WO2001059073A2 - Cytotoxic t lymphocytes activated by dendritic cell hybrids - Google Patents
Cytotoxic t lymphocytes activated by dendritic cell hybrids Download PDFInfo
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- WO2001059073A2 WO2001059073A2 PCT/US2001/004678 US0104678W WO0159073A2 WO 2001059073 A2 WO2001059073 A2 WO 2001059073A2 US 0104678 W US0104678 W US 0104678W WO 0159073 A2 WO0159073 A2 WO 0159073A2
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- 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
- A61K2239/49—Breast
-
- 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
- C12N2510/00—Genetically modified cells
Definitions
- the invention relates to cellular immunology. .
- DCs Dendritic cells
- APC's potent antigen-presenting cells
- Costimulatory signals are generated by interaction between receptor-ligand pairs expressed on the surface of APCs and T cells.
- One exemplary receptor-ligand pair is one of the B7 costimulatory molecules on the surface of DCs and its counter-receptor CD28 or CTLA-4 on T cells (Freeman et al, Science 262: 909-91 1, 1993; Young et al., J. Clin. Invest. 90: 229, 1992; Nabavi et al., Nature 360: 266, 1992).
- DCs are minor constituents of various immune organs such as spleen, thymus, lymph node, epidermis, and peripheral blood.
- DCs represent merely about 1% of crude spleen (Steinman et al., J. Exp. Med. 149:1, 1979) or epidermal cell suspensions (Schuler et al., J. Exp. Med. 161: 526, 1985; and Romani et al., J. Invest. Dermatol. 93: 600, 1989), and 0.1-1% of mononuclear cells in peripheral blood (Freudenthal et al., Proc. Natl. Acad. Sci. USA 87: 7698, 1990).
- the invention features methods of generating effector T lymphocytes (e.g., cytotoxic T lymphocytes (CTL) or helper T lymphocytes (HTL)) in which T lymphocytes are activated by fused cells (also referred to as "fusion cells") produced by fusion between mammalian (e.g., human) dendritic cells and non-dendritic cells (e.g., carcinoma cells) as described below.
- the T lymphocytes can be from, for example, a mammal (e.g., a human) with cancer (e.g., breast cancer) or an infectious disease (e.g., a viral disease such as AIDS).
- the fused cells have the characteristics described below.
- the CTL generated by this method lyse target cells (e.g., autologous tumor cells, pathogen infected cells) by a MHC restricted mechanism.
- Hit secrete helper cytokines that serve to enhance CTL and B-lymphocyte responses after activation by antigenic peptides bound to MHC molecules.
- the invention includes CTL and HTL generated in this manner.
- the invention features methods of identifying peptides recognized by these CTL and HTL. Characterization of these peptides will allow identification of antigens (e.g., tumor associated antigens) that are the target of the immune response.
- compositions for stimulating an immune system contain a plurality of cells including fused cells, each of which fused cells is generated by fusion between at least one mammalian dendritic cell (e.g.. a DC derived from a bone marrow culture or a peripheral blood cell culture) and at least one mammalian non-dendritic cell (e.g., a cancer cell or a transfected cell) that expresses a cell-surface antigen (e.g., a cancer antigen).
- a mammalian dendritic cell e.g. a DC derived from a bone marrow culture or a peripheral blood cell culture
- at least one mammalian non-dendritic cell e.g., a cancer cell or a transfected cell
- a cell-surface antigen e.g., a cancer antigen
- cancer antigen is meant an antigenic molecule that is expressed primarily or entirely by cancer cells, as opposed to normal cells, in an individual bearing the cancer.
- the fused cells in the compositions express, in an amount effective to stimulate an immune system (e.g., to activate T cells), MHC class II molecules.
- B7 and the cell- surface antigen.
- B7 is meant any member (e.g., B7-1 or B7-2) of the B7 family of costimulatory molecules.
- the parental cells used to generate the fused cells can be obtained from a single individual (e.g., a human, a mouse, or a rat). They can also be obtained from different individuals of the same species (e.g., homo sapiens), with matching or non-matching MHC molecules.
- the dendritic cells are autologous or allogeneic Mammalian dendritic cells are fused with mammalian non-dendritic cells expressing a cell-surface antigen in the presence of a fusion agent (e.g., electricity, polyethylene glycol or Sendai virus).
- a fusion agent e.g., electricity, polyethylene glycol or Sendai virus
- the cultured fused cells are separated from unfused parental non-dendritic cells, based on the different adherence properties of the two cell groups.
- the unfused parental dendritic cells do not proliferate, and so die off. Even if they remain present in the therapeutic composition, they will not interfere with the effects of the fused cells.
- the isolated fused cells which typically express (a) MHC class II protein, (b) B7, and (c) the cell-surface antigen on the non-dendritic parental cells, are useful for stimulating an immune system.
- the cells are useful in screening methods or in therapeutic methods directly after fusion or after an in vitro culture step.
- the DC phenotype of a fused cell can be maintained by re-fusing it with at least one additional mammalian dendritic cell.
- the re-fused cells express MHC class II molecules, B7, and the cell-surface antigen of the dendritic parental cells, and are useful for stimulating an immune system.
- compositions of the invention can be administered to an individual (e.g., a human) to stimulate the individual's immune system.
- This individual may need immune stimulation due to infection, or susceptibility to infection, with an intracellular pathogen; cancer; or predisposition to develop cancer.
- the DCs used to generate fused cells can be obtained from this individual. If this individual has cancer, the individual ' s own cancer cells can be used for fusion with his or her own DCs to generate fused cells, which are then administered to the individual. Stimulation of the immune system with DC fusion cells results in enhancement of the individual's immunity against a disease condition characterized by abnormal expression of a cell surface antigen that is also present on the parental non-dendritic partners of the fusion cells.
- Abnormal expression means that the cell surface antigen (i) is not expressed in normal tissue, (ii) is expressed at a much higher level in diseased cells of a given tissue type than in normal tissue of the same type, or (iii) is modified (e.g., phosphorylated) differently in diseased cells of a given tissue type than in normal cells of the same type.
- Enhancement of immunity entails enhancement of the immune system's cell-mediated or humoral functions, or both.
- the invention embodies a method of making a population of cells containing CTL or
- the method includes the steps of: (a) providing a plurality of cells which includes fused cells, each of which fused cells is generated by fusion between at least one mammalian dendritic cell and at least one mammalian non-dendritic cell (e.g., a cancer cell such as a human breast cancer cell) that expresses a cell-surface antigen, wherein at least 5% of the fused cells express, in amount effective to stimulate an immune response, (i) a MHC class II molecule, (ii) B7, and (iii) the cell-surface antigen; and (b) contacting a population of T lymphocytes with the plurality of cells, wherein the contacting causes differentiation of either (i) CTL precursor cells in.
- a mammalian dendritic cell e.g., a cancer cell such as a human breast cancer cell
- the invention also includes a population of cells containing CTL or HTL made by the above procedure and a method of treatment that includes the steps of: (a) identifying a subject having a disease (e.g., a cancer such as human breast cancer) with symptoms that can be decreased by the action of CTL and/or HTL; and (b) administering the above population of cells containing the CTL and or HTL to the subject. Also encompassed by the invention is a method of increasing CTL or HTL in a subject by administering the above population of cells containing CTL or HTL to the subject.
- a disease e.g., a cancer such as human breast cancer
- Also embraced by the invention is a method of testing a peptide for antigenic activity that includes the steps of: (a) providing the above population of cells containing CTL; and (b) contacting a plurality of target cells with the population of cells in the presence of the peptide. Lysis of the plurality of target cells or a portion of the plurality of target cells identifies the peptide as an antigenic peptide that is recognized by CTL within the population of cells.
- the method can include the steps of: (a) providing the above population of cells containing HTL; and (b) containing a plurality of antigen presenting cells with the population of cells in the presence of the peptide.
- a response (e.g., a proliferative response or a cytokine- producing response) of the HTL identifies the peptide as an antigenic peptide that is recognized by HTL within the population of cells.
- the mammalian non-dendritic cells used for producing the fused cells can be cancer cells (e.g., human breast cancer cells).
- peptides identified by this method, or polypeptides of which the peptides are fragments can be administered to the subjects.
- the invention features compositions for stimulating an immune system.
- the invention includes a hybrid cell (or progeny thereof), which is a fusion product of a dendritic cell, e.g., a dendritic cell, and non-dendritic cell.
- the hybrid cell expresses B7 on its surface.
- the hybrid cell also expresses a MHC class I molecule, MHC class II molecule, a costimulatory molecule, and/or an adhesion molecule.
- the dendritic cell fusion partner and the non- dendritic cell are derived from the same species.
- Examples include hybrid cells in which the non-dendritic cell fusion partner expresses a disease-associated antigen such as that derived from a tumor, a bacterium, or a virus.
- the non-dendritic cell is a tumor cell.
- the dendritic cell and said non-dendritic cell are preferably derived from the same individual, e.g., a human patient.
- a hybrid cell is a cell that contains cytoplasmic and/or membrane components from two or more cells.
- the invention also includes a population of activated immune effector cells.
- the cells are activated ex vivo.
- the population contains a T cell and a hybrid cell.
- a substantially pure population of activated, antigen-specific immune effector cells is also within the invention.
- the cells are derived from a coculture of a patient-derived immune cell and a hybrid cell. Effector cells specifically kill autologous tumor cells.
- a method for producing an antigen-specific immune effector cell is carried out by contacting a T cell with the hybrid cell described above.
- the T cell is derived from a variety of sources such as peripheral blood or from a tumor site.
- the contacting step occurs in vivo or ex vivo.
- a method for producing a purified population of activated immune effector cells specific for a target antigen is carried out by contacting a T cell with a hybrid cell for a period of time sufficient to activate said T cell and removing the hybrid cell from said T cell to yield a population of antigen-specific immune effector cells.
- a vaccine which contains a hybrid cell and a pharmaceutically acceptable carrier.
- the vaccine composition contains an activated antigen-specific effector cell, e.g., an effector cell, which is derived from a coculture of a patient-derived immune cell such as a T cell and a hybrid antigen presenting cell.
- the invention also involves an instraspecies hybrid of a dendritic and a non-dendritic cell. This hybrid expresses known and unknown cell antigens from the non-dendritic cells. MHC class I and II molecules, and a B7 costimulatory molecule in an amount effective to stimulate a cytotoxic immune response against the non-dendritic cell antigens.
- the invention also provides a method of making a population of cells comprising activated T cells comprising providing a plurality of cells, at least 5% of which are fused cells generated by fusion between at least one mammalian non-dendritic cells that expresses a cell- surface antigen.
- at least 5% of the fused cells express, in an amount effective to stimulate an immune response, a MHC class II molecule, B7, and the cell-surface antigen.
- a population of T cells is then contacted with this plurality of cells, which causes the activation of the T cells.
- Fig. 1A is a graph showing the results of flow cytometric analysis of the indicated antigens on the surface of DCs (DC), MC38 cells (MC38 MUC1) and fused cells generated by fusion between DCs and MC38/MUC1 cells (FC MUC1 ).
- Fig. IB is a graph showing tumor incidence in female C57BL/6 mice (10 per group) injected subcutaneously with 2 X 10 5 MC38/MUC1 cells (open triangle), 2 X 10 6 DCs mixed with 2 x 10 5 MC38/MUC1 cells (open circle), 2 x 10 5 FC/MUC1 cells (shaded circle), or 5 x 10 5 FC/MUC1 cells (shaded box). Tumor incidence (> 3mm in diameter) was monitored at the indicated days after injection. Similar results were obtained in three separate experiments.
- Fig. 1C is a graph showing [ 3 H] -thymidine incorporation in mixed leukocyte reactions.
- DCs open circle
- MC38/MUC1 cells shaded circle
- FC/MUC1 cells open triangle
- [ 3 H]- Thymidine uptake at 6 h of incubation is expressed as the mean ⁇ s.e.m. of three determinations. Similar results were obtained in three separate experiments.
- Fig. 2A is a graph showing induction of anti-tumor activity by FC/MUC1 in the form of percent tumor incidence.
- Groups of 10 mice were injected subcutaneously twice at 14-day intervals 3 x 10 5 DC (open circle), 3 x 10 5 FC MUC1 (shaded circle), or PBS (open box). After 14 days, the mice were challenged subcutaneously with 2.5 x 10 MC38/MUC1 cells. Tumors >3 mm in diameter were scored as positive. Similar results were obtained in three separate experiments.
- Fig. 2B is a graph showing induction of anti-tumor activity by FC/MUC1 in the form of cytotoxicity.
- FC/MUC1 (shaded circle) or PBS (open box) were challenged with 2.5 x lO 3 MC38/MUC1 tumor cells.
- Splenocytes were isolated at 20 days after challenge and incubated at the indicated effecto ⁇ target ratios with MC38 MUC1 target cells.
- CTL Cytotoxic T lymphocyte
- 2C is a graph showing induction of anti-tumor activity by FC/MUC 1 in the form of percent tumor incidence.
- Mice (8 per group) were injected intravenously and intraperitoneally every other day with mAbs against CD4 + (open box) and CD8" (shaded circle) cells beginning 4 days before the first of two immunizations with FC/MUC 1 and continuing until 4 days before challenge with 5 x 10 5 MC38/MUC1 cells.
- Rat IgG open circle
- Tumors of >3 mm were scored as positive. Similar results were obtained in two separate experiments.
- Fig. 2D is a line graph showing induction of anti-tumor activity by FC/MUC 1 in the form of cytotoxicity.
- Mice were treated as above with mAbs against CD4 + (open box) and CD8 + (shaded circle), or rat IG (open circle), immunized with FC/MUC 1 and then challenged with
- MC38/MUC1 cells Splenocytes were harvested at 20 days after tumor challenge and incubated with MC38/MUC1 cells. CTL activity (mean ⁇ s.e.m.) was determined by the 4-h LDH release assay. Similar results were obtained in three separate experiments.
- Fig. 3A is a graph showing prevention of MC38/MUC1 pulmonary metastases after immunization with FC/MUC 1.
- Groups of 10 mice were inj ected twice with FC/MUC 1 cells or PBS and then challenged after 14 days with intravenous administration of 1 x 10 6 MC38/MUC1 cells. The mice were sacrificed 28 days after challenge. Pulmonary metastases were enumerated after staining the lungs with India ink (Wexler, J. Natl. Cancer Inst. 36: 641-643, 1966).
- Fig. 3B is a graph showing treatment of MC38/MUC1 pulmonary metastases after immunization with FC/MUC 1.
- Groups of 10 mice were injected intravenously with 1 x 10 6 MC38/MUC1 cells or MC38 cells.
- the mice were immunized with 1 x 10 6 FC/MUC1 or FC/MC38 at 4 and 18 days after tumor challenge and then sacrificed after an additional 10 days.
- Pulmonary metastases were enumerated for each mouse. Similar results were obtained in two separate experiments (10/10 mice treated with FC/MUC 1 had no pulmonary metastases in the second experiment).
- Fig. 4A is a series of bi-dimensional flow cytometry histograms showing expression of MUCl and MHC class II on MCF-7 breast cancer cells, human dendritic cells, and fused DC/MCF-7 cells.
- Fig. 4B is a series of photomicrographs showing expression of MUCl (top left) and cytokeratin (CT) (bottom left) in primary human breast cancer cells and of MUCl and MEC class II (top right) and cytokeratin and MHC class II (bottom right) in human DC/primary breast cancer fused cells.
- Fig. 4C is a series of bi-dimensional flow cytometry histograms showing expression of MHC class II and MUC 1 on primary human breast cancer cells (BT), autologous human dendritic cells (DC), and BT DC fused cells.
- Fig. 5 A is a pair of photomicrographs showing clustering of autologous T cells around BT/DC fused cells (right) but not BT cells (left).
- Fig. 5B is a line graph showing the proliferation of T cells in response to stimulation with DC (open circle), autologous BT cells (open box), autologous BT cells mixed with autologous DC (shaded box), or autologous BT/DC fusion cells (shaded circle) at the indicated ratios of T cells to stimulator cells (S).
- Fig. 5C is a line graph showing the proliferation of T cells in response to stimulation by
- Fig. 6A is a bar graph showing the cytolysis of autologous BT target cells by T cells stimulated, in the presence of human IL-2. with autologous DC, autologous BT cells, autologous DC mixed with autologous BT cells (DC + BT), or autologous DC/BT fused cells.
- Fig. 6B is a set of three line graphs showing data obtained with cells from three different breast cancer patients.
- the graphs show the cytolysis of autologous BT target cells by T cells stimulated with either autologous BT cells (open circle) or DC/BT fusion cells (shaded circle).
- Fig. 7A is a pair of bar graphs showing data obtained with cells from two different breast cancer patients.
- the graphs show the cytolysis of autologous BT cells or autologous monocytes (MC) by T cells stimulated with autologous DC/BT fused cells.
- MC autologous monocytes
- Fig. 7B is a bar graph showing the cytolysis, in the absence (solid bars) and presence (hatched bars) of antibody specific for human MHC class I molecules, of autologous BT cells, autologous MC, MCF-7 breast cancer cells, ovarian cancer cells (OVCA), and K562 cells by T cells stimulated with autologous DC/BT fused cells.
- Fig. 8A is a series of flow cytometry histograms showing the expression of a variety of cell surface molecules on human DC.
- ovarian carcinoma cells OVCA
- OVCA/FC OVCA DC fused cells
- Fig. 8B is a series of photomicrographs showing expression of HLA-DR (MHC class II) in DC.
- OVCA, and OVCA/DC fused cells OVCA/FC.
- Fig. 9 is a series of bi-dimensional flow cytometry histograms showing expression of CA-125 (OC-125). MHC class II (MHC II), B7-2, and CD38 on DC.
- OVCA cells autologous OVCA DC fused cells (autologous OVCA/FC), and allogeneic OVCA/DC fusion cells (allogeneic OVCA/FC).
- Fig. 10A is a pair of photomicrographs showing clustering of autologous T cells around OVCA DC fused cells (OVCA/FC) (right) but not OVCA cells (left).
- Fig. 10B is a set of three line graphs showing data obtained with cells from three different ovarian carcinoma patients.
- the graphs show the cytolysis of autologous OVCA target cells by T cells stimulated with either autologous DC (open circle), autologous OVCA cells (open box), autologous OVCA cells mixed with DC (open triangle), or OVCA/DC fused cells (shaded circle).
- Fig. IOC is a bar graph showing cytolysis of autologous OVCA target cells by T cells stimulated with either autologous DC.
- autologous OVCA cells autologous OVCA DC fused cells (OVCA/FC), autologous monocytes (MC).
- Fig. 1 1A is a pair of bar graphs showing data obtained with cells from two different breast cancer patients.
- the graphs proliferative responses the T cells stimulated with autologous or allogeneic DC (solid bar) or OV AC/DC fused cells (hatched bar) produced by fusion of autologous OVCA cells with the autologous or allogeneic DC.
- Fig. 1 IB is a pair of bar graphs showing data obtained with cells from two different breast cancer patients.
- the graphs show the cytolysis of autologous OVCA cells by T cells stimulated with autologous or allogeneic DC (solid bar), OVAC/DC fused cells (hatched bar) produced by fusion of autologous OVCA cells with the autologous or allogeneic DC, or autologous OVCA cells.
- Fig. 12A is a bar graph showing the cytolysis, in the absence (solid bars) and presence (hatched bars) of antibody specific for human MHC class I molecules, of autologous OVCA cells, autologous monocytes (MC). MCF-7 breast cancer cells, allogeneic ovarian cancer cells (Allo-OVCA), and K562 cells by T cells stimulated with autologous OVCA/DC fused cells.
- Fig. 12B is a bar graph showing the cytolysis, in the absence (solid bars) and presence
- the invention features: (1) methods of activating CTL and Hit using fused cells fo ⁇ ned by fusion between DCs and non-dendritic cells: (2) CTL and HTL generated by these methods; (3) methods of treatment involving administration of these CTL and or HTL to subjects having diseases with symptoms that can be decreased by the action of CTL and/or HTL; (4) methods of identifying antigenic peptides recognized by the CTL and/or HTL; and (5) methods of inducing an immune response in a mammal (e.g., a human patient) by administering to the mammal either antigenic peptides identified as in (4), or polypeptide antigens of which such peptides are fragments.
- a mammal e.g., a human patient
- DCs can be obtained from bone marrow cultures, peripheral blood, spleen, or other appropriate tissue of a mammal using protocols known in the art.
- Bone marrow contains DC progenitors, which, upon treatment with cytokines such as granulocyte-macrophage colony- stimulating factor (“GM-CSF”) and interleukin 4 ("IL-4"), proliferate and differentiate into DCs.
- cytokines such as granulocyte-macrophage colony- stimulating factor (“GM-CSF) and interleukin 4 (“IL-4"
- TNF Tumor necrosis factor
- DCs so obtained are relatively immature (as compared to, for instance, spleen DCs).
- GM-CSF/IL-4 stimulated DC express MHC class I and class II molecules.
- these immature DCs are more amenable to fusion than the more mature DCs found in spleen.
- Peripheral blood also contains relatively immature DCs or DC progenitors, which can propagate and differentiate in the presence of appropriate cytokines such as GM-CSF and which can also be used in fusion.
- the non-dendritic cells used in the invention can be derived from any tissue or cancer (e.g., breast cancer, lung, pancreatic cancer, prostate cancer, bladder cancer, neurological cancers, genitourinary cancers, hematological cancers, melanoma and other skin cancers, and gastrointestinal cancers) by well known methods and can be immortalized.
- Non-dendritic cells expressing a cell-surface antigen of interest can be generated by transfecting the non-dendritic cells of a desired type with a nucleic acid molecule that encodes a polypeptide comprising the antigen.
- Exemplary cell-surface antigens are MUCl, ⁇ -fetoprotein, gamma-fetoprotein, carcinoembryonic antigen, fetal sulfoglycoprotein antigen, ferroprotein. placental alkaline phosphatase, and leukemia-associated membrane antigen. Methods for transfection and identifying antigens are well known in the art.
- Fusion between the DCs and the non-dendritic cells can be carried out with well-known methods such as those using polyethylene glycol (“PEG”) Sendai virus, or electrofusion.
- PEG polyethylene glycol
- the ratio of DCs to non-dendritic cells in fusion can vary from 1 : 100 to 1000:1, with a ratio higher than 1 : 1 being preferred where the nondendritic cells proliferate heavily in culture.
- unfused DCs usually die off in a few days in culture, and the fused cells can be separated from the unfused parental non-dendritic cells by the following two methods, both of which yield fused cells of approximately 50% or higher purity, i.e.. the fused cell preparations contain less than 50%, and often less than 30%, unfused cells.
- the post-fusion cell mixtures containing the fused as well as the parental cells may be incubated in a medium containing this reagent for a period of time sufficient to eliminate most of the unfused cells.
- a number of tumor cell lines are sensitive to HAT due to lack of functional hypoxanthine-guanine phosphoribosyl transferasc ("HGPRT"). Fused cells formed by DCs and these tumor cell lines become resistant to HAT, as the DCs contribute functional HGPRT.
- HGPRT hypoxanthine-guanine phosphoribosyl transferasc
- a HAT selection can be performed after fusion to eliminate unfused parental cells. Contrary to standard HAT selection techniques, the HAT selection generally should not last for more than 12 days, since Applicants find that lengthy culturing leads to loss of MHC class II protein and/or B7 costimulatory molecules on the fused cells.
- the second method of separating unfused cells from fused cells is based on the different adherence properties between the fused cells and the non-dendritic parental cells. It has been found that the fused cells are generally lightly adherent to tissue culture containers. Thus, if the non-dendritic parental cells are much more adherent, e.g., in the case of carcinoma cells, the post-fusion cell mixtures can be cultured in an appropriate medium (HAT is not needed but may be added if it slows the growth of unfused cells) for a short period of time. Subsequently, the fused cells can be gently dislodged and aspirated off, while the unfused cells grow firmly attached to the tissue culture containers.
- HAT is not needed but may be added if it slows the growth of unfused cells
- non-dendritic parental cells grow in suspension, after the culture period, they can be gently aspirated off while leaving the fused cells loosely attached to the containers.
- Fused cells obtained by the above-described methods typically retain the phenotypic characteristics of DCs.
- these fused cells express T- cell stimulating molecules such as MHC class II protein, B7-1. B7-2. and adhesion molecules characteristic of APCs such as ICAM-1.
- the fused cells also continue to express cell-surface antigens of the parental non-dendritic cells, and are therefore useful for inducing immunity against the cell-surface antigens.
- the non-dendritic fusion partner is a tumor cell, the tumorigenicity of the fused cell is often found to be attenuated in comparison to the parental tumor cell.
- the fused cells lose certain DC characteristics such as expression of the APC-specific T-cell stimulating molecules, they (i.e., primary fused cells) can be re-fused with dendritic cells to restore the DC phenotype.
- the re-fused cells i.e.. secondary fused cells
- the fused cells can be re-fused with the dendritic or non-dendritic parental cells as many times as desired.
- Fused cells that express MHC class II molecules. B7. or other desired T-cell stimulating molecules can also be selected by panning or fluorescence-activated cell sorting with antibodies against these molecules.
- the fused cells of the invention can be used to stimulate the immune system of a mammal for treatment or prophylaxis of a disease.
- a composition containing fused cells formed by his own DCs and tumor cells can be administered to him, e.g., at a site near the lymphoid tissue.
- the composition may be given multiple times (e.g., three to five times) at an appropriate interval (e.g., every two to three weeks) and dosage (e.g., approximately 10 5 -10 8 , e.g., about 0.5 X 10 6 to 1 X 10 6 , fused cells per administration).
- non-syngeneic fused cells such as those formed by syngeneic DCs and allogeneic or xenogeneic cancer cells, or by allogeneic DCs and cancer cells, can be administered.
- cytotoxic T lymphocytes obtained from the treated individual can be tested for their potency against cancer cells in cytotoxic assays. Multiple boosts may be needed to enhance the potency of the cytotoxic T lymphocytes.
- Example I below demonstrates that fusion cells formed by tumor cells and syngeneic DCs can prevent and treat tumors in animal models.
- Example III further demonstrates that such fusion cells may even activate anergized T cells that are specific for tumor antigens.
- Cells infected with an intracellular pathogen can also be used as the nondendritic partner of the fusion for treatment of the disease caused by that pathogen.
- pathogens include, but are not limited to, viruses (e.g., human iinmunodeficiency virus, hepatitis A, B, or C virus, papilloma virus, herpes virus, or measles virus), bacteria (e.g., Corynebacterium diphtheria, Bordetella petussis).
- compositions containing the appropriate fused cells are administered to an individual (e.g., a human) in a regimen determined as appropriate by a person skilled in the art. For example, the composition may be given multiple times (e.g., three to five times) at an appropriate interval (e.g., every two to three weeks) and dosage (e.g., approximately l O O 8 . and preferably about 10 7 fused cells per administration).
- an individual e.g., a human
- dosage e.g., approximately l O O 8 . and preferably about 10 7 fused cells per administration.
- non-dendritic cells transfected with one or more nucleic acid constructs each of which encodes one or more identified cancer antigens or antigens from a pathogen, can be used as the non-dendritic partner in fusion.
- These antigens need not be expressed on the surface of the cancer cells or pathogens, so long as the antigens can be presented by a MHC class I or II molecule on the fused cells.
- Fused cells generated by DCs and these transfected cells can be used for both treatment and prophylaxis of cancer or a disease caused by that pathogen.
- fusion cells expressing MUCl can be used to treat or prevent breast cancer, ovarian cancer, pancreatic cancer, prostate gland cancer, lung cancer, and myeloma; fusion cells expressing -fetoprotein can be used to treat or prevent hepatoma or chronic hepatitis, where - fetoprotein is often expressed at elevated levels; and fusion cells expressing prostate-specific antigen can be used to treat prostate cancer.
- compositions containing the fused cells so produced are well known in the art.
- Administration of compositions containing the fused cells so produced is as described above.
- the effector cells (CTL and HTL) of the invention can be generated in a number of ways. Basically, lymphocytes (containing CTL and HTL precursor cells), e.g., in peripheral blood mononuclear cells (PBMC), spleen cell, or lymph node cells, tire contacted with a composition containing any of the described fused cells of the invention.
- the contacting can be in vivo (e.g., by injecting the fused cells into the subject), in vitro (e.g., by culturing the cells containing lymphocytes with the fused cells), or by a combination of in vivo and in vitro methodologies (e.g., by in vitro activation of cells from a subject injected with the fused cells).
- the lymphocytes to be activated by the fused cells can be "virgin" T cells (i.e., T cells that have not, prior to being contacted with the fused cells, been exposed to activating antigens expressed by the fused cells) or they can have previously so exposed.
- T cells i.e., T cells that have not, prior to being contacted with the fused cells, been exposed to activating antigens expressed by the fused cells
- they can have previously so exposed.
- the lymphocytes when the lymphocytes are in or from a patient with a tumor or an infectious disease, the lymphocytes will have been exposed to antigens expressed by the tumor or infected cells in the subject prior to the exposure to the fused cells.
- the contacting with the fused cells can be single or multiple, e.g., a single in vitro exposure or multiple in vitro exposures, optionally after one or more in vivo exposures.
- the fused cells used for generation of effector cells can be used untreated or they can be pretreated with an agent (e.g., ionizing radiation or a drug such as mitomycin-C) that abrogates their proliferative capacity. In general, fused cells to be used for in vitro activation will have been pretreated with such an agent.
- the contacting can be performed with or without addition of an exogenous source of any of the cytokines exemplified below.
- the contacting of the precursor cells can result in activation of CTL and or HTL. Testing of the activated cells for CTL and HTL activity can be performed using any of the methods described below.
- CTL CD8+ T cells restricted by MHC class I molecule and the majority of HTL are CD4+ T cells restricted by MHC class II molecules.
- CD4+ CTL restricted by MHC class II molecules and CD8+ HTL restricted by MHC class I molecules have also been described.
- CD4 ⁇ CTL restricted by MHC class II molecules and CD4+ CTL restricted by MHC class I molecules CD8+ CTL also produce, albeit at lower levels than CD4+ helper T cells, a wide range of cytokines. All these effector cell populations, provided they are generating by contacting of T lymphocytes with the fused cells of the invention, are encompassed by the invention.
- the CTL and HTL of the invention can be polyclonal or monoclonal. Methods of cloning CTL and HTL are known in the art.
- effector cells can be used to identify antigens expressed by the non-dendritic cell partners of the fused cells used to generate the effector cells of the invention, by a number of methods used in the art.
- the effector cell-containing cell population is cultured together with a candidate peptide or polypeptide and either an appropriate target cell (where cytotoxicity is assayed) or antigen presenting cell (APC) (where cell proliferation, or cytokine production is assayed) and the relevant activity is determined.
- a peptide that induces effector activity will be an antigenic peptide which is recognized by the effector cells.
- a polypeptide that induces effector activity will be an antigenic polypeptide, a peptide fragment of which is recognized by the effector cells.
- Cytotoxic activity can be tested by a variety of methods known in the art (e.g.. 51 Cr or lactate dehydogenase (LDH) release assays described in Examples I and III-V).
- Target cells can be any of a variety of cell types, e.g., fibroblasts, lymphocytes, lectin (e.g., phytohemagglutinin (PHA), concanavalin A (ConA), or lipopolysaccbaride (LPS)) activated lymphocyte blasts, macrophages. monocytes. or tumor cell lines.
- PHA phytohemagglutinin
- ConA concanavalin A
- LPS lipopolysaccbaride
- the target cells should not naturally express the candidate antigens being tested for antigenic activity, though they could express them recombinantly.
- the target cells should, however, express at least one type of MEC class I molecule or MHC class II molecule (depending on the restriction of the relevant CU), in common with the CTL.
- the target cells can endogenously express an appropriate MHC molecule or they can express transfected polynucleotides encoding such molecules.
- the chosen target cell population can be pulsed with the candidate peptide or polypeptide prior to the assay or the candidate peptide or polypeptide can be added to the assay vessel, e.g., a microtiter plate well or a culture tube, together with the CTL and target cells.
- target cells transfected or transformed with an expression vector containing a sequence encoding the candidate peptide or polypeptide can be used.
- the CTL-containing cell population, the target cells, and the candidate peptide or polypeptide are cultured together for about 4 to about 24 hours. Lysis of the target cells is measured by, for example, release of 5 Cr or LDH from the target cells.
- a peptide that elicits lysis of the target cells by the CTL is an antigenic peptide that is recognized by the CTL.
- a polypeptide that elicits lysis of the target cells by the CTL is an antigenic polypeptide, a peptide fragment of which is recognized by the CTL.
- Candidate peptides or polypeptides can be tested for their ability to induce proliferative responses in both CU and HTL.
- the effector cells are cultured together with a candidate peptide or polypeptide in the presence of APC expressing an appropriate MHC class I or class II molecule.
- APC can be B-lymphocytes, monocytes, macrophages, or dendritic cells, or whole PBMC.
- APC can also be immortalized cell lines derived from B-lymphocytes, monocytes, macrophages, or dendritic cells.
- the APC can endogenously express an appropriate MEC molecule or they can express a transfected expression vector encoding such a molecule.
- the APC can, prior to the assay, be rendered non-proliferative by treatment with, e.g., ionizing radiation or mitomycin-C.
- the effector cell-containing population is cultured with and without a candidate peptide or polypeptide and the cells' proliferative responses are measured by, e.g., incorporation of [ 3 H]-thymidine into their DNA.
- Cytokines include, without limitation, interleukin-2 (IL-2), IFN-, IL-4, IL-5, TNF-, interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-10 (IL-b), interleukin-12 (IL-12), interleukin-15 (IL-15) and transforming growth factor (TGF) and assays to measure them include, without limitation, ELISA, and bio-assays in which cells responsive to the relevant cytokine are tested for responsiveness (e.g., proliferation) in the presence of a test sample.
- cytokine production by effector cells can be directly visualized by intracellular immunofluorescence staining and flow cytometry.
- candidate peptides and polypeptides to be tested for antigenicity will depend on the non-dendritic cells that were used to make the fused cells. Where the non-dendritic cells are tumor cells, candidate polypeptides will be those expressed by the relevant tumor cells. They will preferably be those expressed at a significantly higher level in the tumor cells than in the normal cell equivalent of the tumor cells. Candidate peptides will be fragments of such polypeptides.
- the candidate polypeptide could be tyrosinase or a member of the MART family of molecules; for colon cancer, carcinoembryonic antigen; for prostate cancer, prostate specific antigen; for breast or ovarian cancer, HER2/neu; for ovarian cancer, CA-125; or for most carcinomas, mucin-1 (MUCl).
- MART family of molecules for colon cancer, carcinoembryonic antigen; for prostate cancer, prostate specific antigen; for breast or ovarian cancer, HER2/neu; for ovarian cancer, CA-125; or for most carcinomas, mucin-1 (MUCl).
- the candidate polypeptide will be one expressed by the appropriate infectious microorganism or that expressed by the transfected cells, respectively.
- polypeptides include retroviral (e.g., HIV or HTLV) membrane glycoproteins (e.g., gpl60) or gag proteins, influenza virus neuraiminidase or hemagglutinin, Mycobacterium tuberculosis or leprae proteins, or protozoan (e.g., Plasmodium or Trypanosoma) proteins.
- Polypeptides can also be from other microorganisms listed herein.
- Peptides to be tested can be, for example, a series of peptides representing various segments of a full-length polypeptide of interest, e.g., peptides with overlapping sequences that, in tow, cover the whole sequence. Peptides to be tested can be any length. When testing MHC class I restricted responses of effector cells, they will preferably be 7-20 ( e.g., 8-12) amino acids in length. On the other hand, in MHC class II restricted responses, the peptides will preferably be 10-30 (e.g., 12 - 25) amino acids in length.
- a random library of peptides can be tested. By comparing the sequences of those eliciting positive responses in the appropriate effector cells to a protein sequence data base, polypeptides containing the peptide sequence can be identified. Relevant polypeptides or the identified peptides themselves would be candidate therapeutic or vaccine agents for corresponding diseases (see below).
- Polypeptides and peptides can be made by a variety of means known in the art. Smaller peptides (less than 50 amino acids long) can be conveniently synthesized by standard chemical means. In addition, both polypeptides and peptides can be produced by standard in vitro recombinant DNA techniques, and in vivo genetic recombination (e.g., transgenesis), using nucleotide sequences encoding the appropriate polypeptides or peptides. Methods well known to those skilled in the art can be used to construct expression vectors containing relevant coding sequences and appropriate transcriptional/translational control signals.
- host-expression vector systems can be used to express the peptides and polypeptides.
- Such host-expression systems represent vehicles by which the polypeptides of interest can be produced and subsequently purified, but also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, produce the relevant peptide or polypeptide in situ.
- These include, but are not limited to, microorganisms such as bacteria, e.g., E. coli or B.
- subtilis transformed with recombinant bacteriophage DNA, plasmid or cosmid DNA expression vectors containing peptide or polypeptide coding sequences; yeast, e.g., Saccharomyces or Pichia, transformed with recombinant yeast expression vectors containing the appropriate coding sequences; insect cell systems infected with recombinant virus expression vectors, e.g., baculovirus; plant cell systems infected with recombinant virus expression vectors, e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV), or transformed with recombinant plasmid expression vectors, e.g., Ti plasmids, containing the appropriate coding sequences; or mammalian cell systems, e.g., COS, CHO, BHK, 293 or 3T3, harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells, e.g., metallothionein
- Peptides of the invention include those described above, but modified for in vivo use by the addition, at either or both the amino- and carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant peptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular or mitochondrial uptake.
- blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by methods familiar to artisans of average skill.
- blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety.
- the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
- Peptidomimetic compounds that are designed based upon the amino acid sequences of the peptides or polypeptides.
- Peptidomimetic compounds are synthetic compounds having a three-dimensional conformation (i.e., a "peptide motif) that is substantially the same as the three-dimensional conformation of a selected peptide.
- the peptide motif provides the peptidomimetic compound with the ability to activate T cells in a manner qualitatively identical to that of the peptide or polypeptide from which the peptidomimetic was derived.
- Peptidomimetic compounds can have additional characteristics that enhance their therapeutic utility, such as increased cell permeability and prolonged biological half-life.
- the peptidomimetics typically have a backbone that is partially or completely non- peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based.
- Several types of chemical bonds e.g., ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of protease-resistant peptidomimetics.
- the effector cells can be used in basic research studies of tumor and infection immunology. They can be used in studies, for example, to further elucidate the mechanisms of antigen processing, antigen presentation, antigen recognition, signal transduction in CTL and HTL, and HTL-CTL interactions. In addition to other uses, they can be used as positive or negative controls in appropriate assays. They could also be used for diagnosis.
- the ability of T cells from a test subject to respond to a polypeptide or peptide of the invention would be an indication that the test subject has or is susceptible to a disease associated with expression of the relevant peptide or polypeptide.
- CTL and HTL of the invention would be valuable "positive controls" for an appropriate diagnostic assay.
- the effector cells, polypeptides, and peptides can be used in methods of therapy and vaccination. These methods of the invention fall into 2 basic classes, i.e., those using in vivo approaches and those using ex vivo approaches.
- a polypeptide, peptide or peptidomimetic is administered to a subject by any of the routes listed above. It is preferably delivered directly to an appropriate lymphoid tissue (e.g. spleen, lymph node, or mucosal-associated lymphoid tissue (MALT)).
- lymphoid tissue e.g. spleen, lymph node, or mucosal-associated lymphoid tissue (MALT)
- the subject can have or be suspected of having any of the diseases disclosed herein.
- the immune response generated in the subject by administration of the polypeptide, peptide or peptidomimetic can either completely abrogate of decrease the symptoms of the disease.
- the polypeptide, peptide or peptidomimetic can be administered to a subject as a vaccine, i.e., with the object of preventing or delaying onset of a relevant disease.
- the dosage required depends on the choice of polypeptide, peptide or peptidomimetic, the route of administration, the nature of the formulation, the nature of the patient's illness, and the judgment of the attending physician. Suitable dosages are in the range of 0.1-100.0 g/kg. Wide variations in the needed dosage are to be expected in view of the variety of polypeptides, peptides, or peptidomimetics of the invention available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Ex Vivo Approaches
- populations of cells containing effector cells can be administered to a subject having or suspected of having any of the diseases described herein.
- the lymphoid cells used to generate the effector cells can have been obtained from the subject or a second subject, preferably of the same species, more preferably with no or a single MHC locus (class I or class II) incompatibility with the first subject.
- donor lymphocyte infusion in which allogeneic cells (e.g., PBMC) containing T lymphocytes arc infused into a subject, has been shown to decrease tumor load or even result in full remission in a variety of cancers.
- the therapeutic activity has been attributed to graft-versus-tumor activity of donor T-cells activated by MHC and/or non-MHC alloantigens of the recipient subject.
- the effector function of the cells used for DLI can be enhanced by exposing them (singly or multiply) (e.g., in vitro) to appropriate fused cells of the invention prior to infusion into the recipient subject.
- the fused cells will have been generated from dendritic cells and non- dendritic cells from the recipient subject.
- DLI is usually, but not necessarily, performed after nonmyeloablative bone marrow transplantation. DLI and non-myeloablative bone marrow transplantation methodologies are known in the art.
- lymphoid cells are isolated from the subject, or another subject; and are exposed (e.g., in vitro) to a polypeptide or peptide identified by the method of the invention in the presence of appropriate APC.
- the lymphoid cells can be exposed once or multiply (e.g., 2, 3, 4, 6, 8, or 10 times). T he cytolytic, proliferative. or cytokine-producing ability of the stimulated lymphoid cells can be monitored after one or more exposures. Once the desired level of effector activity is attained, the cells can be introduced into the subject via any of the routes listed herein.
- cells to be used for DLI could, instead of being activated by the fused cells of the invention, be activated by the peptides or polypeptides identified as described above.
- immunoregulatory cytokines e.g., IL-2
- the therapeutic or prophylatic methods of the invention can be applied to any of the diseases and species listed herein.
- Methods to test whether a peptide or polypeptide is therapeutic for or prophylactic against a particular disease are known in the art.
- a test population displaying symptoms of the disease e.g., cancer patients or experimental animals with cancer
- a test effector cell-containing cell population, peptide, or polypeptide is treated with a test effector cell-containing cell population, peptide, or polypeptide. using any of the above described strategies.
- a control population, also displaying symptoms of the disease is treated, using the same methodology, with a placebo. Disappearance or a decrease of the disease symptoms in the test subjects would indicate that the polypeptide or peptide was an effective therapeutic agent.
- effector cell- containing cell populations, polypeptides or peptides of the invention can be tested for efficacy in inducing immune responses or as prophylactic agents, i.e., vaccines. In this situation, prevention of or delay in onset of disease symptoms is tested. Alternatively, the levels of immune responses induced in the experimental arid control groups can be compared.
- Murine (C57BL/6) MC38 adenocarcinoma cells were stably transfected with the DF3/MUC1 cDNA to generate the MC38/MUC 1 cell line (Siddiqui et al., Proc. Natl. Acad. Sci. USA 85:2320-2323, 1988; Akagi et al., J. Immunother. 20:38-47, 1997).
- MC3S, MC38/MUC 1 and the syngeneic MB49 bladder cancer cells were maintained in DMEM supplemented with 10% heat-inactivated fetal calf serum ("FCS"). 2 mM glutamine, 100 U/ml penicillin and 100 :g/ml streptomycin.
- DCs were obtained from bone marrow culture using a method described by Inaba et al.
- B21 -2, anti-la (TIB 229. ATCC); and (5) RB6-8C5, anti-Gr-1 (Pharmingen, San Diego, CA); and then complement.
- the unlysed cells were plated in six-well culture plates in RPMI 1640 medium supplemented with 5% heat-inactivated FCS, 50 :M 2-mercaptoethanol, 1 mM HEPES (pH 7.4), 2 mM glutarnine, 10 U/ml penicillin, 10 :g/ml streptomycin and 500 U/ml recombinant murine GM-CSF (Boehringer Mannheim, Indiana).
- nonadherent and loosely adherent cells were collected and replated in 100-mm petri dishes (10 cells/ml; 8 ml/dish). The nonadherent cells were washed away after 30 mm of incubation and RPMI medium containing (JM-CSF was added to the adherent cells. After 18 hours in culture, the nonadherent cell population was removed for fusion with MC38IMTJCI cells or MC38. Fusion was carried out by incubating cells with 50% PEG in Dulbecco's phosphate buffered saline ("PBS”) without Ca 2+ or Mg 2+ at pH 7.4. The ratio of DCs to tumor cells in the fusion was from 15: 1 to 20: 1.
- PBS Dulbecco's phosphate buffered saline
- the cells were plated in 24-well culture plates in a medium containing HAT (Sigma) for 10-14 days. Because MC38 cells are not very sensitive to HAT, HAT was used to slow the proliferation of, rather than kill, MC38/MUC1 and MC38 cells. MC38/MUC1 and MC3S cells grow firmly attached to the tissue culture flask, while the fused cells were dislodged by gentle pipetting.
- HAT Sigma
- mAb DF3 anti-MUCI
- mAb Ml/42/3.9.8 anti-MHC class I
- mAb M5/1 14 anti-MHC class II
- mAb 16-10A1 anti-B7-l
- mAb GL1 anti-B7-2
- MAb 3E 2 anti-ICA.M-1
- CTL Cytotoxic T cell
- LDH Low Density
- the DCs, MC38/MUC1 and FC/MUC 1 cells were exposed to ionizing radiation (30 Gy) and added to 1 x 10 ⁇ syngeneic or allogeneic T cells in 96-well flat-bottomed cultured plates for 5 days.
- the T cells were prepared by passing spleen suspensions through nylon wool to deplete residual APCs and plated to 90 mm in 100 mm tissue culture dishes. J [H]-thymidine uptake in nonadherent cells was measured at 6 h after a pulse of 1 :Ci/well (GBq/mmoh Du Pont-New England Nuclear. Wilmington, DE). Each reaction was performed in triplicate.
- GK1.5 anti-CD4
- mAb 2.43 anti-CD8
- rat IgG 4 days before the first of two immunizations with FC/MUCI through 4 days before challenge with MC38/MUC1 cells.
- the splenocytes were harvested for flow cytometry and analysis of CTL activity.
- Murine MC38 adenocarcinoma cells were fused to bone marrow-derived DCs.
- MC38 cells that stably express the DF3/MUC1 tumor-associated antigen were first used (Siddiqui et al.. Proc. Natl. Acad. Sci. USA 75: 5132-5136, 1978).
- the fusion cells (FC/MUCI) expressed DF3/MUCI, as well as MHC class I and II, B7-1, B7-2 and ICAM-1.
- Dendritic cells are potent stimulators of primary MLRs; Steinman et at, Proc. Natl. Acad. Sci. U.S.A. 75: 5132-5136, 1978; van Voorhis et al, J. Exp. Med. 158: 174-191, 1983) and induce the proliferation of allogeneic CD8 *T cells in vitro (Inaba et al., J. Exp. Med. 166: 182- 194, 1987; Young et al., J. Exp. Med. 171 : 1315-1332, 1990).
- FC/MUCI cells To characterize in part the function of FC/MUCI cells, their effect in primary allogeneic MLRs was compared with the effect of DC and MC38/MUC1 cells. The results demonstrate that, like DCs, FC/MUCI cells exhibit a stimulatory function in allogeneic MLR. By contrast, MC38/MUC1 cells had little effect on T cell proliferation.
- mice were immunized twice with FC/MUCI cells to assess in vivo function. Tumors developed in mice that had been immunized twice with 10 irradiated MC38/MUC1 cells and subsequently challenged with MC38/MUC1 cells (Table I). In contrast, after immunization with 2.5 X 10 5 FC/MUCI cells, all animals remained tumor-free after challenge with MC38/MUCI cells (Table 1). Control animals immunized with DCs alone or PBS and then challenged subcutaneously with 2.5 X 10 3 MC38 or MC38/MUC1 cells, however, exhibited tumor growth within 10-20 days.
- mice were injected intraperitoneally with antibodies against CD4 + or CD8 + cells before and after immunization with FC/MUCI. Depletion of the respective population by 80-90% was confirmed by flow cytometric analysis of splenocytes. The finding that injection of anti-CD4 and anti-CD8 antibodies increases tumor incidence indicated that both CD4 and CD8 + T cells contributed to antitumor activity (Fig. 2C).
- FC/MUCI cells To determine whether immunization with FC/MUCI cells is effective for the prevention of disseminated disease, a model of MC38/MUC1 pulmonary metastases was used. Immunization with FC/MUC 1 intravenously or subcutaneously completely protected against intravenous challenge with MC38/MUC1 cells. By contrast, all unimmunized mice similarly challenged with MC38/MUC 1 cells developed over 250 pulmonary metastases.
- Leukocytes in buffy coats (or leukopacks) obtained by leukophoresis were fractionated by centrifugation in Ficoll.
- the fraction containing (peripheral blood) mononuclear cells was incubated in a flask containing RPMI 1640 supplemented with 10% fetal calf serum ("FCS") for 30 mm at 37°C.
- FCS fetal calf serum
- FCS fetal calf serum
- the nonadherent cells were incubated in RPMI 1640 supplemented with 20% FCS for 30 mm to 1 hr. after which floating cells were removed and discarded.
- the two samples of loosely adherent DCs were then pooled and cultured in medium containing GM-CSF (lOOOU/ml) and IL-4 (100U/ml) at a density of 10 6 cells/ml for 5-6 days.
- the cells recovered from these cultures were the DCs used in fusion experiments.
- DCs were also obtained from bone marrow stem cell cultures.
- stem cells were placed in a flask containing RPMI 1640 supplemented with 10% FCS. After 30 min of incubation at 37°C, nonadherent cells were washed away. Fresh RPMI 1640 supplemented with 10% FCS was added to the remaining, adherent cells. After overnight incubation, loosely adherent cells were collected and incubated in RPMI 1640/10% FCS medium containing GM- CSF (lOOOU/ml) and IL-4 (lOOU/ml) for 5-6 days. The resultant cells were DCs that were ready for use in fusion.
- Cell fusion was carried out between DCs and human myeloma cells MY5 to produce fused cells DC/MY5. After fusion, the cells were placed in HAT selection for 10-14 days. IL-6 was also added to the culture at 20-50 :g/ml to promote survival of DC/MY5 cells. Procedures for fusion were essentially the same as described in Example 1, supra, except that the fused cells were separated from unfused myeloma cells based upon the higher degree of surface adherence exhibited by the fused cells.
- DC/MY5 cells retained the phenotypic characteristics of their parental cells: DC/MY5 were positively stained by mAbs for HLA-DR, CD38 (a myeloma cell-surface marker), DF3 (a tumor cell-surface marker), CD83 (a DC cell-surface marker), B7-1, and B7-2.
- Mixed lymphocyte reaction (MLR) assays demonstrated that these fused cells were also potent stimulators of T cells.
- a C57B1/6 mouse strain transgenic for human MUCl was established as described by Rowse et al. (Cancer Res. 58:315-321, 1998). 500 ng of tail DNA was amplified by PCR using MUCl primers co ⁇ esponding to nucleotides 745 to 765 and nucleotides 1086 to 1065, respectively, to confirm the presence of MUCl sequences.
- the PCR product was detected by electrophoresis in a 1% agarose gel (Rowse et al., supra).
- MUCl cDNA (Siddiqui et al., Proc. Natl. Acad. Sci. USA 85 :2320-2323, 1988; Akagi et al., J. Immunotherapy 20: 38-47, 1997; Chen et al., J. Immunol. 159:351-359, 1997).
- Cells were maintained in DMEM supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin.
- DC were obtained from bone marrow culture and fused to the carcinoma cells as described in Example I, supra.
- Single cell preparations of spleen and lymph nodes were suspended in RPMI medium supplemented with 10% heat-inactivated FCS, 50 M -mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin.
- the cells were stimulated with 5 U/ml purified MUCl antigen (Sekine et al., J. Immunol, 135:3610-3616, 1985). After 1,3 and 5 days of culture, the cells were pulsed with 1 Ci [ 3 H]thymidine per well for 12 hours and collected on filters with a semi-automatic cell harvester. Radioactivity was quantitated by liquid scintillation.
- Lymph node cells were suspended in complete RPMI medium containing 5 U/ml MUCl antigen. Ten U/ml murine IL-2 was added after 5 days of culture. On days 10 and 15, the cells were restimulated with 5 U/ml MUCl antigen and 1 :5 irradiated (30 Gy) syngeneic spleen cells as APCs. T cell cultures were analyzed after removal of dead cells by Ficoll centrifugation and depletion of residual APCs by passage through nylon wool.
- T cells were stained with FITC-conjugated antibodies against CD3e (145-2C1 1), CD4 (HI 29, 19), CD8 (53- 6.7), TcR (H57-597) and TcR (UC7- 13D5) (PharMingen). After incubation on ice for 1 hour, the cells were washed, fixed and analyzed by F AC SCAN (Becton-Dickinson).
- Microtiter plates were coated overnight at 4°C with 5 U/well purified MUCl antigen. The wells were washed with PBS containing 5% horse serum albumin and then incubated for 1 hour with four-fold dilutions of mouse sera. After washing and incubation with goat anti-mouse IgG conjugated to horseradish peroxidase (Amersham Life Sciences), antibody complexes were detected by development with o-phenylenediainine (Sigma) and measurement in an ELISA microplate autoreader EL310 at an OD of 490 nm.
- vaccines derived from fusions of DC and MC38/MUCI carcinoma cells induce potent anti-tumor immunity.
- FC/MUCI MC38/MUCI carcinoma cells
- the mice were immunized twice with 5x10 5 FC/MUCI and, as controls, with 10 6 i ⁇ adiated MC38/MUC1 cells or PBS. After challenge with 10 6 MC38 or MC38/MUC1 cells, all mice immunized with irradiated MC38/MUC1 cells or PBS developed tumors. By contrast, no tumor growth was observed in mice immunized with FC MUCI.
- CTLs from the FC/MUCI -immunized mice were assayed for lysis of target cells.
- CTLs from MUCl transgenic mice immunized with irradiated MC38/MUCI cells or PBS exhibited little if any reactivity against MC38/MUCI cells.
- CTLs from the mice immunized with FC/MUCI induced lysis of MC38, MC38/MUC1 and MB49/MUC1, but not MB49, cells.
- immunization with FC/MUCI induces immunity against MUCl and other unknown antigens on MC38 cells.
- LNC LNC from mice immunized with i ⁇ adiated MC38/MUC1 cells or FC/MUCI .
- the LNC were stimulated with MUCl antigen in vitro.
- the results demonstrate that LNC from mice immunized with PBS or irradiated MC38/MUC1 cells fail to proliferate in the presence of MUCl antigen.
- LNC from mice immunized with FC/MUCI responded to MUCl with proliferation.
- mice protected against MC38/MUCI tumor exhibited persistent expression of MUCl antigen in normal bronchial epithelium and other tissues that express the transgene (Rowse et al., Cancer Res. 58:315-321, 1998). Also, staining of MUCI- positive tissues with anti-CD4 and anti-CD8 antibodies did not show any T cell infiltration. Reversal of unresponsiveness against a self-antigen in adult mice has potential importance in the field of antitumor immunotherapy. The present example demonstrates that immunization with the DC-tumor fusion cells induces an immune response that is sufficient to achieve rejection of established metastases.
- Example IV Activation of Tumor-specific CTL bv Fusions of Human Dendritic Cells and Breast Carcinoma Cells
- Human MCF-7 breast carcinoma cells (ATCC, Rockville. MD) were grown in DMEM culture medium supplemented with 10% heat-inactivated FCS, 2 mL L-glutamine, 100 U/ml penicillin and 100:g/ml streptomycin.
- Human breast carcinoma cells were obtained with Institutional Review Board approval from biopsies of primary tumors and metastatic lesions of skin, lungs and bone marrow. The cells were separated by incubation in Ca2 + Mg2 + -free Hank's balanced salt solution containing 1 mg/ml collagenase, 0.1 mg/ml hyaluronidase and 1 mg/ml DNase.
- Breast tumor cells were also isolated from malignant pleural effusions by centrifugation and lysis of contaminating red blood cells.
- the breast tumor cells were maintained in RPMI 1640 medium supplemented with 10% heat -inactivated autologous-human serum, 2 mM L- glutamine, 100 U/ml penicillin, 100 :g/ml streptomycin and :g/ml insulin (Sigma).
- PBMC Peripheral blood mononuclear cells
- the PBMC were suspended in RPMI 1640 culture medium supplemented with 10% human serum (Sigma) for 1 h.
- the non- adherent cells were removed and T cells were isolated by nylon wool separation.
- the adherent cells were cultured for 1 week in RPMI 1640 medium/ 10% human serum containing 1000 U/ml GM-CSF (Genzyme) and 500 U/ml IL-4 (Genzyme).
- the GM-CSF/IL-4 stimulated DC expressed MHC class I and II, B7-1, B7-2, ICAM, CD40 and variable amounts of CD83, but not CD 14.
- CD 19, cytokeratin or MUCl Non-adherent and loosely adherent cells were harvested by repeated washes to generate the DC population.
- Firmly adherent monocytes were released from the plates with trypsin.
- DCs were mixed with MCF-7 or primary breast cancer cells at a 10: 1 ratio and incubated in serum-free RPMI 1640 medium containing 50% polyethyleneglycol (PEG) for 5 mm. After slowly diluting with serum-free RPMI 1640 medium, the cells were washed, resuspended in RPMI 1640 medium supplemented with 10%) autologous human serum and 500 U/ml GM-CSF, and incubated at 37°C for 7-14 d.
- serum-free RPMI 1640 medium 50% polyethyleneglycol (PEG) for 5 mm.
- PEG polyethyleneglycol
- PBMC peripheral blood mononuclear cells
- HuIL-2 human interleukin-2
- the stimulated T cells were harvested by nylon wool separation and used as effector cells in CTL assays with cell targets.
- Primary breast tumor cells, monocytes, MCF-7 cells, primary ovarian cancer cells (OVCA) and K562 cells were labeled with 51 Cr for 60 mm at 37°C. After washing to remove unincorporated isotope, the targets (2 x 10 4 ) were cocultured with effector cells for 5 h at 37°C.
- DC from PBMC of patients with metastatic breast cancer were prepared.
- the DCs were initially fused to human MCF-7 breast carcinoma cells.
- Bi-dimensional flow cytometry demonstrated that MCF-7 cells express the MUCl carcinoma-associated antigen and MHC class I, but not MHC class II-B7-1, B7-2 or ICAM.
- DC expressed MHC class I, class II and costimulatory molecules, but not MUCl .
- the resulting heterokaryons coexpressed MUCl and MHC class II. Similar patterns of coexpression of MUCl with B7. 1, B7-2 and ICAM were observed on the fused cells.
- human breast cancer cells were isolated from patients with primary or metastatic tumors for the purpose of making DC fusion cells with them.
- Immunostaining of short term cultures demonstrated that the breast carcinoma cells expressed MUCl and cytokeratin (CT).
- CT cytokeratin
- the breast tumor cells had no detectable expression of MHC class II, costimulatory or adhesion molecules.
- the tumor cells were fused with autologous DC and, after culturing for 7 days, the resulting population was analyzed for the presence of fusion cells. Fusion of the tumor cells to autologous DC resulted in the generation of heterokaryons that expressed both MUCl and MHC class II or cytokeratin and MHC class II.
- the heterokaryons were cocultured with T cells isolated from nonadherent PBMC. As a control, the T cells were also cocultured with autologous tumor cells. While there was no evidence for a T cell response to autologous tumor, the fusion cells stimulated T cell proliferation and the formation of T cell/fusion cell clusters. To assess the specificity of this response, autologous T cells were incubated with DC, irradiated breast tumor cells, a mixture of unfused DC and breast tumor cells, or DC-breast tumor fusion cells. There was little if any T cell stimulation by autologous DC, tumor or a mixture of the two cell types.
- T cells were stimulated for 10 d and then isolated for assaying lysis of autologous tumor cells.
- T cells stimulated with the fusion cells were effective in inducing cytotoxicity of autologous tumor.
- Similar results were obtained with T cells from three breast cancer patients that had been stimulated with autologous DC/breast tumor cell fusions.
- unstimulated T cells that had been cocultured with autologous breast rumor cells failed to mediate significant tumor cell killing.
- Fig. 7A show data obtained with cells from two individual patients.
- Incubation of fusion-stimulated T cells with autologous breast tumor or monocytes demonstrated selectivity for lysis of the tumor cells.
- T cells stimulated with autologous fusion cells demonstrated significant lysis of autologous breast tumor cells, while lysis of MCF-7 cells, primary ovarian cancer cells and NK-sensitive K562 cells was similar to that obtained with autologous monocytes.
- PBMC peripheral blood mononuclear cells
- Ovarian carcinoma (OVCA) cells obtained from primary tumors and malignant ascites were separated from other cells and non-cellular components in Hank's balanced salt solution (Ca ⁇ /Mg "1-1" free) containing 1 mg/ml collagenase, 0.1 mg/ml hyaluronidase and 1 mg/ml DNase.
- the cells were cultured in RPMI 1640 culture medium supplemented with 10% heat-inactivated autologous human serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin until fusion.
- Autologous or allogeneic DC were incubated with the OVCA cells for 5 min at a ratio of 10:1 in serum-free RPMI 1640 medium containing 50% polyethylene glycol (PEG). RPMI 1640 culture medium was then added slowly to dilute the PEG. After washing, the cells were resuspended in RPMI 1640 culture medium supplemented with 10% autologous serum and 500 U/ml GM-CSF for 7-14d.
- PEG polyethylene glycol
- Cytospin cell preparations were fixed in acetone and incubated with MAb OC-125 for 30 min at room temperature. The slides were washed and incubated with biotinylated horse antibody specific for mouse IgG for an additional 30 min. Staining (red color) was achieved with ABC solution (Vector Laboratories, Burlingame. CA). The slides were then incubated with murine antibody specific for human MHC class II for 30 min followed by alkaline phosphatase- labeled anti-mouse IgO. AP-ABC solution (Vector Laboratories) was used to generate a blue counterstain. T cell proliferation assays
- T cells were stimulated with the indicated cell preparations for 1 week in the presence of 20 U/ml HuIL-2.
- the T cells were harvested by nylon wool separation and used as effector cells in CTL assays.
- Autologous OVCA cells, allogeneic OVCA cells, autologous monocytes, MCF-7 breast carcinoma cells and K562 cells were labeled with 51 Cr for 60 mm at 37°C.
- targets (2x10 4 ) were cultured with the T cells for 5 h at 37°C.
- the labeled target cells were incubated with MAb W6/32 (anti-MHC class I) for 30 min at 37°C before addition of the effector cells.
- Supematants were assayed for 3 Cr release in a gamma counter.
- ovarian carcinoma (OVCA) cells fused with autologous and allogeneic DC DC were generated from patients with metastatic ovarian cancer and from normal volunteers.
- Adherent cells were isolated from PBMC and cultured in the presence of GM-CSF and IL-4 for 1 week. The resulting population was subjected to FACS analysis. The DC displayed a characteristic phenotype with expression of MHC class I class II, costimulatory molecules, CD83 and ICAM, but not the DF3/MUC1 or CA-125 carcinoma-associated antigens.
- OVCA 5 cells isolated from a patient with metastatic ovarian cancer expressed MUCl, CA-125, MHC class I and ICAM, but not MHC class II, B7-1, B7-2 or CD83.
- OVCA cells obtained from primary ovarian tumors and from malignant ascites. Fusion of the OVCA cells to autologous DC (OVCA FC) resulted in the generation of heterokaryons (OVCA/FC) that express the CA-125 and MUCl antigens, MHC class II, B7-1, B7-2 and CD83.
- OVCA FC autologous DC
- OVCA/FC heterokaryons
- the pattern of antigen expression was similar when the OVCA cells were fused to allogeneic DC. Immunostaining confirmed that the DC expressed MHC class II and not CA- 125. Conversely, the OVCA cells expressed CA-125 arid not MHC class II. Analysis of the fusion cells (OVCA/FC) demonstrated expression of both antigens.
- Bi-dimensional flow cytometry was used to assess the efficiency of the fusions.
- the OVCA cells expressed CA-125, but not MHC class II, B7-2 or CD83.
- Analysis of OVCA cells fused with autologous DC demonstrated that 32.6% of the population expressed both CA-125 and MHC class II .
- Assessment of CA-125 and B7-2 expression demonstrated that 30% of the autologous OVCA/FC expressed both antigens.
- 10.8% of the autologous OVCA FC population expressed both CA-125 and CD83.
- Fusion of the OVCA cells and allogeneic DC also resulted in cells coexpressing CA-125 and MBC class II, B7-2 or CD83.
- the fusion cells were cocultured with autologous PBMC.
- the experiment was performed with cells from three individual patients and each of the three graphs in Fig. 10B shows data obtained with cells from one of the patients.
- the PBMC were also cultured with autologous OVCA cells.
- the fusion cells but not the tumor cells, stimulated the formation of T cell clusters.
- the T cells were isolated for assessment of cytolytic activity.
- autologous OVCA cells as targets, there was a low level of lysis when assaying T cells that had been incubated with autologous DC, autologous tumor, or a mixture of unfused DC and tumor.
- T cells stimulated with the OVCA/FC were effective in inducing lysis of autologous tumor targets. Similar results were obtained with T cells from the three patients with ovarian cancer. As a control. T cells stimulated with OVCA cells fused to autologous monocytes (OVCA MC) or DC fused to monocytes (DC/MC) had little effect on stimulation of anti-tumor CTL activity.
- OVCA MC autologous monocytes
- DC/MC DC fused to monocytes
- OVCA FC function when the fusion is performed with allogeneic DC, autologous PBMC were stimulated with OVCA cells fused to autologous or allogeneic DC.
- Each of the two graphs in Figs. 1 1 A and 11B shows data obtained with cells from an individual patient.
- the autologous PBMC were also stimulated with unfused DC or OVCA cells.
- Incubation of the T cells with allogeneic DC was associated with greater stimulation than that obtained with autologous DC.
- the results also demonstrate that T cell proliferation is stimulated to a greater extent by OVCA fused to allogeneic, as compared to autologous, DC. Similar findings were obtained with T cells obtained from the two patients.
- T cells After stimulation for 10 d, the T cells were isolated and assessed for lysis of autologous tumor. Stimulation with unfused allogeneic or autologous DC had little if any effect on lytic function compared to that obtained with T cells stimulated in the presence of OVCA cells. By contrast, T cells stimulated with OVCA cells fused to allogeneic DC induced lysis of autologous tumor. Moreover, for both patients, T cells stimulated with OVCA cells fused to autologous or allogeneic DC exhibited induction of CTL activity.
- T cells stimulated with OVCA cells fused to autologous DC were incubated with autologous tumor, autologous monocytes, MCF-7 breast carcinoma cells, allogeneic OVCA cells and NK-sensitive K562 cells.
- the data shown in Figs. 12A and 12B were obtained from CTL assay cultures carried out in the absence (solid bars) or presence (hatched bars) of MAb specific for human MHC class I molecules.
- Incubation of the OVCA/FC stimulated T cells, with autologous tumor or monocytes demonstrated selective lysis of the tumor.
- Preincubation of the targets with an anti-MHC class I antibody blocked lysis of the autologous OVCA cells and had little effect on that obtained for the other cell types in the absence of antibody.
- T cells stimulated with autologous OVCA cells fused to allogeneic DC also demonstrated selective lysis of the autologous tumor.
- lysis of the autologous tumor was abrogated by preincubation of the targets with anti- MHC class I, thereby indicating that recognition of the tumor by the CTL was restricted by MHC class I molecules.
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JP2001558213A JP5054875B2 (en) | 2000-02-11 | 2001-02-12 | Cytotoxic T lymphocytes activated by dendritic cell hybrids |
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AU2001238236A AU2001238236B2 (en) | 1997-04-15 | 2001-02-12 | Cytotoxic t lymphocytes activated by dendritic cell hybrids |
EP01910649A EP1263928B1 (en) | 2000-02-11 | 2001-02-12 | Identification of antigenic peptides by cytotoxic t lymphocytes activated by dendritic cell hybrids |
AU3823601A AU3823601A (en) | 2000-02-11 | 2001-02-12 | Cytotoxic t lymphocytes activated by dendritic cell hybrids |
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Cited By (6)
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WO2005079271A2 (en) * | 2004-02-12 | 2005-09-01 | Tsuneya Ohno | Combined immunotherapy of fusion cells and interleukin-12 for treatment of cancer |
JP2006509830A (en) * | 2002-12-16 | 2006-03-23 | 典也 大野 | Preparation and administration of hybrid cell vaccine for cancer prevention |
EP2848255A1 (en) * | 2001-02-20 | 2015-03-18 | Janssen Pharmaceuticals, Inc. | Artificial antigen presenting cell for preparing CD8 cell suspension for use in the treatment of cancer |
CN105112369A (en) * | 2015-08-25 | 2015-12-02 | 北京康爱瑞浩生物科技股份有限公司 | CTL (cytotoxic T lymphocyte) cytomedicine with continuous antitumor activity and preparation method of CTL cytomedicine |
US9222070B2 (en) | 2001-02-20 | 2015-12-29 | Janssen Pharmaceuticals, Inc. | Cell therapy method for the treatment of tumors |
US9844569B2 (en) | 2013-03-01 | 2017-12-19 | The United State Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods of producing enriched populations of tumor reactive T cells from peripheral blood |
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WO2005084387A2 (en) * | 2004-03-02 | 2005-09-15 | Tsuneya Ohno | Methods and compositions for hybrid cell vaccines for the treatment and prevention of cancer |
JP6230208B2 (en) * | 2007-11-08 | 2017-11-15 | デイナ ファーバー キャンサー インスティチュート,インコーポレイテッド | Stimulation of anti-tumor immunity using dendritic cell / tumor cell fusions and anti-CD3 / CD28 |
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EP2848255A1 (en) * | 2001-02-20 | 2015-03-18 | Janssen Pharmaceuticals, Inc. | Artificial antigen presenting cell for preparing CD8 cell suspension for use in the treatment of cancer |
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US9222070B2 (en) | 2001-02-20 | 2015-12-29 | Janssen Pharmaceuticals, Inc. | Cell therapy method for the treatment of tumors |
JP2006509830A (en) * | 2002-12-16 | 2006-03-23 | 典也 大野 | Preparation and administration of hybrid cell vaccine for cancer prevention |
WO2005079271A2 (en) * | 2004-02-12 | 2005-09-01 | Tsuneya Ohno | Combined immunotherapy of fusion cells and interleukin-12 for treatment of cancer |
WO2005079271A3 (en) * | 2004-02-12 | 2006-12-28 | Tsuneya Ohno | Combined immunotherapy of fusion cells and interleukin-12 for treatment of cancer |
US9844569B2 (en) | 2013-03-01 | 2017-12-19 | The United State Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods of producing enriched populations of tumor reactive T cells from peripheral blood |
US10716809B2 (en) | 2013-03-01 | 2020-07-21 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods of producing enriched populations of tumor reactive T cells from peripheral blood |
US11679128B2 (en) | 2013-03-01 | 2023-06-20 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods of producing enriched populations of tumor reactive T cells from peripheral blood |
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EP1263928A1 (en) | 2002-12-11 |
DE60130634D1 (en) | 2007-11-08 |
DE60130634T2 (en) | 2008-07-17 |
JP5054875B2 (en) | 2012-10-24 |
CA2399432A1 (en) | 2001-08-16 |
WO2001059073A3 (en) | 2002-01-24 |
WO2001059073A9 (en) | 2002-10-24 |
AU3823601A (en) | 2001-08-20 |
ATE374244T1 (en) | 2007-10-15 |
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