EP2723854A1 - Méthode d'activation des mononucléaires du sang périphérique spécifiques - Google Patents

Méthode d'activation des mononucléaires du sang périphérique spécifiques

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Publication number
EP2723854A1
EP2723854A1 EP12729579.8A EP12729579A EP2723854A1 EP 2723854 A1 EP2723854 A1 EP 2723854A1 EP 12729579 A EP12729579 A EP 12729579A EP 2723854 A1 EP2723854 A1 EP 2723854A1
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Prior art keywords
cells
cdlb
preparation
tumour
cell
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EP12729579.8A
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German (de)
English (en)
Inventor
Martin Thurnher
Georg Grünbacher
Oliver Nussbaumer
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Oncotyrol Center for Personalized Cancer Medicine GmbH
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Oncotyrol Center for Personalized Cancer Medicine GmbH
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Priority to EP12729579.8A priority Critical patent/EP2723854A1/fr
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Withdrawn legal-status Critical Current

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    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • 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
    • A61K39/46—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]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • 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
    • 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
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • 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
    • 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
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • 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
    • 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
    • C12N5/0646—Natural killers cells [NK], NKT 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere

Definitions

  • the present invention relates to the activation of specific peripheral blood mononuclear cells.
  • T lymphocytes T cells
  • B lymphocytes B cells
  • T lymphocytes and B cells represent the cells of the adaptive immune system.
  • T cell receptors and B-cell receptors clonotypic receptors
  • T cells with appropriate T cell recep ⁇ tors specific for potentially infectious or dangerous antigens are activated and expanded to be available in large numbers as effector cells for the immediate combat against the infectious or dangerous agent (microbial or tumour cells) . While most of the effector cells subsequently disappear, some of them persist to become memory T cells, which provide protection during re- encounter with the infectious or dangerous agent.
  • the CD4-positive T helper (Th) cells are critical mediators of the cellular immune response against pathogens and tumour cells. For many years, based on cytokine expression profiles, CD4-positive Th cell differentiation has been considered to oc ⁇ cur along a dichotomy of lineages referred to as Thl and Th2.
  • Type 1 cytokines include IL-2, IFN- ⁇ , IL-12 and TNF- ⁇ , while type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13. How ⁇ ever, Th cell research during the last decade has demonstrated that the picture is more complex.
  • Th cells which is required for protection against extracellular pathogens including fungi, was shown to produce IL-17A, IL-17F, IL-22 as well as TNF.
  • IL-17 mediates recruitment of neutrophils and mac ⁇ rophages to infected tissues.
  • Aberrant regulation of Thl7 cells may play a significant role in the pathogenesis of multiple disorders in- eluding inflammatory and autoimmune diseases and possibly also cancer .
  • Adoptive immunotherapy represents a treatment that passively supports the immune system in order to fight diseases, such as cancer and infections.
  • lymphocytes including T cells and NK cells are collected from a patient and expanded by cell proliferation in the laboratory. This increases the number of lymphocytes that are able to kill cancer cells or fight infections. These lymphocytes are adminis ⁇ tered back to the patient to increase the immune system' s capac ⁇ ity to fight the disease.
  • Active immunotherapy attempts to stimulate the host's intrinsic immune response to an infectious disease or cancer. While non-specific active immunotherapy is designed to generally boost the immune system using, for instance, cyto ⁇ kines, specific active immunotherapy (vaccination) attempts the generation of cell-mediated and antibody immune responses di ⁇ rected against specific antigens expressed by the pathogen or cancer cells.
  • a vaccine is a biological composition that is designed to enhance immunity to a particular disease.
  • a vaccine consists of an agent that resembles a disease-causing pathogen or tumour cell. The agent stimulates the patient's immune system to recognize the agent as dangerous, destroy it, and build up immunologic memory, so that the immune system can more easily recognize and destroy any of these threats upon re-encounter.
  • Vaccines can either be prophylactic to prevent or ameliorate the effects of a future infection or therapeutic (vaccines against cancer) .
  • the efficacy of a vaccine can be further en ⁇ hanced by an adjuvant, which can be a pharmacological or immuno ⁇ logical agent that improves the effect of the vaccine.
  • Heat shock proteins are a class of functionally relat ⁇ ed proteins whose expression is increased, when cells are ex ⁇ posed to elevated temperatures or other stress. Extracellular and membrane bound heat-shock proteins, especially Hsp70 are in ⁇ volved in binding antigens and presenting them to the immune system and have thus adjuvant properties.
  • Dendritic cells are the professional antigen presenting cells (APCs) that initiate and regulate a broad repertoire of immune responses (Steinman et al . , Nature (2007), 419-426) .
  • DCs link innate and adaptive immunity.
  • TCR T cell receptor
  • DCs can also activate glycolipid anti ⁇ gen-specific T or NKT cells in a TCR-dependent but CD1- restricted manner (De Libero et al . , Nat. Rev. Immunol. 5 (2005) , 485-96) .
  • DCs interact with and activate ⁇ T cells as well as natural killer (NK) cells, which are important effector cells in innate immune responses against pathogens and tumours .
  • NK natural killer
  • DCs are ideally suited for the induction of immune responses that protect from infectious diseases or cancer.
  • an immunotherapy protocol consists of the following steps:
  • the current limitations of DC-based immunotherapy include the limited efficacy of antigen loading, a lack of physiological maturation protocols that do not exhaust DCs, the limited capac ⁇ ity of DCs to migrate in vivo as well as the fact that the pro ⁇ tocol is time consuming and expensive.
  • the human family of CD1 antigens is expressed on DCs:
  • the human CD1 protein family currently consists of three groups (De Libero et al . , Nat. Rev. Immunol. 5 (2005), 485-96). Based on their sequence similarity CDla, CDlb and CDlc are classified as group 1 CD1 proteins (present in humans but not in mice) .
  • Group 2 consists of CDld only and CDle again represents a separate group.
  • MHC class I-like CD1 pro ⁇ teins comprise a family of antigen-presenting molecules that are capable of presenting nonpeptide lipid and glycolipid antigens to T cells.
  • CDlb+ DCs have been demonstrated to play an important role in infectious diseases and can activate CDlb-restricted T cells with similar glycolipid specificity. Resting monocytes lack group 1 CD1 expression but can be induced to express CD1 proteins during activation in response to GM-CSF suggesting that CD1 proteins are up-regulated, when monocytes differentiate toward DCs in inflammatory lesions. Moreover, af ⁇ ter toll-like receptor (TLR) activation of monocytes, endogenous GM-CSF promotes the differentiation of CDlb+ CD209 (DC-SIGN) - negative DCs.
  • DC-SIGN CDlb+ CD209
  • the group I CD1 proteins in humans were first identified as differentiation markers expressed on immature cortical thymo ⁇ cytes and many monoclonal antibodies (mAb) directed against CD1 antigens have been generated by using thymocytes as an immuno- gen .
  • CD1 is also expressed by leukemic lymphoblasts of T cell lineage from peripheral blood of T-ALL patients and cell lines derived from such cells (MOLT-4, Jurkat) . Down-regulation of CD1 marks acquisition of functional maturation of human thymocytes. So far, CD1 expression could not be detected on normal, non-malignant peripheral T lymphocytes.
  • NK cells Natural kill ⁇ er (NK) cells are innate immune lymphocytes that mediate 2 major functions: recognition and lysis of tumour and virus-infected cells and production of immunoregulatory cytokines.
  • the activa ⁇ tion of an NK cell to kill a target cell is controlled by a com ⁇ plex interaction between activating and inhibitory receptor signals and can be modulated by cytokines.
  • the production of cyto- kines such as interferon (I FN)-Y by NK cells is critical to ear ⁇ ly host defence against a variety of viral, bacterial, and para ⁇ sitic pathogens.
  • I FN interferon
  • Innate immune signals that stimulate NK cell cytokine production are well established and include monokines such as interleukin 12 (IL-12), IL-15, IL-18, and IL- ⁇ and NK receptor ligation.
  • DCs can also activate NK cells.
  • the recipro ⁇ cal impact of this interaction includes NK cell-mediated DC dif ⁇ ferentiation and maturation as well as DC-mediated NK cell acti ⁇ vation .
  • Human NK cells comprise approximately 10% of peripheral blood lymphocytes and are characterized phenotypically by the presence of CD56 and the lack of CD3 (Caligiuri, Blood 112 (2007), 461-469) .
  • the majority (approximately 90%) of human NK cells are CD56 dim and express high levels of FCYRI I I (CD16), whereas a minority (approximately 10%) are CD56 bright and CD16 dim/neg .
  • CD56 bright NK cells constitutively express the high af ⁇ finity IL-2 receptor and low affinity IL-2 receptor and expand in vitro and in vivo in response to low doses of IL-2 but do not themselves make IL-2.
  • ⁇ T lymphocytes which represent less than 10% of human peripheral blood T cells, can be classified according to their expression of TCR variable (V) region segments into V51 T cells and V52 T cells, which also differ in their tissue dis ⁇ tribution and their antigen specificity (Kabelitz et al . , Cancer Res. 67 (2007), 5-8) .
  • V52 T cells recognize phosphoantigens such as the mevalonate pathway-derived isopentenyl pyrophosphate (IPP) .
  • IPP isopentenyl pyrophosphate
  • V51 T cells are considered tissue ⁇ T cells, which are present in intestinal epithelia, in the skin and may be abundant among tumour- infiltrating lymphocytes. V51 T cells recognize MHC class I - chain-related molecule A and B as well as UL-16 binding pro ⁇ teins. Some of the V51 T cells have been reported to recognize CDlc.
  • ⁇ T cells lack CD4 and CD8 expression, which is in accordance with their MHC-non-restricted recognition of uncon ⁇ ventional antigens. Effector function of ⁇ T cells involves strong cytotoxic activity and cytokine production. Although ⁇ T cells are predominantly Thl and produce IFN- ⁇ and TNF- , Th2- type ⁇ T cells have also been described. Moreover, the im ⁇ portance of IL-17-producing ⁇ T cells in models of infectious disease, immune-mediated disease and in injury models is in ⁇ creasingly becoming apparent, ⁇ T cell effector function is controlled by both TCR-dependent and independent mechanisms in ⁇ cluding activating (NKG2D) and inhibitory receptors (CD94) .
  • NVG2D ⁇ cluding activating
  • CD94 inhibitory receptors
  • ⁇ T cells as APCs have recently also been described (Brandes et al . , Science 309 (2005), 264-268) .
  • Short-term activation of ⁇ T cells with small molecular weight non-peptide phosphoantigens induces ⁇ T cell differentiation toward APCs ( ⁇ T cell-APCs) , which are very similar in phenotype and function to monocyte-derived DCs.
  • ⁇ T cell-APCs APCs
  • HMB-PP small microbial compound
  • IPP isopentenyl pyro ⁇ phosphate
  • IPP itself is structurally related to HMB-PP and ubiquitous ⁇ ly present in all living cells including human cells, yet its potency in vitro is much lower.
  • synthetic aminobisphosphonates such as zoledronate (Zometa) or pamidronate (Aredia) that are widely used to treat bone resorption.
  • Increas ⁇ ing evidence suggests that these aminobisphosphonates are not recognized directly by Vy9/V62 T cells as an antigen but may act indirectly, via their effects on the mevalonate biosynthetic pathway, leading to an accumulation of the phosphoantigen IPP.
  • ⁇ T cells can be acti ⁇ vated and expanded by triggering the TCR-associated surface pro ⁇ tein CD3 using a specific monoclonal antibody (signal 1) in com ⁇ bination with a co-stimulatory antibody directed against CD28
  • WO 03/042377 Al discloses expansion of T cells in vitro in the presence of IL-2, yet without presence of an anti-CDlb- positive antibody.
  • Felio et al . JEM 26 (2009), 2497-2505) dis ⁇ closed CDl-restricted adaptive immune responses to Mycobacteria in human group 1 CD1 transgenic mice. Wild type mice have only CDld and do not express the human group 1 CD1 antigens (CDla, CDlb and CDlc) . It was demonstrated that CDl-restricted T cells develop normally in these transgenic mice and that mycobacterial infection or immunization with mycobacterial antigens, which are known to be presented on CD1, activates these CDl-restricted T cells. The stimulation of CDlb-expressing antigen-presenting
  • CD1 self-reactive T cells i.e. T cells that recognize by means of their T cell receptor CDla, CDlb or CDlc without addi ⁇ tion of lipid antigen. It was reported that frequency of such CD1 self-reactive T cells to be unexpectedly high and that these T cells predominantly recognize CDla and CDlc (but not CDlb) . This document, however, does not suggest the use of anti-CDlb
  • US 2006/002927 Al discloses the use of anti-CDl antibodies for the treatment of various disorders.
  • US 2008/254037 Al discloses a method for the treatment of ischemia reperfusion injury by in ⁇ hibiting NK T cells.
  • US 5,679,347 A discloses methods for the detection and isolation of CDl-presented antigens as well as methods of identifying and/or isolating CD1 blocking agents.
  • CDl-presented antigens are nonpolypeptide hydrophobic antigens (lipids, glycolipids) . The methods provided in US 5,679,347 A serve to detect and isolate antigens and to block the presenta ⁇ tion of such antigens.
  • the prior art does not disclose or suggest ac ⁇ tivation of innate and adaptive lymphocytes and for expansion of human T cells, especially ⁇ T cells, and NK cells, using an an- ti-CDlb antibody in a suitable stimulation cocktail to provide e.g. expanded ⁇ T cells.
  • the prior art does not dis ⁇ close or suggest suitable CD56 + cells of such kind.
  • the present invention provides a method for providing a preparation of stimulated cytotoxic T cells and NK cells, comprising the following steps:
  • CDlb-positive (CDlb + ) antigen presenting cells providing a preparation of CDlb-positive (CDlb + ) antigen presenting cells
  • the present invention provides a novel method for the simul ⁇ taneous in vitro (or in vivo) activation of innate and adaptive lymphocytes, which includes the expansion of human T cells, par ⁇ ticularly ⁇ T cells, but also ⁇ T cells and NK cells by cell proliferation as well as the induction of effector functions (cytokine production and cytotoxicity) .
  • the invention also re ⁇ lates to the lymphocytes prepared by such a method and to their use in immunotherapy.
  • Innate and adaptive lymphocytes can easily be prepared from peripheral blood or other sources of CDlb- positive cells and rapidly stimulated to produce important ef ⁇ fector molecules and to exhibit potent cytotoxic activity against pathogens and tumour cells.
  • Tumour cells killed by such activated lymphocytes in vitro represent a pharmaceutical compo ⁇ sition meeting all requirements of a cancer vaccine, which can be loaded onto dendritic cells in vitro or in vivo.
  • T cells particularly ⁇ T cells, and NK cells can be expanded to numbers sufficient to be used in adoptive immuno ⁇ therapy approaches thus representing a pharmaceutical composi ⁇ tion.
  • Lymphocyte activation as induced by this method also re ⁇ sults in the enhancement of, for instance, vaccine-induced pep- tide-specific immune responses, thus serving as a potent adju ⁇ vant (in a pharmaceutical composition) in approaches of antigen- specific vaccination.
  • Lymphocyte activation and concomitant en ⁇ hancement of protein-specific adaptive immune responses can be used in the prevention and treatment of infectious diseases or cancer.
  • the method is also suitable to determine immune compe ⁇ tence of a patient (diagnostic tool) .
  • the invention relates to a method for the simultaneous and potent activation of NK and T cells, which are both major effector cells in the protection against pathogens and tumour cells.
  • the invention further relates to the preparation of large numbers of activated T cells, particularly ⁇ T cells, which represent another subset of effector cells in the defence of pathogens and tumours.
  • the method enables the ex ⁇ pansion of ⁇ T cells by cell proliferation without the drawbacks of bisphosphonates which have intrinsic dose-limiting ef ⁇ fects.
  • the method of the present invention can also be used to kill tumour cells in vitro and generate a highly immunogenic tu ⁇ mour cell extract that by itself meets the requirements of a cancer vaccine, which can be administered to a cancer patient.
  • the highly immunogenic tumour cell extract can be used for the simultaneous antigen-loading and maturation of a patient's dendritic cells in vitro.
  • the antigen-loaded, non- exhausted, mature dendritic cells can then be administered as a tumour vaccine to the cancer patient.
  • the method of in ⁇ vention can be used to enhance protein antigen-specific immune responses thus serving as a potent adjuvant of vaccine antigens.
  • the method according to the present invention comprises the stimulation of CDlb + antigen presenting cells with an anti-CDlb antibody and IL-2.
  • the cells obtained with the method according to the present invention show significant synergistic effects compared to cells which have been treated with anti-CDlb anti ⁇ bodies only (e.g. as disclosed in Theodorou et al . , J. Immunol. 144 (1990), 2518-2523 or as disclosed in Taylor et al . , J. Immu ⁇ nol. 147 (1991), 3794-3802):
  • NK cells natural killer cells
  • Thl7 cytokines are generated, especially IL-17A and IL-22.
  • IL-17A and IL-22 are generated, especially IL-17A and IL-22.
  • These synergistic effects lead to potent inflammatory responses by inducing the production of many other cytokines, chemokines and prostaglandins from many cell types including macrophages. The release of these cytokines attracts other cells including neutrophils.
  • IL-17 function is also essential to a subset of CD4+ T cells therefore called T helper 17 (Thl7) cells, which have been implicated in anti-tumour immunity (Ag- garwal et al . , J. Leukoc. Biol. 71 (2002), 1-8).
  • the CDlb + antigen presenting cells can be any cell which is regarded as CDlb-positive cell (synonymous with "CDlb + cells", "CDlb + antigen presenting cells”), i.e. a cell which expresses CDlb and displays CDlb on the cell surface.
  • CDlb + antigen presenting cells i.e. a cell which expresses CDlb and displays CDlb on the cell surface.
  • CDlb + antigen presenting cells are CD56 + cells and/or peripheral blood mononuclear cells (PBMCs) or a subset thereof, especially a preparation of CD56 + PBMCs. Therefore it is preferred to provide preparation of CD56 + antigen presenting cells, especially CD56 + PBMCs as prepa ⁇ ration of CDlb + antigen presenting cells.
  • PBMCs peripheral blood mononuclear cells
  • CD56 + PBMCs activated according to the present invention represent a preparation, which is enriched in highly cytotoxic lymphocytes with particularly strong antitumor activity (Fig. 6) . Since the method according to the present invention enables the simultaneous activation of T cells and NK cells the final cell preparation comprises activated CD56 + ⁇ T cells, CD56 + ⁇ T cells as well as activated NK cells (which constitutively ex ⁇ press CD56) . Importantly, these subsets have all been reported to exhibit particularly strong cytotoxic activity (Alexander et al., Clin. Cancer Res. 14 (2008), 4232-4240; Pittet et al . , J. Immunol. 164 (2000), 1148-1152; Caligiuri, Blood 112 (2007), 461-469) .
  • PBMCs of healthy donors are provided, however, also PMBCs of patients, e.g. tumour patients, such as renal car ⁇ cinoma patients can be used (see e.g. Fig. 7) .
  • tumour patients such as renal car ⁇ cinoma patients
  • any cell preparation containing CDlb + antigen presenting cells can be used as starting material for the present method.
  • CDlb + antigen presenting cells can be isolated from different tissue (e.g. epidermal cell preparations of dermatitis patients, such as in Taylor et al . , 1991).
  • lymphoid organs such as spleen or lymph nodes can be used as well as tumour tissue.
  • the preferred source of CDlb + cells is, as described above, mononu ⁇ clear cells from human peripheral blood. Therefore, the prepara ⁇ tion of CDlb + antigen presenting cells is preferably obtained from peripheral blood, bone marrow or umbilical cord blood, es ⁇ pecially peripheral blood.
  • the method according to the present preparation may also be performed by provision of a preparation of lymphocytes or monocytes with a low level of CDlb + antigen presenting cells or even without any CDlb + antigen presenting cells being already present. Stimulation of this preparation with anti-CDlb antibody and IL-2 leads to the generation of CDlb + antigen presenting cells which can then be cultivated.
  • a "prepa- ration of CDlb + antigen presenting cells” therefore also includes a preparation comprising lymphocytes or monocytes which can be stimulated with an anti-CDlb antibody and IL-2 to become a prep ⁇ aration of CDlb + antigen presenting cells during stimulation with anti-CDlb antibody and IL-2.
  • the method of preparing the starting cells of the present invention can vary, depending on the source of initial cell.
  • PBMCs or subsets thereof, which contain APCs as well as lymphocytes can vary, depending on the source of initial cell.
  • pe ⁇ ripheral blood, bone marrow, umbilical cord blood may be typical sources of blood for PBMC preparation.
  • the use of peripheral blood is preferable.
  • whole blood can be collected uti ⁇ lizing a vacuum blood tube. Heparin or citric acid may be added to prevent blood coagulation.
  • PBMCs can be directly obtained by use of a component collection system such as leukapheresis . PBMCs can then be separated from whole blood or from leukapheresis prod ⁇ ucts. Any method for separating mononuclear cells may be used. For example, the Ficoll separation method, i.e., a Ficoll-Paque density gradient is frequently used.
  • PBMCs In order to remove plate ⁇ lets, it is preferable to wash the collected PBMCs several times using a culture medium, physiological saline, phosphate buffered saline (PBS) , which may further be supplemented with serum or albumin to prevent unspecific attachment of cells to plastic surfaces .
  • PBS phosphate buffered saline
  • the CD56-positive subset may also be isolated from PBMCs, since this subset is enriched in NK cells as well as T cells and also contains a population of DC-like APCs, which are suitable for the activation of NK cells and T cells T cells (preferred methods are disclosed in Gruenbacher et al . , Blood 114 (2009), 4422-4431; Gruenbacher et al . , Cancer Re. 70 (2010), 9611-9620; Nussbaumer et al . , Blood 118 (2011), 2743-2751).
  • a method to isolate and collect CD56-positive cells utilizing the Magnetic Cell Sorting (MACS; Miltenyi Biotec) is preferred since it is simple and reliable and results in pure populations of CD56- positive PBMCs (>90%) .
  • toll-like receptor (TLR) activation of monocytes endogenous GM-CSF promotes the differentiation of CDlb+ CD209 (DC-SIGN) -negative DCs; therefore, treatment of PBMCs with GM-CSF, toll-like receptor agonists or cytokines such as IL-2, which are known to induce endogenous GM- CSF production, can be used to enhance CDlb expression in PBMCs and thus to provide preparations of CDlb-expressing antigen- presenting cells. Most importantly, these preparations not only contain the CDlb-expressing antigen-presenting cells but also the responding NK cells and T cells including ⁇ T cells.
  • the stimulation of the CDlb + antigen presenting cells according to the present invention is performed by the combined action of the anti-CDlb antibody and IL-2. This combination is much more effective in activation than the stimulation with anti-CDlb antibodies alone.
  • IL-2 belongs to the family of common gamma chain cytokines, which also includes IL-4, IL-7 and IL-15. These cytokines were either much less effective or had no ef ⁇ fect.
  • IL-2 derivatives, such as pegylated IL-2 may be used, e.g. to achieve a depot effect.
  • IL-2 is added at a concentration of 5 to 5000 U/ml, preferably of 10 to 1000 U/ml, especially of 50 to 500 U/ml.
  • the stimulation according to the present invention is achieved by the addition of the combination of anti-CDlb anti ⁇ bodies and IL-2 and subsequent cultivation of the cells which allows the cell preparation to develop the novel phenotype and characteristics.
  • the cells are preferably cultured for a sufficient time at suitable conditions.
  • cultivation is performed for at least 6, preferably for at least 12, especially for at least 16 hours. It is preferred to allow cultivation for about 1 to 3 days, however, also cultivation of at least one week or at least two weeks is possible. Cultivation of more than four weeks or even more than 6 or 8 weeks is not recommendable .
  • Preferred antibody concentrations for in vitro treatment of the cells are from 0.1 to 50 yg/ml, preferably from 0.5 to 20 yg/ml, especially from 1 to 10 yg/ml.
  • Preferred IL-2 concentra ⁇ tions for in vitro treatment of the cells are 1 to 5000 U/ml, preferably 10 to 1000 U/ml, especially from 50 to 500 U/ml.
  • Pre ⁇ ferred temperatures for the in vitro cultivation are 30 to 40°C; preferred CO 2 concentrations are - as for all other parameters - the conditions optimised for the given cells and buffer systems, e.g. 1 to 10%, especially about 5% CO 2 ; for other buffer systems (such as HEPES) , CO 2 concentrations could differ.
  • the preparation of CDlb + antigen presenting cells preferably contains a cell culture medium which allows the cells to develop the desired properties according to the present invention .
  • the cell preparation obtained is preferably washed to remove the stimulating substances attached to the cells and from the cultivation solution and, optionally, also to remove cell products generated during cultivation of the stimu ⁇ lated cells.
  • the stimulating substances or cultivation products such as cytokines
  • the stimulated cytotoxic T cells and NK cells obtained after cultivation are washed with an isosmotic T cell washing solution, especially sa ⁇ line, phosphate buffered saline (PBS) , or other neutrally buff ⁇ ered solutions, especially cell culture media.
  • an isosmotic T cell washing solution especially sa ⁇ line, phosphate buffered saline (PBS) , or other neutrally buff ⁇ ered solutions, especially cell culture media.
  • the stimulated cytotoxic T cells and NK cells may then be finished to an injectable pharmaceutical formulation, especially an intradermally, intravenously, intraarterially or subcutane- ously injectable formulation or a formulation to be injected in ⁇ to a lymph node ( intranodally) .
  • an injectable pharmaceutical formulation especially an intradermally, intravenously, intraarterially or subcutane- ously injectable formulation or a formulation to be injected in ⁇ to a lymph node ( intranodally) .
  • the cultivation of the stimulated cells may be monitored in order to observe an appropriate point in time to end or inter ⁇ rupt cultivation. It is preferred to perform the cultivation at least for a duration until T cells and NK cells start to prolif ⁇ erate. This can easily be observed by measuring cell aggregate generation or cell density via optical microscopy. Usually, the cells start to proliferate after 1 to 3 days of culturing.
  • Preferred culture media are AIM-V ( Invitrogen) , CellGro (CellGenix) , RPMI1640 (Lonza) supplemented with 1-5 % autologous human plasma or human AB plasma.
  • cultivation is per ⁇ formed for 6 h to 30 d, preferably from 12 h to 20 d, especially from 24 h to 10 d.
  • Cultivation may be performed at any tempera ⁇ ture at which cells can be stimulated and treated according to the present invention, e.g. at 30 to 40°C, preferably at 34 to 38°C, especially at 37°C.
  • the generation and expansion of ⁇ T cells is the generation and expansion of ⁇ T cells. Therefore, the number of these cells may be specifically observed during cultivation. Accordingly, the cultivation may be performed for a time to increase the number of ⁇ T cells by a factor of at least 10, preferably of at least 50, especially at least 100.
  • the nature of the anti-CDlb antibody does not seem to be critical as long as it results in binding to the CDlb + cells and allows the cells to take the developments according to the pre ⁇ sent invention, especially increasing the number of ⁇ T cells, secreting IFN- ⁇ and Thl7 cytokines, such as IL-17A and/or IL-22.
  • the anti-CDlb antibody may be a CDlb-binding IgG, IgM, IgA, IgD or IgE antibody or a CDlb-binding fragment thereof, especially an Fab, F(ab' )2 or Fv; or an antibody derivative selected from CDlb-binding bi-, tri- or multispecific antibody, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, or diabody.
  • an antibody refers to any antibody, antibody fragment or antibody derivative whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas , yeast, insect cells or bacteria.
  • one or more antibody vari ⁇ able domains can be incorporated to a given established antibody format or scaffold so as to confer binding specificity for anti ⁇ gen on the structure.
  • Suitable antibody formats are known in the art, such as, chimeric antibodies, humanized anti ⁇ bodies, human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, ho- modimers and heterodimers of antibody heavy chains and/or light chains, antigen-binding fragments of any of the foregoing (e.g. a Fv fragment (e.g., single chain Fv (scFv) , a disulfide bonded Fv) , an Fab fragment, an Fab' fragment, an F(ab')2 fragment), a single antibody variable domain (e.g.
  • a Fv fragment e.g., single chain Fv (scFv) , a disulfide bonded Fv)
  • an Fab fragment e.g., an Fab' fragment, an F(ab')2 fragment
  • a single antibody variable domain e.g.
  • immunoglobulin single variable domain refers to an antibody variable domain (VH, VHH, VL) that specifically binds an antigen or epitope independently of other V regions or domains.
  • An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero- multimer) with other variable regions or variable domains where the other regions or domains are not required for antigen bind ⁇ ing by the single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds antigen inde ⁇ pendently of the additional variable domains) .
  • a "domain anti ⁇ body” or “dAb” is the same as an "immunoglobulin single variable domain” as the term is used herein.
  • a “single immunoglobulin variable domain” is the same as an "immunoglobulin single varia ⁇ ble domain" as the term is used herein.
  • a “single antibody vari ⁇ able domain” or an “antibody single variable domain” is the same as an "immunoglobulin single variable domain” as the term is used herein.
  • An immunoglobulin single variable domain is in one embodiment a human antibody variable domain, but also includes single antibody variable domains from other species such as ro ⁇ dent (for example, as disclosed in WO 00/29004, the contents of which are incorporated herein by reference in their entirety) , nurse shark and Camelid VHH dAbs.
  • Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from spe ⁇ cies including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains.
  • the VHH may be humanized.
  • One preferred anti-CDlb antibody to be used in the present invention is the B-B5 clone, which is an IgGl antibody recogniz ⁇ ing CDlb and CDlc (Dzionek et al . , J. Immunol. 165 (2000), 6037- 6046) . Failure of antibodies recognizing CDlc only to stimulate such immune responses indicates that the specificity of the B-B5 antibody for CDlb is responsible for the functional activity of the B-B5 antibody. Human thymocytes or Jurkat T cells, which both express CDlb, were used as the immunogen in the generation of the B-B5 producing hybridoma clone.
  • thymocytes transiently express CDla, CDlb, and CDlc antigens (Haynes et al . , 1995) .
  • Adult T cells lack these anti ⁇ genes, however, they can re-express these CD1 antigens during ma ⁇ lignant transformation.
  • the T leukemic cell line Jurkat for in ⁇ stance, expresses CDla, CDlb and CDlc. This is why thymocytes and Jurkat T cells can be used as immunogens for CDlb antibody generation.
  • the B-B5 antibody was assigned to the CD1 cluster during Workshop VII (MacDonald et al . , In: Mason D, ed. Leuco ⁇ cyte Typing VII. Oxford, England: Oxford University Press; (2002) , 315-319) .
  • Another preferred anti-CDlb antibody to be used in the pre ⁇ sent invention is the WM25 clone (Favaloro et al . , Immunol. Cell Biol. 65 (1987), 517-527; Favaloro et al . , Disease Markers 4 (1986), 261-70), which is an IgGl antibody recognizing CDlb on ⁇ ly.
  • Human thymocytes were used as the immunogen in the genera ⁇ tion of the WM25 producing hybridoma clone.
  • the WM25 antibody was assigned to the CD1 cluster during Workshop IV (Leucocyte Typing IV (1989) . Oxford University Press) .
  • the present invention also relates to preparation of stimu ⁇ lated cytotoxic T cells, including ⁇ T cells, and NK cells, ob ⁇ tainable by the present method, especially CD56 + cells.
  • the cell preparation according to the present invention comprises specifically advantageous properties, such as the expression of IFN- ⁇ and the Thl7 cytokines, interleukin-17A (IL-17A) and/or inter- leukin-22 (IL-22) .
  • the preparation according to the present invention preferably comprises CD56 + cells in an amount of at least 5 % (of all cells) , more preferred in an amount of at least 10 ⁇ 6 , even more preferred in an amount of at least 30 %, especially in an amount of at least 50%.
  • the amount of CD56 + cells obtained after stimu ⁇ lation and cultivation of the cells according to the present invention usually reflects the amount of the starting cell popula ⁇ tion, i.e. if the starting cell population contained about 10 % of CD56 + cells, the cell population obtained can also contain about 10 % CD56 + cells. If all cells of the starting material are CD56 + cells, the population obtained are also about 100 % CD56 + cells.
  • CD56 + cells are easily and reliably obtainable by standard methods, especially by use of specific and well-established mon ⁇ oclonal antibodies, such as the NKH-1 clone.
  • CD56 is present in the hematopoietic system in three isoforms (120, 140 and 180-220 kD) which differ in the transmembranal and cytoplasmic regions. CD56 + cells can therefore be identified and isolated accurately and efficiently in the hematopoietic system, as disclosed e.g. in Gruenbacher et al . (2010) and Nussbaumer et al . (2011) .
  • the ⁇ T cells obtained by the present method are cytotoxic, i.e. they are able to directly kill tumour cells or co-stimulate the cytotoxicity of other cells such as NK cells against tumour cells (Maniar et al . , Blood. 116 (2010), 1726-1733).
  • Preferred cell preparations according to the present invention have a cy ⁇ totoxicity of at least 10 % of all tumour cells, preferably at least 50 % of all tumour cells, especially at least 80 % of all tumour cells. It is, however, also possible to kill all or al ⁇ most all tumour cells (100 %, 99 %, 98 %, 95 %, 90 %, etc.) by the method according to the present invention.
  • the preparations obtained may also contain NK cells, ⁇ T cells, or mixtures of such cells.
  • NK cells such as bisphosphonates
  • the preparations according to the present invention are specifically advantageous, because known stimulants of ⁇ T cells (such as bisphosphonates ) are not able to perform such ad ⁇ ditional stimulation.
  • the present preparations contain a mixture of NK cells, ⁇ T cells and ⁇ T cells. All three cell populations are contained in CD56 + PBMCs, which are activated by the method according to the present invention.
  • ⁇ T cells co-stimulate the IFN- ⁇ production and cytotoxicity of the NK cells.
  • NK cells activated in this manner can kill tumour cell lines which are otherwise NK cell resistant.
  • the ⁇ T cells significantly contribute to the IFN- ⁇ production (preferably also to the toxicity) .
  • Such syner- gistically potentiating effects have not been possible with the cell preparations disclosed in the prior art.
  • the preparations according to the present invention may be finished to a form which can be pharmaceutically administered, e.g. by providing them in a pharmaceutically acceptable form, preferably by providing (or adding) an isosmotic T cell washing solution, especially saline, phosphate buffered saline (PBS) , lactated Ringer's solution, or other clinically used carrier so ⁇ lutions or suspensions.
  • an isosmotic T cell washing solution especially saline, phosphate buffered saline (PBS) , lactated Ringer's solution, or other clinically used carrier so ⁇ lutions or suspensions.
  • the pharmaceutical preparations according to the present in ⁇ vention may further contain added active substances (i.e. sub ⁇ stances which are not (or not enough) expressed by the cells but actively added to the cell preparation) .
  • Preferred added sub ⁇ stances comprise at least one tumour antigen, especially a prep ⁇ aration of lysed tumour cells; at least one cytokine, especially IL-2; at least one toll-like receptor agonist; a microbiological antigen or mixtures thereof.
  • the preparations according to the present invention prefera ⁇ bly contain 1 to 10 x 10 s ⁇ T cells with antigen-presenting capacity or 1 x 10 s to 1 x 10 8 ⁇ T cells as effector cells per dose.
  • These preparations can be administered once or more to a patient in need thereof. If the present preparation is used as a vaccine, the preparation (dose) is administered preferably at least twice to the patient. Preferably, such vaccination may be performed each 1 to 4 weeks, e.g. for a duration of at least two, at least four or at least six months.
  • the preparation can be administered e.g. each two months or each three months for at least six months or at least twelve months .
  • the preparation according to the present invention may also be stored, e.g. if a given patient should receive more than one administration of the cell preparation at different points in time, or transported. Accordingly, the preparation is preferably stored (or transported) in frozen form, e.g. by using 10% DMSO, 40% serum and 50% RPMI1640 Medium. It is also possible to lyoph- ilise such cells (e.g. for vaccination purposes).
  • the present invention relates to a method for the preparation of a tumour vaccine comprising the following steps:
  • tumour cells especially autologous tumour cells of a cancer patient
  • this method provides a very efficient tumour vaccine. Accordingly, the preparation according to the present invention then resembles not only a general im ⁇ mune stimulant but a tumour vaccine being specific against a certain tumour combined with a very effective adjuvant (i.e. the cells, cytokines and mediators obtained by the present inven ⁇ tion) .
  • a very effective adjuvant i.e. the cells, cytokines and mediators obtained by the present inven ⁇ tion
  • the mix ⁇ ture comprising lysed tumour cells and pro-inflammatory cytokines is contacted for loading and/or maturation of dendritic cells (DCs) in vitro to obtain a DC-based tumour vaccine.
  • DCs dendritic cells
  • the present tumour vaccine can be used for virtually any tumour dis ⁇ ease, especially the most frequent solid tumours, especially colorectal cancer, esophagus cancer, gastrointestinal cancer, gallbladder cancer, lung cancer, breast cancer, oral cancer, ovarian cancer, liver cancer, pancreas cancer, kidney cancer, rectal cancer, stomach cancer, head and neck cancer, cancer of the nervous system, retinal cancer, non-small cell lung cancer, brain cancer, soft tissue cancer, lymphnode cancer, cancer of the endocrine glands, bone cancer, cervix cancer, prostate can ⁇ cer or skin cancer; or a haematological tumour, myelodysplastic syndrome, preferably acquired aplastic anaemia, acute myeloid leukaemia, acute lymphatic leukaemia, Hodgkin lymphoma, non- Hodgkin lymphoma or multiple myeloma.
  • the present invention can also be used for providing a vac ⁇ cine against infectious diseases, especially against a viral, bacterial, fungal or eukaryotic pathogen.
  • the preparation ac ⁇ cording to the present invention can be activated by a microbio ⁇ logical antigen, such as the pathogen itself, by an inactivated preparation of the pathogen or by a specific antigen of the pathogen. All active vaccine concepts known in the art can easi- ly be transformed to the present format according to the present invention.
  • preferred vaccines according to the present invention are therefore vaccines against infections with Yellow Fever virus, Cytomegalovirus, Herpes Simplex, Herpes Zos ⁇ ter, Eastern Equine Encephalitis virus, Hepatitis A, B or C vi ⁇ rus, Hepatitis Delta virus, Dengue virus, and Human Immunodefi ⁇ ciency virus 1 and 2, or more generally, African Swine Fever Vi ⁇ rus, Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Astro- viridae, Baculoviridae, Bimaviridae, Bimaviridae, Bunyaviridae, Caliciviridae, Caulimoviridae, Circoviridae, Coronaviridae, Cystoviridae, Dengue, EBV, HIV, Deltaviridae, Filviridae, Filo- viridae, Flaviviridae, Hepadnaviridae (Hepatitis) ,
  • Rhinovirus Poliovirus
  • Poxviri- dae such as Smallpox or Vaccinia
  • Potyviridae Reoviridae (e.g., Rotavirus), Retroviridae, Rhabdoviridae, Tectiviridae, Togaviridae (e.g., Rubivirus)
  • herpes pox, papilloma, corona, influenza, hepatitis, sendai, Sindbis, vaccinia viruses, west nile, hanta, viruses which cause the common cold; infections with E. faecalis, L. innocua, S. aureus, M. luteus, S. epider- midis, S. pneumoniae, P. aeruginosa, M. hominis or any other bacterial or microbiological pathogen disclosed e.g. in WO 2011/060199 A, and any combination thereof.
  • the present invention relates to a method for in vitro stimulation of white blood cells char ⁇ acterised in by the following steps:
  • This method can be used for diagnostic purposes for the in vitro analysis of blood or other samples from humans, especially human patients (e.g. human patients which are or are planned to be treated with the preparations according to the present inven- tion .
  • human patients e.g. human patients which are or are planned to be treated with the preparations according to the present inven- tion .
  • Preferred phenotypic properties to be measured in this meth ⁇ od are expression levels of one or more cytokines, preferably members of the Thl (IFN- ⁇ , TNF- , IL-12), Th2 (IL-4, IL-5, IL- 13) or Thl7 cytokine family (IL-17A-F, IL-21, IL-22, IL-23) as well as homeostatic cytokines (IL-7, IL-15) or regulatory cyto ⁇ kines (TGF- ⁇ , IL-10) .
  • cytokines preferably members of the Thl (IFN- ⁇ , TNF- , IL-12), Th2 (IL-4, IL-5, IL- 13) or Thl7 cytokine family (IL-17A-F, IL-21, IL-22, IL-23) as well as homeostatic cytokines (IL-7, IL-15) or regulatory cyto ⁇ kines (TGF- ⁇ , IL-10) .
  • Phenotypic analysis also includes the de ⁇ tection and quantification of cellular subsets (dendritic cell subsets, monocyte/macrophage subsets, T cell subsets (including regulatory T cells) , NK cell subsets, granulocyte subsets) .
  • Ex ⁇ pansion of, for instance ⁇ T cells is determined by assessing the frequency (in %) of T cells expressing a ⁇ T cell receptor in the starting population and in the population expanded according to the present invention, which is harvested at the end of the culture period. By considering frequency of ⁇ T cells and total cell numbers and by comparing starting and expanded population the expansion factor can be calculated.
  • the preparation of white blood cells may be obtained from a patient whose immune status has to be monitored.
  • the immune status of the patient may then be de ⁇ fined by comparison with reference values for the properties measured.
  • more than one sample of a patient is subjected to this method during monitoring, e.g. 2, 3, 4, 5, at least 10, at least 20, at least 50, or at least 100 samples.
  • samples from one and the same patient have been taken at different points in time (e.g. before a treatment and after a treatment or in the course of a therapy) .
  • the samples can be taken daily, weekly, monthly, etc..
  • At least two preparations of white blood cells are obtained from a patient at different points in time
  • the same one or more phenotypic properties are measured in the at least two preparations of white blood cells, and the measured phenotypic properties of the at least two prep ⁇ arations are compared with each other to monitor the immune status of this patient.
  • PBMCs may be prepared from peripheral blood of a patient at appropri ⁇ ate time points (before, during and after a certain intervention) and stimulated with anti-CDlb plus IL-2.
  • Cytokine levels as assessed by ELISA or multiplexing technology, activation of individual cell subsets and expansion of, for instance, ⁇ T cells represents a measure of immune competence, which can be correlated with the dosing and duration of the treatment and/or the course of the disease (prognosis) .
  • the present invention relates to a kit for stimulation of white blood cells comprising
  • these immunologic test kits can be provided, which enable the activation and/or expansion of white blood cells.
  • Anti-CDlb antibodies in combina ⁇ tion with IL-2 are part of such test kits in order to induce the activation and/or expansion of NK cells as well as T cells including ⁇ T cells.
  • PBMCs prepared from any donor are stimulated with anti-CDlb antibody plus IL-2 fol ⁇ lowed by a particular read out that may be, but is not restrict ⁇ ed to, a cytokine measurement, phenotypic analysis or cell ex ⁇ pansion (see above) .
  • the present invention also re ⁇ lates to a pharmaceutical composition comprising an anti-CDlb- antibody, preferably for use in immune stimulation, especially for the treatment of infectious diseases or tumour diseases.
  • the method for stimulating cytotoxic T cells and NK cells of the present invention can be performed in vivo by administering the anti-CDlb antibody and optionally IL-2.
  • the (humanized) CDlb antibody can be admixed to any vaccine antigen (either microbial or tumour- associated antigens) .
  • the final vaccine formulation consisting of the antigen preparation and the anti-CDlb antibody (adjuvans) can be administered, typically as an intradermal or subcutante- ous injection.
  • the anti-CDlb antibody can bind to CDlb- expressing APCs and induce immune enhancement analogous to the in vitro embodiments described herein. This immune enhancement generates a stimulatory milieu and favours a potent immune re ⁇ sponse against the vaccine antigen.
  • This pharmaceutical composition comprising an anti-CDlb an ⁇ tibody and optionally IL-2 therefore preferably comprises a vac ⁇ cine antigen, especially a vaccine antigen for vaccination against an infectious disease or a tumour vaccination antigen.
  • Fig. 1 CDlb targeting induces strong IFN- ⁇ responses in the presence of IL-2: exclusion of endotoxin and Fc effects.
  • PBMCs were stimulated for 4 days with anti-CDlb (WM25) in the presence or absence of IL-2 (200 U/ml) .
  • IFN- ⁇ was determined in supernatants on day 4.
  • Polymyxin B (PMB) was used at 60 U/ml to inhibit effects of potential endotoxin (LPS) contaminations of CDlb antibody preparations.
  • PMB polymyxin B
  • LPS potential endotoxin
  • CDlb antibody prepa ⁇ rations were inactivated by boiling (100°C) for 20 min.
  • PBMCs were stimulated with B-B5 IgGl anti-CDlb or an IgGl con ⁇ trol antibody that also binds to APCs (anti-HLA-DR) , or with LPS in the presence or absence of IL-2.
  • IFN- ⁇ was determined in su ⁇ pernatants on day 4.
  • Fig. 2 CDlb targeting induces strong IFN- ⁇ responses in the presence of IL-2: no effect of other homeostatic cytokines.
  • A PBMCs were stimulated for 4 days with anti-CDlb (WM25) in the presence or absence of IL-2 (200 U/ml), IL-4 (200 U/ml), IL-7 (25 ng/ml) and IL-15 (25 ng/ml) .
  • IFN- ⁇ was determined in super ⁇ natants on day 4. Results are mean values of triplicate measure ⁇ ments ⁇ SD.
  • B Microscopic examination of PBMC aggregation in response to anti-CDlb plus IL-2. Increasing cell aggregates are indicative of cell activation and proliferation.
  • PBMCs were stimulated with graded doses of anti-CDlb (B-B5) in the presence of a constant IL-2 concentration.
  • B-B5 PBMCs were stimulated with graded doses of IL-2 in the presence of a constant anti-CDlb concentration.
  • IFN- ⁇ was determined in supernatants on day 4.
  • Fig. 3 IFN- ⁇ production induced by anti-CDlb plus IL-2 de- pends on APCs .
  • PBMCs (1 x 10 6 /ml) or PBMCs depleted of HLA-DR- positive cells were stimulated with IL-2 (100 U/ml) , anti-CDlb (5 yg/ml) or anti-CDlb plus IL-2.
  • IFN- ⁇ was determined in super- natants on day 4.
  • FSC Forward scatter
  • SCC side scatter
  • anal ⁇ ysis of cell preparations confirmed depletion of the APC popula ⁇ tion (top) .
  • Fig. 4 T and NK cells produce IFN- ⁇ in response to stimula ⁇ tion with anti-CDlb plus IL-2.
  • CD56-positive PBMCs (1.5 x 10 6 /ml) were stimulated for 24 h with anti-CDlb (5 yg/ml) plus IL-2 (100 U/ml) .
  • Cells were stained for intracellular IFN- ⁇ and for surface CD3 (all T cells) as well as TCR V52 ( ⁇ T cells only) .
  • A Gating on CD3-positive (all T cells)
  • Fig. 5 Efficient lymphocyte expansion after stimulation with anti-CDlb and IL-2.
  • A ⁇ T cell expansion: CD56-positive PBMCs (1.5 x lOVml) were stimulated with either IL-2 alone (100 U/ml), or with anti-CDlb (5 yg/ml) plus IL-2, or with zoledronate (1 ⁇ ) plus IL-2. Fresh complete medium containing IL-2 was added every 3 days. After 14 days of expansion, cells were harvested, counted and analyzed for TCR V52+ T cell fre ⁇ quency by flow cytometry.
  • Fig. 6 Potent cytotoxicity of PBMCs stimulated with anti- CDlb and IL-2 against human cancer cells.
  • PBMCs (1.5 x 10 6 /ml) were co-cultured with human renal cell carcinoma (RCC) cells (1.5 x lOVml) and were either left untreated or stimulated with IL-2 (100 U/ml) or anti-CDlb (5 yg/ml) alone or with a combina ⁇ tion of anti-CDlb and IL-2.
  • IFN- ⁇ was determined in superna- tants on day 4.
  • B HSP70 was determined in supernatants on day 4.
  • C Microscopic examination was used to monitor the destruc ⁇ tion of the human RCC monolayer.
  • D Supernatants from co- cultures treated with the combination of anti-CDlb and IL-2 were added to immature CD83-negative DCs and up-regulation of the maturation marker CD83 was determined by flow cytometry.
  • Fig. 7 Enhancement of immune responses against a vaccine antigen by targeting of CDlb.
  • PBMCs were either left unstimulated (control) or stimulated for 4 days with the vaccine control antigen KLH (10 yg/ml) , with an- ti-CDlb (WM25) alone or with combinations of KLH and anti-CDlb.
  • A Proliferative responses were assessed as [3H] thymidine in ⁇ corporation.
  • B IFN- ⁇ contents were determined in culture su- pernatants .
  • Fig. 8 Graphic representation of an immunotherapy protocol DC precursor cells are isolated from a patient's blood sample and differentiated into DCs using appropriate differentiation- inducing cytokines (e.g. GM-CSF and IL-4) . After tumour antigen loading and a maturation step using e.g. inflammatory cyto- kines/mediators (TNF- , IL- ⁇ , prostaglandin E2), the mature, antigen-loaded DCs are administered to the patient, which is frequently done by i.d. or i.n. injection. The (repeatedly) in ⁇ jected DCs are expected to induce antigen-specific immune re ⁇ sponses directed against the patient's tumour.
  • cytokines e.g. GM-CSF and IL-4
  • TNF- , IL- ⁇ , prostaglandin E2 e.g. inflammatory cyto- kines/mediators
  • ⁇ jected DCs are expected to induce antigen-specific immune re ⁇
  • Fig. 9 Graphic representation summarising important aspects of the present invention.
  • the schematic presentation summarizes the most important aspects of the invention:
  • lymphocytes including T cells and NK cells.
  • Both lymphocytes and APC produce multiple pro ⁇ inflammatory cytokines.
  • Activated lymphocytes also exhibit po ⁇ tent cytotoxicity against tumour cells.
  • tu ⁇ mor cell destruction (“lysis") tumour antigens and heat shock proteins are released.
  • lysis tu ⁇ mor cell destruction
  • tumour antigens and heat shock proteins are released.
  • the resulting "cocktail” containing tu ⁇ mour antigens, pro-inflammatory cytokines and heat shock pro ⁇ teins meets all requirements of a cancer vaccine.
  • the "cocktail” can also be used for the simultaneous antigen-loading and matu ⁇ ration of non-exhausted DCs.
  • targeting of CDlb on APCs also leads to the expansion of T cells, particu ⁇ larly ⁇ T cells, as well as NK cells by cell proliferation.
  • the expanded T cells and NK cells can be adoptively transferred to the patient, where they can exhibit potent antitumour activity.
  • Targeting of CDlb can strongly enhance pre-existing antigen- specific immune responses, for instance, against a vaccine anti ⁇ gen, and thus serves as potent adjuvant.
  • the measurement of pro ⁇ liferative, cytokine and cytotoxic responses after stimulation with anti-CDlb with or without IL-2 may be used to determine (diagnose) the immune status (immune competence) of healthy per ⁇ sons and patients.
  • Peripheral blood, bone marrow, umbilical cord blood may be typical sources of blood for PBMC preparation. Considering the advantages of accessibility and minimal invasiveness, the use of peripheral blood is preferable. It is preferable to collect a relatively small amount of blood that does not burden the donor. As a method of collection, whole blood can be collected utiliz ⁇ ing a vacuum blood tube. Heparin or citric acid may be added to prevent blood coagulation. In cases where large numbers of cells are required, PBMCs can be directly obtained by use of a compo ⁇ nent collection system such as leukapheresis .
  • PBMCs are then separated from whole blood or from leukapher ⁇ esis products.
  • Any method for separating mononuclear cells may be used.
  • the Ficoll separation method i.e., a Fi- coll-Paque density gradient is frequently used.
  • LSM1077 Lymphocyte Separation Medium PAA, Pasching, Austria
  • Leucosep greyiner bio-one, Frickenhausen, Germany
  • PBMCs peripheral blood mononuclear cells
  • PBS phosphate buffered saline
  • the CD56-positive subset may also be isolated from PBMCs, since this subset is enriched in NK cells as well as ⁇ T cells and also contains a population of DC-like APCs, which are suita ⁇ ble for the activation of T cells and NK cells (Gruenbacher et al., Blood 114 (2009), 4422-4431; Gruenbacher et al . , Cancer Res. 70 (2010), 9611-9620; Nussbaumer et al . Blood (2011), in press) .
  • a method to isolate and collect CD56-positive cells uti- lizing the Magnetic Cell Sorting is preferred since it is simple and reliable and results in pure populations of CD56-positive PBMCs (>90%) .
  • CD56 microbeads are used in combi ⁇ nation with LS columns according to the manufacturer's instruc ⁇ tions (MACS; Miltenyi Biotec, Bergisch-Gladbach, Germany).
  • Example 2 Cell stimulation for NK cell and T cell activation
  • Antibodies against CD1 antigens which are expressed by DCs, are used. Above all, antibodies against CDlb are particularly preferable. Examples include WM-25 IgGl against CDlb and B-B5 IgGl against CDlb/c (Favaloro et al . , 1987; Favaloro et al . , 1986; Dzionek et al . , 2000).
  • the whole PBMCs or the CD56-positive PBMCs obtained are re- suspended preferably at cell numbers of 1.0-1.5 x 10 6 /ml in a cell culture medium such as RPMI-1640 (Lonza, Basel, Switzer ⁇ land), AIM-V (Invitrogen, Carlsbad, CA, USA) or CellGro SCGM (CellGenix, Freiburg, Germany) .
  • the cell culture medium may be further supplemented with autologous or clinical quality heter ⁇ ologous serum or plasma at 1-20%.
  • the culture vessels used which may be a plate, laboratory dish, flask, bag typically used for cell cultivation.
  • An antibody directed against CDlb (clone B-B5 from Diaclone, Besancon, France or clone WM25 from ImmunoKontact via AMS Bio ⁇ technology, Lugano, Switzerland) is added to these cultures.
  • the final concentration of the CDlb antibody in the cell culture to be stimulated is preferably 1-20 yg/ml.
  • a T cell growth factor preferentially interleukin-2 (IL-2, Proleukin, Novartis, Basel, Switzerland), is added to the cultures.
  • IL-2 concentration of 10-1000 U/ml can be used, with 100 U/ml being preferable.
  • NK cells and T cells both ⁇ T cells and ⁇ T cells
  • IFN- ⁇ which is a pro- totypic cytokine of effector cells and crucial for the defence of pathogens and tumours, can be detected intracellularly in T and NK cells and together with various other cytokines during the following days also in the supernatant using cytokine bead arrays (Thl/Th2 or Inflammation CBA; BD Biosciences) or the Human Thl/Th2/Th9/Thl7/Th22 13plex Kit (eBiosciences , San Diego, CA, USA) .
  • the mixture of activated NK cells and T cells can it ⁇ self be considered a pharmaceutical composition, which can be administered to a patient with cancer or an infectious disease for general immune enhancement.
  • the mixture of activated NK cells and T cells can be further processed to be ⁇ come a) a cancer vaccine, b) a "cocktail" for the simultaneous antigen loading and maturation of dendritic cells in vitro, or c) a preparation of expanded T cells, particularly ⁇ T cells, and NK cells.
  • Example 3 Generation of a cancer vaccine
  • tumour cells Stimulation of PBMCs or subsets thereof is performed as de ⁇ scribed above in Example 2 (Cell stimulation for NK cell and T cell activation) and between day 0 and day 10 activated cells are seeded onto tumour cells growing in an appropriate cell cul ⁇ ture vessel at a PBMC to tumour cell ratio of 10:1 to 1:1.
  • the tumour cells may either be autologous, i.e. primary tumour cells derived from the patient to be treated, or represent a permanent cell line derived from another individual but of the same tumour type, i.e. allogeneic tumour cells.
  • tumour cells may be ir ⁇ radiated ( 137 Cs) at a dose of about 30 to 100 Gray, which leaves them intact but prevents growth of tumour cells potentially sur ⁇ viving the attack of the activated NK and T cells.
  • the activated NK cells and T cells in the PBMC preparation will attack, kill and lyse the tumour cells.
  • Tumour cell lysis is monitored by mi ⁇ croscopic inspection and by measuring the release of tumour- associated peptides such as M30 or Hsp70 using specific ELISAs (M30 Apoptosense ELI SA from PEVIVA AB, Bromma, Sweden; HSP70 ELISA from ENZO Life Sciences /Eubio , Vienna Austria) .
  • NK cell and T cell activation is monitored by measuring multiple cytokines at once using cytokine bead arrays (Thl/Th2 or Inflammation CBA; BD Biosciences) or Human
  • Thl/Th2/Th9/Thl7/Th22 13plex Kit (eBiosciences , San Diego, CA, USA) .
  • the tumour cells will be destroyed completely and the resulting tumour cell extract ("cocktail") , which con ⁇ tains a representative mixture of tumour-associated antigens and variety of pro-inflammatory cytokines, can be administered to the cancer patient as a potent, immunogenic cancer vaccine.
  • the "cocktail" will be divided into equal por ⁇ tions (i.e. aliquots) , which can be stored frozen at -20°C to - 180 °C for many months or even years and thawed for the repeti ⁇ tive administration (i.e. vaccination) of the patient.
  • Administration occurs by intradermal or subcutaneous injection, or by injection into a lymph node adjacent to a lesion, or even by direct injection into a lesion.
  • Example 4 Generation of an immunogenic tumour cell extract for simultaneous loading and maturation of DCs in vitro
  • the resulting "cocktail” which contains a representative mixture of tumour-associated antigens and variety of pro-inflammatory cytokines, can be used for sim ⁇ ultaneous loading and maturation of DCs in vitro.
  • in vitro generated DCs at the immature stage (compare Fig. 8; Steinman et al . , 2007) and at a cell density of about 0.5-1.0 x 10 s cells/ml are incubated with appropriate doses of the "cock ⁇ tail", for instance, by removing half of the DC medium and re ⁇ placing it by "cocktail".
  • the DCs use their efficient endocytic mechanisms to pick up, process and present the tumour antigens contained in the "cock ⁇ tail".
  • the abundant pro-inflammatory cytokines contained in the "cocktail” will promote DC maturation, which can be confirmed by measuring the up-regulation of the reliable mat ⁇ uration marker CD83 (clone HB15e-PE, BD Biosciences) using flow cytometry (Gruenbacher et al . , 2009).
  • Mature, antigen-loaded DCs can then be administered as a DC-based tumour vaccine by intra ⁇ dermal or intranodal injection as described previously (Holtl et al., Clin. Can. Res. 8 (2002), 3369-3376).
  • Example 5 Expansion T cells, particularly ⁇ T cells, in vitro
  • PBMCs are prepared from a donor as described in Example 1 and stimulated for 5 days as described in Example 2. Every 2 days, half of the cell culture medium is replaced by fresh medi ⁇ um containing IL-2.
  • the activated cells will form aggregates and T cells, particularly ⁇ T cells, but also NK cells will start to proliferate.
  • the cultures will be split.
  • the cells of a culture well are resus- pended and half of the suspension is transferred to a fresh well. Both wells are filled up to the original volume with fresh medium containing IL-2. Following this protocol, the cells can be expanded for 10 to 20 days.
  • the numbers of ⁇ T cells as de ⁇ termined by multicolour flow cytometry (Gruenbacher et al . , 2009) initially present in PBMC preparations will increase about 10-100-fold using this procedure.
  • the present invention enables the generation of a cell popu ⁇ lation containing activated ⁇ T cells in large quantities and in a simple fashion.
  • These activated ⁇ T cells can directly be used in adoptive immunotherapy and can thus be regarded as a pharmaceutical composition.
  • After washing the expanded cells they can be administered to a patient with, for instance, cancer or an infectious disease. Any washing solution may be used as long as it is isosmotic and suitable for use as a pharmaceutical preparation.
  • the use of Ringer Lactate (Baxter, Deerfield, IL, USA) , physiological saline, PBS or the like is preferable.
  • a clinical grade cytokine such as IL-2 (Proleukin) may al ⁇ so be added.
  • the number of cells administered will be selected depending on the actual purpose. Typically, however, the number of cells is preferably 1-10 x 10 s cells/person, when ⁇ T cells serve as APCs and 1 x 10 8 cells to 1 x 10 10 cells/person, when ⁇ T cells serve as antitumour effector cells, ⁇ T cells can be adminis ⁇ tered by intravenous, intradermal or subcutaneous injection, in ⁇ jected into a lymph node adjacent to a lesion, directly injected into a lesion, given as an intravenous bolus or drip-fed for systemic administration (Holtl et al . , 2002). The preparation can also be injected into an artery next to a lesion.
  • the activated and expanded ⁇ T cells are expected to have strong therapeutic effects by exhibiting direct (cyto ⁇ toxic) and indirect antitumour or anti-pathogen effects (by ex ⁇ hibiting immunomodulatory effects and APC function) .
  • Example 6 Use of anti-CDlb antibody for in vitro stimulation as part of an immunologic test kit
  • PBMCs are prepared from a donor as described in Example 1 and stimulated for 1-5 days as described in Example 2.
  • immunologic test kits are market ⁇ ed, which enable the activation and/or expansion of white blood cells.
  • Anti-CDlb antibodies alone or in combination with other stimuli such as cytokines may be part of such test kits in order to induce the activation and/or expansion of NK cells as well as T cells including ⁇ T cells.
  • PBMCs prepared from any donor would be stimulated with anti- CDlb antibody plus IL-2 (Example 2) followed by a particular read out that may be, but is not restricted to, a cytokine meas ⁇ urement (Example 2), phenotypic analysis or cell expansion (Ex ⁇ ample 5) .
  • the CDlb antibody itself can be considered a pharmaceutical composition.
  • the antibody can be adminis ⁇ tered to patient in vivo with or without a second stimulus such as clinical grade IL-2 (Proleukin) .
  • IL-2 Proleukin
  • the antibody can bind to CDlb-expressing APCs in vivo and induce im ⁇ mune enhancement analogous to the in vitro examples described above (Examples 2 to 5) .
  • Such administration of anti-CDlb anti ⁇ body will lead to the activation of NK and T cells including ⁇ T cells and thus in immune enhancement, which is desirable in the treatment of infectious diseases or cancer.
  • Example 8 CDlb as an adjuvant of a vaccine
  • the (humanized) CDlb antibody could be admixed to any vaccine antigen (either microbial or tu ⁇ mour-associated antigens) .
  • the final vaccine formulation con ⁇ sisting of the antigen preparation and the anti-CDlb antibody (adjuvans) would be administered, typically as an intradermal or subcutanteous injection.
  • the anti-CDlb antibody can bind to CDlb-expressing APCs and induce immune enhancement analogous to the in vitro examples described above (Examples 2 to 5) . This immune enhancement will generate a stimulatory milieu and favour a potent immune response against the vaccine antigen.
  • Example 9 Diagnosing the immune status/immune competence of a patient
  • PBMCs are prepared from a donor as described in Example 1 and stimulated for 1-5 days as described in Example 2.
  • im ⁇ mune status im ⁇ mune competence of a patient is prerequisite for selection of this patient for an immunotherapy regimen since lack of immune competence would result in immunotherapy failure.
  • PBMCs are prepared from peripheral blood of a patient (Example 1) at appropriate time points (before, during and after a certain intervention) and stimulated with anti-CDlb plus IL-2 (Example 2) .
  • Cytokine levels as assessed by ELISA or multiplexing technology, activa ⁇ tion of individual cell subsets and expansion of, for instance, ⁇ T cells (Example 5) represent a measure of immune competence, which can be correlated with the dosing and duration of the treatment and/or the course of the disease (prognosis) .
  • Fig. 1 demonstrates that antibody-mediated targeting of CDlb cooperated with recombinant IL-2 in a highly synergistic fashion to induce the production of very large amounts of IFN- ⁇ by human PBMCs.
  • Antibodies against other CD1 antigens such as the anti- CDla antibodies OKT-6 and HI149 or the CDlc specific antibodies M-T101, L161 failed to induce such cytokine responses in human PBMCs .
  • LPS endotoxin
  • LPS concentra ⁇ tions of the CDlb antibody preparations WM25, B-B5, SN13/K5- 1B8 as determined by the Limulus Amebocyte Lysate (LAL) assay [Limulus Amebocyte Lysate QCL-1000, CAMBREX] were generally low ( ⁇ 10 ng/ml at an antibody concentration of 10 yg/ml) .
  • LPS effects can be prevented by using the LPS-binding agent polymyxin B (PMB) .
  • PMB polymyxin B
  • IgG antibodies may not only generate effects by binding to their specific antigen but also via their Fc ⁇ portion, which can trigger Fc ⁇ receptors expressed on other cells.
  • Octagam hu ⁇ man IgG (Octapharma) was used in all experiments at 50 yg/ml to block Fc Y receptors.
  • an azide-free IgGl anti-HLA-DR antibody was used as an isotype control antibody that also binds to APCs and can thus present its Fc proportion to Fc receptor-expressing cells.
  • IB demonstrates that anti-HLA-DR failed to reproduce the effects observed with anti-CDlb thus excluding the possibility that any IgGl that binds to APCs can trigger such PBMC activation.
  • an Fc Y receptor blocking antibody (anti-CD16) did not re ⁇ cute the response induced by anti-CDlb antibodies unequivocally excluding such non-specific Fc ⁇ effects.
  • Fig. 2 demonstrates that antibodies against CDlb (WM-25, B- B5, SN13/K5-1B8) induced IFN- ⁇ production in cultures of human PBMCs .
  • CDlb antibodies cooperated with IL-2 but not with other members of the common ⁇ chain family of cytokines (IL-4, IL-7, IL-15) to produce very large amounts of IFN- ⁇ (Fig. 2A) .
  • Table 2 the cyto ⁇ kine response was not restricted to IFN- ⁇ but also included the production of IL-17, IL-22, TNF- , IL- ⁇ and IL-6.
  • Fig. 3 illustrates the role of APCs and thus confirms the specificity of anti-CDlb effects.
  • CDlb expression is restricted to APCs, which express high levels of HLA-DR (MHC class II) for presentation of peptide antigens.
  • HLA-DR MHC class II
  • all HLA- DR-positive cells were depleted using HLA-DR microbeads prior to stimulation with anti-CDlb antibody. Depletion of HLA-DR- positive cells eliminates all CDlb-positive cells and thus elim ⁇ inates all potential target cells of the anti-CDlb antibodies.
  • T cells and NK cells are major sources of IFN- ⁇ .
  • T cells CD3-positive cells
  • NK cells CD56-positive cells
  • the concomitant activation of APCs as evidenced by mono ⁇ kine production indicates that mutual activation of lymphocytes and monocytes/DCs occurs during CDlb targeting and IL-2 co-stimulation.
  • Fig. 4 reveals IFN- ⁇ production in various lymphocyte sub ⁇ sets of human PBMCs after stimulation with anti-CDlb antibody in combination with IL-2.
  • intracellular staining of IFN- ⁇ was performed after 24 h of stimulation with anti-CDlb antibody in combination with IL-2.
  • Cells were counterstained for TCRV52 to identify ⁇ T cells and for CD3 to distinguish T cells (CD3+ ) from NK cells (CD3-) .
  • Fig. 4 convincingly shows that anti-CDlb plus IL-2 was very potent in inducing IFN- ⁇ production in all these subsets ( ⁇ T cells, other T cells, NK cells) .
  • Fig. 5 demonstrates that treatment of human PBMCs with anti- CDlb antibody in combination with IL-2 induces massive expansion of ⁇ T cells.
  • PBMCs day 0
  • 5% to 10% of all T cells were ⁇ T cells expressing a V52 TCR, which is the TCR required for the recognition of bisphosphonate-induced phos- phoantigens such as IPP.
  • Treatment with IL-2 alone for 14 days failed to induce V52-positive T cell expansion.
  • V52-positive T cells represented 50% to 70% of all cells (Fig. 5) actually resulting in an about 50 to 100-fold expansion, when the increase in absolute cell number was also considered.
  • ⁇ T cell ex ⁇ pansion becomes less efficient, because protein isoprenylation is inhibited resulting in the attenuation of cell proliferation.
  • bisphosphonates it is a choice between ⁇ T cell proliferation (at lower concentration) and ⁇ T cell effector responses (IFN- ⁇ production, cytotoxicity).
  • ⁇ T cell expansion (Fig. 5B) and IFN- ⁇ production (Fig. 2A) both increase with anti-CDlb concentration representing a clear advantage of anti-CDlb as opposed to bisphosphonates.
  • Fig. 6 shows potent cytotoxicity of human PBMCs activated with anti-CDlb and IL-2 directed against human tumour cells. Then the cytotoxic antitumour effects of PBMCs activated with anti-CDlb plus IL-2 were studied.
  • A-498 cells a well-characterized example of human renal cell carcinoma (RCC) , were cultured in 48-well plates. PBMCs were then added and stimulated with anti-CDlb and IL-2.
  • the "conditioned” culture medium finally contained copious amounts of pro-inflammatory cytokines (Fig. 6A and Table 4), adjuvant heat shock proteins (Fig. 6B) , as well as a variety of tumour antigens present in the tumour lysate (Fig. 6C) thus fulfilling all the requirements of a potent cancer vac ⁇ cine.
  • the "conditioned" culture medium can also be used to treat dendritic cells in vitro.
  • tumour antigens present in the "conditioned" culture medium can be loaded onto dendritic cells also with the support of the endogenous heat shock proteins and b) the pro ⁇ inflammatory cytokines present in the "conditioned" culture me ⁇ dium will induce dendritic cell maturation, which is characterized by the up-regulation of the maturation marker CD83 without the drawbacks of DC exhaustion (Fig. 6D) .
  • anti ⁇ gen-loaded, mature dendritic cells can be generated and subse ⁇ quently administered to the tumour patient for the induction of antitumour immune responses in vivo.
  • Fig. 7 demonstrates that CDlb targeting can enhance antigen- specific, MHC-restricted T cell responses.
  • PBMCs from renal can ⁇ cer patients who had been vaccinated with KLH-loaded DCs (Holtl et al . , 2002), were used.
  • KLH Keyhole limpet hemocyanin
  • the T cell response to KLH elicited by DC vaccination has previ ⁇ ously been shown to be predominated by MHC class II-restricted CD4 + T cells.
  • Postvaccination PBMCs were stimulated with KLH in the presence or absence of anti-CDlb antibody.
  • KLH alone induced a proliferative response confirming KLH-specific immunity in ⁇ quizd by DC vaccination.
  • CDlb targeting alone induced proliferative responses similar to those observed in healthy do ⁇ nors and anti-CDlb enhanced KLH-induced proliferation in a mod ⁇ erately synergistic fashion (Fig. 7A) .
  • CDlb targeting and KLH stimulation alone induced both low amounts of IFN- ⁇ at compara ⁇ ble levels (Fig. 7B) .
  • IL-2 (2500/mI) 558,4 8.016,0 50.5 182 374,7 376,6 with tumor anti-CD 1b (5pg/m! > 1000 9.745,5 87,6 > 250.000 1.008,7
  • GM-CSF granulocyte/macrophage-colony-stimulating factor
  • Method for providing a preparation of stimulated cytotoxic T cells and NK cells comprising the following steps:
  • CDlb-positive (CDlb + ) antigen presenting cells providing a preparation of CDlb-positive (CDlb + ) antigen presenting cells
  • PBMCs peripheral blood mononuclear cells
  • Method according to embodiment 1 or 2 characterised in that cultivation is performed for at least 6, preferably for at least 12, especially for at least 16 hours.
  • charac ⁇ terised in that the preparation of CDlb + antigen presenting cells is obtained from peripheral blood, bone marrow or umbilical cord blood, especially peripheral blood.
  • charac ⁇ terised in that the stimulated cytotoxic T cells and NK cells are washed with an isosmotic T cell washing solution, especially saline and phosphate buffered saline (PBS) .
  • an isosmotic T cell washing solution especially saline and phosphate buffered saline (PBS) .
  • an injectable pharmaceutical formulation espe ⁇ cially an intradermally, intravenously, intraarterially or sub- cutaneously injectable formulation or a formulation to be injected into a lymph node.
  • charac ⁇ terised in that cultivation comprises splitting of the prepara ⁇ tion containing cultured stimulated cytotoxic ⁇ T cells and preferably addition of culture medium and/or further cultivating the preparation.
  • charac ⁇ terised in that the cultivation is performed for a time to in ⁇ crease the number of T cells, especially ⁇ T cells, by a factor of at least 10, preferably of at least 50, especially at least 100.
  • the anti-CDlb antibody is a CDlb-binding IgG, IgM, IgA, IgD or IgE antibody or a CDlb-binding fragment there ⁇ of, especially an Fab, F(ab' )2 or Fv; or an antibody derivative selected from CDlb-binding bi-, tri- or multispecific antibody, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, or diabody.
  • IFN- ⁇ interleukin-17A
  • IL-17A interleukin-17A
  • 21. Preparation according to any one of embodiments 16 to 20, characterised in that it contains added active substances, pref ⁇ erably at least one tumour antigen, especially a preparation of lysed tumour cells; at least one cytokine, especially IL-2; or mixtures thereof. 22. : Preparation according to any one of embodiments 16 to 21, characterised in that it contains 1 to 10 x 10 s ⁇ T cells with antigen-presenting capacity or 1 x 10 s to 1 x 10 8 ⁇ T cells as effector cells per dose.
  • tumour cells especially autologous tumour cells of a cancer patient
  • tumour cells are inhibited in growth, preferably by irradiation, chemotherapy drugs or combinations thereof.
  • Method according to embodiment 27 or 28 characterised in that the preparation of white blood cells was obtained from a patient whose immune status has to be monitored and the method is used for monitoring the immune status of this patient.
  • the same one or more phenotypic properties are measured in the at least two preparations of white blood cells, and the measured phenotypic properties of the at least two prep ⁇ arations are compared with each other to monitor the immune status of this patient.
  • Kit for stimulation of white blood cells comprising
  • a pharmaceutical composition comprising an anti-CDlb- antibody, preferably for use in immune stimulation, especially for the treatment of infectious diseases or tumour diseases.
  • composition according to embodiment 32 char ⁇ acterised in that it comprises a vaccine antigen, especially a vaccine antigen for vaccination against an infectious disease or a tumour vaccination antigen.

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Abstract

Cette invention concerne une méthode de préparation de cellules tueuses naturelles et de lymphocytes T stimulés cytotoxiques (αβ et γδ), ladite méthode comprenant les étapes consistant à : - fournir une préparation contenant des cellules présentant l'antigène CD1b-positif (CD1b+) et des lymphocytes répondeurs (lymphocytes T et cellules tueuses naturelles), - stimuler la préparation de cellules présentant l'antigène CD1b+ et de lymphocytes répondeurs avec un anticorps anti-CD1b et l'interleukine 2 (IL-2), et - cultiver les cellules présentant l'antigène CD1b+ et les lymphocytes répondeurs stimulés dans un milieu de culture pour obtenir des cellules tueuses naturelles et des lymphocytes T stimulés cytotoxiques (αβ et γδ).
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