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WO2004016774A1 - Methode d'amplification des cellules positives inhibitrices du recepteur de la cellule nk - Google Patents

Methode d'amplification des cellules positives inhibitrices du recepteur de la cellule nk Download PDF

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WO2004016774A1
WO2004016774A1 PCT/JP2003/010291 JP0310291W WO2004016774A1 WO 2004016774 A1 WO2004016774 A1 WO 2004016774A1 JP 0310291 W JP0310291 W JP 0310291W WO 2004016774 A1 WO2004016774 A1 WO 2004016774A1
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cells
pbmc
inhibitory
cell receptor
antibody
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Japanese (ja)
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Junji Tanaka
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Kirin Brewery Co Ltd
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Kirin Brewery Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—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
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • 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 A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • 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
    • C12N2501/515—CD3, T-cell receptor complex

Definitions

  • the present invention relates to a method for amplifying inhibitory NK cell receptor positive cells from peripheral blood obtained from a donor.
  • NRR natural killer cell receptor
  • graft-versus-host disease (GVDH) and graft-versus-leukemia response (GVL) effects are the most important issues in allogeneic stem cell transplantation (alio SCT).
  • Peripheral blood mononuclear cell (G-PBMC) grafts mobilized with granulocyte colony-stimulating factor (G-CSF) contain at least 10 times more T cells than normal bone marrow grafts, but allogeneic peripheral blood
  • G-PBMC Peripheral blood mononuclear cell
  • G-CSF granulocyte colony-stimulating factor
  • PBSCT acute graft-versus-host disease after stem cell transplantation
  • CD94 / NKG2 heterodimer is a receptor for H — E nonclassical HLA-I molecule (Braud ', VM et al., Nature 391, 795-799 (1998)).
  • NKG2A which has an immunosuppressive motif (ITIM) with two distinctive tyrosine residues in the intracellular part, can transmit suppressive signals.
  • ITIM immunosuppressive motif
  • NK-like activity and TCR (T cell receptor) -induced dysfunction of CTL (cytotoxic T lymphocyte) expressing inhibitory MR are suppressed by class I recognition by NKR (Phil ips, JH et al., Science 268, 403-405 (1995), Mingari, MC et al., Pro Natl. Acad. Sci.
  • CD158b a specific receptor for HLA-C
  • CD8 + T cells chronic GVHD (cGVHD) patients.
  • increased CD158b expression on T cells was associated with improved clinical symptoms.
  • CD94 / NKG2A on T cells was higher in patients with good prognosis cGVHD than those with poor prognosis (Tanaka, J. et al., Br. J. Haematol. 108, 778-783 ( 2000), Tanaka, J. et al., Bone Marrow Transplant. 26, 287-290 (2000), Tanaka, J. et al., Br. J.
  • An object of the present invention is to provide a method for amplifying suppressive NK cell receptor-positive cells that cause GVL in a recipient but do not cause GVHD, and to provide a method for treating leukemia and tumors.
  • CD94-expressing cells which are inhibitory NK cell receptor-positive cells derived from peripheral blood (G-PC) mobilized by administering G-CSF to a donor, to anti-CD3 monoclonal antibody and IL- Cultured and expanded together with 15 and cultured leukemia cells and patient leukemia They have been found to exhibit cytotoxic effects on diseased cells. Furthermore, the present inventors have found that the amplified CD94-expressing cells suppress the growth of K562 leukemia cells transplanted into N0D / SCID mice, and have completed the present invention.
  • G-PC peripheral blood
  • a method for amplifying inhibitory NK cell receptor-positive cells comprising culturing blood-derived mononuclear cells in the presence of an anti-CD3 antibody and IL-15;
  • (2) a method for amplifying the inhibitory NK cell receptor-positive cells according to (1), which comprises culturing peripheral blood mononuclear cells in the presence of an anti-CD3 antibody and IL-15;
  • G-PBMC G-CSF mobilized peripheral blood mononuclear cells
  • P layer 0 P layer 0
  • a method of amplifying the inhibitory NK cell receptor-positive cells of (1) which comprises culturing cord blood mononuclear cells (CBMC) collected from cord blood in the presence of an anti-CD3 antibody and IL-15;
  • CD94 + or NKG2A + cells in G-PBMC, PBMC or CBMC are amplified 10 times or more compared to before culture.
  • a method for producing an inhibitory NK cell receptor-positive cell comprising:
  • the inhibitory NK cell receptor-positive cells of the present invention were obtained by collecting peripheral blood mononuclear cells (G-PBMC) obtained by administering G-CSF to healthy human transplant donors and recruiting stem cells, using anti-CD3 antibody and IL. -15. In some cases, it can be amplified by culturing with another cytoin.
  • G-PBMC peripheral blood mononuclear cells
  • mobilization of stem cells refers to increasing the number of hematopoietic stem cells in peripheral blood, and is also referred to as “peripherallzation”.
  • the cells used are not limited to G-PBMC, and any blood-derived mononuclear cells collected from human blood can be used.
  • Normal peripheral blood collected without G-CSF or transplanted patients Use peripheral blood mononuclear cells (PBMC) such as peripheral blood mononuclear cells (PBMC) collected from peripheral blood, or cord blood mononuclear cells (CBMC) collected from umbilical cord blood You can do it.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • CBMC cord blood mononuclear cells
  • normal peripheral blood refers to peripheral blood collected from a human who has not undergone transplantation, and peripheral blood from a transplant patient has been collected from a human who has undergone transplantation of bone marrow stem cells, umbilical cord blood stem cells, peripheral blood stem cells, etc. Refers to peripheral blood.
  • the inhibitory NK cell receptor-positive cells express CD94 or NKG2A on the surface, and the expansion of the inhibitory NK cell receptor-positive cells of the present invention is confirmed by whether CD94 or NKG2A-expressing cells are amplified. be able to.
  • Recruitment of stem cells to the peripheral blood of the donor is performed by administering human G-CSF (granulocyte colony-stimulating factor) to the donor.
  • human G-CSF granulocyte colony-stimulating factor
  • recombinant human G-CSF may be administered.
  • Commercially available human G-CSF can be used.
  • the administration may be performed by subcutaneous injection, and G-CSF is administered at a rate of about 10 / ig / kg body weight of the donor every day for several days.
  • the collected peripheral blood can be stored until use by freezing.
  • a blood cell separation device such as CS3000 G-PBMC is collected from the laboratory.
  • G-CSF mobilized donor peripheral blood mononuclear cells G-PBMC or normal peripheral blood or transplant patient peripheral blood (P ⁇ C), or mononuclear cells collected from umbilical cord blood (CBMC)
  • G-PBMC peripheral blood mononuclear cells
  • P ⁇ C normal peripheral blood or transplant patient peripheral blood
  • CBMC umbilical cord blood
  • G-CSF mobilized donor peripheral blood mononuclear cells G-PBMC
  • P ⁇ C normal peripheral blood or transplant patient peripheral blood
  • CBMC umbilical cord blood
  • the anti-CD3 antibody a monoclonal antibody prepared by a known mouse monoclonal antibody preparation method may be used, or a commercially available antibody may be used.
  • commercially available products include UCHT-1 and 0KT3.
  • the anti-CD3 antibody immobilized on a culture vessel For example, the anti-CD3 antibody is adjusted to a concentration of 0.1 to 1.0 Og L with an appropriate buffer in advance, and 5 to 10 mL of the prepared anti-CD3 antibody is placed in a 0.05 L culture vessel, preferably at 4 ° C. Incubate at C for 12-24 hours to immobilize.
  • G-PBM (:, PBM) using the above medium supplemented with IL-15 in a container in which an anti-CD3 antibody is immobilized
  • the density of G-PBMC during culture is 0.5 to 2.
  • O x lo mL preferably 1 x 10 6 / mL.
  • Recombinant human IL-15 may be used as IL-15, and commercially available IL-15 may be used.
  • the concentration of IL-15 added is 1 to 10 ng / mL, preferably 5 ngM.
  • the culture is performed at 37 ° C. for several days, preferably 3 to 5 days, or 5 days or more, preferably 10 days or more.
  • Cytokines may be present in the culture step in addition to the anti-CD3 antibody and IL-15.
  • Cytokines that can be added in addition to the anti-CD3 antibody and IL-15 are not limited, and include basic fibroblast growth factor, IL-11 (interleukin 1), IL-2, IL-13, IL-4, and IL-4.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • erythropoietin CSF-1 (colonizing stimulating factor)
  • SCF Stem cell factor
  • thrombopoietin EGF (epidermal growth factor)
  • TGF-a transforming growth factor-1
  • HB-EGF heparin-binding EGF-like growth factor
  • GDNF vascular endothelial growth factor
  • GDNF vascular endothelial growth factor
  • TNF Tumor growth factor-1 (Daria cell line-derived neurotrophic factor), Mitudokine, TGF- / 3 (Transforming growth factor-1) 3), Baydalican, Activin, BMP (Bone morphogenetic factor), TNF
  • IL-21 Tumor necrosis factor
  • IFN-a / j3 Interferon-1 /
  • IFN-r interferon-1
  • fibronectin laminin, cadherin, integrin, selectin, etc.
  • site power-ins may be natural products or recombinant products, and commercially available products can be used.
  • the concentration of the added site force is preferably from 1 to 25 ng / inL, and particularly preferably 12.5 ng / mL.
  • Other cytodynamics may be added simultaneously with the anti-CD3 antibody and IL-15, or may be added after stimulation culture with the anti-CD3 antibody and IL-15.
  • the culture may be performed for several days, preferably 3 to 5 days, or 5 days or more, preferably 10 days or more. If added after stimulation culture with anti-CD3 antibody and IL-15, stimulate culture with anti-CD3 antibody and IL-15 for several days, preferably 3-5 days, or 5 days or more, preferably 10 days or more.
  • another cytokine may be additionally added, or the medium may be replaced with a medium containing another cytokine.
  • CD94 or NKG2A-expressing cells are amplified can be determined by immunofluorescence staining with a fluorescently labeled anti-CD94 antibody or NKG2A antibody and analyzing by FACS (Fluorescence act ivated cell sorter) or flow cytometry. .
  • FACS Fluorescence act ivated cell sorter
  • G-PBM 15% or more, preferably 20% or more in PBMC or CBMC, More preferably, 25% or more, particularly preferably, 30% or more of cells are CD9.
  • CD94 f or NKG2A + cells 10-fold or more, preferably 20 times or more, in particular amplified preferably 100 times or more.
  • G-PBM CD8 ⁇ cells from PBMC or CBMC were conjugated with FACS or anti-CD8 antibody.
  • the combined immunomagnetic beads may be used to isolate and purify and stimulate with anti-CD3 antibody and IL-15, and optionally with other cytokines. At this time, in addition to stimulation with anti-CD3 antibody and IL-15, the presence of CD14 ⁇ cells is required.
  • CD14 ⁇ cells are isolated, purified and mixed.
  • the CD94 + or NKG2A + cells amplified from the donor G-PBMC, PBMC or CBMC cells by the method of the present invention can be isolated and purified using Cell Souce or anti-CD94 antibody-bound immunomagnetic beads.
  • the present invention relates to G-PBMC, PBMC or CBMC
  • Amplifying the D94 ⁇ or NKG2A + cells also encompasses a method for manufacturing a CD94 ⁇ or NKG2A ⁇ cells comprising said CD94 ⁇ or dishes G2A ⁇ cells isolated and purified.
  • CD94 ⁇ or NKG2A + cells amplified in this manner have cytotoxic activity against tumor cells with no or low expression of HLA class I molecules on the surface or with incompatible HLA class I molecules. However, it has no cytotoxic activity against normal cells that have a high expression level of compatible HLA class I. That is, when donor-derived CD94 + or NKG2A + amplified cells are administered to leukemia or tumor patients, they do not cause GVHD on normal cells and show cytotoxic activity only on leukemia cells and tumor cells.
  • GVL graft versus leukemia
  • GVT graft versus tumor
  • Treatment includes CD94 amplified from G-PBMC, PBMC or CBMC by the method of the invention.
  • CD94 + or NKG2A + cells may be administered to 10 6 to 10 8 per patient patient weight l kg.
  • the administration is performed by, for example, intravenous drip infusion.
  • CD94 ⁇ or NKG2A + cells can damage the patient's leukemia or tumor cells and exert a therapeutic effect.
  • CD94 ⁇ Rui amplified by the method of the present invention are included in the treatment methods also present invention cancer that comprises Azukasuru projecting the NKG2A ⁇ cells to the subject.
  • This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application No. 2002-235601, which is a priority document of the present application.
  • FIG. 1 is a diagram showing induction and amplification of CD94 / NKG2A-expressing cells from donor pre_G and donor G-PBMC.
  • FIG. 2 is a diagram showing induction and amplification of CD94 / MG2A-expressing cells from donor G-PBMC.
  • FIG. 3 is a diagram showing induction and amplification of CD94 / NKG2A-expressing cells from CD8 ⁇ cells purified from donor G-PBMC.
  • FIG. 4 is a diagram showing the amplification rate of CD94 / NKG2A-expressing cells from donor G-PBMC.
  • FIG. 5 is a diagram showing the cytotoxic activity of CD94-expressing cells derived from donor G-PBMC on K562 cells and autologous PHA blasts.
  • FIG. 6 is a view showing the cytotoxic activity of CD94-expressing cells derived from donor G-PBMC on K562 cells, autologous PHA blasts, cells derived from CML-CP patients, and cells derived from CML-BC patients.
  • FIG. 7 is a diagram showing expression of HLA class I antigen in K562 cells.
  • FIG. 8 is a photograph showing the expression of HLA-E in K562 cells.
  • FIG. 9 shows the results of treatment of CD94-expressing cells derived from donor G-PBMC with treated K562 cells and untreated K.
  • Fig. 10 shows treated K562 cells and untreated CD94-expressing cells derived from donor G-PBMC.
  • FIG. 11 is a photograph showing the effect of administering CD94-expressing cells to NOD / SCID mice transplanted with K562 cells.
  • FIG. 12 is a graph showing suppression of proliferation of K562 cells in CD0 / SCID mice by CD94-expressing cells.
  • FIG. 13 is a view showing suppression of proliferation of K562 cells in CD0 / SCID mice by CD94-expressing cells.
  • Example 1 was performed as follows.
  • Peripheral blood stem cell donors were injected subcutaneously with rhG-CSF (Kirin Brewery) at a dose of 10 ig / kg once daily for 4 or 5 days.
  • Leukocyte apheresis was performed from day 4 of rhG-CSF administration.
  • G-PBMC samples were obtained from the first leukocyte apheresis. All samples were stored frozen for simultaneous testing.
  • FITC-conjugated anti-CD3, anti-CD8 mAb and anti-HA, A, B, C mAbs were obtained from Plmrmingeii (USA).
  • Anti-CD3 mAb UCHT-1 was obtained from Ifflnmnotec 0KT3 was obtained from Ortlio Biotech (USA). The fluorescence intensity of the cells was analyzed using a FACS Calibur (Becton Dickinson). Statistical analysis was performed using Student's t-test.
  • D14 ⁇ cell depleted fraction (the percentage of CD14 ⁇ cells, less than 3%), the CD8 cells (percentage of CD8 ⁇ is greater than 90%) and CD94 + cells (CD94 ⁇ proportion of more than 90%) magnetic particles It was obtained by magnetic cell sorting (MACS). The procedure was according to the manufacturer's instructions (Milten yi Biotec GmbH, Germany).
  • G-PBMC-derived CD8 ⁇ cells (500 x l0 3 / mL) of 5 ng / mL in these plates recombinant tut human IL- 15 (R & D system, USA) 37 ° in the presence or absence C.
  • G-PBMC cultures were performed in 24-well plates with or without 0.45 m microporous membrane (FALCON). Purified CD14 + cells (300 ⁇ 10 3 ) from G-PBMC were added directly to the culture or through the membrane.
  • G-PBMC from five different donors were cultured for 10 days under conditions using immobilized anti-CD3 mAb (1 g / mL) and IL-15 (5 ng / mL). At this time, culture was performed at 37 ° C using RPMI 1640 as a medium. After 5 days of culture, fresh medium and IL-15 were added and the cell density was adjusted to 1 ⁇ 10 6 / ml.
  • Figure 1 shows CD94 / NKG2A-expressing cells in PBMC (referred to as pre-G or pre-GPBMC and G-PBMC, respectively) before and after stimulation with anti-CD3 mA and IL-15 for 7 days. The percentages are shown. Open pars indicate pre-G results, solid bars indicate G-PBMC results. 1, 2 and 3 are percentages of cells with CD94 ⁇ / 3 +, 4, 5, and 6 are CD9
  • the percentage of 4 + / 8 ⁇ cells, 7, 8 and 9 represent the percentage of NI (G2A ⁇ / CD3 + cells, and 10, 11, and 12 represent the percentage of NKGZA + A ⁇ cells.
  • 4, 7 and 10 show the results before stimulation with anti-CD3 mAB and IL-15, and 3
  • 6, 9 and 12 show the results before anti-CD3 mAB and I
  • Results after stimulation with L-15, 2, 5, 8 and 11 show the results of stimulation with anti-CD3 mAB alone. As shown in the figure, there was no difference in CD94 / NKG2A expression on T cells among PBMCs obtained from 7 donors before and after administration of G-CSF. However, IL
  • CD94 / NKG2A on T cells from GHPBMC stimulated with immobilized anti-CD3 monoclonal antibody (mAb) in the presence or absence of -15 for 7 days is higher than preG-PBMC won.
  • Figure 2 shows G-PBMC, CD14 removal G-PBMC, CD14 removal G-P ⁇ C with 3 ⁇ 10 5 purified CD14 + cells added and CD14 removal G-PBMC with 3 ⁇ 10 5 shows the proportion of CD94 / NKG2A-expressing cells in anti-CD3 mAb and IL- 15 following stimulation before and stimulation of ⁇ days in pre- G and G-PBMC in the case of adding purified CD 14 1 cells.
  • Open bars indicate results before stimulation with anti-CD3 mAb and IL-15
  • solid bars indicate results after stimulation with anti-CD3 mAb and IL-15
  • diagonal bars indicate CD14 removal G- PBMC results
  • Checked bars indicate CD14-removed G-PBMC with 3 ⁇ 10 5 purified CD14 + cells added.Dotted bars show membranes for CD14-removed G-PBMC. Shown are the results when 3 ⁇ 10 5 purified CD14 ⁇ cells were added via the vial. 94 + / 3 + , 94 + / 8 NKG2A + / 3 ⁇ and N on the graph
  • Figure 3 shows that anti-CD3 mAb and IL-I were obtained when 3 ⁇ 10 5 purified CD14 ⁇ cells were added and when 3 ⁇ 10 5 purified CD14 ⁇ cells were added to CD-removed G-PBMC via a membrane.
  • the percentage of CD94 / NKG2A-expressing cells in purified CD8 ⁇ cells from G-PBMC before and after stimulation at 15 is shown. The meaning of each bar is the same as in FIG.
  • the values indicate the percentage of cells expressing CD94 or NKG2A in pre-G and G-PBMC (mean SD). Significant differences were observed between the following conditions.
  • IL-15 is known as one of CD94 / NKG2A-induced cytokines. Therefore, the results of this example show that IL-15
  • FIG. 4 shows the fold expansion of CD94 / NKG2A-expressing cells 5 and 10 days after stimulation compared to before stimulation with G-PBMCs from 5 different donors. As shown in the figure,
  • CD94 ⁇ / 8 ⁇ cells and NKG2 / ⁇ from G-PBMC obtained from 5 donors after 10 days of culture
  • CD8 ⁇ cells increased 22.1 to 410.0-fold and 121.7 to 2348-fold, respectively, compared to before stimulation.
  • G-PBMCs were stimulated with anti-CD3 antibody and IL-15 at the same time as described above, or simultaneously with anti-CD3 antibody and IL-15. After stimulated culture at -15 for 4-5 days, IL-21 (SIGMA) was added at 12.5 ng / ml and cultured, and the inhibitory NK receptor CD94 / MG2A positive cells could be amplified more efficiently. .
  • SIGMA IL-21
  • CD94 expression was over 40%, NKG2A expression was over 30%, and the amplification rate of CD8-positive cells was 400 to 1,400-fold for CD94 and 500 to 2,600-fold for NKG2A (Table 1).
  • Table 1
  • Example 2 evaluation of cytotoxic activity and RT-PCR were performed as follows. (Evaluation of cytotoxic activity using 4-hour 51 Cr release atsee)
  • CD94 expressing cells were purified by magnetic cell separation (MACS). More than 80% of the cells expressing CD94 co-expressed CD8.
  • 51 Cr-labeled human leukemia cell line K562 al io SCT cytotoxic activity of CD94 expressing cells to the patient before leukemia cells and autologous PHA blasts (5 X 10 3) were test.
  • K562 cells were cultured for 2 days with IFN- ⁇ (0.2 / mD.) HLA-Cw3 signal peptide (VMAPRTLIL; SEQ ID NO: 1) and B15 peptide (VTAPRTVLL; SEQ ID NO: 2) were purchased from KURAB0 (Japan). Was used (purity 95%).
  • First-strand cDNA synthesis was performed using 60 ng RNA, 5 mM MgCl r 1 ol / L MTP, 2.5 iM random 9-mer, and 0.25 U / L AMV reverse transcriptase (Takara RNA PCR Kit, Japan). Then, PCR amplification of the cDNA was performed using a sense primer 5′-CAGCATGAGGGGCTACCCG-3 ′ (SEQ ID NO: 3) for exon 4 of HLA-E and an antisense primer 5′-GTGTGAGGAAGGGGGTCATG-3 ′ (SEQ ID NO: 4). j3 actin
  • FIG. 5 shows the cytotoxic activity of CD94-expressing cells on K562 cells and autologous PHA blasts.
  • the open bars show the results for K562 cells, and the black bars show the results for PHA blasts.
  • the cytotoxic activity of G-PBMC-derived purified CD94-expressing cells against K562, detected by standard 4 hour 51 Cr release was always higher than on autologous PHA blasts.
  • CML-BC chronic myeloid leukemia
  • FIG. 6 shows the cytotoxic activity of CD94-expressing cells amplified from donor G-PBMC on K562 cells, patient leukemia cells and autologous PHA blasts. Open bars indicate results for K562 cells, diagonal bars indicate results for leukemia cells of patients, and checked bars indicate results for autologous PHA blasts.
  • the effector / evening get ratio is 5: 1 and 10: 1, respectively.
  • the left shows 5: 1 and the right shows 10: 1.
  • donor-derived CD94-expressing cells attacked the patient's leukemic cells, but not autologous PHA blasts.
  • the average fluorescence of HLA class I molecules detected by FACS using K562 cells, leukemia cells of patients with CML-CP and CML-BC, PHA blasts 1, PHA blasts 2 and PBMCs of healthy subjects was 21 respectively. 6, 101.8, 108.5, 394.4, 347.8 and 171.1. Therefore, it was found that the cytotoxic activity of CD94-expressing cells was inversely proportional to the expression of HLA class I molecules on target cells.
  • Figure 7 shows K562 cells stained with anti-HLA class I mAb and isotype control mouse IgG GTC after culturing for 2 days with or without IFN-r (0.2 zg / ml). Is shown. The upper figure shows the results of culturing without IFN- ⁇ , and the lower figure shows the results of culturing with IFN- ⁇ added. HLA class I expression is induced on K562 cells by IFN- ⁇ . The average fluorescence of HLA class I molecules on IFN- ⁇ treated K562 was 109.0.
  • FIG. 8 shows the results of detection of j3-actin and HLA-E mRNA in K562 cells by RT-PCR before, one day after, and two days after culturing with IFN-a.
  • Lanes 1, 2 And 3 show the results of jS actin
  • lanes 4, 5 and 6 show the results of HLA-E
  • lanes 1 and 4 show before culture with IFN-a
  • lanes 2 and 5 show 1 day after culture with IFN-a
  • Lanes 3 and 6 show the results after 2 days of culture with IFN- ⁇ .
  • This RT-PCR experiment showed that IFN- ⁇ induced HLA-E mRNA in K562 cells (Ulbreciit, M. et al., J. Immunol. 149, 2945-2953 (1992)).
  • FIG. 9 illustrates the untreated K562, IFN- ⁇ processing ⁇ 562 cells, IFN- ⁇ and HLA-Cw3 peptidyl de (0. 3 mM) cell failure activity of CD94 expressing cells to treatment K562 cells, as well as autologous PHA blasts . Expressed as ⁇ , marrow, ⁇ and ⁇ , respectively. The cytotoxic activity of CD94-expressing cells on IFN-T-treated K562 cells was weaker than that on untreated K562 cells.
  • HLA-Cw3 peptide (0.3 mM), which is a signal sequence of HLA-C and forms a complex with HLA-E as a CD94 ligand, is used to express CD94-expressing cells against these HLA class I-expressing K562 cells. Inhibits impairment activity (Borrego, F. et al., J. Exp. Med. 187, 813-818 (1998)). However, the B15 peptide reported not to interact with CD94 / NKG2A did not have such an inhibitory effect (results for the B15 peptide are not shown in the figure).
  • Figure 10 shows IFN- ⁇ -treated K562 cells, HLA-Cw3 peptide (0.3 mM) and IFN- ⁇ -treated K562 cells, and HLA-Cw3 peptide, anti-NKG2A mAb (10 zg / mL) and IFN- ⁇ -treated K562.
  • 4 shows the cytotoxic activity of CD94-expressing cells on cells. These are indicated by ⁇ , cheats and ⁇ , respectively.
  • the anti-NKG2A mAb (10 zg / inl) canceled the protective effect of HLA class I on the cytotoxic activity of CD94-expressing cells.
  • these CD94-expressing cells could attack K562 without HLA class I molecules, but this damaging activity was blocked by the expression of HLA class I molecules in K562 cells and the HLA class I signal peptide.
  • mice 5-8 week old female N0D / SCID mice were obtained from CLEA (Japan). The breeding was maintained under sterile conditions using a microisolator. K562 cells were suspended in 0.5 mL PBS with or without purified CD94-expressing cells amplified from G-PBMC.
  • NOD / SCID mice were injected subcutaneously on the right side. Attempts to transplant CML blast crisis leukocytes into NOD / SCID also failed to form tumors as did K562 cells. K562 cells were injected subcutaneously into NOD / SCID mice simultaneously with purified CD94-expressing cells derived from G-PBMC.
  • FIG. 11 shows the proliferation of K562 cells in NOD / SCID mice 8 weeks after injection.
  • FIGS. 12 and 13 show the size of tumors when mice were injected subcutaneously with K562 cells alone or with CD94-expressing cells. In Fig.
  • CD94-expressing cells increased the expansion of K562 cells in N0D / SCID mice in a cell number-dependent manner, with CD94-expressing cells: K562 cell ratios of 1 ⁇ 10 1 : 2 ⁇ 10 7 and 2.5 ⁇ 10 5 ⁇ 10 7 When completely, the ratio was 1 ⁇ 10 1 : 5 ⁇ 10 7 and partially inhibited.
  • IL-15 in PBMC mobilized by G-CSF G-PBMC
  • immobilized antibody
  • CD94 / NKG2A Increased expression of CD94 / NKG2A on CD / CD8 ⁇ T cells stimulated with CD3 monoclonal antibody was demonstrated. It was also shown that CD14 ⁇ cells play an important role in inducing CD94 / NKG2A expression on purified CD8 T cells. Thus, G-PBMC-derived CD8 + T cells were able to express CD94 / NKG2A after stimulation. Furthermore, CD94-expressing cells could be amplified about 100-fold from Dona G-PBMC.
  • CD94-expressing cells are deficient in HLA class I molecules.K562 leukemia cells and patient leukemia cells in which HLA class I molecules are reduced compared to PBMCs derived from healthy individuals and autologous PHA blasts But does not attack autologous PHA blasts with greatly increased expression of HLA class I molecules. In vivo analysis showed that these CD94-expressing cells could block K562 cell expansion in NOD / SCID mice. Thus, CD94 inhibitory NKR expressing cells activate the graft versus leukemia effect.
  • Partial HLA-matched bone marrow transplantation greatly amplifies CTLs from donors that express CD158b-inhibitory NKR, suppresses GVHD but does not suppress distinct GVL responses ( Albi, N. et al., Blood 87, 3993-4000 (1996)).
  • Inhibitory NKR positive cells attack class I negative target cells Do not attack the same class I positive cells (Mingar i, MC et al., Proc. Natl.
  • GVL effectors are also important in HLA-incompatible hematopoietic cell transplantation based on the principle of MR incompatibility (Ruggeri, L. et al., Blood 94, 333-339 (1999)).
  • Transgenic expression of CD158b inhibitory NKR suppresses in vivo rejection of H-2 incompatible bone marrow transplants (Cambiag gi, A. et al., Proc. Natl. Acad. Sci.
  • HLA-E specifically binds to peptides from most HLA-A, B, C, and G signal sequences and is upregulated by these peptides (Braud, VM et al. , Nature 391, 795-799 (1998)).
  • HLA class I molecule-expressing K562 cells induced by IFN- ⁇ were used to examine the cytotoxic properties of CD94-expressing cells.
  • the HLA-C signal peptide was found to be induced by IFN- ⁇ in CD94-expressing cells and to suppress cytotoxic activity against K562 cells expressing HLA class I molecules.
  • the anti-NKG2A mAb restores the cytotoxic activity of CD94-expressing cells to K562 cells protected by HLA class I molecules.
  • these HLA class I molecule-dependent cytotoxicity by CD94-expressing cells could not be evaluated in an all or nothing manner. This is because the CD94-expressing cells amplified from these G-PBMCs are not identical to the cloned T cells and have different TCR-Vbeta families, as demonstrated by RT-PCR analysis using the 26TCR_Vi3 primer. by.
  • CD94-expressing cells serve to regulate GVHD and GVL in many patients with different HLA class I types.
  • the cytotoxic properties of inhibitory NKR-expressing cells are key to addressing the question of how to regulate the delicate balance between GVHD and GVL.
  • CD94-expressing cells from these donor G-PBMCs are useful for allogeneic cell therapy to induce GVL effects instead of simple donor lymphocyte infusion without promoting GVHD.
  • Donor G-PBMC would be a source for the expansion of suppressor dish R-expressing cells such as CD94 ⁇ or NKG2Ai cells for allogeneic cell therapy.
  • Example 1 the method of the present invention inhibits G-PMBC
  • CD94-expressing cells that are NK cell receptor-positive cells can be significantly expanded.
  • the D94-expressing cells have cytotoxic activity against cells in which the HLA class I molecule is not expressed or has a low expression level as shown in Example 2, and as shown in Example 3, Can be used to treat leukemia tumors.

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Abstract

L'invention concerne une méthode d'amplification des cellules positives inhibitrices du récepteur de la cellule NK obtenues du sang périphérique d'un donneur. Ladite méthode d'amplification des cellules positives inhibitrices du récepteur de la cellule NK consiste à mettre en culture des cellules mononucléaires sanguines telles que des cellules mononucléaires sanguines périphériques mobilisées G-CSF (G-PBMC), des cellules mononucléaires sanguines périphériques (PBMC) obtenues de sang périphérique normal sans administration de G-CSF, ou obtenues du sang périphérique d'un patient ayant subi une transplantation, ou des cellules mononucléaires sanguines de cordon ombilical obtenues de sang de cordon ombilical en présence d'un anticorps anti-CD3 et IL-15, éventuellement avec une autre cytokine.
PCT/JP2003/010291 2002-08-13 2003-08-13 Methode d'amplification des cellules positives inhibitrices du recepteur de la cellule nk Ceased WO2004016774A1 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1999061617A1 (fr) * 1998-05-29 1999-12-02 Human Genome Sciences, Inc. Interleukine 21 et 22

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061617A1 (fr) * 1998-05-29 1999-12-02 Human Genome Sciences, Inc. Interleukine 21 et 22

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JUNJI TANAKA: "Saibo ryoho no genjo to tenbo NK receptor yose saibo ni ishoku men'eki ryoho", GEKKAN MEDICAL SCIENCE DIRECT, vol. 28, no. 7, June 2002 (2002-06-01), pages 271 - 274, XP002973361 *
TANAKA J. ET AL.: "Expression of inhibitory natural killer cell receptor CD94/NKG2A on CD8 T cells and its immunological significance in granulocyte colony-stimulating factor-mobilized perpheral blood mononuclear cells", BLOOD, vol. 98, no. 11, PT.2, 2001, pages 328B, XP002973360 *

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