US20030119187A1 - Antigen presenting cells, method for their preparation and their use for cancer vaccines - Google Patents

Antigen presenting cells, method for their preparation and their use for cancer vaccines Download PDF

Info

Publication number: US20030119187A1
Authority: US; United States
Prior art keywords: cells; pbmc; cta; adhapi; cancer
Prior art date: 2001-07-30
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Abandoned

Application number

US09/981,239

Other languages

English (en)

Inventor

Rita De Santis

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Sigma Tau Industrie Farmaceutiche Riunite SpA

Original Assignee

Sigma Tau Industrie Farmaceutiche Riunite SpA

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2001-07-30

Filing date

2001-10-18

Publication date

2003-06-26

2001-10-18 Application filed by Sigma Tau Industrie Farmaceutiche Riunite SpA filed Critical Sigma Tau Industrie Farmaceutiche Riunite SpA

2001-10-18 Assigned to SIGMA-TAU INDUSTRIE FARMACEUTICHE RIUNITE S.P.A. reassignment SIGMA-TAU INDUSTRIE FARMACEUTICHE RIUNITE S.P.A. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DE SANTIS, RITA

2003-06-26 Publication of US20030119187A1 publication Critical patent/US20030119187A1/en

2004-07-26 Priority to US10/898,187 priority Critical patent/US20050002920A1/en

Status Abandoned legal-status Critical Current

Links

Images

Classifications

- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
- 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
- A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
- A61K40/41—Vertebrate antigens
- A61K40/42—Cancer antigens
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/52—CD40, CD40-ligand (CD154)
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/59—Lectins
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
- 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
- C12N2510/04—Immortalised cells

Definitions

the present invention relates to the medical field, in particular to products, substances and compositions for use in methods for the treatment of human or animal subjects, more in particular for the diagnosis, treatment, and prevention of cancer.
the present invention relates to cancer vaccines and methods for their preparation.
TAA tumor-associated antigens
TAA can provide multiple immunodominant antigenic peptides recognized by CD8+cytotoxic T lymphocytes (CTL) in the context of specific HLA class I allospecificities (Renkvist N. et al. Cancer Immunol. Immunother. 50:3-15, 2001); furthermore selected TAA, such as for example MAGE (Jager E. et al., J. Exp. Med., 187: 265-270, 1998), NY-ESO-1 (Jager E. et al., J. Exp. Med., 187: 265-270, 1998), SSX (Tureci O, et al.
CTL cytotoxic T lymphocytes
CTA cancer-testis antigens
differentiation-specific antigens expressed in normal and neoplastic melanocytes, such as for example tyrosinase, Melan-A/MART-1, gp100/Pmel17, TRP-1/gp75, TRP-2 (Traversan C., Minerva Biotech., 11: 243-253, 1999);
antigens over-expressed in malignant tissues of different histology but also present in their benign counterpart for example PRAME (Ikeda H. et al., Immunity, 6: 199-208, 1997), HER-2/neu (Traversari C., Minerva Biotech., 11: 243-253, 1999), CEA, MUC-1(Monges G. M. et al., Am. J. Clin. Pathol., 112: 635-640, 1999), alpha-fetoprotein (Meng W. S. et al., Mol. Immunol., 37: 943-950, 2001);
antigens derived from point mutations of genes encoding ubiquitously expressed proteins such as MUM-1, ⁇ -catenin, HLA-A2, CDK4, and caspase 8 (Traversari C., Minerva Biotech., 11: 243-253, 1999);
the cellular elements that are crucial for their effective immunogenicity and efficient recognition by host's T lymphocytes include HLA class I and HLA class II antigens, and co-stimulatory/accessory molecules (e.g., CD40, CD54, CD58, CD80, CD81) (Fleuren G. J. et al., Immunol. Rev., 145: 91-122, 1995).
CTA are particularly suitable therapeutic targets for active specific immunotherapy of cancer patients, because of their limited expression in normal tissues and their known in vivo immunogenicity in living subjects, in particular mammals, humans included (Jager E. et al., J. Exp. Med., 187: 265-270, 1998; Rejnolds S. R. et al., Int. J Cancer, 72: 972-976, 1997).
the heterogeneous expression of specific CTA among neoplastic lesions of different patients limits their biological eligibility to CTA-directed therapeutic vaccination.
malignant lesions of distinct cancer patients can frequently express only selected CTA (Sahin U. et al., Clin.
cancer vaccine which can overcome the drawbacks of the state of the art, in particular poor immunogenicity, in vivo immunoselection, the possibility to practice a cancer vaccine on a wide population of cancer patients, not limited to the specific single targeted CTA, or TAA, and in that the cancer vaccine not be “restricted” to selected HLA class I and/or HLA class II antigens.
CTA Notwithstanding their promising therapeutic profile, CTA, however, show a number of drawbacks, such as that specific CTA so far investigated show a heterogeneous expression within distinct neoplastic lesions, with the co-existence of CTA-positive and ⁇ negative malignant cells; that only selected CTA among the ones so far identified may be expressed on distinct neoplastic lesions, independently from their hystological origin; that threshold levels of expression of specific CTA on neoplastic cells are required for their recognition by CTA-specific CTL and that vaccination against a specific CTA requires an appropriate HLA class I and, for selected CTA also HLA class II phenotype of patients.
CTA CTA-specific immune response in patients affected by malignant diseases of different histology.
Diverse strategies are currently utilized for the in vivo administration of therapeutic CTA in the clinic or for the generation of more powerful vaccinating tools at pre-clinical level (dos Santos N. R. et al., Cancer Res., 60: 1654-1662, 2000; Weber J. et al., Cancer Res., 54: 1766-1771, 1994) as the person expert in the art is aware of.
WO 99/42128 discloses methods for determining the HLA transcription or expression profile of a solid tumor, for selection of appropriate treatments and/or for monitoring progress of the tumor.
the purpose of this reference is to inhibit some isoforms of HLA-G in order to increase the native antitumor response.
the method comprises extracting cells from a tumor sample, lysing them and reacting the lysate with antibodies directed against HLA Class I antigens.
DE 29913522 provides an apparatus for preparing a cancer vaccine by submitting tumor cells extracted from a patient to pressures of 200-9000 bar, in order to kill or damage the cells while leaving their surface intact then reinjecting the cells to the patient.
WO 00/02581 discloses a telomerase protein or peptide, capable of inducing a T cell response against an oncogene or mutant tumor suppressor protein or peptide. Said peptides are used for a cancer vaccine.
WO 00/18933 discloses DNA constructs causing expression of functionally inactive, altered antigens which are unaltered with respect to the efficiency of transcription and translation of DNA, RNA or the generation of antigenic peptides.
the patient affected by cancer is treated by the administration of the RNA or plasmid DNA encoding an altered human cancer associated antigen, in particular PSMA antigen.
autologous dendritic cells that have been exposed in vitro to the RNA or the plasmid DNA are used as vaccine.
WO 00/20581 discloses a cancer vaccine comprising a new isolated MAGE-A3 human leukocyte antigen (HLA) class II-binding peptide.
the peptide can also be used to enrich selectively a population of T lymphocytes with CD4+ T lymphocytes specific the said peptide. Said enriched lymphocytes are also used as cancer vaccine.
WO 00/25813 discloses universal Tumor-Associated Antigen (TAA) binding to a major histocompatibility complex molecule.
the method of treatment comprises administering a nucleic acid molecule encoding the TAA, which is processed by an antigen-presenting cell which activates cytotoxic lymphocytes and kills cells expressing TAA.
TAA Tumor-Associated Antigen
the identification of different TAAs is enabled by a complex computer-aided method synthesis of the computer-designed peptide and biological assays for confirmation of the usefulness of the peptide.
WO 00/26249 discloses fragments of human WT-1 protein or human gata-1 protein. These peptide fragments are used for cancer vaccine through activation of cytotoxic T lymphocytes (CTL).
CTL cytotoxic T lymphocytes
U.S. Pat. No. 6,077,519 provides a cancer vaccine comprising a composition of T cell epitopes recovered through acid elution of epitopes from tumor tissue.
WO 00/46352 provides a cancer vaccine comprising human T lymphocytes that express a functional CD86 molecule.
T lymphocytes are obtained by subjecting T cells to at least two sequential stimuli, each involving at least one activator (an antibody anti CD2, 3 or 28) and a cytokine (interleukine) that stimulates T cell proliferation.
activator an antibody anti CD2, 3 or 28
cytokine interleukine
the cells obtainable according to the method of the present invention, as well as the cellular components thereof whether alone or in combination with said cells, are useful for prevention and treatment, in particular in a mammal, human beings included, of malignancies of different histotype that constitutively express one or more of the multiple tumor associated antigens that are expressed in said cells.
ADHAPI-Cells said cells are briefly named ADHAPI-Cells.
the cells obtainable from the method above mentioned are used in the form of a cancer vaccine.
FIG. 1 shows the proliferation of autologous (aMLR)PBMC (R) stimulated with ADHAPI-Cells/B-EBV or control B-EBV cells (S);
FIG. 2 shows the proliferation of autologous (aMLR)PBMC (R) stimulated with ADHAPI-Cells/PWM-B or control PWM-B cells (S);
FIG. 3 shows the proliferation of autologous (aMLR)PBMC (R) stimulated with ADHAPI-Cells/CD40L-B or control CD40L-B cells (S);
FIG. 4 shows the proliferation of autologous (aMLR)PBMC (R) stimulated with ADHAPI-Cells/PWM ⁇ PBMC or control PWM ⁇ PBMC cells (S);
FIG. 5 shows the proliferation of autologous (aMLR)PBMC (R) stimulated with ADHAPI-Cells/PHA ⁇ PBMC and control PHA ⁇ PBMC;
FIG. 6 shows the proliferation of autologous (aMLR)PBMC (R) stimulated with ADHAPI-Cells/PHA-+PWM ⁇ PBMC or control PHA-+PWM ⁇ PBMC (S);
the cells are collected from a subject, in particular a mammal, more in particular a human.
said human is a cancer patient.
antigen-presenting cells obtainable by the method above described are immune cells.
antigen-presenting cells obtainable by the method above described are non-immune cells.
the cells obtainable according to present invention can express shared immunodominant cancer antigens or can express shared not immunodominant cancer antigens.
cells suitable for the method herein disclosed are:
Epstein-Barr virus-immortalized, DNA hypomethylating agent-treated B-lymphoblastoid cell lines generated from peripheral blood mononuclear cells (PBMC) of cancer patients in advanced stage of disease or healthy subjects (ADHAPI-Cells/B-EBV).
PWM Pokeweed mitogen
CD40 activated, DNA hypomethylating agent-treated B-lymphocytes, generated from B-lymphocytes purified from PBMC of cancer patients in advanced stage of disease or healthy subjects (ADHAPI-Cells/CD40-B).
Pokeweed mitogen (PWM)-activated, DNA hypomethylating agent-treated PBMC generated from purified PBMC of cancer patients in advanced stage of disease or healthy subjects (ADHAPI-Cells/PWM ⁇ PBMC)
CD34+cells fibroblasts, stem cells, fibroblasts and cheratinocytes.
the cells obtainable by the method according to the present invention are suitable for use as agents for the prevention and treatment of malignancies of different histotype that constitutively express one or more of cancer antigens, whether immunodominant or not immunodominant.
vaccinating cells or their cellular components obtainable by the method of the present invention can be used as “reservoir” of pooled cancer antigens to vaccinate patients.
the selected TAA are CTA.
CTA are immunogenic since they include epitopes recognized by HLA class I-restricted CTA-specific CD8+ CTL.
CTA are immunogenic since they include epitopes recognized by HLA class II-restricted CTA-specific CD4+ T lymphocytes.
Selected CTA simultaneously include epitopes presented by HLA class I and by HLA class II antigens; thus, selected CTA can concomitantly induce CD8+ CTL and CD4+ T lymphocytes reactions.
CTA are not expressed in benign tissues with the exception of testis and placenta.
Different CTA can be concomitantly expressed in neoplastic cells of solid and hemopoietic malignancies, providing multiple therapeutic targets that are co-expressed on transformed cells.
Distinct CTA are homogeneously expressed among concomitant and sequential metastatic lesions of given patients.
Distinct CTA can be expressed in malignant tissues of different hystological origin providing common therapeutic targets shared by human neoplasia regardless of their specific hystotype.
Distinct CTA may encode for multiple immunogenic peptides presented in the context of different HLA class I and HLA class II allospecificities.
histone deacetylase inhibitors can sinergize with DNA hypomethylating agents in inducing/up-regulating the expression of CTA, of HLA antigens and of co-stimulatory/accessory molecules on neoplastic cells of different histology.
DNA methylation and histone deacetylation act as synergistic layers for the epigenetic gene silencing in cancer (Fuks F. et al., Nat.
DNA demethylating agents are widely disclosed in the literature, see for example WO 01/29235, U.S. Pat. No. 5,851,773.
a preferred DNA demethylating agent is 5-aza-cytidine or, more preferred, 5-aza-2′-deoxycytidine (5-AZA-CdR).
Antigen presenting cells according to the present invention are suitable for the preparation of cancer vaccines.
said vaccines are autologous vaccines.
said vaccines are allogeneic vaccines.
the cells obtainable according to the method above disclosed may be used both as antigen presenting cells and as in the form of “reservoir” of pooled cancer antigens, whether as cells or cellular components thereof.
the cells and/or the cellular components can be used in a method for generating effector immune cells, said effector immune cells being used for the preparation of a product useful in the well-known adoptive immunotherapy.
the vaccine herein disclosed can be used in combination with a systemic pre-treatment of the cancer patient with a hypomethylating agent, for example decitabine. This embodiment may be performed with an article of manufacture, for example a kit, comprising a vaccine according to the present invention and a pharmaceutical composition suitable for systemic administration of a hypomethylating agent, for example decitabine.
Vaccines can be prepared according to techniques well-know to the person skilled in this art, just resorting to the general common knowledge.
the patent references mentioned in the present description are a sufficient disclosure for the preparation of cancer vaccines, see for example WO 00/25813 or WO 00/46352.
PBMC peripheral blood of cancer patients in advanced stage of disease or healthy subjects.
B-EBV+lymphoblastoid cell lines were generated by incubating PBMC with supernatant from B95.8 marmoset cell line at 37° C. in a 5% CO 2 humidified atmosphere, in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (or human AB serum) and 2 mM L-glutamine.
B-EBV+lymphoblastoid cell lines (7.5 ⁇ 10 5 cells/ml) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (or 10% heat-inactivated human AB serum) and 2 mM L-glutamine at 37° C. in a 5% CO 2 humidified atmosphere, and pulsed four times with 1 ⁇ M 5-aza-2′-deoxycytidine (5-AZA-CdR) every 12 h; then, half of the culture medium was replaced with fresh medium and cultures were allowed to proceed for additional 48 h. Then cells were utilized for experimental procedures and/or frozen under viable conditions. Control cells (B-EBV cells) were cultured under similar experimental conditions but without pulses of 5-AZA-CdR.
ADHAPI-Cells/B-EBV and control B-EBV cells were collected, washed twice with Hanks' balanced salt solution (HBSS) and x-ray treated (75 Gy).
HBSS Hanks' balanced salt solution
x-ray treated 75 Gy.
For aMLR and MLR scalar concentrations (from 1 ⁇ 10 6 cells/ml to 6 ⁇ 10 4 cells/ml) of ADHAPI Cells/B-EBV or control B-EBV cells were added to autologous or allogeneic PBMC (1 ⁇ 10 6 cells/ml) (responder R) in Basal Iscove's medium supplemented with 10% heat-inactivated human AB serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin sulphate, and seeded in 96 well U-bottom plates to a final volume of 200 ⁇ l/well.
PBMC peripheral blood cells were purified by standard Ficoll-Hypaque density gradient centrifugation from heparinized peripheral blood of cancer patients in advanced stage of disease or healthy subjects, and purified B lymphocytes were obtained by conventional E resetting technique utilizing neuraminidase-treated sheep red blood cells.
Purified B-Lymphocytes (1.5 ⁇ 10 6 cells/ml) were added with PWM (3 ⁇ g/ml) and cultured for 48 h at 37° C. in a 5% CO 2 humidified atmosphere in Basal Iscove's medium supplemented with 10% heat-inactivated human AB serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin sulphate.
PWM-activated B-Lymphocytes were pulsed four times with 1 ⁇ M 5-aza-2′-deoxycytidine (5-AZA-CdR) every 12 h; then, half of the culture medium was replaced with fresh medium and cultures were allowed to proceed for additional 48 h. Then cells were utilized for experimental procedures and/or frozen under viable conditions. Control cells (PWM-B) were cultured under similar experimental conditions but without pulses of 5-AZA-CdR.
5-AZA-CdR 5-aza-2′-deoxycytidine
ADHAPI-Cells/PWM-B and control PWM-B cells were collected, washed three times with Hanks' balanced salt solution supplemented with 0.5% ⁇ -methylmannopyranoside, and x-ray treated (30 Gy).
PBMC peripheral blood of cancer patients in advanced stage of disease or healthy subjects.
PBMC peripheral blood mononuclear cells
NIH3T3-CD40L NIH3T3-CD40L
Basal Iscove's medium supplemented with 10% heat-inactivated human AB serum, 2 mM L-glutamine, 2 ng/ml recombinant human (rh) interleukin 4 (rhIL-4), 50 ⁇ g/ml human transferrin, 5 ⁇ g/ml rh insulin, 5.5 ⁇ 10 ⁇ 7 M cyclosporin A (CsA), 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin sulphate (complete medium).
CsA cyclosporin A
PBMC peripheral blood mononuclear cells
PBMC peripheral blood of cancer patients in advanced stage of disease or healthy subjects.
PBMC (1.5 ⁇ 10 6 cells/ml) were added with PWM (3 ⁇ g/ml) and cultured for 48 h at 37° C. in a 5% CO 2 humidified atmosphere in Basal Iscove's medium supplemented with 10% heat-inactivated human AB serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin sulphate.
PWM-activated PBMC were pulsed four times with 1 ⁇ M 5-aza-2′-deoxycytidine (5-AZA-CdR) every 12 h; then, half of the culture medium was replaced with fresh medium and cultures were allowed to proceed for additional 48 h. Then cells were utilized for experimental procedures and/or frozen under viable conditions. Control cells (PWM ⁇ PBMC) were cultured under similar experimental conditions but without pulses of 5-AZA-CdR.
5-AZA-CdR 5-aza-2′-deoxycytidine
ADHAPI-Cells/PWM ⁇ PBMC and control PWM ⁇ PBMC cells were collected, washed three times with Hanks' balanced salt solution supplemented with 0.5% ⁇ -methylmannopyranoside, and x-ray treated (30 Gy).
PBMC peripheral blood of cancer patients in advanced stage of disease or healthy subjects.
PBMC (1.5 ⁇ 10 6 cells/ml) were added with PHA-M (10 ⁇ g/ml) and 100 UI/ml rhIL-2, and cultured for 48 h at 37° C. in a 5% CO 2 humidified atmosphere in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (or in Basal Iscove's medium supplemented with 10% heat-inactivated human AB serum), 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin sulphate (complete medium).
PHA-activated PBMC were pulsed four times with 1 ⁇ M 5-aza-2′-deoxycytidine (5-AZA-CdR) every 12 h; then, half of the culture medium was replaced with fresh complete medium without PHA-M and cultures were allowed to proceed for additional 48 h. Then cells were utilized for experimental procedures and/or frozen under viable conditions. Control cells (PHA ⁇ PBMC) were cultured under similar experimental conditions but without pulses of 5-AZA-CdR.
5-AZA-CdR 5-aza-2′-deoxycytidine
PBMC peripheral blood of cancer patients in advanced stage of disease or healthy subjects.
PBMC (1.5 ⁇ 10 6 cells/ml) were added with PHA-M (10 ⁇ g/ml), PWM (3 ⁇ g/ml), 100 UI/ml rhIL-2 and cultured for 48 h at 37° C. in a 5% CO 2 humidified atmosphere in Basal Iscove's medium supplemented with 10% heat-inactivated human AB serum (or with 10% heat-inactivated autologous serum), 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin sulphate (complete medium).
PHA+PWM-activated PBMC were pulsed four times with 1 ⁇ M 5-aza-2′-deoxycytidine (5-AZA-CdR) every 12 h; then, half of the culture medium was replaced with fresh complete medium without PHA or PWM and cultures were allowed to proceed for additional 48 h. Then cells were utilized for experimental procedures and/or frozen under viable conditions. Control cells (PHA+PWM ⁇ PBMC) were cultured under similar experimental conditions but without pulses of 5-AZA-CdR.
5-AZA-CdR 5-aza-2′-deoxycytidine
ADHAPI-Cells/PHA+PWM ⁇ PBMC and control PHA+PWM ⁇ PBMC were collected, washed three times with Hanks' balanced salt solution supplemented with 0.5% ⁇ -methylmannopyranoside, and x-ray treated (50 Gy).
ADHAPI-Cells as Polyvalent Cellular CTA Vaccines
ADHAPI-Cells represent a totally new and innovative approach, and comprise a number of prominent/remarkable advantages. Among these:
ADHAPI-Cells vs not Genetically-modified Cellular CTA Vaccines
ADHAPI-Cells are new and unique APC vaccines as they concomitantly express multiple/all methylation-regulated CTA; being endogenously synthesised, CTA can directly and simultaneously access both HLA class I and HLA class II antigen processing pathways within ADHAPI-Cells (Jenne L. et al., Trends Immunol., 22:102-107, 2001).
ADHAPI-Cells can concomitantly present immunogenic epitopes of endogenously synthesised CTA both to CD8+ and to CD4+ T autologous lymphocytes; therefore, ADHAPI-Cells can simultaneously induce/amplify a CTA-directed CTL and humoral immune responses. Additionally, ADHAPI-Cells may express and present to host's T cells methylation-regulated CTA that have not been identified and characterized yet (as well as not immunodominant epitopes of known and still unknown CTA).
Eds share major limitations including: i) the unknown fate in vivo of the ex vivo-loaded synthetic CTA peptide(s), of whole synthetic CTA protein or of tumor-derived CTA, which may significantly affect the longevity of antigen presentation to host's immune system; ii) limited amounts of synthetic CTA peptide(s), of whole synthetic CTA protein or of tumor-derived CTA that can be loaded ex vivo onto HLA class I and/or HLA class II antigens of cellular vaccines, which may significantly hamper the immunogenicity of administered CTA; iii) the restriction by the patient's HLA phenotype, and the still relatively limited number of known HLA class I antigens- and even more HLA class II antigens restricted immunogenic epitopes of so far identified CTA; iv) availability of adequate amounts of fresh tumor tissue, that should also be sufficiently representative of the diverse CTA expressed in neoplastic lesions (Jenne L. et al., Trends Immunol., 22:102-107, 2001).
ADHAPI-Cells The expression of endogenously synthesised CTA by ADHAPI-Cells is long lasting; thus, at variance with ex vivo synthetic CTA peptide(s)-pulsed or synthetic CTA whole protein-pulsed or whole tumor cell preparations-pulsed autologous APC vaccines, ADHAPI-Cells can provide a prolonged stimulation in vivo of hosts immune response and with a lower number of administrations to patients. This hypothesis is reinforced by the foreseen possibility to administer ADHAPI-Cells as a viable, not x-ray-treated, cellular vaccines due to their absence of long term tumorigenicity in vivo.
ADHAPI-Cells would undergo physiological death in vivo, they could still act as a “reservoir” of endogenously synthesised CTA peptides and proteins, that could further and efficiently boost the presentation of HLA class I-restricted epitopes of CTA to CD8+ T cells by patient's dendritic cells, through the immunologic mechanism of cross-priming, as well as the presentation of HLA class II-restricted epitopes of CTA to CD4+ T cells, through the well-defined exogenous pathway of antigen processing.
ADHAPI-Cells retain their APC function; in fact, they efficiently stimulate the proliferation and IFN- ⁇ release of autologous and allogeneic PBMC; furthermore, ADHAPI-Cells are in most instances more potent stimulators as compared to their respective control cells. In this respect, it is relevant that in addition to CTA, ADHAPI-Cells may concomitantly express higher levels of HLA class I antigens and/or of different co-stimulatory/accessory molecules as compared to their respective control cells.
ADHAPI-Cells as autologous cellular vaccines, compared to autologous tumor cells that are poorly immunogenic, and do not constitutively express several co-stimulatory/accessory molecules.
ADHAPI-Cells vaccines are generated by fully mature and immunocompetent APC; this aspect overcomes the potential limitation represented by the maturation stage of dendritic cells utilized for the generation of cellular vaccines, which may influence their tolerogenic rather than immunogenic potential.
ADHAPI-Cells vaccines that concomitantly express multiple/all methylation-regulated CTA, is simple, in most cases rapid, does not require cumbersome in vitro cellular manipulations, does not involve genetic manipulations, does not require autologous tumor tissue, and it is highly reproducible both from PBMC of healthy individuals and cancer patients.
ADHAPI-Cells preparations express all investigated CTA that are demethylation-inducible in APC. Due to these characteristics, the generation of ADHAPI-Cells vaccines is easier to standardize and to control for quality (for example by flow cytometry for selected cell surface molecules and RT-PCR for selected CTA) and potency (for example by quantitative RT-PCR for selected CTA). Additionally, compared to other cellular vaccines that to date must be freshly prepared each time they must be administered to patients, thus generating obvious inter-preparations variability (e.
ADHAPI-Cells vaccines once prepared and checked for viability, quality and potency, can be aliquoted, appropriately frozen, and stored under viable conditions until use for therapeutic purposes.
ADHAPI-Cells vaccines represent a practically unlimited source of therapeutic agent for each patient.
treatment with ADHAPI-Cells vaccines is not limited to patients with defined HLA phenotypes; hence, all cancer patients whose neoplastic lesions express one or more CTA can be candidate to treatment with ADHAPI-Cells vaccines, regardless of their HLA phenotype.
one or more of them is generally expressed in most investigated malignancies of different histotype; therefore, vaccination with ADHAPI-Cells is suitable in the large majority of cancer patients.
a significant information is that MAGE, GAGE or NY-ESO-1 are expressed in 96% of human tumors ( Cancer Immunol. Immunother. 50:3-15, 2001).
ADHAPI-Cells vaccines can overcome the immunoselection of CTA-negative tumor variants occurring in the course of treatment against single or few CTA, and overcome the constitutively heterogeneous and sometimes down-regulated expression of distinct CTA occurring in specific neoplastic lesions.
ADHAPI-Cells vaccines are constituted by autologous functional APC that concomitantly express multiple/all known methylation-regulated CTA, and that most likely express still unidentified CTA whose expression is regulated by DNA methylation; furthermore, ADHAPI-Cells vaccines can be utilized in patients affected by CTA-positive tumors of different histotype.
These functional and phenotypic features represent a clear advantage over currently utilized allogeneic tumor cell vaccines (e.g., lysates of whole pooled neoplastic cell lines or their non-purified extracts, shed antigens from pooled neoplastic cell lines).
these tumor cell vaccines may not contain or may contain insufficient amounts of known and of still unknown immunologically-relevant CTA, contain irrelevant cellular components that may compete with CTA for immunological responses, may have increased toxicity being allogeneic, require efficient processing by patients' immune system, and can be utilized exclusively in patients affected by malignancies of the same histologic type.
ADHAPI-Cells vs Genetically Modified Cellular CTA Vaccines
ADHAPI-Cells does not involve the ex vivo genetic manipulations of autologous dendritic cells or of other autologous APC, that are required to produce genetically-modified cellular vaccines expressing selected CTA following transfection or transduction.
a number of limitations affect genetically-modified cellular vaccines; among these are: i) the relative low efficiency of available transfection methodologies; ii) the induction of cellular immune responses against antigens of the viral vectors utilized for cellular transduction, which leads to the destruction of genetically-modified vaccinating cells; iii) the presence of pre-existing or vaccination-induced neutralizing antibodies that interfere with vaccine administration(s); iv) direct effects of viral vectors on the viability, maturation and antigen-presentation ability of transduced cells (Jenne L. et al., Trends Immunol., 22:102-107, 2001).
ADHAPI-Cells TABLE I Recovery of ADHAPI-Cells and control cells

Landscapes

Health & Medical Sciences (AREA)
Life Sciences & Earth Sciences (AREA)
Engineering & Computer Science (AREA)
Biomedical Technology (AREA)
General Health & Medical Sciences (AREA)
Organic Chemistry (AREA)
Chemical & Material Sciences (AREA)
Bioinformatics & Cheminformatics (AREA)
Zoology (AREA)
Wood Science & Technology (AREA)
Genetics & Genomics (AREA)
Biotechnology (AREA)
Public Health (AREA)
Veterinary Medicine (AREA)
Animal Behavior & Ethology (AREA)
Epidemiology (AREA)
Cell Biology (AREA)
Hematology (AREA)
Microbiology (AREA)
Biochemistry (AREA)
General Engineering & Computer Science (AREA)
Oncology (AREA)
Immunology (AREA)
Chemical Kinetics & Catalysis (AREA)
General Chemical & Material Sciences (AREA)
Medicinal Chemistry (AREA)
Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
Pharmacology & Pharmacy (AREA)
Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Micro-Organisms Or Cultivation Processes Thereof (AREA)
Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Peptides Or Proteins (AREA)

US09/981,239 2001-07-30 2001-10-18 Antigen presenting cells, method for their preparation and their use for cancer vaccines Abandoned US20030119187A1 (en)

Priority Applications (1)

Application Number	Priority Date	Filing Date	Title
US10/898,187 US20050002920A1 (en)	2001-07-30	2004-07-26	Antigen presenting cells, method for their preparation and their use for cancer vaccines

Applications Claiming Priority (1)

Application Number	Priority Date	Filing Date	Title
PCT/IT2001/000419 WO2003012085A1 (fr)	2001-07-30	2001-07-30	Cellules presentant l'antigene, procede de preparation et d'utilisation dans les vaccins contre le cancer

Related Parent Applications (1)

Application Number	Title	Priority Date	Filing Date
PCT/IT2001/000419 Continuation WO2003012085A1 (fr)	2001-07-30	2001-07-30	Cellules presentant l'antigene, procede de preparation et d'utilisation dans les vaccins contre le cancer

Related Child Applications (1)

Application Number	Title	Priority Date	Filing Date
US10/898,187 Continuation US20050002920A1 (en)	2001-07-30	2004-07-26	Antigen presenting cells, method for their preparation and their use for cancer vaccines

Publications (1)

Publication Number	Publication Date
US20030119187A1 true US20030119187A1 (en)	2003-06-26

Family

ID=11133712

Family Applications (3)

Application Number	Title	Priority Date	Filing Date
US09/981,239 Abandoned US20030119187A1 (en)	2001-07-30	2001-10-18	Antigen presenting cells, method for their preparation and their use for cancer vaccines
US10/485,351 Expired - Lifetime US7883889B2 (en)	2001-07-30	2002-07-25	Antigen presenting cells, method for their preparation and their use for cancer vaccines
US10/898,187 Abandoned US20050002920A1 (en)	2001-07-30	2004-07-26	Antigen presenting cells, method for their preparation and their use for cancer vaccines

Family Applications After (2)

Application Number	Title	Priority Date	Filing Date
US10/485,351 Expired - Lifetime US7883889B2 (en)	2001-07-30	2002-07-25	Antigen presenting cells, method for their preparation and their use for cancer vaccines
US10/898,187 Abandoned US20050002920A1 (en)	2001-07-30	2004-07-26	Antigen presenting cells, method for their preparation and their use for cancer vaccines

Country Status (17)

Country	Link
US (3)	US20030119187A1 (fr)
EP (1)	EP1412485B1 (fr)
JP (1)	JP2004536615A (fr)
KR (1)	KR100916670B1 (fr)
CN (1)	CN100591762C (fr)
AT (1)	ATE505533T1 (fr)
AU (1)	AU2002334387B2 (fr)
BR (1)	BR0211520A (fr)
CA (1)	CA2453522C (fr)
DE (1)	DE60239741D1 (fr)
DK (1)	DK1412485T3 (fr)
ES (1)	ES2367256T3 (fr)
HK (1)	HK1068913A1 (fr)
HU (1)	HUP0401753A3 (fr)
MX (1)	MXPA04000885A (fr)
PL (1)	PL211907B1 (fr)
WO (2)	WO2003012085A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US20040185563A1 (en) *	2001-07-30	2004-09-23	Rita De Santis	Antigen presenting cells, method for their preparation and their use for cancer vaccines
US20040197343A1 (en) *	2003-02-06	2004-10-07	Dubensky Thomas W.	Modified free-living microbes, vaccine compositions and methods of use thereof
US20040228877A1 (en) *	2003-02-06	2004-11-18	Dubensky Thomas W.	Listeria attenuated for entry into non-phagocytic cells, vaccines comprising the listeria, and methods of use thereof
US20050249748A1 (en) *	2003-12-24	2005-11-10	Dubensky Thomas W Jr	Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof
US20080248066A1 (en) *	2003-02-06	2008-10-09	Cerus Corporation	Modified free-living microbes, vaccine compositions and methods of use thereof
US9895430B2 (en) *	2013-12-27	2018-02-20	SOTIO a.s.	Cryopreservation of apoptotic cancer cells for use in immunotherapy against cancer

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US6982253B2 (en)	2002-06-05	2006-01-03	Supergen, Inc.	Liquid formulation of decitabine and use of the same
DE10329240A1 (de) *	2003-06-24	2005-01-20	Epigenomics Ag	Verfahren zur Analyse des Cytosin-Methylierungsstatus von Cancer-Testis-Antigenen für eine individualisierte Immuntherapie
KR20060129393A (ko) *	2004-01-20	2006-12-15	아이치 프레펙츄러	ＨＬＡ－Ａ2402-제한 Ｅｐ－ＣＡＭ 특이적 ＣＴＬ에인식되는 에피토프/펩티드 및 그 용도
WO2005075646A1 (fr) *	2004-02-03	2005-08-18	Kurume University	Peptide provenant d'une molecule d'antiapoptose induite par un stress (iex-1)
US7250416B2 (en)	2005-03-11	2007-07-31	Supergen, Inc.	Azacytosine analogs and derivatives
WO2007011693A2 (fr)	2005-07-14	2007-01-25	Medistem Laboratories, Inc.	Compositions de cellules souches derivees du placenta, utilisees dans le traitement de cancers
US7700567B2 (en)	2005-09-29	2010-04-20	Supergen, Inc.	Oligonucleotide analogues incorporating 5-aza-cytosine therein
GB0700058D0 (en) *	2007-01-03	2007-02-07	Scancell Aps	Anti-tumor vaccine based on normal cells
US8809050B2 (en) *	2009-12-08	2014-08-19	Wilson Wolf Manufacturing	Methods of cell culture for adoptive cell therapy
AU2012278813B2 (en) *	2011-07-07	2016-09-08	Research Cancer Institute Of America	Systems, methods, and formulations for treating cancer
US8933078B2 (en)	2011-07-14	2015-01-13	Research Cancer Institute Of America	Method of treating cancer with combinations of histone deacetylase inhibitors (HDAC1) substances
PT2750768T (pt)	2011-08-30	2018-12-19	Astex Pharmaceuticals Inc	Formulações de derivados de decitabina
DK2914254T3 (en) *	2012-10-30	2020-03-30	Mei Pharma Inc	Combination therapies to treat chemoresistant cancers
WO2016190940A1 (fr) *	2015-05-22	2016-12-01	Czerniecki Brian J	Fabrication de vaccins multidoses prêts à injecter à base de cellules dendritiques
EP3316685A4 (fr)	2015-07-02	2019-03-13	Otsuka Pharmaceutical Co., Ltd.	Compositions pharmaceutiques lyophilisées
CN105132371B (zh) *	2015-07-24	2016-05-25	北京鼎成肽源生物技术有限公司	体外提呈、激活dc细胞的方法及其应用
US10052372B2 (en) *	2016-05-24	2018-08-21	Tessa Therapeutics Pte Ltd	T cell expansion
US11890292B2 (en)	2017-02-27	2024-02-06	Research Cancer Institute Of America	Compositions, methods, systems and/or kits for preventing and/or treating neoplasms
WO2018170457A1 (fr)	2017-03-17	2018-09-20	Research Cancer Institute Of America	Compositions, méthodes, systèmes et/ou kits pour prévenir et/ou traiter des néoplasmes
MX2020001233A (es)	2017-08-03	2020-07-20	Otsuka Pharma Co Ltd	Compuesto farmaceutico y metodos de purificacion del mismo.
CN107557333B (zh) *	2017-10-16	2018-07-31	北京鼎成肽源生物技术有限公司	一种用于细胞治疗的新抗原呈递细胞的制备方法
EP3710434A4 (fr)	2017-11-17	2021-07-28	Research Cancer Institute of America	Compositions, procédés, systèmes et/ou kits de prévention et/ou de traitement de néoplasmes
KR102319259B1 (ko)	2019-07-26	2021-10-28	정재웅	지반천공기용 확장리더 안전제어장치
WO2021027057A1 (fr) *	2019-08-15	2021-02-18	成都冕康生物科技有限公司	Vaccin à lymphocytes b contre l'antigène du virus d'epstein-barr (veb) et son procédé de préparation
CN114099655A (zh) *	2021-11-16	2022-03-01	苏州大学	一种预防或治疗癌症的疫苗系统及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
WO1996030523A2 (fr) *	1995-03-31	1996-10-03	Hans Wolf	Systeme de presentation d'antigenes fonde sur des particules similaires a des retrovirus
EA003832B1 (ru) *	1997-09-15	2003-10-30	Дженетик Иммьюнити, Ллс.	Ген-доставочный комплекс для чрескожной доставки чужеродного генетического материала в антиген-презентирующие клетки и способы его использования
US6613753B2 (en)	2001-02-21	2003-09-02	Supergen, Inc.	Restore cancer-suppressing functions to neoplastic cells through DNA hypomethylation
WO2003012085A1 (fr)	2001-07-30	2003-02-13	Sigma-Tau Industrie Farmaceutiche Riunite S.P.A.	Cellules presentant l'antigene, procede de preparation et d'utilisation dans les vaccins contre le cancer

2001
- 2001-07-30 WO PCT/IT2001/000419 patent/WO2003012085A1/fr active Application Filing
- 2001-10-18 US US09/981,239 patent/US20030119187A1/en not_active Abandoned
2002
- 2002-07-25 DE DE60239741T patent/DE60239741D1/de not_active Expired - Lifetime
- 2002-07-25 DK DK02791520.6T patent/DK1412485T3/da active
- 2002-07-25 AT AT02791520T patent/ATE505533T1/de not_active IP Right Cessation
- 2002-07-25 ES ES02791520T patent/ES2367256T3/es not_active Expired - Lifetime
- 2002-07-25 CN CN02815007A patent/CN100591762C/zh not_active Expired - Lifetime
- 2002-07-25 BR BR0211520-4A patent/BR0211520A/pt not_active Application Discontinuation
- 2002-07-25 KR KR1020047001217A patent/KR100916670B1/ko not_active Expired - Fee Related
- 2002-07-25 MX MXPA04000885A patent/MXPA04000885A/es active IP Right Grant
- 2002-07-25 WO PCT/IT2002/000488 patent/WO2003012086A1/fr active Application Filing
- 2002-07-25 EP EP02791520A patent/EP1412485B1/fr not_active Expired - Lifetime
- 2002-07-25 PL PL367617A patent/PL211907B1/pl unknown
- 2002-07-25 JP JP2003517263A patent/JP2004536615A/ja active Pending
- 2002-07-25 CA CA2453522A patent/CA2453522C/fr not_active Expired - Lifetime
- 2002-07-25 HU HU0401753A patent/HUP0401753A3/hu unknown
- 2002-07-25 AU AU2002334387A patent/AU2002334387B2/en not_active Expired
- 2002-07-25 US US10/485,351 patent/US7883889B2/en not_active Expired - Lifetime
2004
- 2004-07-26 US US10/898,187 patent/US20050002920A1/en not_active Abandoned
2005
- 2005-02-16 HK HK05101242.9A patent/HK1068913A1/xx not_active IP Right Cessation

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US7883889B2 (en)	2001-07-30	2011-02-08	Sigma-Tau Industrie Farmaceutiche Riunite S.P.A.	Antigen presenting cells, method for their preparation and their use for cancer vaccines
US20040185563A1 (en) *	2001-07-30	2004-09-23	Rita De Santis	Antigen presenting cells, method for their preparation and their use for cancer vaccines
US20040197343A1 (en) *	2003-02-06	2004-10-07	Dubensky Thomas W.	Modified free-living microbes, vaccine compositions and methods of use thereof
US20040228877A1 (en) *	2003-02-06	2004-11-18	Dubensky Thomas W.	Listeria attenuated for entry into non-phagocytic cells, vaccines comprising the listeria, and methods of use thereof
US20080248066A1 (en) *	2003-02-06	2008-10-09	Cerus Corporation	Modified free-living microbes, vaccine compositions and methods of use thereof
US20100068230A1 (en) *	2003-02-06	2010-03-18	Dubensky Jr Thomas W	Modified free-living microbes, vaccine compositions and methods of use thereof
US7691393B2 (en)	2003-02-06	2010-04-06	Anza Therapeutics, Inc.	Listeria attenuated for entry into non-phagocytic cells, vaccines comprising the Listeria, and methods of use thereof
US7695725B2 (en)	2003-02-06	2010-04-13	Aduro Biotech	Modified free-living microbes, vaccine compositions and methods of use thereof
US7833775B2 (en)	2003-02-06	2010-11-16	Aduro Biotech	Modified free-living microbes, vaccine compositions and methods of use thereof
US7927606B2 (en)	2003-02-06	2011-04-19	Aduro Biotech	Modified free-living microbes, vaccine compositions and methods of use thereof
US20050249748A1 (en) *	2003-12-24	2005-11-10	Dubensky Thomas W Jr	Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof
US7842289B2 (en)	2003-12-24	2010-11-30	Aduro Biotech	Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof
US9895430B2 (en) *	2013-12-27	2018-02-20	SOTIO a.s.	Cryopreservation of apoptotic cancer cells for use in immunotherapy against cancer
US10155033B2 (en)	2013-12-27	2018-12-18	SOTIO a.s.	Cryopreservation of apoptotic cancer cells for use in immunotherapy against cancer

Also Published As

Publication number	Publication date
KR100916670B1 (ko)	2009-09-08
EP1412485A1 (fr)	2004-04-28
CN1537161A (zh)	2004-10-13
WO2003012085A1 (fr)	2003-02-13
DE60239741D1 (de)	2011-05-26
US20040185563A1 (en)	2004-09-23
CA2453522A1 (fr)	2003-02-13
CN100591762C (zh)	2010-02-24
BR0211520A (pt)	2004-09-14
DK1412485T3 (da)	2011-08-01
CA2453522C (fr)	2013-04-30
HUP0401753A3 (en)	2011-04-28
ATE505533T1 (de)	2011-04-15
EP1412485B1 (fr)	2011-04-13
AU2002334387B2 (en)	2007-10-04
PL211907B1 (pl)	2012-07-31
ES2367256T3 (es)	2011-10-31
US20050002920A1 (en)	2005-01-06
WO2003012086A1 (fr)	2003-02-13
KR20040021662A (ko)	2004-03-10
HK1068913A1 (en)	2005-05-06
US7883889B2 (en)	2011-02-08
MXPA04000885A (es)	2004-06-03
PL367617A1 (en)	2005-03-07
HUP0401753A2 (hu)	2004-11-29
JP2004536615A (ja)	2004-12-09

Legal Events

Date

Code

Title

Description