which contain the tumor antigen.
  • Figure 1 is an image depicting a method of measuring the proportion of cell populations at various stages in the cell cycle.
  • fluorescence is much weaker when stained with fluorescent dye Hocchsl 33258.
  • the flow cytofluorometry histogram such cells after the division form a "peak" to the left (closer to the origin) than non-proliferating cells having diploid DNA amount (see dark area).
  • the fraction of cells caught in this peak enables the estimation of the percentage of cells that were incubated with bromodeoxyuridine.
  • the abscissa fluorescence intensity in arbitrary units, ordinate: number of analyzed cells.
  • Figure 2 is an image illustrating the suppression of gene expression in cells of the immune system (for example, the gene SOCS1) by transfection of specific short interfering RNA.
  • the effectiveness of suppressing the expression was evaluated by the contents of protein, detectable by enzyme immunoassay of the proteins after electrophorelic separation and immobilization on a nitrocellulose membrane.
  • Left lane molecular weight markers
  • lane Average Socsl protein in control cells
  • right-hand lane protein Socsl in cells previously transfected with short interference RNA, anti-SOCSI.
  • Figure 3 is a series of graphs illustrating that transfection of anti-FAS and anti- CTLA4 short interfering RNA dramatically increases the proportion of proliferative capacity of T lymphocytes.
  • Upper histogram phytohemagglutinin stimulation of proliferation of T lymphocytes of cancer patients.
  • Average histogram the same, but after transfection mixture of anti-FAS and anti-CTLA4 short interfering RNA.
  • Lower histogram the same, but after transfection to study the non-specific short interfering RNA genes.
  • the abscissa fluorescence intensity in arbitrary units, ordinate: number of analyzed cells.
  • Figure 4 is an image depicting that transfection of anti-FOXP3 short interfering RNA increases the proportion of proliferating T lymphocytes.
  • Upper histogram phytohemagglutinin stimulation of proliferation of T lymphocytes of cancer patients.
  • Lower histogram the same as upper histogram, with the addition that after transfection, anti-FOXP3 short interfering RNA was used.
  • the abscissa fluorescence intensity in arbitrary units, ordinate: number of analyzed cells.
  • Figure 5 is an image depicting that addition of a lysate of tumor cells can suppress the proliferation of T lymphocytes, and transfection of anti-SOCSI short interfering RNA can abolish this effect.
  • Upper histogram phytohemagglutinin stimulation of proliferation of T lymphocytes of cancer patients. Average histogram: the same as the upper histogram, after adding the lysatcs of tumor cells. Lower histogram: the same, but at the point after the combined action of tumor cell lysate and transfection of anti-SOCSI short interfering RNA.
  • the abscissa fluorescence intensity in arbitrary units, ordinate: number of analyzed cells.
  • the present disclosure presents compositions and methods useful to overcome the immunosuppressive state of immune system cells due to a temporary suppression of gene expression that may limit the proliferative capacity of T lymphocytes.
  • the disclosure presents compositions and methods for overcoming suppression of the immune system by temporarily switching off the expression of one or more genes including FAS, CTLA4, ⁇ 3, S0CS1, CD200, CDS I (PECA -1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM-1, TIM- 2, TIM-3, and ⁇ - ⁇
  • the properties of the cells can be manipulated.
  • suppression of dendritic cells to the induction of tolerance when interacting with lymphocytes increases the viability and immunogenicity of such dendritic cells.
  • this suppression can be used to overcome the immunosuppressive properties of T lymphocytes, preventing their activation.
  • suppression of gene expression of one or more of FAS, I I ..VI. SOCS1 and/or FOSP3 genes can be used to prepare such dendritic cells.
  • IL-2 interleukin-2
  • dendritic cells the ability of dendritic cells, depending on the variety of circumstances, to effect not only activation of T lymphocytes and activation of the immune response, but also the state of tolerance of T lymphocytes and associated with this immunosuppression; and 2) states of immunosuppression that were previously developed in cancer patients, combined with the inability of T lymphocytes to proliferate in response to antigenic stimulation.
  • a new composition e.g., a cell-based vaccine
  • a method of treatment for infectious diseases including, but not limited to, hepatitis C, hepatitis B, papilloma virus, AIDS.
  • Modified to suppress expression refers to a cell that has been treated in any manner to suppress the expression of one or more genes.
  • the suppression can be temporary, for any length of time, or can be permanent.
  • the suppression may partially decrease the expression of the target gene, or it may completely shut off the expression of the target gene.
  • Suppression of the expression of desired genes begins before administration of the vaccine to a subject. Suppression of expression may or may not continue after administration of the vaccine to a subject.
  • a dendritic cell may be modified to suppress the expression of SOCS1 by transfecting the dendritic cell with siRNA specific to suppress the expression of the SOCS 1 gene.
  • DCs Dendritic cells
  • the dendritic cel ls are large cells (1 5-20 microns) round, of oval or polygonal shape, with off-center located nucleus, numerous branched processes of the membrane.
  • Dendritic cel ls express a set of surface molecules characteristic of other antigen presenting cel ls: receptors for cel l wall components and nucleic acids of m icroorganisms, including the receptors for complement components and toll-like receptors, the molecules of class II major histocompatibi lity complex (MHC); costimulatory molecules CD40, B7 Y 2 (CD80, CD86), 137 -DC, B7-H 1 ; intercel lular adhesion molecules (ICAM- 1 ). Dendritic cells can be produced easily from peripheral blood monocytes and can effectively present the antigen of T-lymphocytes. To date, many studies on the modulation of immune response in patients with chronic infectious diseases and cancer using dendritic cel ls primed with antigen were done.
  • Antigen refers to substances that cause specific to them cellular or humoral immune response.
  • Monocytes refers to a large group of mature single-core agranu locytic leukocytes 12-20 microns in d iameter with an eccentrically located polymorph ic nucleus with loose chromatin network, and azurophi lic granules in the cytoplasm. Monocytes have an unsegmented nucleus, and arc the most active phagocytes in peripheral blood. The cells have an oval shape with a large bean-shaped, chromatin-rich nucleus and a large cytoplasm having many lysosomes. Normal ly, monocytes comprise from 3% to 1 1 % of the total number of blood leukocytes.
  • I n an embod iment, there are approximately 450 monocytes in ' l ul of blood. Monocytes are resident i n the blood for 2-3 days, after which they go into the surrounding- tissues, where, having reached maturity, they become tissue macrophages or dendritic cells.
  • T-lymphocytes or “T-cells” refer to lymphocytes, the developing in the thymus of mammals from precursors, prc-thymocytes, derived from the red bone marrow. In the thymus T-lymphocytes di fferentiate, acquiring T-cell receptor (TCR) and surface markers. They play an important role i n adaptive, that is, acquired immune response. Provide recognition and destruction of cel ls bearing foreign antigen, enhances the action of monocytes, N -cells, as well as take part in the switching of immunoglobulin isotypes from early immunoglobulin IgM, the later production of IgG, IgE, IgA in B-cells.
  • TCR T-cell receptor
  • Activated T-lymphocytes refers to T-lymphocytes exposed to inflammatory cytokines and agents that stimulate their proliferation (phytohemagglutinin) for the implementation of Thl response as well as T-lymphocytes obtained as above where the expression of genes CTLA4 and/or FAS and/or FOXP3 has been suppressed.
  • Composition for treatment generally refers to a therapeutic cell vaccine (also referred to herein as an “immunogenic composition”) designed to treat a particular disease or disorder, prepared on the basis of the loading antigenic (immunogenic) material on dendritic cells and phytohemagglutin-activated T lymphocytes, used directly as such or in an amplified version - in conjunction with the temporary arrest of the genes SOCS1, FAS , CTLA4, and FOXP3, for example, by introducing into cells of siRNA or any modified form of siRNA, for example chemically modified form of siRNA, including asymmetric siRNAs and self-deliverable siRNA.
  • RNA Small interfering RNA
  • siRNA refers to short double-stranded RNA molecules capable of RNA i nter ferencc.
  • Asymmetric siRNA refers to siRNA with single stranded overhangs either on the 3' or 5' end of the duplex, or combination of both.
  • Modified siRNA refers to chemically modified siRNA enabling more specific , less toxic and longer lasting gene knockdown effect than unmodified siRNA.
  • Chemical modifications include but not limited to 2'-flouro, phosphorothioates (PS), 2'-0-methyl (2'OMe) RNAs, locked nucleic acids (LNAs), neutral methoxyethyl (MEA), phosphoramidales, cationic ⁇ , ⁇ -dimethylethylenediamine (DMED), morpholinos.
  • “Self-deliverable” siRNA refers to chemically modified siRNA that penetrates cells without additional transfection agents, formulations, delivery vehicles and transfection procedures.
  • RNA interference also referred to as born RNA interference, RNAi
  • RNAi refers to small RNA molecules mediating the suppression of gene expression at the stage of transcription, translation or degradation of mRNA.
  • Small interfering RNA small interfering RNA, siRNA
  • siRNA small interfering RNA
  • RNAi a useful tool for studies using cell cultures and in living organisms, as synthetic double-stranded RNA introduced into cells, causing suppression of specific genes. For example, RNA interference is used to systematically "turn off genes in the cells.
  • Gene expression refers to a process in which genetic information from genes (DNA nucleotide sequence) is transformed into a functional product (RNA or protein).
  • Immunosuppression refers to suppression of immunity of an organism.
  • SOCSl refers to the gene coding a protein called a suppressor of cytokine signaling 1. This protein is a negative regulator of activation of macrophages and plays an important role in regulating autoimmune reactions involving dendritic cells.
  • the suppression of gene expression by SOCSl breaks the tolerance of the immune system to its own antigens and may enhance the immune response.
  • FasR a receptor on the cell surface
  • CTL4 refers to a gene that encodes Cytotoxic T-Lymphocyte Antigen 4 - a protein of immunoglobulin family whose appearance on the surface of activated T- lymphocytes leads to a suppression of cellular proliferation.
  • FOXP3 refers to forkhead box P3, a gene that encodes the coding transcriptional regulator of T-lymphocytes that is necessary for the formation and functioning of regulatory cells that negatively regulate immune response.
  • Dendritic cells are a heterogeneous cell population with characteristic morphology and a widespread distribution in tissues, including blood. The cell surface of dendritic cells is unusual, with characteristic veil-like buds. Mature dendritic cells are usually identified as CD3-, CD1 lc I , CD 19-, CD83 -I-, CD86 + and HLA-DR +. Dendritic cells process and present antigens and stimulate the activation of T-cells and T-cell memory. Dendritic cells have a high ability to effectively present antigens to T-cells as major histocompatibility complex (MHC), and contribute to the initiation of the immune response by releasing cytokines that stimulating the activity of lymphocytes and macrophages.
  • MHC major histocompatibility complex
  • an immunogenic composition is provided.
  • the immunogenic composition is a cell-based vaccine.
  • an immunogenic composition is provided for use as an individual therapeutic cellular vaccine.
  • the immunogenic composition is used as a cellular vaccine in conjunction with one or more other compositions, complementary to conventional treatment.
  • a cellular vaccine is used in conjunction with one or more other compositions known in the art to be useful for treating a particular disease or disorder.
  • a cellular vaccine set forth herein is administered in conjunction with chemotherapeutic therapy.
  • a cellular vaccine set forth herein is administered in conjunction with therapy for hepatitis C.
  • a cellular vaccine is used in conjunction with one or more other compositions previously unknown in the art to be useful for treating a particular disease or disorder.
  • a cellular vaccine is administered as a sole therapeutic agent to a patient having cancer and/or an infectious disease.
  • the cellular vaccine is administered in a dosing regimen that is similar to a dosing regimen for a previously-known therapeutic agent for the particular cancer or infectious disease.
  • a cellular vaccine as described generally herein may be administered to patient having hepatitis C, wherein the cellular vaccine is administered once a week for a time course of five weeks.
  • an immunogenic composition comprising dendritic cells modified to suppress the expression of at least one of the genes selected from the group consisting of PAS, CTLA4, FOXP3, SOCSI, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TTM-1 , TIM-2, TIM-3, and TIM-4.
  • an immunogenic composition comprising T cells that were previously activated for Thl response and further comprising dendritic cells modified to suppress the expression of at least one of the genes selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECA -1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein FIuR, amphiregulin (A REG), TIM-1, TIM-2, TIM-3, and ⁇ -4.
  • the genes selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECA -1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein FIuR, amphiregulin (A REG), TIM-1, TIM-2, TIM-3, and ⁇ -4.
  • the T cells are modified to suppress of the expression of at least one genes selected from the group consisting of CTL4, FAS, FOXP3 CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM-1, TIM-2, TIM-3, and T1M-4,
  • an immunogenic composition comprising T cells modified to suppress the expression of at least one of the genes selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein FluR, amphiregulin (AREG), TIM-1, TIM-2, TIM-3, andTIM-4.
  • the genes selected from the group consisting of FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECAM-1), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein FluR, amphiregulin (AREG), TIM-1, TIM-2, TIM-3, andTIM-4.
  • the number of cells administered in a vaccine is an effective amount of cells for bringing about the desired treatment.
  • an effective amount of cells is that amount which can slow down or stop the progression of the cancer.
  • an effective amount of cells is that amount which can send the cancer into remission, and in other cases, even cure the cancer.
  • an effective amount of cells is that amount which can slow down or stop the progression of the in ectious disease.
  • an effective amount of cells is that amount which can send the partially or wholly eliminate the infectious disease in the patient. Based on the disclosure herein, the skilled artisan will understand how to determine the effective amount of cells in a cellular vaccine.
  • the effective amount may comprise dendritic cells, T cells, or a combination of both.
  • an effective amount of cells for treating hepatitis C is an amount of dendritic cells, T cells, or a combination of both, wherein aftcr ' administration of a vaccine comprising the effective amount of such cells to a patient having hepatitis C, the level of hepatitis C in the patient is diminished and/or eliminated.
  • the administration of the cellular vaccine improves the condition of the patient and/or partially or completely eliminates the virus from the patient's body.
  • dendritic cells in an immunogenic composition are present at a concentration of about 5xl0 3 to about 5xl0 7 cells/ml.
  • the T cells in an immunogenic composition are present at a concentration of about Ixl O 7 cells/ml.
  • the T-lymphocytes in an immunogenic composition are prepared from the monocytes from a subject's blood.
  • compositions disclosed herein can be used by introducing into cells short interfering RNAs that temporarily switch off genes, suppressing the immune response.
  • siRNAs are directed to one or more of the genes FAS, CTLA4, FOXP3, SOCSl, CD200, CD31 (PECAM-l), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphircgulin (A EG), TJM-1, TIM-2, TIM -3, and ⁇ -4.
  • enhanced efficacy of the disclosed methods and compositions may be obtained by employing siRNA techniques to obtain a temporary shutdown of certain genes that are limiting the development of the immune response in the patient.
  • the cells in a cellular vaccine or immunogenic composition have some degree of suppression of expression of one or more genes selected from the group consisting of FAS, CTLA4, FOXP3, SOCSl, CD200, CD31 (PECAM-l), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TlM-1, TIM-2, ⁇ -3, and T1M-4.
  • the suppression may be in dendritic cells, T cells, or both.
  • one cells or both cells may have suppression of one or more genes.
  • the same genes may be suppressed in both cells.
  • a cell may have the expression of all of FAS, CTLA4, FOXP3, SOCSl, CD2'00, CD31 (PECAM-l), Cylindromatosis gene (CYLD), Fas ligand (FasL), RNA-binding protein HuR, amphiregulin (AREG), ⁇ -1, TIM-2, ⁇ -3, and TIM -4 suppressed.
  • the suppression is a partial suppression.
  • the suppression is nearly total suppression.
  • the suppression is 100%) suppression. The level or degree of suppression of any such gene is measured in comparison to the level of expression of the same gene in the absence of any suppression of the gene.
  • one or more of such genes is suppressed at least 5%>, at least 1 ()%>, at least 1 5%, at least 20%>, at least 25%., at least 30% » at least 35%, at least 40%), at least 45%, at least 50%., at least 55%, at least 60%>, at least 65%, at least 70%., at least 75%., at least 80%., at least 85%, at least 90%, at least 95%., at least 96%., at least 97%., at least 98%., at least 99%., and 100%.
  • one or more of such genes is suppressed from about 1 %. to about 99%., from about 5%. to about 95%., from about 1 0%.
  • one or more of such genes is suppressed about 5%>, about 10%>, about 1 5%, about 20%), about 25%>, about 30%>, about 35%., about 40%), about 45%, about 50%), about 55%., about 60%), about 65%,, about 70%>, about 75%, about 80%), about 85%, about 90%, about 95%), about 96%, about 97%, about 98%, about 99%., and 1 00%..
  • a method of treating cancer and/or an infectious disease comprising administering to a patient in need thereof a composition comprising dendritic cel ls modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCS 1 , CD200, CD31 (PECA - 1 ), Cyl indromatosis gene (CYLD), Fas l igand (FasL), ⁇ -binding protein FIuR, amph iregul in (A EG), ⁇ - 1 , TIM -2, TIM-3, and TIM-4.
  • a method of treating cancer/or an infectious disease comprising adm in istering to a patient in need thereof a composition comprising: dendritic cells modi fied to suppress the expression of at least one gene selected from the group consisting of I AS, CTLA4, FOXP3, SOCS 1 , CD200, CD3 1 (PECAM- 1 ), Cyl indromatosis gene (CYLD), Fas l igand (FasL), RNA-binding protein Hull, amphiregul in (AREG), TIM- 1 , TIM-2, TIM-3, and TIM-4, and T-lymphocytes that were activated for Th- J type response.
  • dendritic cells modi fied to suppress the expression of at least one gene selected from the group consisting of I AS, CTLA4, FOXP3, SOCS 1 , CD200, CD3 1 (PECAM- 1 ), Cyl indromatosis gene (CYLD), Fas l igand (FasL), RNA-binding
  • the T-lymphocytes were modified to suppress at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, CD200, CD3 1 (PECAM- 1 ), Cylindromatosis gene (CY LD), Fas l igand (FasL), RNA-binding protein Hull, amphiregulin (AREG), TIM- 1 , TI M-2, TIM-3, andTIM-4.
  • at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, CD200, CD3 1 (PECAM- 1 ), Cylindromatosis gene (CY LD), Fas l igand (FasL), RNA-binding protein Hull, amphiregulin (AREG), TIM- 1 , TI M-2, TIM-3, andTIM-4.
  • a method of treating cancer/or an infectious disease comprising adm inistering to a patient in need thereof a composition comprising T cells modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCS 1 , CD200, CD31 (PECAM- 1 ), Cyl indromatosis gene (CYLD), Fas l igand (FasL), RNA-binding protein HuR, amph iregul in (AREG), TIM- 1 , TIM-2, T1M-3, and TI M -4.
  • a composition comprising T cells modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3, SOCS 1 , CD200, CD31 (PECAM- 1 ), Cyl indromatosis gene (CYLD), Fas l igand (FasL), RNA-binding protein HuR, amph iregul in (AREG), TIM- 1 , TIM-2
  • a method of making of a composition for treatment of canccr/or an in lectious disease comprising preparing dendritic cel ls modified to suppress the expression of at least one gene selected from the group consisting of FAS, CTLA4, FOXP3.
  • the suppression of gene expression can be achieved by any means known in the art.
  • suppression of gene expression is achieved using short interfering RNA techniques.
  • the suppression of expression of a gene is achieved by transfecting dendritic cells and/or T-lymphocytes with the respective correspond ing short interfering double-stranded RNAs.
  • transfection of the cells with leiitivi rus, targeting a gene of interest can be used to suppress expression of the target gene.
  • the gene selected for suppression of expression is a gene that would otherwise place the cel l (e.g., dendritic cell, T cell) in an i mmunosuppressive state.
  • a gene is CT.LA4, FAS FOXP3, CD200, CD3 1 (PECAM- 1 ), Cylindromatosis gene (CYLD), Fas l igand (FasL), RNA-binding protein HuR, amphiregulin (AREG), TIM- 1 . TI M-2, TIM-3, and TIM-4, among others.
  • the expression of one or more of such genes is suppressed to overcome the immunosuppressive state.
  • RNAi used to suppress gene expression was the Ki-RNA transfection (ON- ' f ARGETplus SMARTpool, Dharmacon).
  • the RNAi was used at a concentration of 100 nM / ml in IX transfection buffer (Dharmacon) was performed using lipofectaminc (Dharmacon-FECT), in combination with clectroporalion (350V, 50 microseconds). Given the possible toxicity of lipofectamine, each experiment included a negative control transfection without ki-RNA.
  • Immunodetection Assay For the immunnodeteclion assay (ELISA), the level of expression of proteins was assayed by fixing the cells of interest with 4% formaldehyde (PBS) for 10 minutes, followed by incubation in 5.1% solution of BSA (bovine serum albumin (Biolot)), in phosphate-buffered saline (PBS), for 1 hour. After each treatment, the cell suspension was washed three times with PBS.
  • PBS formaldehyde
  • BSA bovine serum albumin
  • PBS phosphate-buffered saline
  • T lymphocytes To assess changes in immune status of mononuclear cells, the "reaction blast” transformation of T lymphocytes was used. The ability off lymphocytes to respond to the impact of proliferation with phytohemagglutinin was assayed. To this end, mononuclear leukocytes isolated from peripheral blood were incubated 4 days at 37 °C in RPMI-1640 medium supplemented with 10% of human serum and 10 (ig / ml PHA. It is well known that under these conditions, T lymphocytes enter into the proliferative phase of the cell cycle in massive numbers. In the case of the presence of specific immunosuppression, a decrease in the proportion of lymphocytes capable of proliferation was observed.
  • lymphocytes passing through the cell cycle in the preceding 4 days was assessed. Fluorescence quenching showed the dye Hoechst 33258 binding to cellular DNA.
  • the essence of the method used is that for cells which have incorporated bromodeoxyuridine during DNA replication, fluorescence is much weaker when staining with the fluorescent dye l-Ioechst 33258.
  • the flow cytometry histogram of the cells after division shows a "peak" to the left (closer to the origin) than the peak for non-proliferating cells (i.e., those having diploid DMA amount) ( Figure 1).
  • the fraction of cells caught in this peak allows estimation of the percentage of cells that shared incubation time with bromodeoxyuridine.
  • signal transduction via the T-cell receptor is not enough for inducing the proliferation of T- cells, and the interaction with helper cells is required, just as in the case of the blast transformation reaction, where the peripheral blood monocytes are playing the role of these helper cells.
  • the model used can be regarded as a close analogue of the normal process of activation of T cell responses, based not only on activation via the T cell receptor, but also because it requires other co-stimulating signals received in the interaction of T lymphocytes and antigen presenting cells.
  • T lymphocytes are usually about 60% of the original mononuclear blood cells used in the reaction of blast transformation, and by dividing the number of partitions of cells is doubled (assuming that the incubation time of cells carry no more than one division), the maximum possible share of the last cell cycle of f cells, presented in a flow-cytometry histogram, will not exceed 75%.
  • the actual proportion capable of activating T lymphocytes is substantially less, resulting in significantly fewer turns and share the descendants of this division, represented by reducible cytometric histograms as a peak with fluorescence intensity less than non-proliferating cells in phase Gl. According to our estimates of the average healthy person, it varies from 30 to 40%.
  • the percentage of activated T cells may be substantially lower, due to a lack of cells capable of proliferation under such conditions, which may be regarded as a state of i m m u n o s u p p re s s i o n .
  • RNA interference methods were used. Short interfering RNA (siRNA), homologous to the studied genes in mononuclear cells, was transfected by clcctroporation of RNA preparations associated with lipofectamine. Transfected and control cells were activated with phytohemagglutinin. On the 4th day after transfection, flow cytometry was used to estimate the amount of cells proceeding through the cell cycle. The suppression of gene expression was revealed in a comparison, by electrophoresis, of the decrease in level and/or presence of the control content of the cells, versus the codin of proteins. Electrophoresis was followed by immunob lotting or immunofluorescence detection of the cell in situ. Suppressing the expression of genes studied on the example of the gene SOCS1, presented in Figure 2.
  • CTLA4 cytotoxic T-lymphocyte antigen 4
  • FAS antigens i.e., cell surface receptors that induce apoptosis during their
  • Another way to suppress the proliferation of T lymphocytes may be through modulation of their interaction with the regulatory CD4 + CD25 + FOXP3 cells belonging to a specialized subpopulalion off cells, which suppress the immune system and thus participate in maintaining homeostasis and tolerance to their own antigens.
  • Regulatory T cells modulated the expression of wishbone-head transcription factor Foxp3 (forkhead box p3).
  • Foxp3 wishbone-head transcription factor Foxp3
  • Figure 4 illustrates that the inhibitory effect of regulatory CD4 i CD25 I FOXP3 cells can make a significant contribution to limiting the proliferative activity of phytohemaggkitinin stimulated T lymphocytes. Suppressing the expression of FOXP3 leads to a marked increase in the proportion of dividing cells.
  • the use of short interfering RTMA as set forth herein to specifically suppress the expression of the gene SOCS1 was able to overcome the inhibitory effect of lysates of tumor cells.
  • a rejuvenating effect may be due to a variety of factors in lysates of tumors that may inhibit the proliferation of T lymphocytes.
  • Example 2 Methods of treating viral infection.
  • a therapeutic cell vaccine is prepared and used as follows:
  • monocytes are obtained from blood and are cultivated at 37 ° C in RPMI1640 medium supplemented with 10%
  • growth factors are added as follows: 1) granulocyte-macrophage colony-stimulating factor in the concentration of 3000 U / ml, (as is possible to use pharmacological agents "Leukomax” and “Sargramostim”) and 2), intcrlcukin 4 (IL4) in a concentration of 500 U / ml, which can be replaced by interlcukin 15 or interferon alpha.
  • antigenic material is added to dendritic cells, mainly in the form of fragments of viral protein 1MS3 and / or core protein (in case of hepatitis C) /or the combination of a tumor lysate with antigens (in the case of tumors) or another specific antigens in other cases.
  • antigenic material is injected into the dendritic cells by electric charge (e I ectropo rat i o n ) ., '
  • proinflammatory signals are added: a conditioned medium derived from autologous mononuclear cultivation, or trace amounts of bacterial lipopolysaccharide (0.2 microgram per ml), or a mixture of cytokines TNFa + I Lib.
  • HLA-DR emergence of a large number of surface markers characteristic of dendritic cells (HLA-DR, HLA-ABC, CD80, CD83) is determined using a fluorescent microscope or flow cytometry technique.
  • the activated T-lymphocytes are also prepared from the same portion of blood, for which the mononuclear leukocytes are isolated by centrifugation in a density gradient by a standard procedure, and T-lymphocytes from the cell mixture are activated to proliferate by adding phytohemagglutinin (20 micrograms / ml).
  • the procedure is carried out as standard blast transformation of T-lymphocytes, except that for the primary stimulation of Thl-cell immune response, the bacterial lipopolysaccharide (0.2 micrograms per ml) is added to the cells.
  • the siRNA specific for the genes FAS, CTLA4 and FOXP3 is injected into cells by clcctroporation prior to before adding phytohemagglutinin.
  • T-lympocytes The remainder of the procedure of preparing the T-lympocytes is conducted in any manner known in the art.
  • the dendritic cells loaded with antigen and T-lymphocytes activated to implement the Thl response are combined together in 1.5 ml of medium in which they are cultured, and injected into the patient mainly paravertebral ly in the interscapular region, intradermally, in the form of lemon-peel in 2 or 3 points on the back.
  • the preferential treatment of chronic hepatitis lasts for 5 weeks with weekly blood sampling and preparation of portions of dendritic cells loaded with antigen, mainly gene-engineered fragments of protein NS3 and/or core protein.
  • a standard treatment of hepatitis such as the one using interferon and ribavirin, may be employed.
  • the standard polymerase chain reaction (PCR) is used to estimate the virus titer in the blood. If necessary, the treatment can be repeated.
  • a similar approach can also be used to treat other viral infections, particularly hepatitis B, herpes and papilloma virus infections.
  • the antigen can be used in standard commercial vaccine against hepatitis B, herpes simplex virus and papilloma virus.

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Abstract

La présente invention concerne la médicine et l'immunologie et elle peut être utilisée pour améliorer l'efficacité de vaccins immunothérapeutiques cellulaires par l'intermédiaire d'une manipulation de l'expression génique dans les cellules vaccinales. La présente invention concerne une composition immunogène spécifique comprenant des cellules dendritiques modifiées pour réprimer l'expression d'au moins un gène choisi dans le groupe constitué par FAS, CTLA4, FOXP3, SOCS1, CD200, CD31 (PECAM-1), le gène de la cylindromatose (CYLD), le ligand de Fas (FasL), la protéine liant l'ARN HuR, l'amphiréguline (AREG), TIM-1, TIM -2, TIΜ-3 et TIM-4. La présente invention a également pour objet des procédés de production de la composition comprenant des cellules dendritiques modifiées, ladite composition pouvant être utilisée pour le traitement du cancer ou de maladies infectieuses.
PCT/RU2012/000101 2011-02-14 2012-02-14 Amélioration de l'efficacité de vaccins immunothérapeutiques cellulaires par une répression génique dans les cellules dendritiques et les lymphocytes t à l'aide de sirna WO2012112079A1 (fr)

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