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WO1995016775A1 - Fusions de cellules tumorales et procedes d'utilisation de telles fusions de cellules tumorales - Google Patents

Fusions de cellules tumorales et procedes d'utilisation de telles fusions de cellules tumorales Download PDF

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WO1995016775A1
WO1995016775A1 PCT/US1994/014297 US9414297W WO9516775A1 WO 1995016775 A1 WO1995016775 A1 WO 1995016775A1 US 9414297 W US9414297 W US 9414297W WO 9516775 A1 WO9516775 A1 WO 9516775A1
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cell
tumor
tumor cell
cells
fusion product
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Yajun Guo
Mark L. Tykocinski
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    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • 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
    • A61K40/13—B-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • 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
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/05—Immunological preparations stimulating the reticulo-endothelial system, e.g. against cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • 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
    • A61K2239/53—Liver

Definitions

  • the present invention relates to products and methods useful for tumor immunotherapy.
  • tumors that arise de novo are poorly immunogenic, thereby escaping host antitumor responses (Hewitt et al. , 33 Br. J. Cancer 241, 1976) .
  • Methods that have been described for enhancing tumor immunogenicity include: (1) using mutagen or drug treatment (Van Pel and Boon 79 Proc. Natl. Acad. Sci. USA 4718, 1982 and Frost el al. , 159 J. EXP. Med. 1491, 1984) ; (2) transfecting with a foreign gene encoding an exogenous antigen such as influenza hemagglutinin (Fearon et al. , 38 Cancer Res .
  • Antigen-presenting cells provide molecular signals including signals mediated by APC-derived soluble cytokines and APC-derived cell surface costimulators such as: (1) B7 (Linsley et al. , 87 Proc. Natl. Acad. Sci. U.S.A. 5031, 1990) ; (2) ICAM-I (van Seventer et al. , 144 J. Immunol. 4579, 1990) ; (3) VCAM-I (van Seventer et al . , 174 J. EXP. Med. 901, 1991) ; (4) LFA-3 (van Seventer et al., 21 Eur. J. Immunol.
  • a tumor cell once appropriately modified through genetic manipulation, can itself function as an APC (Chen et al . , 71 Cell 1093, 1992; Townsend et al. , 259 Science 368, 1993; Tykocinski &_ IIan, 259 Science 94, 1993) .
  • the present invention provides products and methods useful for enhancing tumor immunogenicity. More specifically, the present invention provides a cell fusion product, and methods of using the cell fusion product to enhance tumor immunogenicity.
  • the general usefulness of this technology relates to the prevention and treatment of various diseases, including cancer.
  • the disease may be present in any animal, including a human.
  • This technology further embraces a wide range of utilities including inducing the production of antibodies in vitro.
  • the present invention may also be used to induce the production of antibodies in a variety of animals, including humans.
  • the immunogenicity of a tumor cell can be remarkably enhanced by fusing a membrane of a tumor cell to a membrane from a another cell with greater immunogenic potential than the tumor cell.
  • the phrase "cell fusion partner" is used to describe any other cell with a greater immunogenic potential than the tumor cell.
  • Membrane extracts or whole cells may be fused in the present invention.
  • fusion of a membrane from a tumor cell with a membrane from a cell fusion partner changes the capacity of the tumor cell to activate specific T-cell responders in the host immune system so that an immune response can be mounted against that tumor cell.
  • introduction of a hybrid cell comprising a tumor cell fused to a cell fusion partner not only reduces tumor growth, but also the growth of normal tumor cells (i.e. , non-fused tumor cells) within the same host.
  • the cell fusion partner contributes relevant tumor-specific antigens to the hybrid cell and that the cell fusion partner contributes cell surface costimulators, soluble cytokines, MHC molecules, and other undefined molecular factors to the hybrid cell. These contributions in aggregate result in a highly antigenic and immunogenic phenotype for the hybrid cell.
  • the hybrid cell can be used as an effective cellular vaccine.
  • this proposed theory is not meant to act as a limit to any alternative theories or mechanisms of carrying out the invention.
  • the present invention is based upon the finding that enhanced immunogenicity can be induced by fusing a cell membrane from a tumor cell with a cell membrane from a cell fusion partner, preferably one capable of effective antigen presentation and T-cell activation.
  • a cell fusion partner preferably one capable of effective antigen presentation and T-cell activation.
  • the full complement of molecular factors that are normally produced by APC's and that are required for effective T-cell activation are combined with the full set of potential tumor antigens associated with a particular tumor.
  • fusion partners it is possible to tailor the nature of the anti-tumor T- cell immune response.
  • the invention features methods for inducing immunity against a tumor cell by providing to a patient a cell fusion product.
  • cell fusion product is meant a cell membrane from a tumor cell fused to a cell membrane from another cell that has a greater immunogenic potential than the tumor cell.
  • One type of cell fusion product is a hybrid cell.
  • hybrid cell is meant a cell which is derived from the fusion of two parental cells, and it is either the direct fusion product or a daughter cell that is derived by cell division from the original fusion product .
  • a hybrid cell contains one or more molecular components of each of its parental cells.
  • parental cell is meant either component of the hybrid cell and includes a tumor cell or another cell with greater immunogenic potential than a tumor cell.
  • step of "providing” is meant any method that results in the presence of a cell fusion product in a patient .
  • the step of "providing” can be performed in a variety of ways including either administering a cell fusion product that was formed ex vivo or fusing the membranes in vivo.
  • immunological is meant the state of being refractory to a specific disease, which is mediated by the immune system or a state of not being susceptible to the invasive or pathogenic affects of potentially infective microbes or to the affects of potentially toxic antigenic substances.
  • immune response is meant the response of the whole or part of an immune system of an organism. This response could include the activation of cellular or humoral systems, including B-cells, and T-cells.
  • tumor is meant a collection of cells, usually dysfunctional, due to abnormal proliferation. Benign tumors are not life threatening, e.g., warts. Malignant tumors are potentially lethal cancers. All tumor types may be treated using the methods of the present invention, since cell fusion is not dependent upon any particular cell phenotype.
  • the tumor cell may be autologous, heterologous, cultured, primary, or metastatic.
  • a tumor cell is from the patient to be treated, or from another patient having a common major histocompatibility phenotype.
  • primary is meant that a tumor cell from the organ of tumor origin in the patient to be treated is used. It also means a primary cultural cell, as distinct from a cell line.
  • metal is meant that the tumor cell is proliferating at sites distant from the organ of tumor origin.
  • heterologous is meant that a tumor cell from another patient is used. Clinicians and others skilled in the art are able to identify patients in need of treatment using procedures that are well known and routine in the art . Procedures for obtaining a turner cell from such a patient and for culturing such a cell are also well known and routine in the art.
  • patient is meant any animal, including a human, with a tumor.
  • fusing is meant a process whereby the cell membranes are combined into a single membrane and a cell fusion product is formed.
  • the fusing may be performed by directly injecting the cell fusion partner into a tumor mass in a patient. This may further involve identifying a patient in need of tumor therapy, obtaining a tumor cell from the patient, and culturing the tumor cell.
  • the fusion step may involve more than two fusion partners.
  • the fusion step may also involve the use of a chemical fusogen, such as polyethylene glycol, or electrofusion.
  • immunogenic potential is meant the capacity to activate specific T-cell responders in the immune system or the ability to raise an immune response in an animal, preferably a human. It is commonly found that tumor cells have poor immunogenic potential relative to APCs.
  • anti-tumor response is meant any response that measurably reduces the size of a tumor, including the complete destruction of the tumor.
  • membrane extract an extract of a cell enriched for membranes, but not necessarily containing only membranes. Such an extract is chosen because it will have the antigenic and immunogenic properties necessary to induce an immune response to the tumor cell in vivo or ex vivo.
  • a cell membrane extract may be derived from an autologous or heterologous tumor cell or another cell with greater immunogenic potential than the tumor cell.
  • the tumor cell or cell fusion product may be treated, for example by irradiation, to reduce its ability to proliferate.
  • membrane is meant a sheet, usually about 10 nm thick and normally composed of a bimolecular layer of lipid and protein, enclosing or partially enclosing a cell, organelle, or vacuole.
  • Cell fusion products may be formed using less than an entire membrane, for example, portions of membranes may be used.
  • the invention features methods for inducing immunity against a tumor cell by providing to a patient either a tumor cell fused to an APC or the fused membranes of such cells.
  • a conventional APC include an activated B-cell, a dendritic cell, a macrophage, an activated T-cell, or an endothelial cell.
  • Methods for identifying other cell fusion partners that can confer enhanced immunogenicity to a tumor cell are defined herein.
  • Preferred candidate cells are those for which there is evidence of immunogenic potential.
  • the invention features methods for inducing immunity against a tumor cell by providing to a patient a tumor cell fused to an activated B-cell with an artificial adhesin.
  • artificial adhesin is meant a genetically engineered molecule that is expressed through gene transfer, or through protein transfer is exogenously coated, on the surface of a cell and thereby promotes adhesion to another cell expressing a molecule on its surface that can bind to the engineered molecule.
  • artificial adhesins include a glycosyl-phoshatidylinositol-modified polypeptide or a biotin-lipid conjugate, or other compounds with equivalent properties.
  • the invention features methods for inducing immunity against a tumor cell by providing to a patient a T-cell activated by contact with a cell fusion product.
  • the T-cell may be part of an immunoselected subset such as CD8-positive T-cells.
  • T-cells are known to be critical mediators of anti-tumor immunity.
  • T-cells are activated by cells collectively referred to as "antigen-presenting cells” (APC) .
  • APC antigen-presenting cells
  • the diverse cell types that comprise this category share the ability to present antigens, via their major histocompatibility molecules, to the T-cell receptors on antigen-specific T-cells.
  • the present invention discloses the capacity of a cell fusion product to stimulate anti-tumor T-cells.
  • This capacity can be utilized not only for in vivo stimulation of T-cells, but also for ex vivo stimulation of T-cells.
  • Ex vivo stimulation of a T-cell using a cell fusion product can be used as a means of amplifying T- cells with tumor specificity prior to infusion of such T- cells into patients .
  • Methods are well known in the art for delivering T-cells into patients.
  • Methods are well known in the art for deriving T-cells from the peripheral blood of cancer patients or isolating infiltrating T-cells directly from tumors and nonspecifically amplifying their cell numbers using reagents such as interleukin-2.
  • T-cells contacting such T-cells with a cell fusion product offers a means for selectively amplifying the tumor-specific T- cells out of the mixed T-cell populations at these sites. Once • amplified, the T-cells can be re-infused into a patient. It should be evident from this that the same patient can be coordinately treated with a cell fusion product, as an active vaccine, along with ex vivo amplified T-cells, as a passive vaccine. This combined treatment maximizes therapeutic effects and is advantageous for immunosuppressed patients .
  • the invention features a method for identifying a cell fusion partner that can be fused to a tumor cell to generate a hybrid cell with greater immunogenicity than the tumor cell.
  • This method involves fusing a tumor cell with a candidate cell and determining the immunogenicity of the resulting cell fusion product.
  • Such methods can be based in vivo or ex vivo. Clinicians and others skilled in the art are able to determine the immunogenicity of a cell using procedures that are well known and routine in the art.
  • the invention features a method for fusing a tumor cell with another cell.
  • This method involves expressing an artificial adhesin on either the tumor cell, the other cell, or both.
  • the method also involves combining the cells with a fusogenic agent.
  • fusogenic agent is meant any compound that increases the ability of a membrane from a tumor cell to fuse with a membrane from another cell that has greater immunogenic potential than said tumor cell.
  • the present invention discloses that selectivity can be conferred to a cell fusion process by inducing relevant paired cells to adhere to each other prior to addition of a fusogenic agent.
  • a pre- adhesion step By combining such a "pre- adhesion" step with subsequent fusion at low cellular densities (generally below IO 5 cells/ml) , more efficient fusion can be achieved.
  • use of low cell density is not a required parameter in this invention.
  • a preferred method for achieving such pre- adhesion is through the use of an artificial adhesin that has been delivered to a relevant cell surface by any one of a number of gene and/or protein transfer methods .
  • alternative methods that do not involve artificial adhesins can be used to achieve pre-adhesion.
  • a heterobifunctional antibody can be used to adhere a tumor cell and an APC. The adherence-inducing method should not perturb the antigenicity and immunogenicity of the hybrid cell to be used as a membrane source for immun
  • the invention features an immunogenic cell fusion product.
  • a membrane from a tumor cell is fused to a membrane from a another cell with greater immunogenic potential than the tumor cell.
  • the immunogenic cell fusion products of the present invention are distinct from hybridomas used for monoclonal antibody production where a cultured myeloma cell is fused with a splenocyte.
  • the immunogenic cell fusion product of the present invention is also distinct from non-immunogenic hybrid cells used in routine laboratory experiments. However, the methods described above for inducing immunity against a tumor cell can utilize these hybridoma and hybrid cells.
  • the membrane of a tumor cell isolated from a patient is fused with a membrane from another cell, such as an APC, with greater immunogenic potential than the tumor cell.
  • a primary tumor cell is involved in the fusion process .
  • myelomas which have the ability to produce antibodies the tumor cell of the present invention need not be able to make antibodies.
  • the cell fusion products of the present invention generally exclude the use of a myeloma cell fused to a splenocyte.
  • One or more of the cells that generate the cell fusion product, or alternatively the cell fusion product itself, may be molecularly modified prior to administration to a patient.
  • Molecular modifications can be directed toward any one of a number of functional endpoints, including enhancing the fusion process, promoting selective adhesion between the parental cells, altering the in vivo tissue targeting properties of the cell fusion products, and further enhancing the immunogenicity of the cell fusion product above and beyond the enhanced immunogenicity that would otherwise be conferred to the hybrid cell by the parental cell.
  • a cell fusion partner may be modified prior to fusion with a tumor cell in order to increase the immunotherapeutic efficacy of the resulting hybrid cell. This modification can be effected by alternative methods, including gene or protein transfer.
  • proteins that can be expressed or inhibited in an APC include, a cell surface costimulator, a soluble cytokine, a selectin, an adhesin, a major histocompatibility complex protein, and a coinhibitor (e.g., CD8) .
  • foreign protein is meant a protein that is not normally expressed in a particular cell.
  • natural cell surface molecule is meant any molecule that is naturally found on the surface of a particular cell.
  • One molecular modification entails the coating of one or more of the parental cells with artificial adhesins that promote selective adhesion between the two cell types prior to the fusion event.
  • a tumor cell or another cell may be modified to enhance its fusion potential.
  • the tumor cell can be modified in vivo or in vitro prior to cell fusion.
  • the tumor mass may be modified in vivo, or the other cell may be modified in vitro, or both.
  • the useful jLn vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular cellular compositions employed, and the specific use for which these cellular compositions are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, will be within the ambit of one skilled in the art .
  • the present invention also provides kits including materials used in cell fusion.
  • the invention generally features generation of a fusion product, e.g., through cell fusion, to provide a reagent suitable for immunization against a tumor, either in a prophylactic or treatment procedure, and features methods for identifying the optimal cell fusion partner to be fused with a tumor cell derived from such a tumor.
  • tumor cell immunogenicity may be enhanced by fusion with another cell with greater immunogenic potential than the tumor cell.
  • the use of cell fusion provides substantial advantages for practicing tumor cell engineering and enhancing tumor cell immunogenicity. Advantages of cell fusion over gene transfer include, but are not limited to, the following:
  • the present invention obviates the need for decoding the precise molecular signaling systems for individual tumor cell:T-cell combinations.
  • the present invention bypasses the general lack of understanding of the composite set of antigenic peptides and costimulatory molecules required for effective anti-tumor T-cell responses.
  • cell fusion is applicable to diverse tumor cell types. Unlike gene transfer, cell fusion is not dependent upon the proliferative potential of the tumor cell, and hence, can be applied to a variety of tumor types which grow poorly in primary culture.
  • cell fusion is a relatively rapid process and does not impose a burden of excessive cell culturing, shortening the interval between biopsy and treatment .
  • Certain gene transfer-based immunotherapeutic strategies require selection for stable transfectants. This can be a time consuming process and complicates the clinical practice of such methods and imposes a delay period between biopsy and treatment .
  • Cell fusion as a means of enhancing tumor immunogenicity according to the present invention, may be combined with other known methods for enhancing tumor immunogenicity, including those cited herein, for example, expression of an exogenous gene in a tumor cell or inhibition of an endogenous gene in a tumor cell. Indeed, it is believed appropriate to combine the therapies or methods described herein with other methods to enhance the immunogenicity of a tumor cell .
  • Fig. 1 shows schematic diagrams of the pM-CSF- ' GPI/REP4 ⁇ . and pM-CSFR/REP7j ⁇ expression constructs which encode members of an artificial adhesion pair.
  • the M- CSF-DAF chimeric sequences are depicted in the multiple cloning site of pREP4 ⁇ .
  • the M-CSF receptor coding sequence is depicted in the pREP7S vector.
  • EBV oriP Epstein-Barr virus origin of replication
  • EBNA-1 EBV nuclear antigen- 1
  • RSV 3'LTR Rous sarcoma virus 3' long terminal repeat promotor
  • PA SV40 polyadenylation/termination sequences
  • hph hygromycin-B resistance gene
  • Amp ampicillin- resistance gene.
  • the present invention addresses the need for conferring a complex phenotype, immunogenicity, to diverse types of tumor cells .
  • Methods are provided for conferring this complex phenotype to tumor cells ex vivo and in vivo.
  • methods are provided for using modified tumor cells to generate T-cells to be used for cell transfer.
  • Yet other methods are provided for enhancing adhesion between a tumor cell and another cell, as a prelude to fusion of the two cells.
  • the present invention entails the fusion of a tumor cell to another cell.
  • Methods designed to promote intercellular adhesion can be combined with conventional non-selective cell fusion methods in order to selectively target and enhance the cell fusion process.
  • nonselective fusion agents that are routinely used to fuse cells will not only induce tumor cell:APC fusions, but also tumor cell:tumor cell and APC:APC fusions as undesired byproducts.
  • a cell fusion method for minimizing these undesired fusion events and simultaneously maximizing the desired tumor cell:APC fusion events. This is accompanied by artificially promoting adhesion between a tumor cell and an APC, without influencing self-adhesiveness, and adding fusing agents or performing electrofusion when adherent tumor cell:APC conjugates are at low cell densities. The lower cell densities would be less favorable to fusion between nonadherent cells of the same cell type.
  • a combined adhesion/fusion method for generating tumor cell:APC hybrids can comprise any one of a number adhesion and fusion component methods.
  • a method for altering the adhesive properties of cells that is particularly well- suited for the adhesion/fusion method has been developed. This method is based upon the use of a class of molecules that can be designated by the term "artificial adhesins. " Specifically, in one example, a glycosyl- phosphatidylinositol (GPI) -modified variant of the cytokine macrophage colony stimulating factor (M-CSF) , designated M-CSF-GPI, was expressed on the surface of human bone marrow stromal cells.
  • GPI glycosyl- phosphatidylinositol
  • M-CSF-GPI cytokine macrophage colony stimulating factor
  • M-CSF decay- accelerating factor expression construct
  • Cell:cell binding assays established that this artificially membrane-tethered cytokine functioned as a potent cellular adhesin, allowing for enhanced binding to M-CSF receptor-expressing cellular transfectants.
  • Antibody blocking analyses confirmed the M-CSF:M-CSF-receptor dependence of the enhanced intercellular binding. This capacity to direct the cellular interactive repertoire of selected cells can in principle be applied to other cell types and other molecular pairs to be used in cell-based therapies.
  • Intercellular adhesion is mediated by homotypic and heterotypic molecular interactions at membrane interfaces .
  • cell surface molecules that have been assigned functions as natural adhesins in regulatory interactions between cells. Since natural adhesins have frequently been found to be multifunctional, they often cannot be used as neutral molecules for altering cellular adhesive properties.
  • natural adhesins by virtue of being transmembrane hydrophobic peptide-anchored molecules, can only be expressed on cell membranes by gene transfer. Therefore, methods are needed to artificially modify adhesiveness between cells in a neutral way and without necessarily using gene transfer.
  • GPI anchors for adhesins stems from the fact that purified GPI-modified proteins, by virtue of their amphophilic properties, can be readily reincorporated into cell membranes in the presence of low, non-lytic concentrations of non-ionic detergents such as NP-40 (Medof et al . , 160 J. Exp. Med. 1558, 1984) or even in the absence of detergent.
  • non-ionic detergents such as NP-40 (Medof et al . , 160 J. Exp. Med. 1558, 1984) or even in the absence of detergent.
  • cells can be coated with purified GPI- modified polypeptides, representing a form of "protein paint,” bypassing the requirement for gene transfection into the cell whose surface is being molecularly engineered. Delivery of exogenous polypeptides to cells by such a protein transfer approach circumvents problems associated with gene transfer, particular in the case of primary, nontransformed cells which in general cannot be easily transfected.
  • the immunostimulatory and/or effector properties of cells used in cell-based therapies such as tumor cell:APC hybrids, APCs, immunogenic tumor cells, or T-cells, could be selectively enhanced by increasing their adhesive properties in a selective way.
  • a preferred protein transfer method for coating cells with artificial adhesins involves the use of GPI- modified proteins. Methods for performing protein transfer using GPI-modified proteins have been described.
  • Another preferred protein transfer method for coating cells with artificial adhesins entails the use of a chimeric polypeptide in which an adhesin polypeptide sequence, for example, M-CSF, is linked to a streptavidin sequence.
  • an adhesin polypeptide sequence for example, M-CSF
  • streptavidin sequence Methods for using prokaryotic expression systems to quantitatively produce such polypeptide- streptavidin chimeras are known to those familiar with the art.
  • the cell of interest is pre-coated with biotin.
  • a useful method for pre-coating cells with biotin is through the use of biotin-lipid conjugates which can be used to pre- coat cells to high biotin densities (up to IO 7 biotin molecules/cell) .
  • the cells can be chemically biotinylated using standard cellular biotinylation procedures.
  • a chimeric adhesin-streptavidin polypeptide is added to a pre-biotinylated cell, in order to generate a cell expressing the artificial adhesin at its surface. It is generally not avidin on one cell and biotin on a second cell that are being used to bring two cells together, but instead avidin and biotin are usually simply being used on the same cell surface to deliver an adhesion to that cell's surface. Then, the same process may be applied to a second cell again, potentially using an avidin-biotin complex at that cell surface.
  • a tumor cell can be coated with one member of an artificial adhesin pair, and a conventional APC can be coated with the other member of the pair.
  • a conventional APC can be coated with the other member of the pair.
  • cell populations of each of the cell types are combined, intercellular conjugates form, pairing a tumor cell with an APC.
  • an electric current is applied, or a chemical fusing agent is added, to the mixed cell population, adherent cells within conjugates preferentially fuse. Preferential fusion can be further promoted by keeping the mixed cell population at a low cell density. This will minimize the formation of extraneous tumor cell:tumor cell and APC:APC hybrids with no immunotherapeutic potential.
  • a preferred method for fusing cells comprises the use of polyethylene glycol as a fusing agent.
  • Other methods of fusing cells can be used, including electrofusion or use of viruses or viral components, for example, Sendai virus, that promote cell fusion.
  • Example 1 Hepatocarcinoma fused to an activated B-celi lose their tumorigenicitv Activated B-cells are effective APCs (Lanzavechia, 140 Nature 1985, 1985; Ron, 138 J. Immunol. 2848, 1987; and Kurt-Jones, 140 J. Immunol . 3773, 1988) .
  • BERH-2 is a chemical carcinogen-induced rat hepatocellular carcinoma cell line from the Wistar rat. BERH-2 grows rapidly and forms tumors in the liver of syngeneic animals.
  • BERH-2 cells were fused with activated B-cells in an attempt to enhance the immunogenicity of BERH-2 cells.
  • the data provided herein indicates that the hybrid cells, designated BERH-2- B, became more immunogenic and less tumorigenic than the tumor cell .
  • Example IA Fusion and selection of BERH-2-B cells
  • Activated B-cells were obtained from the spleens of rats injected 14 days earlier with bovine serum albumin in complete Freud's adjuvant.
  • BERH-2 cells were fused with these purified activated B-cells using polyethylene glycol, using a standard B-cell hybridoma fusion protocol.
  • the fused cells, designated BERH-2-B were selected by panning, first with a rabbit anti-BERH-2 antiserum and second with a rabbit anti-rat B-cell antiserum.
  • the rabbit anti-BERH-2 and rabbit anti-rat B- cell antisera were prepared by immunizing rabbits subcutaneously with either BERH-2 hepatoma or purified B- cells from Wistar rats in complete Freud's adjuvant. Activated B-cells were purified by panning with plates coated with purified goat anti-rat Ig antibody. After repeat boosting during two months, antiserum was collected and purified by protein G-sepharose chromatography. Finally, the antiserum was repeatedly absorbed with either BERH-2 hepatoma cells or rat B-cells.
  • Example IB Expression and characterization of antigens Expression of class I MHC, class II MHC, B7, ICAM-1 and FA-1 on BERH-2 cells, activated B-cells and BERH-2-B hybrid cells were assessed. Cells were washed with phosphate-buffered saline (PBS) and stained with monoclonal antibody to rat MHC class I (OX-18) , MHC class II (OX-6) , ICAM-1 (LA 29) or LFA-1 (WT.l) as primary antibody. To stain for rat B7, we used a chimeric protein, CTLA-4-Ig. Cells were incubated with the antibodies or chimeric protein for 30 minutes on ice.
  • PBS phosphate-buffered saline
  • a mouse anti-human CD3 monoclonal antibody (GH3, IgG2b) and a chimeric human CD44-Ig protein were used as negative controls. Cells were washed three times. FITC-goat anti- mouse Ig or FITC-labeled rabbit anti-human Ig was used as a secondary antibody and added for another 30 minutes on ice. After washing, samples were fixed and analyzed in a FACScan flow cytometer.
  • Parental and hybrid tumor cells were phenotyped by immunocytochemical staining and flow cytometry.
  • the parental BERH-2 cells expressed low levels of class I MHC antigen and ICAM-1, but lacked class II MHC antigen, LFA-1 and the costimulator B7. All four hybrid BERH-2-B cell lines displayed increased class I MHC expression.
  • BERH-2-B hybrid cell lines expressed MHC class II antigen, ICAM-1, LFA-1 and B7. These BERH-2-B cell lines have stably expressed both tumor and B-cell antigens for more than five months.
  • Example 1C Comparison of tumoricrenicity of parent and hybrid cells The tumorigenicity of parental BERH-2 and hybrid
  • BERH-2-B cells were compared, and the survival data shows enhanced animal survival for syngeneic animals injected with BERH-2-B hybrid .tumor cells as compared to BERH-2 tumor cells.
  • Two groups of female Wistar rats (ten/group) were injected intrahepatically with 2 x 10 s BERH-2 cells or 2 x IO 6 BERH-2-B hybrid cells. All animals injected with BERH-2 parental cells developed liver tumors and died within 60 days. In contrast, the BERH-2-B injected rats remained tumor-free for more than 180 days. While the four hybrid cell lines lost their ability to form tumors in syngeneic rats, they were able to grow and form tumors in nude mice.
  • a series of tumor cure experiments were next performed to show that immunization with BERH-2-B cells could also eradicate an established hepatoma.
  • One set of fourteen rats were injected intrahepatically with 2 x IO 6 parental BERH-2 cells. Ten days later, eight of the injected rats were immunized with a subcutaneous injection of 5 x IO 6 BERH-2-B hybrid cells. These rats survived for more than 120 days.
  • rats injected both times with parental BERH-2 cells all died within 42 days.
  • a second set of rats were surgically implanted with a small fragment of BERH-2 hepatoma intrahepatically.
  • Fourteen rats were intrahepatically implanted with a small fragment (0.3 mm x 0.5 mm) of BERH-2 tumor.
  • Two days later, eight of the animals were injected subcutaneously with 5 x 10 s BERH-2-B hybrid cells.
  • the other six rats were injected subcutaneously with same number of BERH-2 cells.
  • Ten days later, a subset of the tumor-implanted animals were injected with BERH-2-B cells, the remaining control rats were injected with parental BERH-2 cells.
  • Example 2B Determination of type of T-cell mediation
  • mice Female Wistar rats (Groups A, B, C, D) were treated with purified anti-rat CD4 (0X38) , or anti-CD8 (OX-8) or a control mouse anti-diethylthiamine pentaacetic acid monoclonal antibody. Each animal received 500 ⁇ g of the purified antibody intravenously twice per week for three weeks.
  • peripheral blood lymphocytes were obtained from individual treated rats and stained with monoclonal antibodies to CD4 or CD8 to verify the depletion of CD4 + or CD8 + cells, respectively.
  • Treatment with anti-CD4 monoclonal antibody depleted more than 95% of the CD4 + cells, and treatment with anti-CD8 monoclonal antibody depleted close to 95% of the CD8 + cells; treatment with control antibody did not alter the number of CD4 + and CD8 + cells.
  • All rats were injected intrahepatically with 5 x 10 s BERH-2-B tumor cells.
  • Example 2D Groups E-G
  • Rats were first immunized with BERH-2-B cells and then depleted of CD4+ or CD8+ cells 14 days later.
  • Female Wistar rats (Groups E, F, G) were first immunized with 2 x IO 6 BERH-2-B cells subcutaneously. Two weeks after immunization, animals were treated with anti-CD4, or anti-CD8 or control antibody, and the effectiveness of the depletions was verified by immunofluorescence and flow cytometry. Three days after the last injection of monoclonal antibody, all animals received 5 x 10 s BERH-2 cells intrahepatically. These experiments have been repeated twice with comparable results.
  • CD4-or CD8-depleted rats were then challenged with BERH-2 cells. Tumors developed in CD8- depleted, but not in CD4-depleted rats. This indicates that whereas both CD4 + and CD8 + cells are necessary for the induction of protective anti-tumor immunity, once the immune response has been induced, CD8 + cells are sufficient alone to mediate tumor cell destruction alone. These results contrast with those reported previously for murine melanoma cells transfected with the B7 costimulator gene where CD4 + cells were not required for induction of the anti-tumor immune response (Chen, 71 Cell 1093, 1992; Townsend, 259 Science 368, 1993) .
  • Example 2E Tumor-specificity of immunity
  • NBT-II is a bladder carcinoma that grows rapidly in syngeneic Wistar rats. Immunization with BERH-2-B hybrid cells prevented the growth of the parental BERH-2 cells. However, immunization with BERH-2-B was unable to inhibit the growth of NBT-II cells.
  • the specificity of the anti-BERH-2 immune response elicited by BERH-2-B hybrid tumor cells was documented.
  • Female Wistar rats were injected with 2 x IO 6 BERH-2-B cells subcutaneously. Two weeks after immunization, one group of the rats were injected with 5 x IO 6 BERH-2 cells intrahepatically. Another group of rats were injected with 5 x IO 6 NBT-2 rat bladder carcinoma cells (obtained from the American Tissue Type Collection) .
  • Tumor protective immunity induced with BERH-2 tumor cells fused with activated B cells does not require in vitro selection.
  • Three groups of rats (8/group) were injected subcutaneously with 5 x IO 6 BERH-2 cells, 5 x IO 6 BERH-2 cells mixed with 5 x IO 6 activated B cells, or 5 x IO 6 BERH-2 cells fused with 5 x IO 6 activated B cells in the presence of PEG. Fused cells were washed three times with PBS, resuspended in PBS and injected subcutaneously. Two weeks later, all groups of rats were challenged with 5 x IO 6 BERH-2 tumor cells intrahepatically.
  • BERH-2 tumor cells were fused with activated B- cells. After fusion, cells were washed and injected into syngeneic rats with in vitro selection. The efficiency of the fusion in such experiments ranged form 30% to 50%. As controls, BERH-2 tumor cells mixed with activated B-cells without PEG were injected subcutaneously. All animals were then challenged with the parental BERH-2 cells intrahepatically.
  • hybrid BERH-2-B cells may express other molecules that are essential for the activation of anti- tumor T-cells. This may include, but is not limited to, soluble cytokines . Production of cytokines by hybrid tumor cells may be important in the elicitation of host immune responses.
  • Example 3 Glycosylphosphatidylinositol (“GPI" ) -modified cytokine can function as an artificial adhesin
  • GPI Glycosylphosphatidylinositol
  • pREP4o two EBV vector variants designated pREP4o. and pREP73 were used, both of which share a transcriptional cassette in which the RSV 3' LTR promoter, a multiple cloning site, and the SV40 late polyadenylation/termination signal are linked in tandem.
  • M-CSF-GPI An expression construct for M-CSF-GPI was generated as follows.
  • the 1.8 kb M-CSF coding region fragment (Xhol - EcoRI) of p3ACSFRl (Fig. 1) was inserted into the corresponding sites of pBluescript (pBT, Stratagene, Inc.) .
  • This generated a GPI-anchored variant of M-CSF, pM-CSF/BT was cut with Ncol, filled-in with the pollk, and subsequently cut with BamHI.
  • An M-CSF receptor EBV episomal expression construct was generated as follows : A . Okb EcoRI fragment of pc-fms 102 was subcloned into the EcoRI site of pBluescript (Stratagene, Inc.) to generate pM-CSFR/BT. The 3.6 kb BamHI fragment of this subclone, containing the entire M-CSFR coding region, was subcloned in a sense orientation into the BamHI site of pREP7/S to generate pM- CSFR/REP7/3. The overall experimental strategy of this study was to use stable gene transfer to modify the adhesive properties of cells.
  • the paired cellular targets chosen were the human SV40 large T-immortalized bone marrow stromal cell line KM-102 and the human myeloid leukemia cell line K562. Previous work with both the KM-102 and K562 lines have shown them to be efficient transfection targets with EBV expression vectors.
  • M-CSF-GPI an artificial GPI- modified variant of M-CSF
  • natural M-CSF coding sequence is linked in-frame to the GPI signal sequence of human DAF, into KM-102 stromal cells.
  • Indirect immunofluorescence staining demonstrated a high level surface expression of M-CSF epitopes on the pM-CSF GPI/REP40! hyg R KM-102 transfectants.
  • M-CSF epitope was detectable on the surface of nontransfected KM-102 cells or on KM-102 cells transfected with the irrelevant EBV episome pRSVCAT ⁇ /220.2.
  • pM-CSFR/REP73 and pM-CSF GPl/REP4o were introduced into K562 and U937 cells, respectively, by lipofection.
  • the KM-102 stromal cell line was kindly provided by K. Harigaya and maintained in McCoy's 5a medium (Gibco, Inc.) supplemented with 10% heat- inactivated fetal bovine serum (FBS) (M.A.
  • pM-CSF-GPI/REP4o_ was introduced into KM-102 cells by lipofection. Briefly, cells were grown to 50% confluence in six-well plates, and washed twice with PBS and once with Opti-MEM (Gibco) . Cells were then incubated for 5-8 hours at 37°C with 1 ml Opti-MEM, containing 10 ⁇ g DNA and 30 ⁇ g lipofectin, before adding 1 ml of complete medium containing 20% FBS.
  • M-CSFR expression was confirmed by indirect immunofluorescence and flow cytometry (FACS) , using a rat monoclonal anti-M-CSFR primary antibody (Oncogene Sciences) and FITC-conjugated rabbit anti-rat IgG secondary antibody (Miles ICN) .
  • K562 leukemic cells were transfected with human M-CSF receptor (M-CSFR or c-fms) episomal expression construct, designated pM-CSFR/REP73.
  • M-CSFR human M-CSF receptor
  • c-fms c-fms episomal expression construct
  • PIPLC phosphatidylinositol-specific phospholipase C
  • PIPLC cleavage was performed by incubating 1 x 10 s cells with 1 unit of PIPLC (Boehringer Mannheim Biochemicals) at 37°C for one hour in RPMI 1640 medium containing 10%FBS and 0.01% sodium azide. Cleavage was assessed using an anti-DAF antibody and flow cytometry. In light of this finding, pM-CSF-GPl/REP4 ⁇ ! was additionally transfected into the U937 cell line which is known to produce PIPLC-sensitive GPI anchors. PIPLC susceptibility of M-CSF and endogenous DAF on these cells was assessed. Nontransfected U937 cells did not express M-CSF on their surface. The pM-CSF-GPl/REP4o.
  • U937 transfectants expressed high levels of cell surface GM- CSF U937 transfectants expressed high levels of cell surface GM- CSF.
  • the tethered M-CSF could be specifically cleaved with PIPLC to an extent similar to that seen with endogenous GPI-anchored DAF protein.
  • the PIPLC cleavage yielded removal of surface M-CSF epitope to the same extent as DAF. This indicates that the pM-CSF-GPI/REP4o ⁇ construct generates a GPI-anchored form of M-CSF.
  • KM-102 transfectants would bind preferentially M-CSFR + cellular targets.
  • Cell:cell binding between adherent KM-102 cells and M-CSFR + nonadherent K562 targets cells was enhanced approximately three-fold when M-CSF-GPI was present on the surface of the KM-102 cells.
  • Intercellular adhesion between adherent KM-102 transfectants and nonadherent K562 transfectants was measured using a cell:cell binding assay.
  • K562 target cells either nontransfected (none) or expressing the M-CSF receptor (PM-CSFR/REP73) or CAT (pRSVCAT/220.2) , were added to the wells and allowed to bind to the KM-102 cells.
  • the number of K562 target cells which remained adherent following an inverted centrifugation was calculated by measuring the specific activity of the target cells added.
  • a significant increase in adherence between pM-CSFR-REP4 ⁇ . transfected KM-102 and pM-CSFR-REP37 transfected KM-102 was noted whereas no increase in adhesion was noted for other combinations .
  • Nontransfected K562 cells (none) or K562 cells stably expressing the M-CSFR (pM-CSFR/REP7j ⁇ ) or CAT
  • pRSVCAT/220.2 were allowed to bind to KM-102 cells expressing GPI-anchored M-CSF (pM-CSF-GPI/REP4o.) .
  • Blocking antibodies were added to the appropriate cells, as indicated, 30 min. prior to adding the cells to the wells.
  • Anti-M-CSF polyclonal anti-M-CSF antibody
  • anti- M-CSFR monoclonal anti-M-CSF receptor (c-fms) antibody
  • an anti-TRF monoclonal anti-transferrin receptor antibody.
  • Normal rabbit serum (NRS) was added, as indicated, to prevent F receptor cross-linking.
  • Cell:cell binding assays employed a modification of a published method (McClay et al. , 78 Proc. Natl. Acad. Sci USA 4975, 1981) . Briefly, 3 x IO 4 nontransfected or transfected KM-102 cells were placed in wells of a polyvinyl, flat-bottom 96-well plate (Dynatech Laboratories) with 0.1 ml complete medium per well, and the cells were incubated at 37°C for two days. Wells were pretreated with fetal bovine serum for two hours to promote attachment of the KM-102 cells. The KM-102 cells were generally 60-80% confluent at the time of the cell:cell binding assay. The K562 target cells, labeled with 35 S-methionine, were washed, resuspended in complete medium at 5 x 10 s cells per ml, and 0.1 ml was added directly to the wells.
  • the plates were incubated at 37°C for 2.5 hours to allow for maximal binding. Medium was added to each well to produce a positive meniscus, and then plates were carefully sealed with adhesive plate sealers (Dynatech Labs, Inc.) . The plates were inverted and centrifuged for 10 minutes at room temperature using Sorvall micro-plate carriers. A relative centrifugal force (RCF) of 900 x g was used for most experiments. Post-centrifugation, still inverted plates were flash-frozen at -80°C, and the bottoms of each well, containing the stromal cells and bound targets, were cut off and placed in scintillation vials for counting. The number of K562 cells bound per well was calculated as follows:
  • Each group represents the means of at least triplicate samples. Representative KM-102 stromal cell wells were harvested and counted to ensure that equivalent cell numbers were present in each well.
  • antibodies were added directly to cell suspensions or to wells (as indicated) and incubated at room temperature for 30 minutes just prior to the addition of target cells to the wells.
  • the final concentrations of antibodies used in the blocking studies were: rabbit anti-human M-CSF polyclonal antibody (Genzyme) , lO ⁇ g/ml; rat anti-c-fms/CSF-1 receptor
  • an artificial GPI-modified variant of a model cytokine M-CSF when anchored at the cell surface, can augment cellular binding to M-CSF receptor-bearing tumor cells.
  • High level expression of both the M-CSF-GPI and M-CSFR molecules on their respective cells could be efficiently obtained via gene transfer using episomal expression vectors.
  • the M-CSF-dependence of the effect was verified by anti-M-CSF and anti-M-CSF receptor antibody blocking.
  • the invention is not limited to these variants since GPI- anchored variants of multiple ligands other than M-CSF could be used as alternative artificial adhesins.
  • M-CSF:M-CSFR pair was GPI-anchored.
  • the M-CSFR component could also be GPI-anchored. Together, this would permit coating one cell with a ligand:GPI (e.g., M-CSF:GPI) chimera and a second cell with a receptor:GPI (e.g., M-CSFR:GPI) chimera and thereby selectively enhancing adhesiveness between the respective cells via protein transfer.
  • a ligand:GPI e.g., M-CSF:GPI
  • receptor:GPI e.g., M-CSFR:GPI
  • the pre-adhesion step entails the mixing of three components, namely, a tumor cell, a second cell with greater immunogenic potential, and artificial adhesin molecules. After a co-incubation period, generally lasting greater than 20 minutes, the mixture is centrifuged at 400g for 5-10 minutes in appropriate tissue culture medium supplemented with antibiotic and resuspended in the same medium. The cell suspension is kept at room temperature for 30 minutes with gentle stirring. Subsequently the mixture is spun through a sterilized isotonic sucrose (320 mOsm) solution containing 2 mM sodium phosphate buffer, pH 7.2 (400g for 5-10 minutes) . The cell pellet comprising tumor cell:second cell conjugates is gently suspended in 1 ml of isotonic sucrose buffer.
  • fusion is carried out.
  • a preferred method for fusing the heterologous cells in conjugates is electrofusion. Methods for performing electrofusion are well-known to those familiar with the art. Of note, most electrofusion protocols comprise two-steps: the induction of membrane-membrane contact followed by application of the fusogenic pulse. The dielectrophoresis method is used in most experiments to achieve the first step of congregating cells before fusion-inducing electric pulses are applied.
  • a preferred method according to the present invention entails omitting the dielectrophoresis step for congregating cells non- selectively, and instead applying the fusogenic pulse to heterologous cell conjugates that have been pre-adhered with artificial adhesins.
  • the fusogenic pulse can be applied with a high voltage generator, and alternative instruments are commercially available. Though it is preferable to use conjugates at low conjugate densities
  • Typical electrofusion conditions use five square wave pulses of 2.5 kV/cm and 5 microsecond duration, at a controlled temperature of 35C and with intervals of 15 sec between pulses to permit dissipation of Joule heat. Optimization of fusion conditions for particular cell types can be readily performed. (Less optimal adhesins are those described by Tsong and Tomita, 220 Methods in Enzymology 238, 1993) .

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Abstract

L'invention concerne des produits et des procédés permettant d'induire une réponse immune contre une cellule tumorale en administrant un produit de fusion cellulaire à un patient. L'expression 'produit de fusion cellulaire' signifie qu'une membrane cellulaire d'une cellule tumorale fusionne avec une membrane cellulaire d'une seconde cellule qui possède un potentiel immunogène supérieur à celui de la cellule tumorale.
PCT/US1994/014297 1993-12-14 1994-12-14 Fusions de cellules tumorales et procedes d'utilisation de telles fusions de cellules tumorales Ceased WO1995016775A1 (fr)

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US7361332B2 (en) 1995-03-17 2008-04-22 The Regents Of The University Of California Treating tumors using implants comprising combinations of allogeneic cells
US6805869B2 (en) 1996-06-12 2004-10-19 Shanghai Cp Guojian Pharmaceutical Co., Ltd. Cellular vaccines and immunotherapeutics and methods for their preparation
US6277368B1 (en) 1996-07-25 2001-08-21 The Regents Of The University Of California Cancer immunotherapy using autologous tumor cells combined with cells expressing a membrane cytokine
US7264820B2 (en) 1996-07-25 2007-09-04 The Regents Of The University Of California Cancer immunotherapy using autologous tumor cells combined with cells expressing a membrane cytokline
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EP0825256A3 (fr) * 1996-08-23 2001-04-04 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Cellule hybride et son utilisation pour préparer un médicament pour induire une réponse immune contre une tumeur
WO1998011202A1 (fr) * 1996-09-10 1998-03-19 Medical University Of South Carolina Hybrides semi-allogenes de cellules servant de vaccins preventifs et curatifs pour le cancer et le sida
US6063375A (en) * 1996-09-10 2000-05-16 Medical University Of South Carolina Semiallogeneic cell hybrids and related methods for treating cancer
US6207147B1 (en) 1996-10-11 2001-03-27 The Regents Of The University Of California Cancer immunotherapy using tumor cells combined with mixed lymphocytes
US7601342B2 (en) 1997-04-15 2009-10-13 Dana Farber Cancer Institute Cell fusions and methods of making and using the same
US6652848B1 (en) 1997-04-15 2003-11-25 Dana Farber Cancer Institute, Inc. Dendritic cell hybrids
EP1071333A4 (fr) * 1998-03-20 2005-02-23 Genzyme Corp Induction de l'immunite contre des autoantigenes tumoraux
US7541184B2 (en) 2000-02-24 2009-06-02 Invitrogen Corporation Activation and expansion of cells
US7572631B2 (en) 2000-02-24 2009-08-11 Invitrogen Corporation Activation and expansion of T cells
WO2003015813A3 (fr) * 2001-08-16 2004-06-17 Cellvax Vaccins antitumoraux
EP1284144A1 (fr) * 2001-08-16 2003-02-19 Cellvax Vaccin anti-tumoral
WO2013106895A1 (fr) * 2012-01-20 2013-07-25 Fernando Thome Kreutz Vaccin à base de cellules cancéreuses autologues
US9320785B2 (en) 2012-01-20 2016-04-26 Fernando Thome Kreutz Autologous cancer cell vaccine
CN112195173A (zh) * 2019-11-26 2021-01-08 李洪江 一种心肌细胞和肿瘤细胞融合的方法

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