Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/145—Orthomyxoviridae, e.g. influenza virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/46—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
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
  • C12N2502/1121—Dendritic cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
  • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
  • C12N2710/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to the field of immunology and treatment of infectious diseases and more specifically to a method of activation of antigen specific T cells and the T cells produced by said method.
• immunotherapy can be broadly defined as the administration of effector cells of the immune system for the treatment of disease.
• the primary function of the cellular immune system is to afford protection against pathogens, including acute and persistent infections.
• T cells recognize infected cells and prevent onset of disease by killing these target cells.
• the interplay of pathogens and the immune system is complex, as demonstrated by chronic infections developing in the presence of specific T cells, whereby the pathogens obviously could evade T-cell surveillance.
• T cells The ability of T cells to detect virtually any pathogenic invader is granted by its extraordinarily diverse receptor repertoire, which allows the T-cell pool to recognize a vast number of peptides upon presentation by major histocompatibility complex (MHC) molecules. Still, signaling through the T-cell receptor (TCR) (signal 1 ) is not sufficient for adequate T-cell activation, as costimulatory molecules provide indispensable signals for proliferation, survival, and differentiation (signal 2). In fact, naive T cells that only receive signal 1 without signal 2 are rendered anergic
• signals 1 and 2 are required for full T-cell activation, and the strength of these signals shapes the size of the ensuing T-cell pool.
• full differentiation into effector T cells is generally dependent on a third signal, which is supplied by the antigen-presenting cell (APC) in soluble form and provides instructive signals for the type of effector T cell that is required.
• APC antigen-presenting cell
• This 'three-signal' concept depicts a model for the activation of naive T cells and the subsequent formation of effector T cells.
• the immune system provides a plethora of diverse costimulatory molecules and these various types of signal 2 and 3 all contribute in their own unique manner to the quality of the T-cell response.
• Costimulatory signals and soluble forms of signal 3 can act on particular aspects of T-cell activation, such as survival, cell cycle progression, type of effector cell to be developed, and differentiation to either effector or memory cell.
• DCs mature antigen-presenting dendritic cells
• helper signals has multiple effects on DCs, including the upregulation of costimulatory molecules, the secretion of cytokines, and the upregulation of several antiapoptotic molecules, all of which cumulatively potentiate the ability of DCs to optimally activate cognate T cells, especially CD8 + T cells.
• helper lymphocytes may also express or secrete factors that directly affect T cell survival, cell cycle progression, type of effector cell to be developed, and differentiation to either effector or memory cell.
• Persistent or chronic viral infections such as HIV, HBV, or HCV, or the herpes viruses CMV and EBV
• HIV HIV
• HBV herpes virus
• CMV herpes viruses
• EBV herpes viruses
• the balance between the virus and the host immune response is crucial.
• the immune system keeps the virus in check, and the virus counters by evading the immune response to avoid clearance, ultimately tipping the balance in favor of the virus and causing disease in many cases.
• the continuous presence of the viral antigen renders virus-specific T cells to become dysfunctional.
• HIV human immunodeficiency virus
• AIDS acquired immunodeficiency syndrome
• CD27(+) and CD27(-) T cells that concurrently are triggered via CD27 exhibit more resistance to apoptosis, interleukin 2 production, and proliferation than CD27(-) T cells.
• CD27(+) T cells After transfer back into an HIV-infected patient, autologous HIV-specific CD27(-) T cells rapidly disappear, but CD27(+) T cells derived from the same clone persist at high frequency. It is therefore suggested that the CD27-CD70 interaction in HIV infection may provide CD27(+) CD8(+) T cells with a survival advantage and compensate for limiting or absent CD4(+) T help to maintain the CD8 response. (Ochsenbein et al., Exp Med. 2004 Dec 6;200(1 1 ): 1407-17)
• T-cell therapy involves the transfer of effector T cells to restore specific T-cell responses in the host.
• Adoptive cell transfer therapy is the administration of ex vivo activated and expanded autologous virus-reactive T cells.
• the present invention relates to an in vitro method for priming of antigen specific CD4+ and/or CD8+ T cells suitable for administration to a patient having a viral infection.
• the method comprises co-culturing target T cells from the patient to be treated, dendritic cells, infectious material or infection associated proteins or peptides and lymphocytes.
• the lymphocytes are sensitized against MHC class I and/or MHC class II antigens on antigen presenting cells (APCs).
• APCs antigen presenting cells
• the lymphocytes are sensitized by way of a mixed leukocyte reaction.
• the present invention also relates to the antigen specific CD4+ and/or CD8+ T cells obtainable by the method and uses thereof.
• Figure 1 illustrates that lymphocytes that have been sensitized against MHC antigens expressed on irradiated allogeneic peripheral blood mononuclear cells (PBMCs) in a conventional MLR markedly enhance the expression of CD70 on mature monocyte- derived DCs which are autologous to the irradiated PBMCs that were used as stimulator cells during the initial sensitizing MLR.
• PBMCs peripheral blood mononuclear cells
• Figure 2 illustrates that gamma-irradiated lymphocytes that have been sensitized against MHC antigens expressed on irradiated allogeneic PBMCs in a conventional MLR markedly enhance the expression of CD70 on mature monocyte-denved DCs which are autologous to the irradiated PBMCs that were used as stimulator cells during the initial sensitizing MLR,
• Figure 3 illustrates that coculture of lymphocytes that have been sensitized against MHC antigens expressed on irradiated allogeneic PBMCs in a conventional MLR for 6 days with monocyte-derived DCs which are autologous to the irradiated PBMCs that were used as stimulator cells during the initial sensitizing MLR induce substantial IL-12 production.
• Figure 4 illustrates that co-culture of lymphocytes that have been sensitized against MHC antigens expressed on irradiated allogeneic PBMCs in a conventional MLR for 6 days with monocyte-derived DCs which are autologous to the irradiated PBMCs that were used as stimulator cells during the initial sensitizing MLR induce substantial IFN-gamma production.
• Figure 5 illustrates that co-culture of lymphocytes that have been sensitized against MHC antigens expressed on irradiated allogeneic PBMCs in a conventional MLR for 6 days with monocyte-derived DCs which are autologous to the irradiated PBMCs that were used as stimulator cells during the initial sensitizing MLR induce substantial IL-2 production.
• Figure 6 illustrates that co-culture of a) irradiated lymphocytes that have been sensitized against MHC antigens expressed on irradiated allogeneic PBMCs in a conventional MLR with b) monocyte-derived DCs which are autologous to the irradiated PBMCs that were used as stimulator cells during the initial sensitizing MLR, induce a higher number of CD27-expressing alloreactive CD8+ T cells from a third-party donor.
• Figure 7 illustrates that addition of irradiated lymphocytes that have been sensitized against MHC antigens expressed on irradiated allogeneic PBMCs in a conventional MLR to monocyte-derived DCs which are autologous to the irradiated PBMCs that were used as stimulator cells during the initial sensitizing MLR, reduces the number of apoptotic (Annexin-V positive) alloreactive CD8+ T cells from a third-party donor.
• Figure 8 illustrates that addition of irradiated lymphocytes that have been sensitized against MHC antigens expressed on irradiated allogeneic PBMCs in a conventional MLR to monocyte-derived DCs which are autologous to the irradiated PBMCs that were used as stimulator cells during the initial sensitizing MLR, induce a stronger (6- fold) secondary proliferative response in alloreactive CD8+ lymphocytes that are re- stimulated with B-cells that are autologous to the DCs used during primary stimulation
• Figure 9 illustrates that addition of irradiated lymphocytes that have been sensitized against MHC antigens expressed on irradiated allogeneic PBMCs in a conventional MLR to monocyte-derived DCs which are autologous to the irradiated PBMCs that were used as stimulator cells during the initial sensitizing MLR, induce a modest (3- fold) increase of IFN-gamma production by alloreactive CD8+ lymphocytes
• Figure 10 illustrates the frequencies of virus-specific CD8 positive cells (specific for CMV, EBV or influenza A virus respectively) after expansion with a) activated allogeneic cells and antigen-loaded (a, b and c), expansion with antigen-loaded autologous DCs but without activated allogeneic cells (d, e and f) and expansion with activated allogeneic cells with unloaded autologous DCs (g, h and i).
• the singular forms "a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
• the term “about” is used to indicate a deviation of +/- 2 % of the given value, preferably +/- 5 %, and most preferably +/- 10 % of the numeric values, where applicable.
• the term "antigen-specific” relates to the specific recognition/binding by a unique T cell receptor (TCR) of a short unique peptide sequence presented on a self MHC molecule.
• the term "priming” and “activation” relates to a programmed activation process that occurs in a naive antigen-specific T cell that become stimulated by antigen-presenting cells with or without concurrent presence of "helper” cells.
• responder cells relates to different lymphocyte subpopulations, including, but not limited to, T cells, NK cells and NKT cells which respond to co-cultured allogeneic PMBCs by activation and/or
• lymphocyte subpopulations including T cells, NK cells and NKT cells which have been pre-activated by co-cultured allogeneic cells, including PBMCs.
• target cells relates to CD4+ or CD8+ T cells that become stimulated by either allogeneic or autologous APCs or antigen- presenting autologous APCs.
• Sites of patient lymphocyte (target cell) collection can, for example, be peripheral blood or bone marrow.
• the term "mature” in relation to monocyte- derived DCs relates to their expression of "maturity-markers” , including, but not limited to, CD40, CD86, CD83 and CCR7 that is induced by the stimulation of immature DCs with microbial products such as LPS or inflammatory mediators such as TNF-alpha and/or IL-1 beta.
• Immature DCs are cells characterized by high endocytic activity and low T-cell activation potential. Immature DCs constantly sample the surrounding environment for pathogens such as viruses and bacteria. Immature DCs phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they
• Mature DCs probably arise from monocytes, white blood cells which circulate in the body and, depending on the right signal, can turn into either DCs or macrophages.
• the monocytes in turn are formed from stem cells in the bone marrow.
• Monocyte-derived DCs can be generated in vitro from peripheral blood monocytes.
• non-proliferative of a cell is used to indicate that the cell has been rendered incapable of cell division to form progeny.
• the cell may nonetheless be capable of response to stimulus, or biosynthesis and/or secretion of cell products such as cytokines.
• Methods of making cells nonproliferative are known in the art. Preferred methods of making cells non-proliferative are treatment with anti-proliferative drugs such as mitomycin C, or irradiation, such as gamma irradiation. Cells that have been fixed or permeabilized and are incapable of division are also examples of non-proliferative cells.
• mixed lymphocyte reaction mixed lymphocyte culture
• MLR mixed lymphocyte culture
• MLC mixed lymphocyte culture
• MLR mixed lymphocyte culture
• MLC are used interchangeably to refer to a mixture comprising a minimum of two different cell populations that are allotypically different. At least one of the allotypically different cells is a lymphocyte.
• the cells are cultured together for a time and under suitable conditions to result in the stimulation of the lymphocytes.
• a frequent objective of an MLR is to provide allogeneic stimulation such as may initiate proliferation of the lymphocytes; but unless indicated, proliferation during the culture is not required. In the proper context, these terms may alternatively refer to a mixture of cells derived from such a culture.
• treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of clinical pathology. Desirable effects include preventing occurrence or recurrence of disease, alleviation of symptoms, and diminishment of any direct or indirect pathological consequences of the disease, lowering the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
• antigen-presenting cells comprise PBMCs, monocytes, B-cells or monocyte-derived DCs from the patient to be treated or from an unrelated blood donor expressing at least one MHC class II antigen that is identical with MHC class II antigens expressed on APCs from the patient to be treated.
• a "chronic infection” is a long lasting persistent infection that evades the immune system of its host.
• a chronic infection can be caused by a bacteria or a virus.
• viruses causing chronic infections are human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV).
• culturing refers to the in vitro propagation of cells or organisms in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical
• a suitable culturing medium can be selected by the person skilled in the art and examples of such media are RPMI medium or Eagles Minimal Essential Medium (EMEM).
• MHC major histocompatibility complex
• HLA complex The proteins encoded by the MHC complex are known as "MHC molecules" and are classified into class I and class II MHC molecules.
• Class I MHC molecules include membrane heterodimeric proteins made up of a chain encoded in the MHC associated non-covalently with p2-microglobulin. Class I MHC molecules are expressed by nearly all nucleated cells and have been shown to function in antigen presentation to CD8+ T cells. Class I molecules include HLA-A, -B, and-C in humans. Class I molecules generally bind peptides 8-10 amino acids in length. Class II MHC molecules also include membrane heterodimeric proteins.
• Class II MHCs are known to participate in antigen presentation to CD4+ T cells and, in humans, include HLA-DP, -DQ, and DR. Class II molecules generally bind peptides 12-20 amino acid residues in length.
• MHC restriction refers to a characteristic of T cells that permits them to recognize antigen only after it is processed and the resulting antigenic peptides are displayed in association with either a self class I or self class 1 1 MHC molecule.
• vacuna immunogenic composition
• immunogenic composition refers to a compound or composition that is capable of conferring a degree of specific immunity when administered to a human or animal subject.
• a "cellular vaccine” or “cellular immunogen” refers to a composition comprising at least one cell population, which is optionally inactivated, as an active ingredient.
• the immunogens, and immunogenic compositions of this invention are active, which mean that they are capable of stimulating a specific immunological response (such as an anti-virus antigen or anti-virus response) mediated at least in part by the immune system of the host.
• the immunological response may comprise antibodies, immunoreactive cells (such as helper/inducer or cytotoxic cells), or any combination thereof, and is preferably directed towards an antigen that is present on a virus towards which the treatment is directed.
• the response may be elicited or restimulated in a subject by administration of either single or multiple doses.
• a compound or composition is "immunogenic” if it is capable of either: a) generating an immune response against an antigen (such as a viral antigen) in a naive individual; or b) reconstituting, boosting, or maintaining an immune response in an individual beyond what would occur if the compound or composition was not administered.
• a composition is immunogenic if it is capable of attaining either of these criteria when administered in single or multiple doses.
• the present invention relates to the production of allo-sensitized allogeneic
• ASLs antigen-specific T cells
• DCs dendritic cells
• the present invention is based on in vitro studies using peripheral blood
• PBMCs mononucelar cells
• the present invention relates to an in vitro method for priming of antigen specific CD4+ and/or CD8+ T cells suitable for administration to a patient having a viral infection.
• the method comprises co-culturing target T cells from the patient to be treated with DCs, preferably monocyte-derived DCs, autologous or allogeneic infectious material or infection associated proteins or peptides and lymphocytes sensitized against MHC class I and/or MHC class II antigens on antigen presenting cells (APCs).
• APCs antigen presenting cells
• Said APCs preferably being allogeneic with respect to the lymphocytes.
• the DCs may be autologous or allogeneic.
• the present invention also provides a method of preparing a T cell population for use in adoptive immunotherapy comprising T cells engineered (by viral transduction, transfection, electroporation or other methods of introducing genetic material) to express a T cell receptor or a chimeric T cell receptor that recognize the target antigen; activating these engineered T cells with antigen-loaded DCs in the presence of anti-CD3 antibodies and sensitized lymphocytes; expanding these target cells in culture; and reintroducing these cells back into the patient. More specifically, an in vitro method for priming of antigen specific CD4+ and/or CD8+ T cells suitable for administration to a patient having a viral infection wherein the CD4+ and/or CD8+ T cells have been genetically manipulated.
• the method comprises co-culturing viral antigen receptor transfected target T cells from the patient to be treated with DCs (preferably monocytes-derived), anti-CD3 antibodies and lymphocytes sensitized against MHC class I and/or MHC class II antigens on antigen presenting cells (APCs).
• DCs preferably monocytes-derived
• APCs antigen presenting cells
• Target T cells can be transformed with T cell receptor (TCR) coding genes or alternatively through the use of a chimeric antigen receptor (CAR) that is capable of relaying excitatory signals to T cells in a non-MHC-restricted manner.
• TCR T cell receptor
• CAR chimeric antigen receptor
• These hybrid proteins composed of an extracellular antigen recognition domain fused to an intracellular T-cell activation domain, may therefore be used in patients regardless of their human leukocyte antigen genotype.
• the target T cells Prior to transfection, are preferably pre-stimulated with anti-CD3 antibodies in order to optimize subsequent transduction. Any suitable CAR can be used in the present invention.
• the CAR ligand should be expressed by the virus. Selecting and preparing a suitable CAR is within the skills of the person skilled in the art.
• Transduction of TCR- or CAR-coding genes into T cells can be performed by using any suitable method known to the skilled person, such as viral or non viral vectors (see for example Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed., vol. 1 -3, Cold. Spring Harbor Laboratory, Cold Spring Harbor, NY).
• viral vectors include retro, lenti, adeno, adeno-associated viral vectors and non-limiting non-viral vector systems include electrotransfer of plasmids and transposon/transposase systems.
• Lymphocytes from the patient that are to be expanded may originate from the peripheral blood or the bone marrow.
• the viral infection may be a chronic viral infection.
• Non-limiting examples of viruses causing a chronic viral infection are HIV, HBV, HCV, CMV and EBV.
• Patients having a chronic viral infection have inactive virus specific CTLs and are therefore a suitable target for the inventive method priming antigen specific Th1 cells or CTLs.
• Inactive HIV specific CTLs are present in HIV patients in a high concentration.
• the inactive CTLs express the CD 70 receptor, CD27, in a high degree making the primed antigen specific Th1 cells or CTLs of the present invention particularly suitable for administration to HIV patients.
• the ASALs are responder cells obtained from a mixed leukocyte reaction that are cultured together with DCs and target cells. When the target T cells are genetically manipulated, also anti-CD3 antibodies are added to the culture.
• the ASALs are selected from the group consisting of peripheral blood lymphocytes, including CD4 + T cells, CD8 + T cells and natural killer (NK) cells.
• the target cells are CD4 + and/or CD8 + T cells that preferably are autologous to the DCs and the DCs are preferably monocytes-derived.
• the monocyte-derived DCs are loaded with virus material or virus associated proteins or peptides or virus derived antigens.
• CD27 + CD8 + T cells represent potentially more effective CTLs (cytotoxic T cells) for adoptive immunotherapy since they can provide an antigen-driven autocrine signal for proliferation.
• helper-independent CD8 T cells would not require exogenous help in the form of IL-2 or CD4+ T cells to survive and expand.
• the present invention provides methods for treating an immune- mediated disease by providing a subject with a CD8 + T cell population that is programmed for strong cytotoxic activity in the absence or reduced presence of additional cytokines, such as IL-2, or CD4 + T cells.
• the methods are particularly useful for ex vivo expansion of cytolytic, virus-specific CD8 + T cells, but may also be used for expansion of virus-specific CD4 + T cells.
• the percentage of cytolytic antigen-specific CD8+ T cells expressed as percentage of the total number of CD8+ T lymphocytes is preferably at least about 5%, more preferably at least about 10%, more preferably at least about 15%, more preferably at least about 20%, even more preferably at least about 25%, even more preferably at least about 30% and most preferably at least about 35%.
• the method of the present invention relates to an in vitro method for priming of antigen specific CD4+ and/or CD8+ T cells suitable for administration to a patient having a viral infection, wherein said method comprising the following steps: a) culturing non-proliferating antigen presenting cells together with peripheral blood mononuclear cells that are allogeneic with respect to the non-proliferating antigen presenting cells,
• lymphocytes including but not limited to CD4 + T cells, CD8 + T cells and/or natural killer (NK) cells from step a) with mature DCs from step b).
• the monocyte-derived DCs are obtained by first culturing monocytes in a
• composition comprising GM-CSF and IL-4 for about 2-7 days, preferably about 5 days to obtain immature DCs and subsequently add a second composition that enables the immature DCs to become mature DCs by culturing for at least about 12 to 72 hours and preferably about 24-48 hours.
• the second composition comprises components that allow the immature DCs to become mature monocyte-derived DCs that can be used to activate CD4+ and CD8+ T cells.
• the second composition comprises TNF alfa, IL-1 beta, interferon gamma, interferon beta and a TLR3 ligand, such as poly-l:C (Mailliard et al., Alpha-type-1 polarized DCs: a novel immunization tool with optimized CTL-inducing activity. Cancer Res. 2004;64:5934- 5937.).
• the second composition comprises interferon gamma, a TLR 3 and/or a TLR 4 ligand and a TLR7 and/or a TLR 8 ligand and/or a TLR9.
• a TLR 3 ligand is poly-l:C
• of a TLR7/ 8 ligand is R848, and of a TLR9 ligand is CpG.
• MLR mixed leukocyte reaction
• PBMCs peripheral blood mononuclear cells
• lymphocytes from one of the individuals are inactivated (usually by treatment with toxins, such as mitomycin or irradiation, such as gamma irradiation) thereby allowing only the untreated remaining population of cells to proliferate in response to foreign histocompatibility antigens.
• the antigen presenting cells used in the MLR are selected from the group consisting of PBMCs and monocytes-derived DCs.
• the monocytes-derived DCs are from the patient or from a healthy donor, i.e. can be autologous or allogeneic.
• infectious material or infection associated proteins or peptides are selected from the group consisting of killed viral particles from the patient, allogeneic viral particles of the same type as the viral infection of the patient and isolated and purified viral proteins or peptides. Isolated and purified viral proteins or peptides are well known to the skilled person.
• the viral material is viral proteins loaded into the monocytes-derived DCs by transfection with mRNA encoding the viral protein.
• the cells T cells from the patient to be treated, monocyte-derived dendritic cells, virus material or virus associated proteins or peptides and lymphocytes sensitized against MHC class I and/or MHC class II antigens on antigen presenting cells (APCs) that are allogeneic with respect to the lymphocytes
• APCs antigen presenting cells
• anti-CD3 antibodies, exogenous IL-2, IL- 7, IL-15, anti-IL-4 and/or IL-21 are added to the cell culture in order to optimize cell proliferation and survival. It is also possible to restimulate the primed antigen specific CD4+ and/or CD8+ T cells by culturing said cells together with new DCs, new sensitized lymphocytes and optionally addition of anti-CD3 antibodies and/or exogenous IL-2, IL-7, IL-15, anti-IL- 4 and/or IL-21 to the cell culture.
• the present invention also relates to an immunogenic composition obtainable by the method described above as well as the antigen specific CD4+ and/or CD8+ T cells obtainable by the method described above.
• the antigen specific CD4+ and/or CD8+ T cells are suitable for administration to a patient and preferably have at least one of the following features:
• the present invention also relates to the antigen specific CD4+ and/or CD8+ T cells obtained by the inventive method for use as a medicament as well as for use in the manufacture of a medicament.
• the present invention relates to the use of antigen specific CD4+ and/or CD8+ T cells obtainable by the method of the invention or as defined above for use in the treatment of a viral infection, such as a chronic viral infection, or for eliciting an anti-infectious immunological response in a human as well as for the manufacture of a medicament for the treatment of a viral infection, such as a chronic viral infection, or for eliciting an anti-viral immunological response in a human.
• the CD4+ and/or CD8+ T cells can be administered after the first stimulation or alternatively after restimulation. In one embodiment the CD4+ and/or CD8+ T cells are administered in combination with a therapeutic anti-viral vaccine.
• T cell populations prepared according to the methods described herein and known in the art can be used in such methods.
• Antigen-primed T cells undergo increased proliferation and decreased apoptosis upon re-stimulation when exposed to ASALs during their initial DC-mediated priming in vitro.
• methods for enhancing secondary T cell responses upon vaccination if adoptively transferred back to the patient before and/or during vaccination are also contemplated by the present invention.
• the present invention also provides methods for improving viral vaccine therapy.
• Many virus express foreign antigens that can potentially serve as targets for destruction by the immune system.
• Viral vaccines generate a systemic virus-specific immune response in a subject that comprises both humoral and cellular components. The response is elicited from the subject's own immune system by administering a vaccine composition subcutaneously, intramuscularly or per-orally.
• the antibodies or immune cells bind the virus antigen and lyse the viruses.
• T cell-responsiveness upon vaccination of patients afflicted with a viral infection Adoptive transfer of preactivated apoptosis-resistant virus-specific T cells with high proliferative potential before, or at the time of vaccination, may therefore enhance vaccine-mediated immune responses in vivo.
• composition according to the invention can also be administered in combination with a therapeutic anti-viral vaccine.
• the cells obtainable by the method of the invention can be administered directly to an organism, such as a human, to increase proliferation and survival of antigen- specific T cells during their activation.
• Administration of these cells, often with pharmaceutically acceptable carriers, is by any of the routes normally used for introducing a cell into ultimate contact with a mammal's blood or tissue cells.
• Formulations suitable for parenteral administration such as, for example, by intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes and carriers include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
• Intravenous administration is the preferred method of administration for the CD4+ and/or CD8+ T cells of the invention.
• the dose of the CD4+ and/or CD8+ T cells administered to a patient should be sufficient to enhance the immune response in the patient.
• cells are administered to a patient in an amount sufficient to elicit an effective immune response to the virus antigen and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease.
• An amount adequate to accomplish this is defined as a "therapeutically effective dose”.
• the dose will be determined by the activity of the cells produced and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
• the physician needs to evaluate progression of the disease and the induction of immune response against any relevant virus antigen.
• Encompassed by the present invention is any combination of the above disclosed embodiments.
• Non-adherent cells were removed and the remaining adherent monocytes, were cultured in AIM-V medium supplemented with recombinant human GM-CSF and IL-4 (R&D Systems, Abingdon, UK; both at 1 ,000 U/mL) for 4-6 days.
• Maturation of immature DC was induced by supplementing the culture media with IFN-a (3,000 U/mL), IFN-Y (1 ,000 U/mL), TNF-a (50 ng/mL), IL-1 ⁇ (25 ng/mL) (all from R&D Systems) and p-l:C (Sigma-Aldrich; 20 pg/mL) during the last 24 hours of incubation.
• the mature DC populations all contained more than 70 % CD83+ DCs as determined by FACS analysis.
• lymphocytes markedly enhance the expression of CD70 on mature monocyte-derived DCs which are autologous to the irradiated PBMCs used in the MLR.
• gamma-irradiated allogeneic lymphocytes similarly enhance the expression of CD70.
• CD8+ lymphocytes were isolated (using negative selection with antibody-coated magnetic beads) after coculture of DCs, irradiated allosensitized lymphocytes and PBMCs (allogeneic to the DCs) for 6 days and subsequently restimulated with B-cells (autologous to the DCs used during primary stimulation) and stained for expression of CD27 and Annexin-V. Subsequent analysis was performed with FACS.
• addition of allosensitized lymphocytes during primary stimulation substantially increased expression of CD27 when the CD8+ cells restimulated with B-cells.
• FIG. 9 shows that addition of allosensitized lymphocytes during primary stimulation substantially increased production of IFN-gamma by CD8+ cells after restimulation.
• Example 5 ASALs increase the frequency of antigen-specific CD8+ T cells generated following primary in vitro stimulation
• Materials & methods Peripheral blood mononuclear cells (PBMC) from healthy blood donors were isolated using a density medium and typed for HLA-A2 with monoclonal antibodies and flow cytometry, according to standard procedures, and frozen. After thawing PBMC were incubated in plastic wells to enrich monocytes by plastic adherence and after rinsing off the non-adherent cells incubation was performed in a cell culture medium containing GM-CSF and IL-4 for 5 days.
• PBMC Peripheral blood mononuclear cells
• the immature DC were incubated with a cytokine cocktail (containing IL-1 ⁇ , TNF- ⁇ , IFN- ⁇ , and INF- ⁇ ) and a mix of overlapping MHC class l-binding peptides of antigens from CMV, EBV and influenza A-virus (PX14 ProMix CEF pool (standard); a peptide library from Proimmune LTD, Oxford, UK. The use of this mix is not limited to one HLA type.) for 48 hours (maturation of DC and pulsing with antigenic peptides).
• a cytokine cocktail containing IL-1 ⁇ , TNF- ⁇ , IFN- ⁇ , and INF- ⁇
• a mix of overlapping MHC class l-binding peptides of antigens from CMV, EBV and influenza A-virus PX14 ProMix CEF pool (standard); a peptide library from Proimmune LTD, Oxford, UK.
• PX14 ProMix CEF pool standard
• the cells were re- stimulated with pulsed DC (as described above) and after 1 hour a pentamer staining was performed, and after 24 hours an assay for IFN-y-forming cells (ELISpot PLUS for Human lnterferon- ⁇ , Mabtech, Sweden) was performed according to instructions by the manufacturer. The number of virus-specific cells was measured as IFN-y-spots per 10 6 cells added.
• virus-specific CD8 positive cells specific for CMV, EBV or influenza A virus respectively.
• Table 1 Shows the frequency of virus-specific cells (expressed as IFN- ⁇ - spots per 10 6 cells) after expansion with activated allogeneic PBMC.

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