EP1185626A2 - A method for the transfer of antigens to dendritic cells - Google Patents

A method for the transfer of antigens to dendritic cells

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Publication number
EP1185626A2
EP1185626A2 EP00936788A EP00936788A EP1185626A2 EP 1185626 A2 EP1185626 A2 EP 1185626A2 EP 00936788 A EP00936788 A EP 00936788A EP 00936788 A EP00936788 A EP 00936788A EP 1185626 A2 EP1185626 A2 EP 1185626A2
Authority
EP
European Patent Office
Prior art keywords
antigen
cells
dcs
antigens
dendritic cells
Prior art date
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Withdrawn
Application number
EP00936788A
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German (de)
English (en)
French (fr)
Inventor
Catia Traversari
Vincenzo Russo
Claudio Bordignon
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Genera SpA
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Genera SpA
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Application filed by Genera SpA filed Critical Genera SpA
Publication of EP1185626A2 publication Critical patent/EP1185626A2/en
Withdrawn legal-status Critical Current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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • 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
    • 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
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464486—MAGE
    • 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
    • A61K39/464496—Fusion proteins originating from gene translocation in cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/05—Adjuvants
    • C12N2501/052—Lipopolysaccharides [LPS]
    • 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
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Definitions

  • the present invention relates to a method for antigen transfer to dendritic cells, comprising cocultivation of dendritic cells with cells transfected with, or expressing the, relevant antigens.
  • DCs Dendritic cells
  • Human DCs have a number of unique features and properties. They have been identified by their typical morphology, by the high membrane density of HLA class II and costimulatory molecules, and by the expression of a unique pattern of cell surface molecules. Functionally, DCs have the ability to endocytose and concentrate soluble proteins in the HLA class II compartment. They are strong stimulators for allogeneic T-lymphocytes in mixed lymphocyte reactions (MLR) and have the unique property of priming cord blood naive T-cells.
  • MLR mixed lymphocyte reactions
  • Human DCs can be generated in vitro from CD34+ cells in response to granulocyte/macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNFc-) .
  • GM-CSF granulocyte/macrophage-colony stimulating factor
  • TNFc- tumour necrosis factor alpha
  • An alternative source of DCs is represented by the monocyte fraction of peripheral blood mononuclear cells (PBMCs) cultured in the presence of GM-CSF and interleukin 4 (IL- 4) .
  • PBMCs peripheral blood mononuclear cells
  • IL- 4 interleukin 4
  • DCs genetically engineered for constitutive expression of a given antigen could provide an important advantage over antigen-pulsed DCs in terms of stable expression of the target gene product. This could overcome the limitation potentially represented by the rapid intracellular degradation of the HLA class I-peptide complexes during the different phases of ex vivo isolation, manipulation, and in vivo administration.
  • adenoviral vectors for transfer of the tumour antigens m DCs from monocytes mainly causes immunization to the vector viral components, making the subsequent uses of the vector less efficient.
  • the method of the invention comprises cocultivation of DCs with "donor" cells expressing on their surface or within the cytoplasm the desired antigens, both of recombmant and natural origin.
  • donor cells in which the expression of the antigen is obtained by transfection with plasmide vectors, or by transduction with viral or retroviral vectors, according to the invention comprise, but are not limited to: primary cultures of tumour and non-tumour mammal cells or continue cell lines, fibroblasts or m general cells which can be efficiently transfected, such as HeLa, COS, NIH3T3 , CHO or HEK-293 cells (ATCC No. CRL 1573), D cells or monocyte cultures. Particularly preferred are NIH3T3 mouse fibroblasts.
  • transfection methods are based on the use of suitable plasmid expression vectors, viral or retroviral, which contain the concerned gene under control of a viral or cell-constitutive promoter (sucn as CMV, SV40, HSV TK, etc.) for high efficiency expression.
  • Said vectors can also contain a gene for the selection of transduced or transformed cells (Neomycm, erbamycm, TK, etc.) .
  • the methods for the transfection or transduction of the "donor cells” are conventional and comprise, for example, microm] ection, electroporation, lipofection, precipitation of DNA with calcium phosphate or the use of DEAE dextran, the infection or other conventional procedures as described for example m Sambrook et al .
  • the method of the invention allows transfer m DCs of intact cytoplasmic and surface antigens, which can be either recombmant, such as those used for labeling and selecting the transfected / ⁇ LNGFr cells as described in
  • antigens which can also be expressed the recombmant form m the donor cells include, but are not limited to: "tumour antigens" or antigens specifically expressed by tumor cells, such as the members of the MAGE (Melanoma Associated Antigen) or BAGE family, Melan-A, gplOO, Mart-1, PSA (Prostate Specific Antigen) , MUC-1, or foetal or embrionic antigens, such as AFP (a-Foetoprotem) , or CEA (ChorioEmb ⁇ onic Antigen) , or oncogens such as HER2/neu, or oncogens derived form "normal” gene mutation, such as p53, c-ras, or the ldiotype of the V region of lmmunoglobulms hyperproduced in lymphomas, or still antigens coded by virus known as the etiological agents of neoplasias, such as EBV (Epstem-Barr Virus)
  • transfection can be performed using all the tumour and non tumour antigens known to date, particularly histocompatibility antigens, antigens of melanoma, carcinoma, sarcoma cells, or from lymphomas, epitheliomas , etc.
  • Donor cells expressing natural antigens are for example tumour cells from biopsies of patients, or cells expressing heterologous histocompatibility antigens, against which tolerance has to be induced, such as those of an organ or bone marrow trasplant recipient, or the cells of the organ to be transplanted (liver, lung, pancreas, heart) for inducing tolerance in the host.
  • the histocompatibility antigens are transferred to the cell surface of the DC and then they result co-expressed with those naturally coded by the DC.
  • Dendritic cells obtainable according to the invention, loaded with the heterologous histocompatibility antigens and optionally treated with suitable cytokmes, can be used for modulating GVHD (Graft Versus Host Disease) , or in the control of the graft rejection of a transplanted organ, or for controlling autoimmmune reactions.
  • GVHD Gel Versus Host Disease
  • the antigen can be transferred to DCs by subjecting the transfected or transduced cells to treatment with pro-apoptotic agents, such as radiations (UV, rays X), or cytokmes such as TNF- ⁇ , or molecules such as actmomycm-D, or they are subjected to interaction with FasL or with antibodies capable of activating a pro- apoptotic cascade (anti-Fas, FasL antibodies, etc.).
  • pro-apoptotic agents such as radiations (UV, rays X), or cytokmes such as TNF- ⁇ , or molecules such as actmomycm-D
  • FasL antibodies capable of activating a pro- apoptotic cascade
  • the mtracellular antitumour antigen is uptaken by the DCs cells thanks to their ability to phagocyte apoptotic bodies.
  • the phagocyted antigen material is then mtracellularly processed by DCs and displayed on the cell surface m form of complexes with HLA class I and/or II peptides
  • surface antigens an efficient transfer of the intact molecule from the donor cells to DCs is obtained without inducing apoptosis
  • the contact between DCs and donor cells is sufficient DCs obtainable according to the method of the invention can be used for adoptive immunotherapy protocols, namely for the ex-vi vo expansion of effector cells, using the manipulated DCs as cells presenting the antigen, or for active immunotherapy strategies, re-mfusing the manipulated DCs into the patient.
  • the manipulated DCs according to the invention can further be used for the identification and the use, for example in tumour therapy, of novel epitopes directly generated by DCs processing of the complete antigen.
  • the antigens can be expressed in the donor cells by transfection with cDNA coding for the complete antigen, or with cDNAs from "libraries" (for example tumour cells libraries), therefore allowing transfer to DCs, by the method of the invention, and their natural processing through sub-cellular compartments of DCs.
  • the dendritic cells obtainable according to the invention can therefore be used for activating HLA class I or II-restricted antigen-specific cytotoxic T cells.
  • cocultivation conditions of DCs with cells transfected with, or expressing at their surface, the antigens are conventional. Typically, cocultivation is mainteined from 12 to 48 hours, preferably about 24 hours, optionally in the presence of polybrene or of other agents promoting cell fusion.
  • the methods for the stimulation of lymphocytes with the DCs loaded with the antigens according to the invention are also conventional. For example, lymphocytes from neoplastic patients are cocultured with the irradiated DCs in IMDM medium (Iscove's Modified Dulbecco Medium) containing 10% human serum. Some days after the first stimulation, the medium is added with a concentration of 10 U/ml of interleukin 2.
  • IMDM medium Iscove's Modified Dulbecco Medium
  • Example 1 Transfer to DCs of the lmmunoqenic fusion protein TN (Herpes Simplex thvmidine kmase and neomycin phosphotransferase) and of / ⁇ LNGFr surface marker
  • Packaging cell line SFCMM2 already described (Bonini et al . Science 276, 1719-1724 (1997), produces a retroviral vector coding for the fusion protein (TN) , containing the herpes simplex virus thymidme kmase (HSV-Tk) and the neomycin phosphotransferase (NeoR) and the ALNGFr surface marker . All the cell lines were cultured RPMI 1640 supplemented with 2 mM L-glutamme, antibiotics and 10% foetal calf serum (FCS) .
  • TN fusion protein
  • HSV-Tk herpes simplex virus thymidme kmase
  • NeoR neomycin phosphotransferase
  • FCS foetal calf serum
  • DCs were isolated from either hestriased fresh whole blood (5-6x10 ) , or leukocyte-enriched buffy coats were allowed to adhere to T25 flasks. After 1 h at 37°C, the non-adherent cells were removed and the adherent cell layer was cultured m RPMI 10% FCS supplemented with LPS 10 ⁇ g/ml, GMCSF 800 U/ml , IL-4 100 U/ml , 2 mM L-glutamme, 50 mM 2S-ME.
  • DCs were transduced using two different protocols: 1- by cocultivation with a monolayer of irradiated (100 Gys) packaging cells in the presence of polybrene (4 ⁇ g/ml) . After 72 hours, DCs were harvested, and seeded in fresh medium. 2- by substituting the culture medium- ith the packaging line cell-free re rovirus-containing supernatant. Three consecutive cycles of infection were performed every 24 hours.
  • the percentage of infected cells obtained with the two procedures was evaluated 48 hours after the last exposure to retrovirus, by flow cytometry for ALNGFr expression performed with the mAb 20.4 (ATCC, Rockville, MD) .
  • the results, reported m la show a very high surface marker DCs transduction efficiency, ranging from 50 to 90% and independent of the procedure utilized.
  • the transduction procedure did not alter the immunophenotype, nor the stimulatory capacity of DCs, as shown example 3 where, by measurement of the capability of DCs to induce a TN- specific cytotoxic response m peripheral blood lymphocytes, transgene TN transfer to CDs was monitored.
  • the transfer mechanism of A NGFr surface marker was evidenced by using the 3T3 -TN/ ALNGFr line for coculture of DCs, according to the protocol described in example 1, namely a cell line transduced using the transgene and expressing the cell surface marker / ⁇ LNGFr , but packaging defective and unable to produce any vector particle.
  • the 3T3-TN/ ALNGFr line was derived by transduction of NIH/3T3 fibroblasts (devoid of gag-pol env genes necessary for producing intact retroviral particles) with the retroviral vector produced by packaging cell lines SFCMM2.
  • Peripheral blood lymphocytes (PBLs 1 ! (2x10 ) from a Thymid o Kmase immune patient (TK-immune), were stimulated vitro with autologous irradiated (50 Gys) DCs previously cocultered with the SFCMM2 vector irradiated as described m example 1 Lytic activity of the effector cells was tested after one round of stimulation. All the stimulations were performed m IMDM containing 10% human serum. Three days after the 1st stimulation, a final concentration of 10 U/ml of IL2 was added to each culture. Lytic activity of the effector cells was tested, eight days after the last restimulation, a 4 hours cytolytic chromium release assay against appropriated target cells.
  • DCs transduced by cocultivation showed an ability to induce a strong immune response against the TN transgene (figure 2a), whereas DCs transduced by cell-free vector- containing supernatants failed to elicit any detectable response.
  • M3-CSM packaging cells were labeled with the PKH-26 red fluorescent dye (Sigma), irradiated and added to a 2-days culture of DCs. Uptake of apoptotic cells by DCs was tested by flow cyto etry and confocal microscopy analyses of the DCs population after 24 and 48 hours. Apoptotic cell death was assayed using Annexin and propidium iodide, according to the manufacturer's instructions. Negative controls were performed on the non-irradiated packaging cells. The obtained results proved that about 30% of DCs took up apoptotic bodies within 24 hours, with the proportion of positive cells increasing over time. The presence of fluorescent bodies within the cytoplasm of DCs was confirmed by confocal microscopy. Appropriate control experiments with unirradiated packaging cells confirmed the complete dependence of the phenomenon by irradiation
  • TIL Tumour infiltrating lymphocytes
  • DCs obtained from a HLA-Bw4 negative donor were exposed to melanoma cells expressing the HLA—Bw4. After 24 hours, DCs were analyzed by flow cytometry for the expression of the relevant histocompatibility antigene HLA and were found to have acquired the HLA-Bw4 allele (figure 4a) . Similar results were reproduced for the HLA-A2 system (figure 4b) .
  • a confocal microscopy analysis was performed. The fusion between cell membranes of DCs and donor cells was clearly evidenced under the microscope. Said analysis gives evidence that cell-to-cell contact is a prerequisite for transduction of surface molecules to DCs.

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EP00936788A 1999-05-28 2000-05-24 A method for the transfer of antigens to dendritic cells Withdrawn EP1185626A2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
ITMI991180 1999-05-28
IT1999MI001180A IT1312570B1 (it) 1999-05-28 1999-05-28 Metodo per il traferimento di antigeni a cellule dendritiche.
PCT/EP2000/004712 WO2000073415A2 (en) 1999-05-28 2000-05-24 A method for the transfer of antigens to dendritic cells

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JP (1) JP2003501019A (it)
KR (1) KR20020013563A (it)
CN (1) CN1187442C (it)
AU (1) AU772703B2 (it)
CA (1) CA2375349A1 (it)
HK (1) HK1047131A1 (it)
IL (1) IL146777A0 (it)
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US6506596B2 (en) 2000-06-01 2003-01-14 Anna-Lena Spetz-Holmgren Method of DNA transfer
CN100392074C (zh) * 2003-10-15 2008-06-04 上海海欣生物技术有限公司 树突状细胞肿瘤疫苗及其制法和用途
KR20160045151A (ko) * 2005-04-08 2016-04-26 아르고스 쎄라퓨틱스 인코포레이티드 수지상 세포 조성물 및 방법

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IT1261847B (it) * 1993-09-01 1996-06-03 San Romanello Centro Fond Metodo per marcare cellule eucariote.
EP0765386B1 (en) * 1994-06-14 2014-12-10 The Board Of Trustees Of The Leland Stanford Junior University Methods for in vivo t cell activation by antigen-pulsed dendritic cells
US5871728A (en) * 1995-03-31 1999-02-16 University Of Pittsburgh Method of regulating dendritic cell maturation
DK0904786T3 (da) * 1997-08-22 2005-03-21 Science Park Raf S P A Tumorvaccination under anvendelse af autologe eller HLA-relaterede antigenpræsenterende celler (APC) transduceret med et tumorantigen og et fremmed antigen, som kan fremkalde en immunreaktion
JP2002504322A (ja) * 1998-02-20 2002-02-12 ザ ロックフェラー ユニバーシティー 樹状細胞に対するアポトーシス細胞介在抗原提示

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CA2375349A1 (en) 2000-12-07
KR20020013563A (ko) 2002-02-20
IT1312570B1 (it) 2002-04-22
JP2003501019A (ja) 2003-01-14
AU772703B2 (en) 2004-05-06
CN1187442C (zh) 2005-02-02
ITMI991180A1 (it) 2000-11-28
AU5215900A (en) 2000-12-18
CN1360628A (zh) 2002-07-24
IL146777A0 (en) 2002-07-25
HK1047131A1 (zh) 2003-02-07
WO2000073415A3 (en) 2001-03-01
WO2000073415A2 (en) 2000-12-07

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