EP0941309A1 - Adjuvant cellulaire - Google Patents

Adjuvant cellulaire

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Publication number
EP0941309A1
EP0941309A1 EP97913011A EP97913011A EP0941309A1 EP 0941309 A1 EP0941309 A1 EP 0941309A1 EP 97913011 A EP97913011 A EP 97913011A EP 97913011 A EP97913011 A EP 97913011A EP 0941309 A1 EP0941309 A1 EP 0941309A1
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EP
European Patent Office
Prior art keywords
cells
interferon
antigen
csf
day
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP97913011A
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German (de)
English (en)
Inventor
Thomas Luft
Kenneth Pang
Elizabeth Le Thomas
Jonathan Simon Cebon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research New York
Original Assignee
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research New York
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Application filed by Ludwig Institute for Cancer Research Ltd, Ludwig Institute for Cancer Research New York filed Critical Ludwig Institute for Cancer Research Ltd
Publication of EP0941309A1 publication Critical patent/EP0941309A1/fr
Withdrawn legal-status Critical Current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
    • 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
    • A61K39/4615—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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
    • 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
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]
    • 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
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)

Definitions

  • This invention relates to a cellular adjuvant and to a method of production thereof.
  • the invention relates to activation of dendritic cells to mature, functional dendritic cells for use as a cellular adjuvant in treatment of neoplastic disease.
  • DC are usually identified by their typical morphology, characterized by the presence of dendrites or membrane processes, and their characteristic surface antigen expression pattern (positive for CDla, CDllc, CD40, CD80, CD83, CD86, HLA-A,B,C and HLA-DR, and negative for mature leukocyte markers of other lineages) (Macatonia et al , 1991; Szaboles et al , 1995; Caux et al , 1992).
  • DC can be purified from peripheral blood (Steinman and Young, 1991) . They can also be grown in culture from peripheral blood mononuclear cells (PBMC) in the presence of cytokines such as granulocute/macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) (Romani et al , 1994; Inaba et al , 1992).
  • PBMC peripheral blood mononuclear cells
  • cytokines such as granulocute/macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) (Romani et al , 1994; Inaba et al , 1992).
  • GM-CSF granulocute/macrophage-colony stimulating factor
  • IL-4 interleukin-4
  • Another technique for growing DC involves harvesting CD34 + cells from umbilical cord blood, bone marrow or cytokine-mobilized peripheral blood progenitor cells and culturing these cells in GM-CSF and tumour necrosis factor-alpha (TNF- ⁇ ) with or without IL-4 (Szabolcs et al , 1995; Caux et al , 1992; Romani et al , 1994; Santiago et al , 1992; Mackensen et al , 1995; Siena et al , 1995; Herbst et al , 1996; Strunk et al , 1996) .
  • SCF stem cell factor
  • FCS foetal calf serum
  • Caux et al 1992; Mackensen et al , 1995; Siena et al , 1995
  • pooled human serum Santiago Schwarz et al , 1992; Siena et al , 1995; Santiago Schwarz et al , 1995
  • DC that differentiate from cultured CD34 + cells would be expected to take up and process proteins derived from serum present in the medium.
  • donor serum may also constitute an infectious risk for patients if DC are to be used clinically, and the use of xenogeneic serum is usually considered to be undesirable.
  • serum contains growth factors, and thus its presence might interfere with the study of the role of cytokines in DC differentiation and maturation, even though this may not be of great relevance to ultimate production of a cellular adjuvant for clinical use.
  • DC which function as potent APC for both allogeneic T lymphocytes and autologous, peptide-specific CTL, using CD34 + progenitors under serum-free conditions.
  • CD34 + progenitors under serum-free conditions.
  • the invention provides a method of inducing the maturation of dendritic cells in vi tro, comprising the step of culturing mononuclear cells in a serum-free medium in the presence of a Type I interferon following an initial phase of growth in the presence of
  • the mononuclear cells are CD34 + haematopoietic progenitor cells.
  • the cells which are cultured may be peripheral blood progenitor cells, for example obtained by leukapheresis, or bone marrow progenitor cells. More preferably the starting cell population is also enriched for CD34 + cells prior to culture.
  • the cells are cultured for 14 to 35 days, more preferably from 14 to 28 days, and even more preferably for 14 to 17 days.
  • GM-CSF, TNF- ⁇ are present in the culture medium for the whole period of culture, and preferably the Type I interferon is present commencing at day 13 of culture.
  • IL-4 is present commencing at day 6, or is present for the whole period of culture.
  • Any Type I interferon may be used, for example interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , or consensus interferon (Infergen; Amgen, Inc., Thousand Oaks, California) .
  • the interferon is IFN- ⁇ 2a, IFN- ⁇ 8 or IFN- ⁇ .
  • peripheral blood mononuclear cells are to be used, it may be advantageous to pre-treat the patient or animal from which the cells are to be obtained with a cytokine such as G-CSF or GM-CSF prior to leukapheresis in order to mobilise bone marrows stem cells into the circulation. Enrichment of between 10- and 1000-fold of stem cells in the circulation is desirable. In the absence of such priming, there may be insufficient stem cells in the circulation, and bone marrow cells are then preferably used.
  • a cytokine such as G-CSF or GM-CSF
  • the cells may also be cultured in the presence of stem cell factor (SCF) .
  • SCF stem cell factor
  • SCF stem cell factor
  • Maturation of the dendritic cells may be monitored using morphological markers such as the presence of dendritic processes at the cell surface, and the presence of immunological markers such as CD40, CD80, CD83, CD86, and CMRF44.
  • the matured dendritic cells produced by the method of the invention are non-phagocytic, and have potent antigen-presenting activity. They are capable not only of stimulating multiplication of allogeneic lymphocytes in the mixed leukocyte reaction (MLR) , but also of stimulating autologous, peptide-specific cytotoxic T lymphocytes in the mixed leukocyte peptide culture assay system (MLPC) . Therefore in a second aspect the invention provides a method of enhancing the antigen-presenting capacity of dendritic cells, comprising the step of exposing dendritic cells to a Type I interferon. Any Type I interferon can be used, as described above. Interferon can be administered in vivo, in order to activate either the recipient's own resident dendritic cells, including
  • interferon is administered in vivo it may be delivered at the site of dendritic cell administration, as set out below or may be administered in such a way as to spread systemically through the body.
  • the interferon it may be given in conjunction with other cytokines which induce proliferation of DC; for example interferon may be given in conjunction with, ie sequentially or simultaneously with, GM-CSF or GM-CSF and IL-4.
  • dendritic cells are exposed to interferon in vi tro, as described above.
  • the invention provides a method of preparation of a cellular adjuvant for treatment of a neoplastic disease or a disease caused by an infectious agent, comprising the steps of maturing dendritic cells in vi tro as described above, and exposing the matured dendritic cells to an antigen derived from the neoplastic cells (ie. a tumour-associated antigen) or from the infectious agent, respectively.
  • the infectious agent may be a bacterium, a virus, a yeast or a parasite.
  • the antigen may be a protein, peptide, polysaccharide, or nucleic acid.
  • the dendritic cells may be transfected with the antigen, or may be incubated in vi tro with the antigen.
  • the cellular adjuvant of the invention may be used in the treatment of a neoplastic disease, in order to provoke or stimulate an anti-tumour response.
  • the invention provides a method of treatment of neoplastic disease, comprising the step of administering an effective dose of the cellular adjuvant according to the invention to a patient in need of such treatment .
  • matured dendritic cells according to the invention may be administered to the patient together with the antigen. Any suitable route of administration may be used, for example
  • the dendritic cells are autologous, ie. from the patient.
  • the neoplastic cell antigen may also be derived from the patient's own tumour cells.
  • the cellular adjuvant of the invention may be used in the treatment of disease caused by an infectious disease, using an antigen derived from the infectious agent as discussed above.
  • the method of the invention may also be used to generate clones of activated tumour-associated T cells.
  • the invention provides a method of producing a population of activated tumour-associated T cells, comprising the steps of harvesting dendritic cells and lymphocytes from peripheral blood or bone marrow of a patient suffering from a tumour, maturing dendritic cells according to the method described above, and culturing the dendritic cells and the lymphocytes, either separately or together, in the presence of a tumour-associated antigen, and optionally in the presence of a cytokine.
  • the cytokine is a type I interferon, more preferably interferon- ⁇ .
  • This aspect of the invention also provides a method of treatment of a patient suffering from a tumour, comprising the step of administering an effective dose of dendritic cell-activated, tumour-associated T cells to a patient in need of such treatment.
  • this aspect of the invention provides a method of treatment of a pathological condition, comprising the step of administering an effective dose of mature dendritic cells having the functional and antigenic characteristics as defined herein to a mammal in need of such treatment, together with or subsequently to adminstration of an antigen associated with the condition.
  • the pathological condition may be a neoplastic disease, in which case the antigen is derived from neoplastic cells alternatively the condition may be an infectious disease, and the antigen is derived from the infectious agent .
  • the tumour antigen is a cancer-associated peptide such as Melan A, tyrosinase, GPlOO, or an antigen of the MAGE family, or a member of the family of cancer antigens, such as ESO-I or a cancer testis antigen detectable by the SEREX method.
  • the antigen may alternatively be derived from cancer cells or may be autologous, irradiated tumour cells, optionally in conjunction with GM-CSF.
  • Another important aspect of the invention provides a method of using a Type I interferon as an adjuvant for peptide-based anti-tumour vaccination strategies.
  • a Type I interferon is administered locally or systemically at the same time as, or 1 to 3 days after vaccination of a patient in need of such treatment with a tumour peptide or tumour cell lysate, optionally in conjunction with GM-CSF.
  • this aspect of the invention provides a method using a type I interferon as an adjuvant for vaccination against an infectious agent.
  • the infectious agent is a bacterium, a virus, a yeast or a parasite.
  • Figure 1 shows a comparison of antigen expression and morphology of cultured cells after 14 and 28 days of culture
  • Figure 2 shows the immunophenotype of DC after 28 days in serum-free culture. Analysis of DC by flow cytometry at day 28.
  • Figure 3 shows the assessment of antigen- presenting function of mature DC.
  • Thymidine incorporation after 5 days was measured and divided by the Thymidine uptake of responder cells alone to calculate the stimulation index.
  • TNF- ⁇ (20 ng/mL) .
  • Figure 8 shows the morphology of sorted CDla + DC and CDla " cells at day 14 A) Early DC stages showed temporary plastic adherence. Phenotype of CDllb righc DC at day 14, 24 hours after sorting. Original magnification (OM) xl80.
  • Ill mononuclear, non-phagocytic cell. The bar indicates 1.8 ⁇ m.
  • Figure 9A shows the DC surface phenotype on days 14 and 28 by flow cytometry.
  • CD34 + cells were cultured in GM-CSF, TNF-a and IL-4 and analyzed by flow cytometry after 14 days and 28 days. Cultures were gated to examine only the large cells (20% of total cells) .
  • Figure 10 shows the differentiation of subpopulations sorted at day 13.
  • CDllb + cultured until day 18 in GM-CSF, TNF- ⁇ and IL-4.
  • Figure 11 shows the allostimulatory capacity of DC cultured in GM-CSF, TNF- ⁇ and IL-4 for days 14 and 28 using mixed leukocyte reaction
  • the allostimulatory capacity of 14 individual unsorted DC cultures was directly compared after 14-15 days ( ⁇ ) and 28 days (T) using the same allogeneic responder cells for both time points.
  • DC were cultured in GM-CSF,
  • TNF- ⁇ and IL-4 irradiated and co-cultured with 10 5 PBMC in triplicate wells. 3 H-thymidine uptake was measured after 5 days . Thymidine incorporation of responder cells alone was 1600 ⁇ 350 cpm. Results are shown as the mean ⁇ SE of 14 individual experiments (* p ⁇ 0.05, ** p ⁇ 0.01).
  • Figure 12 shows the superior effectiveness of mature Dendritic Cells expressing low levels of CDllb in stimulating CD8+ peptide-specific lymphocytes, compared to immature DC expressing high amounts of CDllb.
  • Figure 13 shows the effect of addition of human serum to cultures containing GM-CSF, TNF- ⁇ and IL-4 on the expression of CDla and CD86. Cultures were split 3 days before analysis and human serum was added into one half culture . A) Normal development of serum-free cultures: accumulation of a population of large sized, CDla+ DC which did not express CD86.
  • Figure 14 shows the screening for cytokines for activating effects on DC.
  • CD34 + cells were cultured in 96 well plates in GM-CSF, TNF- ⁇ and IL-4 (GTI) .
  • cytokines were added at the concentrations shown in Table 3.
  • Cultures were analyzed by flow cytometry at day 17.
  • the proportion of activated DC (HLA-DR +++ and/or CD86 + ) was calculated referring to the mean expression in multiple control cultures (GTI) as 100%. SDs were calculated for all protocols and a no-difference interval of two standard deviations above control levels of expression was chosen as a cut-off level for increased expression (shaded area) . Only human serum and IFN- ⁇ 2a were capable of inducing an increase >2SD in the proportion of activated DC in culture.
  • Figure 15 shows the concentration dependent effect of type I IFNs on DC maturation.
  • Figure 16 shows the induction of CD83 expression in DC cultures by IFN- ⁇ 2a.
  • Progenitor cells were cultured under standard serum-free conditions. On day 14 the culture was split and IFN- ⁇ 2a (1000 U/ml) was added into one half. FACS analysis was performed on day 17.
  • Vertical gate indicates fluorescence of 98% of cells with control antibody.
  • GT GM-CSF (50 ng/mL)+TNF- ⁇ (20 ng/mL).
  • GTI GM-CSF, TNF- ⁇ +IL-4 (500 U/mL) ) .
  • Figure 19 shows the allostimulatory capacity of DC activated by IFN- ⁇ in the same six cultures as in Figure 18.
  • Figure 20 shows the effect of IFN-a on migration and activation of skin-derived dendritic cells.
  • the serum-free medium X-Vivo 20 was purchased from BioWhit aker, Walkersville MD. Cell lines were grown in RPMI 1640 (Trace Biosciences, Melbourne, Australia) supplemented with 20 mM HEPES, 60 mg/1 penicillin G,
  • GM-SCF (10-200 ng/ml) (Schering-Plough, Sydney, Australia), SCF (100 ng/ml) (AMGEN, Thousand Oaks, CA) .
  • IL-4 (100-1000 U/ml) (Schering-Plough New Jersey, NJ) .
  • the following commercial monoclonal antibodies (mAb) were purchased: FITC-conjugated IgGl isotype control; OKT6, anti-CDla; PE-conjugated T4, anti-CD4; B3 , anti-CD22; IL-2RI, anti-CD25; MY9, anti-CD33; NKH1, anti-CD56;
  • AICD 58, anti-CD58; (Coulter Corp., FL) ;
  • FMC 17 anti-CD14 ; WMG1 , anti-CD15 ; FMC 63 , anti-CD19 ; Bl , anti-CD20 , UCHL1 , anti-CD45RO ; FMC 71 , anti-CD45RA; ICAM-1 , anti -CD54 .
  • the influenza matrix peptide residues 57 -68 : GILGFVFTL was a gift from Dr. P. Romero, Ludwig Institute for Cancer Research, Lausanne, Switzerland.
  • BM Cell Line Human bone marrow
  • leukapheresis harvest samples were obtained from normal donors and from patients of the Department of Medical Oncology and Clinical Haematology, Royal Melbourne Hospital, Melbourne.
  • Patients with non-Hodgkin' s lymphoma or solid tumours received stem cell-mobilizing chemotherapy and granulocyte colony- stimulating factor (G-CSF) as part of their treatment.
  • G-CSF granulocyte colony- stimulating factor
  • Rib segments removed during thoracotomy from patients with lung cancer were obtained from the Department of Surgery, Austin Hospital, Melbourne.
  • the human T2 cell line (Hosken and Bevan, 1990) was kindly provided by Dr. P. Romero, Ludwig
  • Mononuclear cells were prepared by density centrifugation on Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) . Using 14 gauge needles, rib segments were flushed with RPMI to mobilize BM cells. Residual red cells were lysed with red cell lysis buffer (7.79 g/L NH 4 C1, 0.037 g/L EDTA, and 1 g/L NaHC0 3 ) and CD34 + cells were separated with the MACS CD34 progenitor cell isolation kit (Miltenyi Biotec, Sunnyvale, CA) (Lansdorp, 1989) .
  • red cell lysis buffer 7.79 g/L NH 4 C1, 0.037 g/L EDTA, and 1 g/L NaHC0 3
  • CD34 + cells were separated with the MACS CD34 progenitor cell isolation kit (Miltenyi Biotec, Sunnyvale, CA) (Lansdorp, 1989) .
  • CD34 + fraction Purity of the CD34 + fraction was assessed by flow cytometry using HPCA-2 mAb (Lansdorp, 1989) and was consistently over 96%.
  • Cells (10 6 ml-4 x 10 6 ml) were cultured in 100 ⁇ l X-Vivo 20 with cytokines in 96 well microcultures (Nunc) . 50-100% fresh medium and cytokines were added every 2 to 3 days .
  • Proliferating confluent cultures were transferred with an Eppendorf pipette into progressively larger tissue culture plates (48 well plates (Falcon) , 24 well plates (Nunc) , 12 well plates (Flow Lab. VA) and 6 well plates (Nunc) ) .
  • Cytocentrifuge preparations were prepared by applying 10 4 cells to glass slides and spinning for 10 min at 300 rpm (Shandon, Cytospin 2). These were air-dried and stained with May-Grunwald/Giemsa stain.
  • PBMC Peripheral blood mononuclear cells
  • Betaplate scintillation counter (Wallac) .
  • DC or autologous PBMC (CD34 " fraction) of HLA-A2 + patients were pulsed with influenza matrix peptide (10 ⁇ g/ml) in the presence of ⁇ 2 -microglobulin (2.5 ⁇ g/ml, Sigma Aldrich, NSW) in X-Vivo 20 for 45 min at 37°C. Stimulators were washed and co-incubated with 10 6 autologous PBMC (CD34 " fraction) as responders in X-Vivo 20, 10% pooled human A serum and 10-20 U/ml IL-2 (Pepro Tech, Rocky, NJ) .
  • cultures were established in hanging drops 11 in Terasaki plates (Nunc, 20 ⁇ l drops containing approx. 10 5 cells per drop) . After 7 days, cultures were transferred into 96 well flat-bottomed plates and re-stimulated with peptide-pulsed, irradiated, autologous PBMC at day 7 and 14. After 3 to 4 weeks, cultures were examined for
  • CD8 + lymphocytes by staining with anti-CD8 mAb, and flow cytometry and 51 Cr release assays were performed, depending upon growth and expansion of responder cells.
  • responder cells from MLPC were assessed for peptide-specific lysis.
  • T2 cells were labeled with 100 ⁇ Ci of Na( 51 Cr)0 4 (Du Pont NEN) with 10 ⁇ l of W6/32 ascites for 2 hours at 37°C on a rotating stand. After 3 washes in RPMI, cells were incubated with 2.5 ⁇ g/ml ⁇ 2 -microglobulin ⁇ 1 ⁇ g/ml peptide in X-Vivo 20 for 1 hour at 37°C, washed once, and re-suspended in RPMI with 10% FCS at 10 4 cells/ml.
  • K562 is a Natural Killer cell (NK) -target and was added to inhibit nonspecific lysis by contaminating NK cells. l,000 51 Cr-
  • Cytokine deprivation and phagocytosis Bulk DC cultures and sorted DC were washed, then cultured in X-Vivo 20 with 10% human serum for 5 days in order to assess the stability of their phenotype in the absence of exogenous cytokines and their ability to phagocytic Candida albicans cells.
  • adherent PBMC were obtained by density centrifugation as described above from blood samples of normal volunteers and incubated for 1 hour at 37°C in X-Vivo 20 with 20% human serum (HS) . Non-adherent cells were removed, and remaining adherent cells incubated in the same medium for another 5 days .
  • FIG. 1 shows a brief summary of the phenotypic and morphological events which occur during days 14 and 28.
  • a proportion (10-25% of bulk culture cells) expresses CDla, a MHC-like surface antigen known to be expressed on epidermal Langerhans cells.
  • CDla a MHC-like surface antigen known to be expressed on epidermal Langerhans cells.
  • DC-associated markers like CD83 and CD86 are only expressed on a minority of the cells and gradually increase until day 28. This process is referred to herein as DC maturation .
  • Figure 1A shows the up-regulation of CD83 and CD86 and down-regulation of CDla on a subpopulation of large cells between days 14 and 28.
  • Figure IB depicts the corresponding morphological changes .
  • Panel I shows cells at day 14 which included a small percentage of DC which adhered to plastic surfaces (arrows) and were detached here by pipetting. At day 28, most DC were non-adherent and displayed a round phenotype with fine dendrites, as shown in panel II .
  • Phenotype of Mature DC at day 28 The surface antigen expression pattern of these mature large cells is shown in Figure 2.
  • the cells showed high expression of HLA-A,B,C, HLA-DR, CD40, CD54, CD58 and
  • CD86 S BSTITUTE SHEET RULE25
  • CD86 S BSTITUTE SHEET RULE25
  • CD86 did not express T and B cell markers such as CD2 , CD3 , CD5, CD8, CD19 and CD20, Fc receptors such as CD16 and CD32, nor monocyte-associated markers such as CD14 and CD45RA.
  • T and B cell markers such as CD2 , CD3 , CD5, CD8, CD19 and CD20, Fc receptors such as CD16 and CD32, nor monocyte-associated markers such as CD14 and CD45RA.
  • CD80, CD83, CD86, CMRF44 as well as adhesion molecules (CDlla, CDllc, CD54, CD58) are expressed, whereas markers of other lineages are lacking.
  • DC were tested in MLR and MLPC assays .
  • MLPC assays were performed using an HLA-A2 -restricted peptide (GILGFVFTL) derived from the influenza matrix protein ( Figure 3B) .
  • GILGFVFTL HLA-A2 -restricted peptide
  • Figure 3B HLA-A2 -restricted peptide
  • DC were sorted according to cell size to avoid blocking of functional receptors by antibodies (CDla, CD80, CD86) .
  • effector cells of one MLPC were sorted into CD8 + and CD8 " cells. Only the CD8 + fraction had the ability to lyse peptide-pulsed target cells.
  • DC are characterized functionally as non-phagocytic, potent antigen-presenting cells (APC) , stimulating allogeneic lymphocytes in the MLR, and autologous peptide-specific cytotoxic T-lymphocytes in the MLPC.
  • APC potent antigen-presenting cells
  • GM-CSF, TNF- ⁇ and/or IL-4 were titrated over a range of concentrations.
  • DC development was quantified using the expression of CDla at day 14 and of CD86 at day 28. Day 14 Although not sufficient to stimulate
  • IL-4 no CD14 " , CDllb +++ , CDla + cells developed in significant numbers during the first 2 weeks (not shown) .
  • CD34 + cells Different sources of CD34 + cells were compared. These included leukapheresis harvests, BM aspirates and rib fragments from normal donors as well as from cancer patients. The yield of CD34 + cells/ml leukapheresis product was recorded for 19 samples (8 patients) . Yields ranged from 2 x 10 " ' to 5 x 10 ' , with a median of 1.9 x 10 CD34 + cells per ml leukapheresis product. Table 1 shows overall cell yields and the proportion of CDla + DC obtained at day 16 from 12 leukapheresis harvests, 4 rib segments, and 3 normal BM samples, all performed during one 3 month period. In 4 cases, total cell numbers did not exceed the number of seeded CD34 + cells.
  • CD34 + HPC were cultured for 16 days in the presence of GM-CSF (10 ng/mL), TNF- ⁇ (20 ng/mL) and IL-4 (500 U/mL) , at which time cells yields were assessed and CDla expression evaluated. Yields are calculated based upon a starting inoculum of 10 CD34 + HPC LH
  • BM bone marrow
  • nBM normal bone marrow
  • CDla + cells expressed low levels of CD4 but no DC activation markers such as CD80, CD83, CD86 or CMRF44 (not shown). They were positive for CD4, CDllb, CDllc, CD13 , CD33, CD58 and CD54 (not shown). Between days 3-15, cultures contained a significant proportion of loosely adherent cells with dendritic morphology ( Figure 8) . It was noted that depletion of non-adherent cells from these cultures removed CDla + cells (not shown) . At day 14, 3 cultures were sorted into CDla + and CDla " cells. A significant proportion of CDla + DC showed Birbeck Granules
  • BG (20-40%) ( Figure 8) .
  • BG were never seen in CDla " cells.
  • the CDla " cells were a heterogeneous population including phagocytic cells and immature cells
  • HLA-DR The expression of HLA-DR was gradually lost as these cells matured.
  • the myeloid marker CD13 was likewise expressed on all small cells (not shown) .
  • CDla + cells sorted on day 14 contained Birbeck Granules (see Figure 8) .
  • this population spontaneously matured into non- adherent APC with up-regulation of HLA-A,B,C, HLA-DR, costimulatory molecules (CD80, CD86) and DC lineage associated antigens (CD83, CMRF44) .
  • down regulation of CDllb and CDla was seen ( Figure 9A; ——— ) .
  • the loss of these antigens was slow and was complete between days 28-40.
  • No Birbeck Granules were found in activated DC.
  • This late phase (dl4-28) now referred to as phenotypic maturation, was associated with a loss of adherence to plastic (not shown) .
  • FIG. 9B shows the CDllb expression in one representative culture at three different time points. Most large sized cells expressed high levels of CDllb at day 16. At day 28, 50% of the large cells had down regulated CDllb to low levels, whereas 50% still expressed high levels of this antigen (cells were gated for size as indicated by the horizontal line and for antigen expression by the dotted line) .
  • the small cell population remaining after 28 days was HLA-DR " . 50% of these were granulocytes on the basis of CD15 expression.
  • IL- - 8 100 ng/ml Genentech Inc., CA, gift of
  • IGF-1 (CR3) 50 ng/ml Dr. R. Whitehead, LICR
  • IGF-1 50 ng/ml Dr. R. Whitehead, LICR
  • FLT-3L 40 ng/ml Genzyme Corp., Cambridge, MA LPS: Sterotype 0111:134 100 ng/ml Sigma, St. Louis, MO w 3. BSA 1% Sigma, St. Louis, MO m
  • Cytokines were added on day 14 and cultures were analyzed by flow cytometry for the presence of activated (HLA-DR bri9ht , CD86 + ) DC on day 17.
  • Each set of experiments included control cells grown in standard conditions in GM-CSF, TNF- ⁇ and IL-4 ("GTI") .
  • GTI TNF- ⁇ and IL-4
  • the percentage of activated DC in these control cultures on day 17 was referred to as 100%. Standard deviations were calculated for all experiments to control for the variations between individual experiments.
  • the results are summarized in Figure 14, in which twice the mean of all SD was used to define the no-difference interval (grey) .
  • HS human serum
  • H-LPS Zetapor filter to remove LPS
  • IFN- ⁇ 2a were capable of increasing the percentage of activated DC above 2 SD of the control.
  • CD34 + progenitor cells were cultured in GM-CSF (40 ng/ml), TNF- ⁇ (20 ng/ml) and IL-4 (500 U/ml) for 16 days and labelled with monoclonal antibodies against CDllb and the DC associated markers CD83, CD86 and CMRF-44 prior to sorting. Flow profiles were gated for large sized cells. A second group of cells was cultured under the same conditions, but IFN- ⁇ (1000 U/ml) was added daily between days 13 and 16. CD83, CD86 and CMRF44 expression was up- regulated, whereas CDllb expression decreased.
  • IFN- ⁇ accelerated DC maturation.
  • FIG. 18 shows the results of 6 individual experiments . Cultures were split on day 14 and IFN- ⁇ 2a (1000 U/ml) was added daily for 3 days into one half. Cultures exposed to IFN- ⁇ 2a contained 23+3.4% CD86 + DC, and these cells showed significantly increased allostimulatory capacity (*p ⁇ 0.05), as shown in Figure 19A. Cultures without IFN- ⁇ contained 9 ⁇ 1.5% CD86 + DC on day 17 ( Figure 18) . These results were confirmed by an MLR using DC sorted on day 18. DC exposed to IFN- ⁇ 2a for 3 days were more stimulatory than control DC, as shown in
  • FIGS 17A and 17B show the effect of IFN- ⁇ addition between days 13-16 on expression of CD80 and CD86 in DC cultures.
  • type I IFNs were the only cytokines that could stimulate DC maturation, it appeared likely that autocrine or paracrine production of IFN was responsible for DC maturation in our serum-free cultures. This hypothesis was tested by assaying culture supernatants for IFN activity. Supernatants were collected at different time between days 14 and 30. IFN activity corresponding to 12 ⁇ 2 IU/ml (range 8-25 IU/ml) was detected in 10 samples from cultures of 6 patients. In supernatants from cultures of 5 other patients, IFN-like activity was not detectable. These data suggest that type I interferons can be produced by the cells in these cultures, and may act as autocrine or paracrine factors to regulate the final stages of DC maturation.
  • Example 15 IFN- ⁇ Activates the Migration of Skin- Derived DC
  • Dendritic Cells x 10 3 time 24 hours 48 hours
  • Figure 20A shows the cumulative DC number of one representative experiment. DC migration was observed in 6 of 10 experiments. Furthermore, migrating DC exposed to IFN- ⁇ showed similar CDla, CD83 and CD86 expression, but accelerated expression of CD80 compared to the cells which migrated in the absence of exogenous IFN- ⁇ ( Figure 2 OB) . In addition to the effects of IFN- ⁇ on in vi tro derived DC, we have shown that IFN- ⁇ activated migration of resident DC from split skin samples floating in serum-free medium. IFN-a accelerated CD80 expression on these cells similar to its effects on progenitor-derived DC. These results suggest that the adherent, immature DC in our serum-free cultures are similar to skin derived DC in response to type I IFN as well as in phenotype.
  • This study describes a method for growing mature and functionally potent DC from human CD34 + hematopoietic progenitor cells in the absence of serum.
  • Three cytokines reported previously to support DC growth in the presence of serum (GM-CSF, TNF- ⁇ and IL-4) were studied.
  • CD54, CD58 CD54, CD58
  • costimulatory molecules CD40, CD80, CD86
  • DC-associated molecules CD83, CMRF44
  • the cells raised in this study represented terminally differentiated, functionally potent DC.
  • PBMC peripheral blood mononuclear cells
  • a similar behaviour of committed DC was recently reported in response to M-CSF (Szabolcs et al , 1996) .
  • these mature DC could be distinguished from macrophages because they were non-phagocytic (Sallusto and Lanzavecchia, 1994) , and remained so in the presence of human serum.
  • DC-cultures were at least 30 times more potent stimulators of allogeneic PBMC proliferation and of the expansion and activation of autologous, peptide-specific CTL.
  • IL-4 had a potent effect on differentiation of DC.
  • human serum inhibited DC differentiation, which underlines the value of this serum-free culture system obtaining DC under defined conditions.
  • Foetal calf serum which is mainly used by groups working with progenitor-derived DC contains xenoantigens and the risk of introducing infectious agents make these protocols less desirable for clinical studies.
  • Autologous human serum does not appear to be a better alternative because of its adverse effects on DC differentiation.
  • GM-CSF and TNF- ⁇ were required for DC development under serum-free conditions.
  • TNF- ⁇ was necessary in the early culture period but had an additional effect on optimal DC development beyond the first 5 days of culture.
  • IL-4 had a potent effect on the differentiation pathway of DC, increasing CDla expression at day 14 and CD86 expression at day 28. When IL-4 was added during the first week and removed after day 7, the cultures already contained precursors committed to a CD14 ⁇ lineage that differentiated into CD83 + DC.
  • IL-4 is produced in serum-free cultures, leading to the development of mature DC from CD14 + precursors.
  • the possibility of an IL-4 independent pathway and the effect of early IL-4 addition to CD14 ⁇ progenitors and precursors can now be examined.
  • GM-CSF, TNF- ⁇ and IL-4 were necessary for DC maturation, but did not appear to be sufficient for optimal DC generation under serum-free conditions. Other factors must be important for both proliferation and maturation of these cells. In the presence of serum DC yields are 2-6 fold higher, suggesting that serum contains additional DC growth and/or maturation factor (s) . Secondly, a prolonged time course (21-28 days) was required for DC maturation when compared to previous reports, in which DC were generated in serum containing cultures after 6 days (Szabolcs et al , 1996). This permissive effect of serum on DC growth in vitro was reported previously (Reid et al , 1992).
  • the density dependence of DC production implies that cells in the culture may be providing additional autocrine or paracrine factors to enhance growth.
  • the time course of increase in cell numbers in serum-free cultures indicates the presence of proliferating CD33 + progenitors in the serum-free cultures. This suggests that a more efficient expansion is possible.
  • the early drop in viable cell number together with the exclusive survival of CD33 + cells suggests a selective cell death of non-myeloid cells.
  • CD14 + monocytic cells
  • CD14 " CDla + Langerhans cells
  • CD14 " CDla + peripheral blood derived DC
  • CD34 + cells differentiated to an intermediate stage which is CD14 " , CDla + , and positive for Birbeck Granules. This is consistent with the Langerhans cell phenotype.
  • IFN-like activity was produced in most spontaneously maturing serum-free DC cultures.
  • IFN- ⁇ activated migration of resident DC from split skin samples floating in serum-free medium.
  • IFN- ⁇ accelerated CD80 expression on these cells similar to its effects on progenitor-derived DC.
  • type I IFN can enhance the effect of TNF- ⁇ in the induction of this process. This suggests that in addition to the multiple immunomodulatory effects, type I IFNs may also regulate immune responses at the level of the antigen- presenting cell.
  • Type I interferons have a well established role in the response to infections with viruses (Hertzog et al , 1991; van den Broek et al , 1995; Hwang et al , 1995) .
  • Immunomodulatory effects include the promotion of Thl responses by inhibition of IL-4 and IL-5 secretion (Demeure et al , 1994; Belardelli, 1995), increase in IFN- ⁇ producing cells (Brinkmann et al , 1993) and effects on IgG production (Romani et al , 1989).
  • Our studies show a novel mechanism, that type I interferons also mediate effects by inducing maturation and activation of DC. This may help to explain the autoimmune phenomena associated with the use of IFN in hepatitis and cancer patients (Hertzog et al , 1991; Ronnblom et al , 1991; Gisslinger et al , 1992; Preziati et al , 1995) .
  • IFN- ⁇ is therefore a useful candidate as a vaccine adjuvant in clinical trials using tumor antigens as vaccines.
  • this serum-free system should assist the further study of events associated with DC activation as well as providing clinical opportunities using interferon- activated DC as cellular adjuvants.
  • IFN- ⁇ may be used as an adjuvant to anti-tumour vaccination strategies (eg. in conjunction with peptides like MelanA, Tyrosinase, GPlOO and the MAGE family, or together with autologous, irradiated tumour cells with and without GM-CSF) .
  • the intratumoural injection of IFN- ⁇ should be reconsidered, since tumours contain DC which do not induce anti-tumour responses . This may be because intratumoural DC are inhibited from maturation and migration by tumour-produced inhibitors (such as soluble neutralizing IFN- ⁇ receptors. Such inhibitors could therefore be blocked by appropriate agents, such as specific antibodies, so as to permit maturation of these DC.
  • autoimmune phenomenon are associated with inteferon- ⁇ injections. This may likely be caused by activated DC presenting self-antigens. The inhibition of this pathological condition by inhibiting interferon may therefore be considered.
  • Verhasselt V., Buelens, C, Willems, F., De Groote, D., Haeffner-Cavaillon, N. and Goldman, M. J. Immunol., 1997 _158 2919-2925

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Abstract

Cette invention, qui a trait à une technique de maturation de cellules dendritiques in vitro dans un milieu non sérique, porte également sur des méthodes thérapeutiques faisant usage de ces cellules dendritiques adultes.
EP97913011A 1996-11-27 1997-11-27 Adjuvant cellulaire Withdrawn EP0941309A1 (fr)

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