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WO2014164464A1 - Cellules souches cancéreuses ovariennes de haute pureté pour immunothérapie autologue active - Google Patents

Cellules souches cancéreuses ovariennes de haute pureté pour immunothérapie autologue active Download PDF

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Publication number
WO2014164464A1
WO2014164464A1 PCT/US2014/022494 US2014022494W WO2014164464A1 WO 2014164464 A1 WO2014164464 A1 WO 2014164464A1 US 2014022494 W US2014022494 W US 2014022494W WO 2014164464 A1 WO2014164464 A1 WO 2014164464A1
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Prior art keywords
csc
cells
population
biomarkers
cell
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English (en)
Inventor
Andrew N. CORNFORTH
Gabriel Nistor
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NeoStem Oncology LLC
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California Stem Cells Inc
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Priority claimed from PCT/US2013/053850 external-priority patent/WO2014028274A1/fr
Priority to HK16108669.5A priority Critical patent/HK1220616A1/zh
Priority to CA2905288A priority patent/CA2905288A1/fr
Priority to JP2016500982A priority patent/JP2016512423A/ja
Priority to KR1020157028644A priority patent/KR20150139529A/ko
Priority to US14/775,313 priority patent/US20160058855A1/en
Application filed by California Stem Cells Inc filed Critical California Stem Cells Inc
Priority to CN201480027224.XA priority patent/CN105339000A/zh
Priority to AU2014249346A priority patent/AU2014249346A1/en
Priority to EP14778635.4A priority patent/EP2968531A4/fr
Publication of WO2014164464A1 publication Critical patent/WO2014164464A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • 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
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes

Definitions

  • the present disclosure relates to ovarian carcinoma stem cells, immunogenic compositions derived therefrom, and methods of making and using same.
  • Ovarian cancer is thought to have the origins in the epithelia lining the ovaries, hence the adenocarcinoma histological characteristics.
  • Ovarian cancer stem cells have been previously described as a subpopulation or "side population" (SP) that is selected from the main population (MP) in the SKOV3 and A224 cell lines, expressing stem cell marker genes (Oct4 and Nanog), transporter genes (ABCG2, ABCC4, ABCB 1 ) and CD markers (CD44, CD24, CD 177), with potential to differentiate into cancers, with different histologies, suggesting the pluripotent character of stem cells. Isolation of such cells was accomplished using the exclusion of the fluorescent DNA-binding dye Hoechst 33342
  • Specific cancer stem cell populations can be the origin of neoplasms and can be a source of recurrence of a cancer that had been treated. Also, subpopulations of cancer stem cells in a tissue can, when exposed to certain signals, restart the growth cycle and produce cells that can reestablish the tumor.
  • the cancer stem cell niche is dormant until proper signaling triggers the re-entry in the proliferation cycle. Re-entry signals can originate from local events such as trauma, cell damage, microorganism aggression (viral, bacterial or fungal), or mediated by local growth factors, cytokines or intercellular communication. Also, hormones can modulate stem cells in tissue-specific niches.
  • Neoplasms can result in perturbation of the above functions.
  • Neoplasms can result from such perturbations, and these include random mutations that influence control over the cell cycle.
  • Mutations leading to cancer vary from individual to individual. Such variability is observed between those who suffer from one type of cancer, such as one breast cancer patient versus another breast cancer patient, as well as between different types of cancer, such as ovarian cancer versus melanoma. In ovarian cancer, damage to the TP53 gene has been identified in the majority of the cases, however this mutation is not always reflected in the tumor cell phenotype. [0005] Inconsistent markers are also associated in various proportions with the tumor cells and used more or less successfully to trigger an immune-response.
  • Such therapies using tumor- associated antigens employ various proteins or peptides such as CA125, Her-2, Muc-1 , Neu, NY- ESO-1 , or tumor derived heat shock proteins (HSP).
  • Autologous immune therapies employ a patient's own tumor tissue to sensitize the immune system and attach the cancerous cells. Lysates or whole cell approaches are administrated alone or with adjuvants have been used to enhance the immune response to tumors.
  • ovarian carcinoma cancer stem cells (CSC)
  • CSC ovarian carcinoma cancer stem cells
  • immunogenic compositions comprising OV-CSC-loaded dendritic cells for the treatment of ovarian carcinoma.
  • an immunogenic composition comprising dendritic cells activated ex vivo by tumor antigens derived from the population of purified ovarian carcinoma (OV) cancer stem cells (CSC) disclosed herein.
  • the tumor antigens comprise cell extracts of the OV-CSC.
  • the tumor antigens comprise lysates of the OV- CSC.
  • the tumor antigens comprise intact OV-CSC.
  • the tumor antigens comprise messenger RNA transfected into the dendritic cells ex vivo.
  • the intact OV-CSC are rendered non-proliferative.
  • the intact OV-CSC are rendered non-proliferative by irradiation.
  • the intact OV-CSC are rendered non-proliferative by exposure of the OV-CSC to a nuclear or protein cross-linking agent.
  • the immunogenic composition further comprises a pharmaceutically acceptable carrier and/or excipient.
  • the immunogenic composition further comprises an adjuvant.
  • the adjuvant is granulocyte macrophage colony stimulating factor.
  • immunogenic composition comprises activated dendritic cells and OV-CSC.
  • the OV-CSC are in the form of OV-CSC spheroids, early OV-CSC, mixed OV-CSC, or EMT-OV-CSC.
  • a method of treating ovarian carcinoma in a subject in need thereof comprising administration of an immunogenic composition disclosed herein to the subject.
  • the immunogenic composition is administered in a plurality of doses, each dose comprising about 5-20x10 6 cells.
  • the dose comprises about 10x10 6 cells.
  • the dose is administered weekly for 2-5 doses, followed by monthly for 3-6 doses.
  • the subject receives from 6-10 doses of immunogenic composition.
  • an immunogenic composition disclosed herein an OV- CSC disclosed herein, or an OV-CSC cell line disclosed herein in the manufacture of a medicament for the treatment of ovarian carcinoma.
  • an immunogenic composition disclosed herein an OV- CSC disclosed herein, or an OV-CSC cell line disclosed herein for the treatment of ovarian carcinoma.
  • OV ovarian carcinoma
  • CSC cancer stem cells
  • the method comprising: acquiring a sample of OV; dissociating the cells of the sample, and in vitro culturing the dissociated cells in a defined medium on a nonadherent substrate, wherein the defined medium is serum free and is supplemented with at least one growth factor that acts through the mitogen activated protein kinase (MAPK) pathway, thereby forming OV-CSC spheroids; wherein at least 80% of the cells in the OV-CSC spheroid population express two or more of the biomarkers EpCAM, CA-125, MUC-1 , CD1 17, He-4, ALDH, CD133, CD24, and Ki-67.
  • At least 80% of the cells in the OV-CSC spheroid population further express one or more of the biomarkers CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, vimentin, CK8, CK18, AFP, testosterone, TGF3R, EGFR, TAG-72, CD46, CD44, ABCG2, Slug/Snail, nestin, and TP53.
  • at least 90% of the cells in the OV-CSC spheroid population express two or more of the biomarkers EpCAM, CA-125, MUC-1 , CD1 17, He-4, ALDH, CD133, CD24, and Ki-67.
  • the method further comprises culturing the OV-CSC spheroids in a defined medium on an adherent substrate, wherein the defined medium is serum free and is supplemented with at least one growth factor that acts through the MAPK pathway, thereby forming a population of early OV-CSC, wherein at least 80% of the cells in the early OV- CSC population express two or more of the biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17, and Ki-67. In another embodiment, at least 80% of the cells in the early OV-CSC population further express one or more of the biomarkers CA-125, MUC-1 , TGF3R, and CD24. In yet another embodiment, at least 90% of the cells in the early OV-CSC population express two or more of the biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17, and Ki-67.
  • the method further comprises culturing the OV-CSC spheroids in a defined medium on an adherent substrate, wherein the defined medium contains serum, thereby forming a population of mixed OV-CSC, wherein at least 80% of the cells in the mixed OV-CSC population express two or more of the biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18, and Ki-67.
  • the defined medium further comprises at least one growth factor that acts through the MAPK pathway.
  • the defined medium is a low calcium defined medium.
  • At least 80% of the cells in the mixed OV-CSC population further express one or more of the biomarkers CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, testosterone, TGF3R, EGFR, TAG- 72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin, and TP53.
  • at least 90% of the cells in the mixed OV-CSC population express two or more of the biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18, and Ki-67.
  • the method further comprises culturing the OV-CSC spheroids in a defined medium on an adherent substrate, wherein the defined medium contains serum and is supplemented with at least one growth factor that acts through the MAPK pathway, thereby forming a population of epithelial to mesenchymal transitioned (EMT)-OV-CSC, wherein at least 80% of the cells in the EMT-OV-CSC population express two or more of the biomarkers NCAM, Slug/Snail, CD24, and Twist.
  • EMT epithelial to mesenchymal transitioned
  • At least 80% of the cells in the EMT- OV-CSC population further express one or more of the biomarkers CA-125, MUC-1 , CD133, Nanog, CD1 17, N-cadherin, CD44, and vimentin. In another embodiment, at least 90% of the cells in the EMT-OV-CSC population express two or more of the biomarkers NCAM, Slug/Snail, CD24, and Twist.
  • the method further comprises culturing the OV-CSC spheroids, the mixed OV-CSC, or the EMT-OV-CSC in a defined medium on an adherent substrate, wherein the defined medium is serum free and is supplemented with at least one growth factor that acts through the MAPK pathway, thereby forming a population of early OV-CSC, wherein at least 80% of the cells in the early OV-CSC population express two or more of the biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17, and Ki-67.
  • At least 80% of the cells in the early OV-CSC population further express one or more of the biomarkers CA-125, MUC-1 , TGF3R, and CD24. In yet another embodiment, at least 90% of the cells in the early OV-CSC population express two or more of the biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD17, and Ki-67.
  • the method further comprises culturing the OV-CSC spheroids, the early OV-CSC, or EMT-OV-CSC in a defined medium on an adherent substrate, wherein the defined medium contains serum and is supplemented with at least one growth factor that acts through the MAPK pathway, thereby forming a population of mixed OV-CSC, wherein at least 80% of the cells in the mixed OV-CSC population express two or more of the biomarkers AFP, CK7, CK19, EpCAM, E-cadherin, Nanog, FoxA2 HNF4a, and ABCG2.
  • the defined medium further comprises at least one growth factor that acts through the MAPK pathway.
  • the defined medium is a low calcium defined medium.
  • at least 80% of the cells in the mixed OV-CSC population further express one or more of the biomarkers CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, testosterone, TGF3R, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin, and TP53.
  • at least 90% of the cells in the mixed OV-CSC population express two or more of the biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18, and Ki-67.
  • the method further comprises culturing the OV-CSC spheroids, the early OV-CSC, or mixed OV-CSC in a defined medium on an adherent substrate, wherein the defined medium contains a serum source and is supplemented with at least one growth factor that acts through the MAPK pathway, thereby forming a population of EMT-OV-CSC, wherein at least 80% of the cells in the EMT-OV-CSC population express two or more of the biomarkers NCAM, Slug/Snail, CD24, and Twist.
  • At least 80% of the cells in the EMT-OV-CSC population further express one or more of the biomarkers CA-125, MUC-1 , CD133, Nanog, CD1 17, N-cadherin, CD44, and vimentin. In another embodiment, at least 90% of the cells in the EMT-OV-CSC population express two or more of the biomarkers NCAM, Slug/Snail, CD24, and Twist.
  • the defined media is any media described in Table 2, any media from a combination of Table 2 and Table 3, any media from a combination of Table 2, Table 3, and Table 4, or any media from a combination of Table 2 and Table 4.
  • the growth factor is one or more of fibroblast growth factor (FGF), epidermal growth factor (EGF), or activin A.
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • the FGF is basic FGF (bFGF).
  • the defined medium is not supplemented with activin A.
  • the defined medium is supplemented with an agonist of activin A, in an amount effective to prevent spontaneous differentiation of OV stem cells.
  • the media comprises an antagonist of activin A, and the antagonist is follistatin or an antibody that specifically binds to activin A.
  • the medium is not supplemented with an antioxidant.
  • the antioxidant is superoxide dismutase, catalase, glutathione, putrescine, or ⁇ -mercaptoethanol.
  • the medium is supplemented with glutathione.
  • the adherent substrate is configured to adhere to, and optionally to collect, anchorage dependent cells, such as fibroblasts.
  • the non-adherent substrate is an ultralow adherent polystyrene surface.
  • the adherent substrate comprises a surface coated with a protein rich in RGD tripeptide motifs.
  • OV-CSC are OV-CSC spheroids, early OV-CSC, mixed OV-CSC, or EMT-OV-CSC.
  • an OV-CSC cell line prepared by any of the method of disclosed herein.
  • the OV-CSC are OV-CSC spheroids, early OV-CSC, mixed OV- CSC, or EMT-OV-CSC.
  • method of stimulating an immune response against ovarian carcinoma in a subject in need thereof comprising administration of an immunogenic composition disclosed herein, OV-CSC cells disclosed herein, or an OV-CSC cell line disclosed herein.
  • OV-CSC cells disclosed herein or a OV-CSC cell line disclosed herein in the manufacture of a medicament for the treatment of ovarian carcinoma.
  • OV-CSC cells disclosed herein or a OV-CSC cell line disclosed herein for the treatment of ovarian carcinoma.
  • FIG. 1 is a flow chart of the process of isolation, expansion, and harvest of ovarian carcinoma (OV) cancer stem cells (OV-CSC) from an excised tumor (solid boxes and arrows) or from a small sample such as a needle biopsy or peritoneal lavage (dashed boxes and arrows) into spheroids.
  • OV-CSC ovarian carcinoma cancer stem cells
  • FIG. 2 depicts an established tumor cell line (OVCAR3) that produce irregular cell agglomerates in non-adherent conditions, thus demonstrating a higher degree of differentiation (phase contrast 20x)
  • FIG. 3 depicts patient-derived ovarian cancer stem cells that produce typical spheres in non-adherent conditions, thus demonstrating the presence and expansion of the cancer stem cells.
  • FIG. 4A-D depicts a patient-derived ovarian cancer stem cell culture labeled for cancer stem cell biomarkers EpCAM and NCAM (FIB. 4A).
  • the cells were plated in 5% serum containing media, 10 ng/mL bFGF, and 10 ng/mL EGF.
  • FIG. 4B depicts a red channel image of the cells of FIG. 4A reflecting the NCAM-positive cells.
  • FIG. 4C depicts a green channel image of the cells of FIG. 4A demonstrating loss of EpCAM in a phenomenon associated with an epithelial to mesenchymal transition (EMT) of the cells.
  • EMT epithelial to mesenchymal transition
  • FIG. 4D depicts a blue channel image for bisbenzimide nuclear staining.
  • FIG. 5A-D depicts a patient-derived ovarian stem cancer cell culture labeled for EMT biomarkers Slug/Snail and CD1 17 (FIG. 5A). The cells were plated in 5% serum containing media, 10 ng/mL bFGF, and 10 ng/mL EGF.
  • FIG 5B depicts a red channel image indicating that the majority of the cells of FIG. 5A are positive for Slug/Snail.
  • FIG. 5C depicts a green channel image indicating that the cells of FIG. 5A are positive for CD1 17.
  • FIG. 5D depicts a blue channel image for bisbenzimide nuclear staining.
  • FIG. 6A-C depicts a patient-derived ovarian cancer stem cell culture labeled for cancer stem cell biomarker nestin (FIG. 6A).
  • the cells were plated in 5% serum containing media, 10 ng/mL bFGF, and 10 ng/mL EGF.
  • FIG. 6B depicts a red channel image indicating that the majority of cells are positive for nestin. This phenomenon is associated with EMT.
  • FIG. 6C depicts a blue channel image for bisbenzimide nuclear staining.
  • FIG. 7 depicts a patient-derived ovarian cancer stem cell culture in a media containing 5% FBS on gelatin (phase contrast, 10x)
  • FIG. 8 depicts a patient-derived ovarian cancer stem cell culture in a media containing 5% FBS, 10 ng/mL bFGF, and 10 ng/mL EGF on gelatin (phase contrast, 10x)
  • FIG. 9 depicts a patient-derived ovarian cancer stem cell culture in serum-free media on gelatin (phase contrast, 10x)
  • FIG. 10 depicts a patient-derived ovarian cancer stem cell culture in serum-free media containing 10 ng/mL bFGF, and 10 ng/mL EGF on gelatin (phase contrast, 10x)
  • FIG. 1 1 depicts a small colony of patient-derived ovarian cancer stem cells in serum- free media containing 10 ng/mL bFGF, 10 ng/mL EGF, and 5 ng/mL activin A (phase contrast, 40x). Small cells with large nuclei that represents about 90% of the cell size can be observed in the compact colony suggesting very early cancer stem cells, (embryonic stem cell like, "early" OV- CSC).
  • FIG. 12A-D depicts a patient-derived ovarian cancer stem cell culture labeled for ovarian cancer biomarkers CA125 and MUC-1 (FIG. 12A).
  • the cells were plated in a serum-free media containing 10 ng/mL bFGF and 10 ng/mL EGF.
  • FIG. 12B depicts a red channel image of the cells of FIG. 12A that shows more than 90% of the cells are positive for CA125.
  • FIG. 12C depicts a green channel image of the cells of FIG. 12A indicating that the majority of cells are positive for MUC-1 at various intensities.
  • FIG. 12D depicts a blue channel image for bisbenzimide nuclear staining.
  • FIG 13A-D depicts a patient-derived ovarian cancer stem cell culture labeled for ovarian cancer biomarker CK8 and proliferation marker Ki67 (FIG. 13A).
  • the cells were plated in a serum-free media containing 10 ng/mL bFGF and 10 ng/mL EGF.
  • FIG. 13B depicts a red channel image of the cells of FIG. 13A demonstrating intense proliferation (positive for Ki67 marker).
  • FIG. 13C depicts a green channel image of the cells of FIG 13A that shows the majority of the cells are positive for CK8.
  • FIG. 13D depicts a blue channel image for bisbenzimide nuclear staining.
  • FIG 14A-D depicts a patient-derived ovarian cancer stem cell culture labeled for cancer stem cell biomarkers EpCAM and NCAM.
  • the cells were plated in a serum-free media containing 10 ng/mL bFGF and 10 ng/mL EGF.
  • FIG. 14B depicts a red channel image of the cells of FIG. 14A that shows faint peri-nuclear expression of NCAM in some of the cells.
  • FIG. 14C depicts a green channel image of the cells of FIG. 14A that depicts that the majority of the cells are positive for EpCAM and demonstrating the epithelial nature of the cells.
  • FIG. 14D depicts a blue channel image for bisbenzimide nuclear staining.
  • FIG. 15A-D depicts a patient-derived ovarian cancer stem cell culture labeled for cancer stem cell biomarkers CD44 and nestin (FIG. 15A).
  • the cells were plated in a serum-free media containing 10 ng/mL bFGF and 10 ng/mL EGF.
  • FIG. 15C depicts a green channel image of the cells of FIG. 15A indicating that the majority of the cells are positive for CD44.
  • FIG. 15B depicts a red channel image of the cells of FIG. 15A indicating some positive cells for nestin, mostly peri-nuclear.
  • FIG. 15D depicts a blue channel image for bisbenzimide nuclear staining.
  • FIG. 16A-C depicts a patient-derived ovarian cancer stem cell culture labeled for the cancer stem cell biomarker CD133 (FIG. 16A).
  • the cells were plated in a serum-free media containing 10 ng/mL bFGF and 10 ng/mL EGF.
  • FIG. 16B depicts a green channel image of the cells of FIG. 16A indicating that the majority of the cells are positive for CD133.
  • FIG. 16C depicts a blue channel image for bisbenzimide nuclear staining.
  • the present disclosure provides a cell population obtained from human ovarian carcinoma (OV) tumors that consist mainly of high purity cancer stem cells.
  • the purity of the cell population is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% cancer stem cells.
  • These cancer stem cells are ovarian carcinoma progenitors and have the capacity of continuous self-renewal and differentiation to a certain level.
  • the disclosure also concerns a method to produce a purified population of OV-derived stem cells, for further use as an antigen source for autologous immune therapy of cancer.
  • the present disclosure uses the high purity OV stem cell population for genetic analysis to identify unique changes that drive the formulation of personalized medicines.
  • the present disclosure provides a novel cell line that is modified in vitro, where this modification enhances the immune stimulatory characteristics of the OV.
  • the OV cell line is an improvement over similar technologies using crude tumor preparations, as it provides a superior antigenic signal to noise ratio.
  • the cell line lacks contaminant cell populations, such as fibroblasts, that could alter or diminish the in vitro or in vivo applications.
  • the exemplary cell line of the present disclosure is also used for manufacturing of a drug for treating OV.
  • the term "derived from,” in the context of peptides derived from one or more cancer cells, encompasses any method of obtaining the peptides from a cancer cell or a population of cancer cells.
  • the cancer cell can be broken, for example, by a homogenizer or by osmotic bursting, resulting in a crude extract.
  • Peptides, oligopeptides, and polypeptides of the crude extract can be exposed to dendritic cells, followed by processing of the peptides by the dendritic cells.
  • derived from also encompasses intact cancer cells, where the cancer cells are living, or where the cancer cells have been treated with irradiation but are still metabolically active, or where the cancer cells have been treated with a cross-linking agent and therefore still comprise the peptides.
  • “Derived from” also includes mixtures of cancer cell debris, free cancer cell proteins, and irradiated cancer cells, that therefore are derived from the cancer cells.
  • “Derived from” also includes isolation or amplification of messenger RNA from the cancer cells, or cancer stem cells, for use in transfecting dendritic cells ex vivo to enable antigen presentation.
  • administering refers without limitation to contact of an exogenous ligand, reagent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition to the subject, cell, tissue, organ, or biological fluid, and the like.
  • administering can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods.
  • Administration can refer to in vivo treatment of a human or animal subject.
  • Treatment of a cell encompasses contact of a reagent with the cell, as well as contact of a reagent with a fluid, where the fluid is in contact with the cell.
  • Administration also encompasses in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell.
  • Effective amount encompasses, without limitation, an amount that can ameliorate, reverse, mitigate, prevent, or diagnose at least one symptom or sign of a medical condition or disorder. Unless dictated otherwise, explicitly or by context, an "effective amount” is not limited to a minimal amount sufficient to achieve a desired outcome nor limited to the optimal amount sufficient to achieve the desired outcome.
  • Biomarkers include blood counts, metabolite levels in serum, urine, or cerebrospinal fluid, tumor cell counts, cancer stem cell counts, tumor levels. Tumor levels can be determined by the Response Evaluation Criteria In Solid Tumors (RECIST) criteria (Eisenhauer, et al. (2009) Eur. J. Cancer. 45:228-247). Expression markers encompass genetic expression of mRNA or gene amplification, expression of an antigen, and expression of a polypeptide. Clinical parameters include progression-free survival (PFS), 6- month PFS, disease-free survival (DFS), time to progression (TTP), time to distant metastasis (TDM), and overall survival, without implying any limitation.
  • PFS progression-free survival
  • DFS disease-free survival
  • TTP time to progression
  • TDM time to distant metastasis
  • a composition that is "labeled” is detectable, either directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemical, isotopic, or chemical methods.
  • useful labels include 32 P, 33 P, 35 S, 14 C, 3 H, 125 l, stable isotopes, epitope tags fluorescent dyes, electron-dense reagents, substrates, or enzymes, e.g., as used in enzyme-linked immunoassays, or fluorettes (disclosed in US 6,747, 135 which is incorporated by reference herein for all it discloses regarding fluorettes).
  • a biopsy of OV comprising acquiring a biopsy of OV, dissociating the cells of the biopsy, in vitro culturing the dissociated cells in a defined medium on a substrate, wherein the defined medium is supplemented with at least one growth factor that acts through the mitogen activated protein kinase (MAPK) pathway to yield a population of purified spheroids, or single cell preparations of OV stem cells.
  • MPK mitogen activated protein kinase
  • At least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cancer stem cells in the population of purified OV-CSC express one or more of the biomarkers ATP-binding cassette sub-family G member 2 (ABCG2; GenBank Accession Number AAG52982.1 ), CD133, CD24, CD44, CD46, CD1 17, cytokeratin 18 (CK18), cytokeratin 8 (CK8), alpha fetoprotein (AFP), epithelial cell adhesion molecule (EpCAM; GenBank Accession Number NP_002345.2), Ki-67, Nanog (GenBank Accession Number NM_024865.2, NP_079141 .20), N- cadherin, neural cell adhesion molecule (NCAM; CD56), Oct3/4 (GenBank Accession Number NP_002692.2; NP_976034.4; NP_001 167002.1 ; NP_068812.10), Slug (SNAI2)/Snail (
  • the OV-CSC do not substantially express any of carcinoembyronic antigen (CEA), follicle stimulating hormone (FSH), alpha human chorionic gonadotropin (aHCG), beta human chorionic gonadotropin ( ⁇ -ICG), and desmin.
  • CEA carcinoembyronic antigen
  • FSH follicle stimulating hormone
  • aHCG alpha human chorionic gonadotropin
  • ⁇ -ICG beta human chorionic gonadotropin
  • desmin desmin.
  • the term “substantially” refers to cells, or populations of cells, in which the indicated markers are expressed on less than 20% of the cells. In other embodiments, the biomarkers are expressed on less than 15% of the cells, less that 10% of the cells, or less than 5% of the cells.
  • FIG. 1 A flow chart of the formation of the disclosed cell populations is presented in FIG. 1 .
  • spheroids refers to spherical aggregates of cancer stem cells formed by culture of cancer cells in serum-free medium. The ability to form spheroids is a characteristic of cancer stem cells.
  • At least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cells in the OV-CSC spheroid population express two or more of the biomarkers EpCAM, CA-125, MUC-1 , CD1 17, He-4, ALDH, CD133, CD24, Ki-67, CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, vimentin, CK8, CK18, AFP, testosterone, TGF3R, EGFR, TAG-72, CD46, CD44, ABCG2, Slug/Snail, nestin, and TP53.
  • At least about 80% of the cells in the OV-CSC spheroid population express two or more of the biomarkers AFP, CK7, CK19, EpCAM, E-cadherin, Ov1 , and OV6. In another embodiment, at least about 90% of the cells in the OV-CSC spheroid population express two or more of the biomarkers EpCAM, CA-125, MUC-1 , CD117, He-4, ALDH, CD133, CD24, and Ki-67.
  • the spheroid population can be further expanded into one of three different subpopulations by altering culture conditions such as media composition and substrate.
  • culture conditions such as media composition and substrate.
  • the characteristics of the bulk tumor, spheroid, early, mixed, and EMT OV-CSC populations are presented in Table 1.
  • Table 1 Summary of the conditions used to produce OV cell populations from bulk OV tumors
  • Monolayer with 14 days or EMT-OV-CSC At least two of Adherent Proper spindle or and longer (mesenchymal NCAM, culture, antigenic irregular -like) Slug/Snail, Twist, bFGF, signal shaped cells CD24 Expansion
  • any of the early OV-CSC, mixed OV-CSC, or EMT-OV-CSC populations can be obtained from OV-CSC spheroids, early OV-CSC, mixed OV-CSC, or EMT-OV-CSC by changing the media and conditions as disclosed in Table 1 .
  • the OV-CSC spheroids are further cultured on an adherent substrate in the presence of activin A, FGF, and a serum-free media (selection media) to yield colonies with small cells referred to herein as an "early" population of OV-CSC which have characteristics of embryonic stem cells, and at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cells in the early OV-CSC population express two or more of biomarkers EpCAM, CD 133, CD44, Nanog, Sox2, Oct3/4, CD1 17, and Ki-67.
  • At least about 80% of the cells in the early OV-CSC population express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, Ki-67, CA-125, MUC-1 , TGF3R, and CD24. In another embodiment, at least about 90% of the cells in the early OV-CSC population express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, and Ki-67.
  • the OV-CSC spheroids are further cultured on an adherent substrate under low calcium conditions in a serum-containing (expansion media) to yield colonies mixed with a monolayer wherein the cells have heterogeneous morphologies.
  • the culture medium optionally includes EGF.
  • These cells are referred to herein as a "mixed" population of OV-CSC which have a mixed differentiation profile, and at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cells in the mixed OV-CSC population express two or more of biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18, and Ki-67.
  • At least about 80% of the cells in the mixed OV-CSC population express two or more of biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18, Ki-67, CA19-9, HER2/neu, NCAM, ganglioside CD2, estrogen receptor alpha, testosterone, TGF3R, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin, and TP53.
  • at least about 90% of the cells in the mixed OV-CSC population express two or more of biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18, and Ki-67.
  • the OV-CSC spheroids are further cultured on an adherent substrate in the presence of FGF and a serum-containing media (expansion media) to yield a monolayer of spindle- or irregularly-shaped cells referred to herein as mesenchymal-like OV-CSC or ⁇ -OV-CSC" (epithelial to mesenchymal transitioned [EMT] cancer stem cells).
  • mesenchymal-like OV-CSC or ⁇ -OV-CSC epithelial to mesenchymal transitioned [EMT] cancer stem cells.
  • EMT epithelial to mesenchymal transitioned
  • loss of the expression of a biomarker refers to undetectable expression or expression in 40% (or less) of the cells, expression in 30% (or less) of the cells, expression in 20% (or less) of the cells, or expression in 10% (or less) of the cells.
  • the EMT process is characterized by the increase in the expression of at least one, or all, of the mesenchymal biomarkers Slug/Snail, Twist, CD44, NCAM, N-cadherin, and vimentin to at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells in the population expressing the biomarker(s) of interest.
  • At least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cells in the EMT-OV-CSC population express two or more of the biomarkers NCAM, Slug/Snail, CD24, and Twist.
  • at least about 80% of the cells in the EMT-OV-CSC population express two or more of the biomarkers NCAM, Slug/Snail, CD24, Twist, CA-125, MUC-1 , N-cadherin, CD44, vimentin, CD33, Nanog, and CD1 17.
  • at least about 90% of the cells in the EMT-OV-CSC population express two or more of the biomarkers NCAM, Slug/Snail, CD24, and Twist.
  • the cells express one or more of the indicated biomarkers.
  • the cells express two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of the indicated biomarkers.
  • the cells express 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 of the indicated biomarkers.
  • Biomarkers by a single cell, by a population of cells, or by a population of cells located in a specific structure such as a monolayer or a spheroid can be determined by measuring expression of the polypeptide form of the biomarker or the mRNA form of the biomarker.
  • Polypeptide expression can be measured using a labeled antibody, while nucleic acid expression can be measured by hybridization techniques, are available to the skilled artisan.
  • Biomarkers that are not polypeptides or nucleic acids, such as oligosaccharides or small molecule metabolites, can also be measured by methods available to the skilled artisan.
  • the tumor samples can be fresh or frozen, are dissociated by mechanical and/or enzymatic treatment, or are cultivated directly with minimal mechanical fragmentation.
  • a non-adherent substrate is any biocompatible material with anti-biofouling properties or a coating with anti-biofouling properties (reduces accumulation of cells on a wetted surface) applied to a common culture surface.
  • the coating can be applied using coating agents such as amino-silanes. If there is a non-adherent or anti-biofouling substrate, this substrate can be used for about 0-25 days, such as 0-21 days, 5-20 days, 5-10 days, 10-20 days, or any time period between zero and 25 days..
  • the adherent substrate can be one that is rich in RGD (Arg-Gly-Asp) tripeptide motifs (e.g., collagen, gelatin, MATRIGEL®).
  • An adherent substrate is a surface that is configured to adhere to, and to collect, anchorage dependent cells.
  • the substrate can be an adherent substrate that is configured to adhere to and to collect anchorage dependent cells that are fibroblasts.
  • RGD peptides can also be grafted on polymeric backbones such as polystyrene, hyaluronan, poly-lactic acid, or combinations thereof.
  • the backbone can further carry proteoglycans.
  • the proteoglycans can carry growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), activin A or follistatin.
  • a non-adherent substrate can cause fast and efficient enrichment of the cultures with cancer stem cells.
  • a non-adherent substrate may be used when a large enough sample is provided, for example a surgically excised tumor, so that purification of OV-CSC can begin immediately. If the sample is very small, such as needle aspirate or peritoneal lavage, and nonadherent culture is not feasible, an adherent culture may be used for initial expansion, followed by a purification step on a non-adherent substrate, then followed by another expansion under adherent conditions.
  • FIG. 1 dashexin, adherent substrate, then followed by another expansion under adherent conditions.
  • a first period of culture is provided on an adherent substrate, followed by a second period of culture on a non-adherent substrate. Also provided is a first period of culture on a non-adherent substrate, followed by a second period of culture on an adherent substrate.
  • Periods can be, for example, one half day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, and the like, or any range thereof, such as 2-4 days, or 8-10 days, and so on.
  • the cycle can repeat such as an adherent culture followed by a non-adherent culture followed by an adherent culture, etc.
  • the cycle can repeat such as a non-adherent culture followed by an adherent culture, followed by a non-adherent culture, etc.
  • the defined medium is supplemented with at least one growth factor that acts through the mitogen activated protein kinase (MAPK) pathway.
  • the growth factor is one or both of FGF and EGF, or an analogue thereof.
  • the FGF is basic fibroblast growth factor (bFGF).
  • the defined medium is supplemented with activin A.
  • the defined medium is not supplemented with activin A.
  • a defined medium supplemented with an agonist of activin A in amount effective to prevent spontaneous differentiation of OV stem cells.
  • the defined media is supplemented by one or all of bone morphogenic protein (BMP) 2, 4, or 7.
  • BMP bone morphogenic protein
  • OV-CSC cell line that is unique to each patient obtained from the patient's primary ovarian tumor, that (a) carries stem cell characteristics of self-renewal and pluripotency and the ability to differentiate; and (b) that carries a unique genomic cancerous signature in the majority of the cells, such as more than 50%.
  • nucleic acids, gene products, polypeptides, and peptide fragments where identity can be reasonably established by a trivial name alone. Also encompassed, are nucleic acids, gene products, polypeptides, and peptide fragments, based on a particular GenBank Accession No., where the nucleic acid, polypeptide, and the like, has at least 50% sequence identity, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity sequence identity, to that of the GenBank No. where the biochemical function, or physiological function are shared, at least in part, or alternatively, irrespective of function.
  • an immune response to cancer in a subject is stimulated with one of the compositions disclosed herein.
  • the immune response that is stimulated comprises one or more of CD4 + T cell response, CD8 + T cell response, and B cell response.
  • the CD4 + T cell response, CD + T cell response, or B cell response can be measured by ELISPOT assays, by intracellular cytokine staining (ICS) assays, by tetramer assays, or by detecting antigen-specific antibody production, according to assays that are known by persons of ordinary skill in the art.
  • the immune response can comprise a survival time such as a 2-year overall survival (OS), and where the 2-year overall survival is at least 60%.
  • OS 2-year overall survival
  • An immune response in a patient can also be assessed by endpoints that are used in oncology clinical trials, including objective response (RECIST criteria), overall survival, progression-free survival (PFS), disease-free survival, time to distant metastasis, 6-month PFS, 12-month PFS, and so on.
  • RECIST criteria objective response
  • PFS progression-free survival
  • disease-free survival time to distant metastasis
  • 6-month PFS 6-month PFS
  • 12-month PFS 12-month PFS
  • dendritic cells stimulated ex vivo with the OV-CSC, or antigens derived therefrom, for use in therapy of ovarian carcinoma.
  • immunogenic compositions such as vaccine compositions, comprising dendritic cells loaded with (exposed to) the OV-CSC ex vivo.
  • the dendritic cells and tumor cells are from the same human subject (autologous) although embodiments where the dendritic cells and OV cells are from different subjects are within the scope of the present disclosure.
  • Dendritic cells can be loaded with OV tumor cell antigens comprising whole cells, cell lysates, cell extracts, irradiated cells or any protein derivative of an OV tumor cell, such as a OV- CSC.
  • Dendritic cell immunogenic compositions can be prepared, and administered to a human subject by one or more routes of administration as are known to persons of ordinary skill in the art.
  • the OV-CSC cells are irradiated, or otherwise treated to prevent cell division, prior to loading with the dendritic cells.
  • Alternatives to radiation include nucleic acid cross-linking agents that prevent cell division.
  • a method that uses of the OV-CSC population, as disclosed above, as a source of antigen for autologous immune therapy, for example, where the OV-CSC are inactivated by a radiant energy (e.g., gamma, UV, X), temperature (e.g., heat or cold), or chemical (e.g., cytostatic, aldehyde, alcohol) methods, or combinations thereof.
  • the OV-CSC are used as a source of antigen for ex vivo activation of dendritic cells.
  • the present disclosure provides prepared OV cells (OV-CSC), provides DC loaded with the prepared OV cells, and provides immunogenic compositions (or vaccines) comprising dendritic cells loaded the prepared OV cells.
  • an immunogenic composition of the present disclosure can comprise DC loaded with OV spheroids, loaded with a population of cells that comprises spheroids, loaded with a population of cells that was derived from spheroids and that were expanded on an adherent surface prior to loading on DC, loaded with spheroids that were subjected to homogenization or sonication prior to loading on DC, loaded with a population of expanded cells that were subjected to homogenization or sonication prior to loading on DC, and so on.
  • the DC are loaded with early OV-CSC, mixed OV-CSC, or EMT-OV-CSC.
  • Also disclosed herein is a population of OV-CSC that is capable of stimulating an effective immune response against a cell expressing at least one OV-specific antigen, wherein the OV-CSC population is contacted with at least one dendritic cell, wherein the OV-CSC population is processed in vivo or ex vivo by the dendritic cell, and wherein an effective immune response occurs in the subject in response to administration of the at least one dendritic cell to a subject.
  • An immune stimulatory amount of the disclosed compositions is, without limitation, an amount that increases ELISPOT assay results by a measurable amount, that increases ICS assay results by a measurable amount, that increases tetramer assay results by a measurable amount, that increases the blood population of antigen-specific CD4+ T cells by a measurable amount, that increases the blood population of antigen-specific CD8+ T cells by a measurable amount, or where the increase is by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2.0- fold, 3.0-fold, and the like, when compared to a suitable control.
  • a suitable control can be a control composition, where dendritic cells are not loaded with OV cells, or are not loaded with peptide derived from OV cells.
  • the disclosure also provides pharmaceuticals, reagents, kits including diagnostic kits, that wherein the pharmaceuticals, reagents, and kits, comprise dendritic cells (DC), antibodies, or antigens. Also provided are methods for administering compositions that comprise at least one dendritic cell and at least one antigen, methods for stimulating antibody formation, methods for stimulating antibody-dependent cytotoxicity (ADCC), methods for stimulating complement- dependent cytotoxicity, and methods and kits for determining patient suitability, for determining patient inclusion/exclusion criteria in the context of a clinical trial or ordinary medical treatment, and for predicting response to the pharmaceutical or reagent.
  • DC dendritic cells
  • kits comprise dendritic cells (DC), antibodies, or antigens.
  • methods for administering compositions that comprise at least one dendritic cell and at least one antigen methods for stimulating antibody formation, methods for stimulating antibody-dependent cytotoxicity (ADCC), methods for stimulating complement- dependent cytotoxicity, and methods and kits for determining patient suitability, for determining patient inclusion/exclusion criteria in the context of
  • compositions, reagents, and related methods, of the disclosure encompass CD83 positive dendritic cells, where CD83 is induced by loading with IFN-gamma-treated, or untreated, cancer cells.
  • CD83 is induced by at least 2%, at least 3%, at least 4%, 6%, 7%, 8%, 9%, 10%, and the like.
  • excluded are DC reagents, or DC-related methods, where CD83 on dendritic cells is not detectably induced by loading with IFN-gamma.
  • a kit which includes all of the reagents for generating OV-CSC spheroids, early OV-CSC, mixed OV-CSC, and/or EMT-OV-CSC from tumor samples according to the methods disclosed herein and/or reagents for characterizing the OV-CSC spheroids, early OV-CSC, mixed OV-CSC, and/or EMT-OV-CSC, and instructions for generating and/or characterizing OV-CSC spheroids, early OV-CSC, mixed OV-CSC, and/or EMT-OV-CSC.
  • the kit additionally, or alternatively, includes reagents and instructions for isolating dendritic cells, for loading the dendritic cells with OV-CSC, and/or for administering the DC-OV composition to a subject.
  • the ovarian carcinoma (OV) stem cell population of the present disclosure can originate from fresh or frozen samples of patient tumor.
  • the tumor sample can be a biopsy or a lavage of the peritoneal cavity containing OV cells.
  • OV stem cells are isolated from needle biopsies and from the lavage fluid.
  • the tumor sample may be transported in a generic buffered media with a pH of about 7.4 (+/- 0.6) such as RPMI, DMEM, F12, Williams, or combinations containing a protein source such as animal or human serum in concentrations from 0 to 100% or albumin at concentrations from 0 to 0.5% or macromolecules that ensure a physiological osmotic pressure.
  • a generic buffered media with a pH of about 7.4 (+/- 0.6) such as RPMI, DMEM, F12, Williams, or combinations containing a protein source such as animal or human serum in concentrations from 0 to 100% or albumin at concentrations from 0 to 0.5% or macromolecules that ensure a physiological osmotic pressure.
  • a protein source such as animal or human serum in concentrations from 0 to 100% or albumin at concentrations from 0 to 0.5%
  • macromolecules that ensure a physiological osmotic pressure.
  • natural or artificial macromolecules are, but not limited to, hyaluronan, de
  • An antibiotic such as penicillin, streptomycin, gentramicyn in an optional combination with an antifungal such as amphotericin B, FUNGIZONE® (Life Technologies, Carlsbad, CA), can be used in the media to provide antimicrobial properties and reduce the risk of contamination during transportation.
  • the tumor sample can be kept below a metabolic active state by reducing the media temperature to 2 to 30°C, thus allowing the viability maintenance for a limited time (between 0 to 72 hours) before processing.
  • Packaging e.g., insulated packaging
  • the solid tumor tissue is then processed by mechanical dissociation using a sharp blade or tissue grinder device into small, less than 1 mm (on any dimension) fragments.
  • the solid tissue is optionally further processed by enzymatic dissociation.
  • a variety of enzymes can be used to isolate single cells. Nonspecific proteolytic enzymes such as trypsin and pepsin can be used successfully. Targeting minimal cell membrane damage specific enzymes, including collagenase, dispase, elastase, hyaluronidase, or combinations thereof, may be used in the disclosed methods.
  • Deoxyribonuclease (DNAse) can be used to degrade the free DNA from cell detritus responsible for unwanted stickiness of the cell preparation. After dissociation, the cells in suspension are washed from the excess enzyme and debris by straining through a 50-100 ⁇ mesh and repeated centrifugation in a buffered saline (PBS, HBSS) or cell culture media.
  • PBS buffered saline
  • the single cell suspension described above is transferred in culture conditions that promote isolation, expansion of the stem cells and suppression of the differentiated and/or normal cells. This is accomplished by the congruence of the physical conditions, chemical environment, and manipulations.
  • the cell suspension is exposed to a non-adherent (anti-biofouling) substrate that does not allow cell attachment.
  • Mature cells are commonly anchorage dependent and are rapidly eliminated when a proper adherent substrate is not provided.
  • An anti-biofouling substrate can employ commercial products such as ultralow adherent flasks (Corning, Corning, NY), polymers with natural hydrophobic properties (polyvinyl, polyethylene, polypropylene, fluoro-polymers) or coating with natural carbohydrate polymers such as agar-agar, starch, and the like.
  • the cancer stem cells will aggregate and/or clonally expand in spheroid formations that contain high purity cancer stem cells.
  • FIG. 3 A culture of cancer stem cell aggregates is shown in FIG. 3 having easily identifiable spheroid structures of various sizes. The mature cells will remain isolated and non-adherent. A differential gravitational separation can be used to select the larger spheroids from single cells, by simply allowing a timed vertical sedimentation or a short time low force centrifugation (less than 100xG).
  • the selection method described is designed to accomplish the following: (a) eliminate of anchorage dependent cells that are, in general, mature, normal cells; (b) promote the clonal expansion in small clumps or spheroids of the young, stem cells that are anchorage independent; (c) promote the local autocrine activity as a result of clonal expansion of the stem cells; and (d) eliminate the autocrine source of activin A that is secreted by normal fibroblasts or ovarian cells.
  • integrins cell-surface proteins
  • Homophilic integrins expressed on the cell's surface ensure that cells of the same type "stay together".
  • Spheres are formed directly from enzyme digest which is a single cell suspension at the very beginning of a culture, or can be formed from frozen sample or an existing attached culture at any time.
  • the enzyme digest seeding result in this spherical formations that incorporate the cells with the specific surface properties.
  • Fibroblasts for example, are not incorporated into spheroids and are removed from a culture during gravitational feeding.
  • the media used lacks molecules that promote adhesion in order to prevent the non-specific agglomeration of the cells not having homophilic proprieties and to prevent the adhesion to the culture vessel surfaces.
  • Such cell adhesion molecules CAMs
  • CAMs cell adhesion molecules
  • a media composition which is serum- free is suitable for culture of non-adherent spheroids.
  • supplements to the media may include any hormones, nutrients, mineral, and vitamins that are required for supporting growth and maintenance, or other desired aspects of cell physiology and function.
  • Spheres of cells can be characterized in terms of biomarker expression by way of fixing and staining with labeled antibodies, followed by viewing with confocal microscopy. Biomarkers may also be measured by other immunochemistry methods, e.g., flow cytometry. Spheres can be prepared, for example, from suspensions obtained from fresh tumors, or from cells adapted to grow as adherent cells. The morphology of spheres, for example, large and irregular versus tiny and compact, may be influenced by the choice of medium.
  • a cell population adherent to the anti-biofouling coating can be isolated based on aberrant activation of sonic hedgehog signaling mediated by protein kinase B (AKT) and focal adhesion kinase (FAK) signaling. These phenomena can be enhanced by modifications of the membranes induced by enzymes such as metalloproteases or enzymes used in dissociation (trypsin/collagenase). Such cell population can be associated with rapid proliferative and invasive tumors. Methods for assessing normal or aberrant activation of the sonic hedgehog signaling are available and known to persons of ordinary skill in the art.
  • the defined media that is used to isolate the OV stem cells promotes cell survival and is specifically formulated for selection.
  • the media is rich in carbohydrates and lipids but has minimal amount of protein (0.1 % - 3% albumin or 1 %-5% serum). It contains not more than 1.5 mMol total calcium, does not contain inorganic iron compounds; rather, iron is completely bound to a transporter such as transferrin.
  • the media is provided with an excess of essential and nonessential amino acids and essential lipids (alpha-linolenic and linoleic acids) (Table 4).
  • the media does not contain activin A and may contain an activin A receptor blocker such as follistatin.
  • the media does not contain antioxidants such as superoxide dismutase (SOD) or catalase, but contains thiolic antioxidants such as glutathione.
  • the culture medium contains a low level of calcium, 0.1-1.5 mM, for example 10-150 mg/L in the form of calcium chloride.
  • the culture media consists in a basal formulation such as DMEM, F12, Williams, RPMI, Lebovitz supplemented with proteins (in certain formulations), amino acids, antioxidants, energetic substrate (glucose, galactose, L-glutamine), vitamins (B12), hormones (thyroid hormones, insulin) and growth factors (FGF, EGF) as depicted in Table 2.
  • DMEM basal formulation
  • F12 fetal growth factor
  • Williams RPMI
  • Lebovitz supplemented with proteins (in certain formulations), amino acids, antioxidants, energetic substrate (glucose, galactose, L-glutamine), vitamins (B12), hormones (thyroid hormones, insulin) and growth factors (FGF, EGF) as depicted in Table 2.
  • the protein can be albumin in concentration of 0.1-0.5%, fetal bovine serum (FBS) 0.5%-20%.
  • FBS fetal bovine serum
  • the protein can be substituted with macromolecules such as dextrans, hyaluronan, poly-vinyl alcohol in concentration ranging from 0.1 % to 0.5%.
  • the composition of such media is listed in Table 2, Table 3, and Table 4. The supplements are added into the media and mixed for feeding the cell cultures.
  • the media can be replaced in a three day a week schedule (e.g., Monday - Wednesday - Friday), or more frequently, e.g., every other day or daily, if the expansion is fast.
  • a continuous feed or a micro-batch feed bioreactor can be used in the expansion phase.
  • the media contains growth factors that act through the MAPK pathway such as FGF and EGF.
  • the concentration of these growth factors can vary between 0.1 to 100 ng/mL, commonly around 10 ng/mL. Table 2. Basal media com osition options for cancer stem cells:
  • the lipid mix is made by o/w emulsions using
  • the media is supplemented with FGF at about 0.1 to 100 ng/mL, at about 0.5-50 ng/mL, at about 1-40 ng/mL, at about 2-30 ng/mL, at about 3-20 ng/mL, at about 5- 15 ng/mL, at about 6-14 ng/mL, at about 7-13 ng/mL, at about 8-12 ng/mL, at about 9-1 1 ng/mL, or at about 10 ng/mL.
  • FGF FGF
  • FGF is present in the media at about 5 ng/mL, at about 6 ng/mL, at about 7 ng/mL, at about 8 ng/mL, at about 9 ng/mL, at about 1 1 ng/mL, at about 12 ng/mL, at about 12 ng/mL, at about 14 ng/mL, or at about 15 ng/mL.
  • the media is supplemented with EGF at about 0.1 to 100 ng/mL, at about 0.5-50 ng/mL, at about 1-40 ng/mL, at about 2-30 ng/mL, at about 3-20 ng/mL, at about 5-15 ng/mL, at about 6-14 ng/mL, at about 7-13 ng/mL, at about 8-12 ng/mL, at about 9-1 1 ng/mL, or at about 10 ng/mL.
  • EGF at about 0.1 to 100 ng/mL, at about 0.5-50 ng/mL, at about 1-40 ng/mL, at about 2-30 ng/mL, at about 3-20 ng/mL, at about 5-15 ng/mL, at about 6-14 ng/mL, at about 7-13 ng/mL, at about 8-12 ng/mL, at about 9-1 1 ng/mL, or at about 10 ng/mL.
  • EGF is present in the media at about 5 ng/mL, at about 6 ng/mL, at about 7 ng/mL, at about 8 ng/mL, at about 9 ng/mL, at about 1 1 ng/mL, at about 12 ng/mL, at about 12 ng/mL, at about 14 ng/mL, or at about 15 ng/mL.
  • SOD superoxide dismutase
  • the use of antioxidants can have both positive and negative consequences. Cancer stem cells are far more tolerant than normal cells to free radicals and glycolytic metabolism. Therefore in suboptimal cultures such as high density, infrequent media replacement, high concentration of metabolites in the media, it is most likely that the normal sensitive cells to be eliminated first.
  • antioxidants such as catalase and inhibitors of SOD are added to the culture medium and in other embodiments, these compounds are omitted from the culture media.
  • the activin/follistatin system can be used to isolate very early cancer stem cells.
  • the addition of activin A can select a subpopulation of activin A-resistant OV stem cells.
  • Follistatin is used to block the activin A receptors and prevent spontaneous differentiation of the OV stem cells, especially when large numbers of cells that endogenously secrete activin A are present, such as fibroblasts and normal ovarian cells.
  • the use of follistatin has no effect if the cells are insensitive to activin A or in high purity OV stem cell populations where follistatin can be secreted endogenously.
  • Activin A is a protein that is a member of the transforming growth factor-beta (TGF- beta) superfamily. When added or included in culture medium, activin helps maintain stem cell pluripotency and self-renewal. However, activin A promotes maturation and differentiation of young ovarian cells and cancer cells that are receptive. Therefore, an initial goal is in vitro fast expansion of the tumor that also sustains the proliferation of cancer stem cells by creating a proper autocrine environment in the culture. Although activin A may select a subpopulation of very young cancer stem cells, such conditions applied early in the manufacturing will greatly delay the expansion given the very low concentration of the hepatic cancer stem cells in the bulk. For example, a "fast expansion” is an expansion that results in the media in the culture vessels having obvious signs of consumption (change of pH for example) and the number of cells is visibly higher every day reflected by increased confluence.
  • TGF- beta transforming growth factor-beta
  • activin A is preferably omitted and not added, because it will slow down the culture growth. For some applications the interest is to obtain a very early stem cell population and the use of the activin A will select that cell population. Therefore, in one embodiment, an activin A-containing expansion is initiated and a first composition is administered to a subject comprising the activin A-activated cultured cells, followed by the isolation of the activin A-insensitive cells in an activin-A free culture and administering this second composition comprising the activin A free cultured cells to the subject.
  • the media is supplemented with activin A at about 0.01 to 10 ng/mL, at about 0.05-9 ng/mL, at about 0.1-8 ng/mL, at about 0.5-7 ng/mL, at about 1-6 ng/mL, at about 1-5 ng/mL.
  • activin A is present in the media at about 0.5 ng/mL, at about 0.7 ng/mL, at about 0.9 ng/mL, at about 1 ng/mL, at about 1.25 ng/mL, at about 1.5 ng/mL, at about 1.75 ng/mL, at about 2 ng/mL, at about 2.25 ng/mL, at about 2.5 ng/mL, at about 2.75 ng/mL, at about 3 ng/mL, at about 3.5 ng/mL, at about 4 ng/mL, at about 4.5 ng/mL, at about 5 ng/mL, at about 6 ng/mL, at about 7 ng/mL, at about 8 ng/mL, at about 9 ng/mL, or at about 10 ng/mL.
  • the media is supplemented with an antagonist of activin A, such as, but not limited to, follistatin or an antibody that specifically binds to activin A.
  • an antagonist of activin A such as, but not limited to, follistatin or an antibody that specifically binds to activin A.
  • the media is supplemented with follistatin at about 0.1 to 100 ng/mL, at about 0.5-50 ng/mL, at about 1-40 ng/mL, at about 2-30 ng/mL, at about 3-20 ng/mL, at about 5-15 ng/mL, at about 6-14 ng/mL, at about 7-13 ng/mL, at about 8-12 ng/mL, at about 9-1 1 ng/mL, or at about 10 ng/mL.
  • follistatin at about 0.1 to 100 ng/mL, at about 0.5-50 ng/mL, at about 1-40 ng/mL, at about 2-30 ng/mL, at about 3-20 ng/mL, at about 5-15 ng/mL, at about 6-14 ng/mL, at about 7-13 ng/mL, at about 8-12 ng/mL, at about 9-1 1 ng/mL, or at about 10 ng/mL.
  • follistatin is present in the media at about 5 ng/mL, at about 6 ng/mL, at about 7 ng/mL, at about 8 ng/mL, at about 9 ng/mL, at about 1 1 ng/mL, at about 12 ng/mL, at about 12 ng/mL, at about 14 ng/mL, or at about 15 ng/mL.
  • the combination of mitogens e.g., FGF/EGF
  • activin A e.g., FGF/EGF
  • adherent substrate may result in an increase in the proliferation of normal cells such as fibroblasts or stellate cells.
  • mitogens e.g., FGF/EGF
  • activin A e.g., FGF/EGF
  • adherent substrate may result in an increase in the proliferation of normal cells such as fibroblasts or stellate cells.
  • stroma constituted by cells with nourishing or encapsulating properties (e.g., fibroblasts, stellate cells).
  • the colonies of OV are progressively observed to develop along and spatially displace the stroma in the course of the next few days to weeks of cell culture.
  • the media used in this method is the combination of the formulations described in Tables 2, 3 and 4.
  • FGF and EGF cause proliferation of OV stem cells in any differentiation status including the very early ones.
  • activin A is in the cell culture medium, the activin A is permissive for (allows) proliferation exclusively of the very early OV stem cells that are insensitive to activin A. If the OV stem cells become sensitive, the proliferation will be stopped or reduced by activin A.
  • Insensitivity to FGF and EGF is not common and there are no natural blockers. Insensitivity to activin A can be mediated by follistatin, a natural blocker of the activin receptor. Follistatin can be secreted by the same tumor cell or by cells surrounding the tumor.
  • Activin A is typically secreted by the cells surrounding the tumor, therefore it is possible that the expansion of the tumor is dependent on the surrounding cells (inhibiting) and by the tumor (promoting the expansion).
  • the lack of receptor for activin A a characteristic of the very early, undifferentiated cancer stem cells can prevent the control of the tumor by the surrounding tissue.
  • the in vitro cultures will contain embryonic stem cell-like colonies. These colonies may be surrounded by stromal cells, that can be normal fibroblasts, differentiated tumor cells, or mesenchymal transitioned tumor cells.
  • the present disclosure provides method for preparing OV-CSC where the total culturing time including time required for manipulations such as changing media, replating, centrifugation, and sedimenting, is less than five months, less than four months, less than three months, less than two months, less than one month, less than 150 days, less than 120 days, less than 90 days, less than 60 days, less than 30 days, or less than 150 days (+/-20 days), less than 120 days (+/-20 days), less than 90 days (+/-20 days), less than 60 days (+/-20 days), less than 30 days (+/-20 days).
  • the present disclosure can exclude any method for preparing cancer stem cells, and any population of cancer stem cells prepared by that method, where time required for manipulation is greater than one of the time-frames disclosed above. Also provided is a time in adherent culture that is indicated by one of the above time-frames. Also provided is a time in non-adherent culture that is one of the above time-frames. Moreover, provided is a combined time in adherent culture and in non-adherent culture that is identified by one of the above time-frames.
  • EMT Epithelial to mesenchymal transition
  • Tumors of epithelial origin are known to regress or trans-differentiate into a mesenchymal state.
  • Epithelial phenotypes are immobile, contribute to volume growth of the tumor limited to the originating tissue and are typically more differentiated.
  • EMT occurs, the cells gain mobility and produce adjacent tissue infiltration and distant metastases.
  • the transitioned cell also gains a stem cell-like phenotype, with the ability to replicate and differentiate resulting in a new tumor (metastasis) in the host tissue with characteristics of the originating (primary) tumor.
  • the tumor cells gain additionally immunosuppressive ability, drug pump and radioresistance.
  • the media composition and the physical selection method promote the EMT phenomenon in vitro.
  • the advantage of using an EMT transitioned population as an immunogen is in prevention of tumor recurrences.
  • the antigenicity of EMT cancer cells could enable the immune system to recognize and destroy mobile cancer cells that cause metastasis. In the process of metastasis these cells travel in very low number, seed the host tissue, revert to an epithelial phenotype (MTE transition), grow and form a new tumor that has similar characteristics with the primary tumor.
  • MTE transition epithelial phenotype
  • the conditions necessary to cause in vitro EMT are spheroid formation in serum free media, stimulation with bFGF, stimulation with BMP2, 4, or 7, then plating on adherent substrate containing RGD (Arg-Gly-Asp) peptide motifs (e.g., collagen, gelatin, etc).
  • RGD Arg-Gly-Asp
  • the EMT-OV-CSC subpopulation is obtained by culturing OV spheroids, or early OV or mixed OV, under culture conditions as described in Table 1 and FIG. 1.
  • OV-CSC can generally refer to OV-CSC spheroids, early OV-CSC, mixed OV-CSC, or EMT-OV-CSC.
  • OV stem cell selection is used when the number of sample cells is small.
  • a small number of viable cells obtained from a tumor is less than 10x10 6 viable cells after enzymatic dissociation.
  • a small sample refers to a sample obtain for example from a needle biopsy or peritoneal lavage, in contrast to a sample obtained from an excised tumor, which is typically not considered a small sample and weighs at least 0.5 to 5-10 grams. Core biopsies are done with 18 or 16 or 14 gauge needles, resulting in 5-50 mg samples. A relatively new procedure called a vacuum assisted biopsy is also done with an 1 1 gauge needle, and a vacuum assisted device (VAD).
  • VAD vacuum assisted device
  • An 1 1 gauge probe paired with a vacuum-assisted device typically picks up 94 mg with each core sample.
  • the 14 gauge needle with vacuum assistance typically picks up 37 mg, but only 17 mg when paired with an automated biopsy gun.
  • FIG. 1 dashed arrows and boxes
  • cells obtained from the tumor sample are transferred, before or after dissociation, to an adherent substrate containing RGD (Arg-Gly-Asp) rich compounds (e.g., collagen, gelatin or MATRIGEL®) and in the presence of a selection (serum-free) culture media described herein.
  • the selection method described is designed to (a) promote initial clonal expansion of the individual cancer stem cells that are present in low number, and (d) promote the local autocrine activity as a result of clonal expansion of the stem cells.
  • Adherent substrates are RGD rich proteins such as collagen or gelatin.
  • the substrate can be constructed by attaching the protein or peptide to various materials such as polystyrene polycarbonate, cyclic olefin copolymer or glass.
  • the RGD peptide can be grafted on polymeric backbones such as hyaluronic acid, polylactic acid and combinations.
  • Such polymers can be further enhanced with carrier terminations for growth factors such as proteoglycans (e.g., heparin sulfate, chondroitin sulfate, keratin sulfate, and so on).
  • the cell culture surface can be used directly or using coating agents such as aminosilanes.
  • a coating is a compound that has adherent property (substrate) for the cells and is applied on top of the growth vessel's material. It can be a natural compound such as collagen or gelatin and also can be constructed of a more synthetic polymer having the mentioned radicals/terminations.
  • a coating agent (glue, such as silanes) can be used to improve the adherence of the coating to the culture vessel material (for example to glass). Silanes alone can be used if they contain the desired radicals or terminal groups.
  • tissue samples such as needle biopsies containing 10 3 to 10 6 cells, can be expanded in 3-4 weeks to about 10 8 cells.
  • the OV-CSC can be propagated and expanded indefinitely, as an additional characteristic of stem cells.
  • the OV-CSC can be partial or totally differentiated. If the stem cell expansion conditions are removed, the OV stem cells can slow down or stop the proliferation, and change morphology and phenotype to a more differentiated cell type.
  • the morphology can become flat, epitheloid or stelate having multiple nuclei - a characteristic of the more mature ovarian cells or stelate cells.
  • the adherent cultures can be dissociated in single cell suspension and transferred to non-adherent (anti-biofouling) conditions to remove the anchorage dependent differentiated cells. After 2-3 days, the stem cells tend to aggregate and clonally expand in small spheroids that based on differential sedimentation can be separated from the single cells. The spheroids can be re- plated in adherent conditions and further propagated. This method will purify the culture stem cell content if the cultures are overtaken by differentiated cells or normal cells such as fibroblasts, from 1-30% to 90-99% stem cell content. The method can be repeated as many times needed in order to restore stem cell purity.
  • Small spheroids generally have the dimensions of between 0.1 mm and 2 mm.
  • the size distribution, in terms of number of cells per small spheroid, is generally between 10 cells and 10,000 cells.
  • the shape of a small spheroid can be spherical or oval, and can also occur as conglomerates of spherical or oval structures.
  • a patient-specific OV-CSC cell line can be used to identify the genomic mutation responsible for the neoplastic transformation when compared with normal tissue from the same patient.
  • the genomic mutation may not be expressed in every stage of differentiation. Some regulatory proteins, or transcription factors, are only temporary expressed and may disappear during maturation, resulting in a malformed cell but with normal proteins. Identification of a cell population that is maximally expressing the mutation and exposing this population to the immune system could be a major advantage of using cancer stem cells as an antigen source for immune- therapy [0134]
  • a personalized formulation can be created for a cancer treatment, for example a small molecule, a DNA sequence, antisense RNA or combinations.
  • Such cell lines can be further used to create screening plates (96 wells for example) for drug discovery. Multiple lines from various patients can be combined in a single plate to address variability between individuals.
  • Ovarian carcinoma cancer stem cells may retain some properties of the originating tissue such as secretion of proteins, growth factors and hormones (functional tumors). These properties can be exploited given the immortal characteristics of the cell lines, to produce "self" proteins that can be used for the same patients (for example albumin, transforming growth factor (TGF), insulin, glucagon, DOPA etc).
  • TGF transforming growth factor
  • DOPA DOPA
  • the cells can be introduced in small bioreactors and the secretion product collected, purified and stored for the same patient use. This method is particularly advantageous that the patient will not develop immune resistance such as the more traditional biosimilars.
  • the individual OV-CSC cells obtained from the patient can be used to produce an antigen for immune therapy.
  • the advantage of using the purified stem cell line resides in a better signal to noise ratio.
  • the more mature cells from the tumor may have compensatory mechanisms that can mask the antigenicity and could be not identified by the immune system.
  • the OV-CSC can be used alive, mitotically inactive, nonviable or fragmented.
  • Various methods can be used to modify the cells for optimal antigen exposure: a radiant energy (e.g., gamma, UV, X), temperature (e.g., heat or cold), or chemical (e.g., cytostatic, aldehyde, alcohol) or combinations.
  • the present disclosure encompasses reagents and methods for activating dendritic cells (DCs), with one or more immune adjuvants, such as GM- CSF, a toll-like receptor (TLR) agonist, e.g., CpG-oligonucleotide (TLR9), imiquimod (TLR7), poly(l:C) (TLR3), glucopyranosyl lipid A (TLR4), murein (TLR2), flagellin (TLR5), as well as an adjuvant such as CD40 agonists, e.g., CD40-ligand, or the cytokine, interferon-gamma, prostaglandin E2, and the like.
  • TLR toll-like receptor
  • TLR9 CpG-oligonucleotide
  • imiquimod TLR7
  • TLR3 poly(l:C)
  • TLR4 glucopyranosyl lipid A
  • TLR2 murein
  • flagellin TLR
  • the present disclosure encompasses ex vivo treatment of DCs with one or more of the above adjuvant reagents, or in addition, or alternatively, administration of the adjuvant to a human subject, animal subject, or veterinary subject.
  • the immune system encompasses cellular immunity, humoral immunity, and complement response.
  • Cellular immunity includes a network of cells and events involving dendritic cells, CD8 + T cells (cytotoxic T cells; cytotoxic lymphocytes), and CD4 + T cells (helper T cells).
  • Dendritic cells acquire polypeptide antigens, where these antigens can be acquired from outside of the DC, or biosynthesized inside of the DC by an infecting organism.
  • the DC processes the polypeptide, resulting in peptides of about ten amino acids in length, transfers the peptides to either MHC class I or MHC class II to form a complex, and shuttles the complex to the surface of the DC.
  • TLR dendritic cell's toll-like receptors
  • Humoral immunity refers to B cells and antibodies.
  • B cells become transformed to plasma cells, and the plasma cells express and secrete antibodies.
  • Naive B cells are distinguished in that they do not express the marker CD27, while antigen-specific B cells do express CD27.
  • the secreted antibodies can subsequently bind to tumor antigens residing on the surface of tumor cells. The result is that the infected cells or tumor cells become tagged with the antibody. With binding of the antibody to the infected cell or tumor cell, the bound antibody mediates killing of the infected cell or tumor cell, where killing is by NK cells.
  • NK cells are not configured to recognize specific target antigens, in the way that T cells are configured to recognize target antigens, the ability of NK cells to bind to the constant region of antibodies, enables NK cells to specifically kill the cells that are tagged with antibodies.
  • the NK cell's recognition of the antibodies is mediated by Fc receptor (of the NK cell) binding to the Fc portion of the antibody. This type of killing is called, antibody-dependent cell cytotoxicity (ADCC).
  • ADCC antibody-dependent cell cytotoxicity
  • NK cells can also kill cells independent of the mechanism of ADCC, where this killing requires expression of MHC class I to be lost or deficient in the target cell.
  • the disclosure encompasses administration of cancer stem cell antigens, or administering dendritic cells loaded with cancer stem cell antigens, where the antigens stimulate the production of antibodies that specifically recognize one or more of the cancer stem cell antigens, and where the antibodies mediate ADCC.
  • the phrase, loaded with antigens refers to the ability of the dendritic cell to capture live cells, to capture necrotic cells, to capture dead cells, to capture polypeptides, or to capture peptides, and the like.
  • the tumor antigens comprise cell extracts of the OC-CSC, lysates of the OC-CSC, or intact OC-CSC cells.
  • the tumor antigens comprise messenger RNA transfected into the dendritic cells ex vivo.
  • Capture by cross-presentation is encompassed by the present disclosure. Also encompassed is the use of antigen-presenting cells that are not dendritic cells, such as macrophages or B cells.
  • delayed type hypersensitivity response can be used to distinguish between immune responses that mainly involve cellular immunity or mainly involve humoral immunity.
  • a positive signal from the delayed type hypersensitivity response indicates a cellular response.
  • compositions and methods where tumor cells are inactivated, e.g., by radiation, nucleic acid cross-linkers, polypeptide linkers, or combinations of these.
  • Cross-linking is the attachment of two chains of polymers molecules by bridges, composed of either an element, a group, or a compound that join certain carbon atoms of the chains by primary chemical bonds.
  • Cross-linking occurs in nature in substances made up of polypeptide chains that are joined by the disulfide bonds involving two cysteine residues, as in keratins or insulin, trivalent pyridinoline and pyrrole cross-links of mature collagen, and cross-links in blood clots which involve covalent epsilon-(gamma-glutamyl)lysine cross-links between the gamma- carboxy-amine group of a glutamine residue and the epsilon-amino group of a lysine residue.
  • Cross-linking can be artificially effected in proteins, either adding a chemical substance (cross-linking agent), or by subjecting the polymer to high-energy radiation.
  • Cross- linking with fixatives and heat-induced aggregation has been shown to enhance immune responses as well as completely inhibit proliferation.
  • Substances that may be used to cross-link proteins on the surface, and therefore prevent proliferation, of OV-CSC include, but are not limited to, 10% neutral-buffer formalin, 4% paraformaldehyde, 1 % glutaraldehyde, 0.25-5mM dimethyl suberimidate, ice-cold 100% acetone or 100% methanol. Additionally, combinations of 1 % glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer solution may also be used.
  • Formaldehyde and glutaraldehyde have both been shown to induce the activation of T helper type 1 and type 2 cells.
  • heat induced aggregation of antigens was also shown to enhance the in vivo priming of cytotoxic T lymphocytes.
  • Cross-linking of antigens by 3,3'- dithiobis(sulfosuccinimidylpropionate) results in increased binding of antigens to dendritic cells and the cross-linked antigens are processed through the proteosomal pathway for antigen presentation.
  • formalin fixed ovarian carcinoma tumor cells have been used in clinical trials with no evidence of proliferation.
  • whole OV-CSC are fixed with cross-linking agents, and then used as the antigen source in combination with the dendritic cells.
  • the nucleic acids of the cells are cross-linked.
  • An exemplary nucleic acid alkylator is beta-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester.
  • Exemplary cross-linkers such as psoralens, often in combination with ultraviolet (UVA) irradiation, have the ability to cross-link DNA but to leave proteins unmodified.
  • the nucleic acid targeting compound can be 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen (S-59).
  • Cells can be inactivated with 150 ⁇ psoralen S-59 and 3 J/cm 2 UVA light (FX 1019 irradiation device, Baxter Fenwal, Round Lake, IL).
  • the inactivation with S-59 with UV light is referred to as photochemical treatment, where treatment conditions can be adjusted or titrated to cross-linked DNA to the extent that cell division is completely prevented, but where damage to polypeptides, including polypeptide antigens, is minimized.
  • Cells can be suspended in 5 mL of saline containing 0, 1 , 10, 100, and 1000 nM of psoralen S-59. Samples can be UVA irradiated at a dose of approximately 2 J/cm 2 .
  • Each sample can then transferred to a 15 mL tube, centrifuged, and the supernatant removed, and then washed with 5 mL saline, centrifuged and the supernatant removed and the final pellet suspended in 0.5 mL of saline. See U.S. Pat. 7,833,775 and 7,691 ,393, which are incorporated herein by reference for all they disclose regarding inactivation of cells.
  • the ability to divide can be tested by the skilled artisan by incubating or culturing in a standard medium for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least two months, at least three months, at least four months, and so on.
  • Cell division can be assessed by stains that reveal chromosomes, and that reveal that cell division is, or is not, taking place. Cell division can also be measured by counting cells. Thus, where the number of cells in a culture plate remains stable for a period of two weeks, one month, or two months, and so on, it can reasonably be concluded that the cells cannot divide.
  • the dendritic cell immunogenic composition is administered subcutaneously (SC).
  • SC subcutaneously
  • each dose ranges from about 5-20 million loaded DCs, repeated in a series of 6-10 doses.
  • the doses are administered every five days, every week, every 10 days, every other week, or every third week for two, three, four, five or six doses, followed by administration of doses every two weeks, every three weeks, every four weeks, every month, every five weeks, or every 6 weeks for two, three, four, five or six doses additional doses for a total of 6-10 doses.
  • the first four injections are given every week for a month, and then once a month for the next 4 injections.
  • administration is once a week for 3 weeks then once a month for 5 months for a total of 8 administrations.
  • Each dose comprises about 5-20x10 6 loaded DCs, about 5-17x10 6 loaded DCs, about 6-16x10 6 loaded DCs, about 7-15x10 6 loaded DCs, about 7-14x10 6 loaded DCs, about 8-13x10 6 loaded DCs, about 8-12x10 6 loaded DCs, or about 9-1 1 x10 6 loaded DCs.
  • each dose comprises about 8x10 6 loaded DCs, about 9x10 6 loaded DCs, about 10 x10 6 loaded DCs, about 1 1 x10 6 loaded DCs, or about 12x10 6 loaded DCs.
  • the loaded DCs comprise a mixture of DCs and residual OV-CSCs which have not been taken up by the DCs.
  • the administered dose comprises a mixture of these cells and the dose reflects this mixture.
  • the loaded DCs are administered with a pharmaceutically acceptable carrier or excipients.
  • the pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier or excipient be one which is chemically inert to the loaded DCs and one which has no detrimental side effects or toxicity under the conditions of use.
  • excipient or carrier will be determined in part by the particular therapeutic composition, as well as by the particular method used to administer the composition.
  • the formulations described herein are merely exemplary and are in no way limiting.
  • the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include, but are not limited to, saline, solvents, dispersion media, cell culture media, aqueous buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM
  • an adjuvant is given simultaneously with every dose.
  • the cell dose is suspended in a carrier containing an adjuvant.
  • an adjuvant is administered, but not with every single dose.
  • the adjuvant is GM-CSF.
  • dendritic cells e.g., autologous or allogeneic dendritic cells
  • cancer stem cell antigens as a cell lysate, acid elution, cell extract, partially purified antigens, purified antigens, isolated antigens, partially purified peptides, purified peptides, isolated peptides, synthetic peptides, or any combination thereof.
  • the dendritic cells are then administered to a subject, for example, a subject having OV, or a control subject not having OV.
  • dendritic cells are contacted with, injected into, or administered, by one or more of a route that is subcutaneous, intraperitoneal, intranodal, intramuscular, intravenous, intranasal, inhaled, oral, by application to intestinal lumen, and the like. Additionally, the immunogenic compositions can be administered directly to the site of a tumor or metastasis.
  • Example 1 Generation of cancer stem cell lines from ovarian tumor samples
  • Samples of ovarian tumors from 3 patients were obtained during surgical procedure after proper consenting. The samples were transported to the tissue processing facility in media containing antibiotics and maintained at a temperature of 4-10°C during transportation.
  • the dissociated cells were washed in media, counted and frozen in aliquots for later use in RPMI media containing 15% FBS and 10% DMSO.
  • the typical aliquots contain 10-30x10 6 cells and the expected viability at thaw is 40% to 75%.
  • the aliquots can be utilized for later purification and expansion of the cancer stem cells.
  • the spherogenic property of the cancer stem cells was used to isolate cancer stem cells.
  • Two different media as shown in Table 5 consisting of a literature described composition (Lit- M) and a proprietary formulation (CSC-M) were used to assess the spherogenic properties of the tumor lines.
  • OVCAR-3, SKOV-3 and TOV-21 G commercial cell lines and the enzyme digested tumors from patients were thawed from master cell bank. After a count and viability evaluation, 50,000 viable cells/cm 2 from each line were seeded in appropriately labeled ultra-low adherent flasks (Corning).
  • the cultures were grown for 7 days and media replaced every second day or Monday-Wednesday-Friday schedule.
  • the cell suspension was collected in 50 ml_ tubes, centrifuged at 150 rcf for 3 minutes, and the supernatant replaced with fresh CSC-M or Lit-M media.
  • CSC-E medium is expansion media and is serum containing
  • CSC-M is serum-free selection medium. Both CSC-E and CSC-M are based on the basal media of Tables 2-4.
  • the spheroids were dissociated first with collagenase/hyaluronidase mix for 5-10 minutes and gently pipetting, then the enzyme was removed by centrifugation and replaced with media.
  • the cells were expanded for 2 passages following the M-W-F feeding schedule, re-plating each time at a density of 10,000 cells/cm 2 .
  • the cells were transferred into 96 well plates for immunocytochemistry analysis at a density of 5,000-10,000 cells/well. After 2 days the plates were fixed with 4% paraformaldehyde and stained for the following markers: Nanog, Nestin, Sox2, CD133, CD1 17, NCAM, EpCAM, Slug/Snail, CD24, CA-125, ALDH, CD46, CEA, He-4, Muc-1 , CD44, and HER-2.
  • the commercial cell lines did not form typical cancer stem cell spheroids, in either Lit-M or CSC-M media. Instead they formed loose cell agglomerations with irregular shapes (FIG. 2).
  • the patient tumors formed typical, compact, spherical structures reassembling stem cell spheroids (FIG. 3) that progressively expanded over the 7 days.
  • cultures After transferring the cells in adherent conditions, cultures expanded slowly reassembling a monolayer of epithelial cells. Some of the cells displayed fast growing phenotypes of small cuboidal cells and some shaped as large epithelial cells with vacuolated cytoplasm or small, spindles reassembling mesenchymal cells.
  • the commercial cell lines expanded faster in the CSC-E media as compared to Lit-E condition.
  • the patient lines expanded slower in the identical conditions, distressed when passaged showing cytoplasm vacuoles, detachment from substrate and extensive cell death.
  • the typical in vitro cell culture is a cyclic process in which cells are plated to a lower density, allowed to grow until reaching confluence, then dissociating and seeding again at low density on larger surface (passaging).
  • OV-CSCs are sensitive to physical and enzymatic manipulation during passaging manifested by differentiation and extensive cell death and epithelial to mesenchymal transition (EMT).
  • EMT epithelial to mesenchymal transition
  • ASI active specific immunotherapy
  • the single expansion is avoided to save the culture space in the incubators and stimulate the cell proliferation by periodic enzymatic dissociation and avoidance of contact inhibition.
  • the cells were plated at various low densities of 5000, 10,000, and 15,000 cells/cm 2 and allowed to expand for 4 days. Cultures were fed on a Monday-Wednesday- Friday feeding schedule with CSC-E media (Table 5) that contained only 5% FBS instead of 15% in the initial formulation. At the end, the cells were counted and analyzed for phenotype.
  • a method can be used to expand the cells to the desired number by initially seeding at low density and allowing the cultures to growth until confluence is reached without repeated passaging before the final harvesting. Additionally, the media containing 5% FBS caused EMT, according to the ICC profile (Table 7) just like the higher concentration (15%) of serum in the previous experiments (Table 6).
  • Example 4 Determination of cell culture conditions for selection of early ovarian cancer stem cells
  • FBS-containing media causes differentiation of the cancer stem cells and EMT.
  • This phenomenon may be caused by factors contained in the serum such as bone morphogenic protein (BMP4).
  • BMP4 bone morphogenic protein
  • the cells appear larger, with an epithelial look and slower doubling rate that is typical for the well-differentiated carcinoma. If growth factors are added to this media, EMT is observed.
  • growth factors include FGF, EGF, VEGF, HGF, PDGF, activin A, and TGFbeta.
  • the KGM-GOLDTM basal media is formulated with low calcium for epithelial cell expansion.
  • STEMBLAST ® is a serum free formulation used for the cultivation of embryonic stem cells that contains 5 ng/mL activin A.
  • MATRIGEL ® is a complex extracellular matrix containing laminin, collagen and proteoglycans.
  • KGM-GOLDTM Very slow or not growing, Very slow or not growing, Very slow or not many debris many debris growing, many debris
  • epithelial cells Minimal small cuboidal cells and Few differentiated, debris large nucleus. Few large epithelial cells.
  • MATRIGEL inhibited the growth of tumor cells in all media or growth factor conditions.
  • the tissue culture plastic (plasma treated) displayed less adherence of the cells at initial plating, however did support adequate expansion of the tumor cells in all conditions.
  • Gelatin (0.1 %) coating allowed very good initial adherence and supported fast expansion of the tumor cells.
  • KGM-GOLDTM did not support a fast expansion of the tumor cells, regardless of the substrate or presence of growth factors, caused senescence of the ovarian cancer cells that appeared with large cell bodies and vacuolated cytoplasm.
  • the CSC-E media containing 5% FBS supported fast expansion of the cells only in the presence of bFGF and EGF (FIGs. 7 and 8), while the serum free version supported the fast expansion regardless of the presence of the growth factors (FIGs. 9 and 10). It appears the animal serum contain factors that are inhibiting the fast expansion of the ovarian tumor cells, effect that is compensated by the addition of growth factors that are ligands to receptor tyrosine kinases (FGF, EGF). Other ligands to receptor tyrosine kinases (RTK) such as VEGF, HGF, PDGF may have the same effect on the tumor cell expansion.
  • FGF receptor tyrosine kinases
  • RTK receptor tyrosine kinases
  • STEMBLAST supported good expansion only in the presence of the growth factors. The association of EGF, FGF and activin A in STEMBLAST® caused a very similar morphology to embryonic stem cell cultures with small cell bodies and large nucleus that occupies the majority of the cell and minimal cytoplasm (FIG. 1 1 ).
  • the ovarian cancer epithelial markers marker CA125 was found on more than 90% of the cells (FIG. 12 B).
  • MUC-1 another ovarian cancer epithelial marker was found in about 60% of cells with various intensity of expression (FIG. 12C).
  • Ovarian cancer cytokeratin marker CK8 was present in more than 80% of the cells (FIG. 13B).
  • the culture conditions promoted the expansion of cancer stem cells, demonstrated by the presence in high percentage (better than 75%) of cells expressing EpCAM (FIG. 14C), CD44 (FIG. 15C), and CD133 (FIG. 16).
  • the cultures show a rapid expansion by the high level expression of the Ki67 proliferative marker (FIG. 13B) on the majority of the cells.
  • a serum free media with minimal amount of or ligands for RTK or low concentration animal serum in media supplemented with ligands for RTK can maintain and expand optimally a population of ovarian cancer stem cells.
  • ligands for RTK such as FGF, EGF
  • Increasing the amount of serum and decreasing the RTK ligands will cause differentiation of the CSC.
  • Increasing the serum concentration and increasing the RTK ligand concentration will cause EMT.
  • a serum free media containing RTK ligand and activin A can sustain a very early stem cell status, embryonic stem cell-like.
  • the antigen source is autologous tumor cells from continuously proliferating, self- renewing cells derived from the patient's fresh tumor tissue. These cells have the characteristics of tumor stem cells. At all times in the surgical and pathology setting, biopsies are handled with strict adherence to sterility protocols to ensure that samples are sterile.
  • the pathologist obtains fresh tissue from biopsy of the patient's tumor. Using sterile scalpels and forceps, the specimen is cut into 10 mm slices and transferred to the transport tubes containing transport media, working quickly to avoid specimen drying. Specimens are shipped by overnight courier to the manufacturing facility within 48 hours of surgical resection.
  • samples are dissociated into single cell suspensions in a clean room and placed in cell culture conditions designed to enrich for and proliferate the OV- CSC.
  • normal cells such as lymphocytes, stromal cells and connective tissue are eliminated.
  • the enriched proliferating OV-CSC tumor cells, TC
  • TC vapor phase liquid nitrogen storage
  • PBMCs peripheral blood mononuclear cells
  • Elutriation is a process by which monocytes are purified from other lymphocytes in order to enrich for cells that can be turned into antigen presenting cells or dendritic cells.
  • the elutriated monocytes are incubated with the cytokines GM-CSF and interleukin-4 (IL-4) for six days.
  • the purified tumor cell product is removed from cryostorage, thawed and combined with the dendritic cells for 18-24 hours. This process results in "antigen loading" of the DC.
  • the final product is either entirely DC or may contain some residual irradiated TC (which is considered permissible), and is referred to as DC-TC.
  • the combined dendritic cell/tumor cell mixture is collected, cryopreserved to retain viability of the dendritic cells and stored in vapor phase liquid nitrogen.
  • the batch Upon completion of the quality controls assays and release of the autologous cell therapy product, the batch is shipped to the treatment facility under vapor phase liquid nitrogen conditions. After arrival, the cell therapy product is stored under vapor phase liquid nitrogen conditions until prepared for administration.
  • This study is a Phase II, double-blind, randomized, single center trial of ovapuldencel- T (autologous dendritic cells loaded with irradiated autologous OV-CSC in GM-CSF) vs. autologous peripheral blood mononuclear cells In GM-CSF (MC) as a component of maintenance or secondary therapy in patients with stage III or IV epithelial ovarian, fallopian tube or primary peritoneal carcinoma after debulking surgery and adjuvant chemotherapy.
  • the purpose of the study is to compare overall survival (OS) from the date of randomization for patients treated with ovapuldencel-T to patients treated with autologous blood mononuclear cells in GM-CSF control.
  • Ovapuldencel-T is prepared according to the methods of Example 5.
  • Performance status must be an ECOG score of 0 or 1 at the time of enrollment for ASI treatment.
  • Inclusion criteria include histologic diagnosis of epithelial ovarian, fallopian tube or primary peritoneal carcinoma; advanced (metastatic, stage III or IV) epithelial ovarian, fallopian tube or primary peritoneal carcinoma and a candidate for surgical debulking to obtain fresh viable tumor tissue for efforts to establish a short-term tumor cell line; age > 18 years; each patient must be aware of the neoplastic nature of her disease process and must willingly consent to the manipulation of tumor tissue for efforts to establish a tumor cell line; and patients must have the ability and willingness to travel to the treatment center for administration of ASI treatment.
  • Exclusion criteria includes ECOG performance status greater than 2; known positive for hepatitis B or C or HIV; pregnant or lactating women; underlying cardiac disease associated with myocardial dysfunction that requires active medical treatment, or unstable angina related to atherosclerotic cardiovascular disease, or under treatment for arterial or venous peripheral vascular disease; diagnosis of any other invasive cancer which is considered to be life-threatening within the next five years, and/or taking anti-cancer therapy for cancer other than ovarian (such as continuation of hormonal therapy for prostate or breast cancer diagnosed more than five years earlier); active infection or other active medical condition that could be eminently life-threatening, including active blood clotting or bleeding diathesis; active central nervous system metastases at the time of treatment; known autoimmune disease, immunodeficiency, or disease process that involves the use of immunosuppressive therapy; a low malignancy potential tumor; or received another investigational drug within 28 days of the first dose.
  • Patients will be stratified into: (1 ) platinum-resistant, based on progression during adjuvant chemotherapy or detectable disease at the conclusion of adjuvant therapy; or (2) platinum-sensitive, with no evidence of disease (NED) at the conclusion of adjuvant therapy per elevated blood CA-125 and/or other tumor markers and/or detection of disease by physical examination or imaging.
  • NED no evidence of disease
  • Ovapuldencel-T will be administered in 500 micrograms of GM-CSF once a week for three weeks and then every three to four weeks at the time subsequent maintenance or secondary therapy is given, for up to a total of 8 doses of vaccine therapy given over four to six months, depending on the timing of the maintenance or secondary therapy.
  • the primary study objective is to compare overall survival (OS) from the date of randomization for patients treated with ovapuldencel-T to patients treated with autologous blood mononuclear cells in GM-CSF control.
  • the primary endpoint of this trial is death from any cause with the metric of overall survival (OS) from the date of randomization.
  • Progression-free survival (PFS) is a secondary endpoint and is calculated as the time from the date of randomization for treatment until subjective tumor progression or death. Progression is subjectively defined by the treating physician, and is based on tumor marker levels (e.g. CA-125) and/or imaging. Secondarily, PFS and OS are defined from the date of debulking surgery.
  • All patients who qualify for participation in the study will be followed for up to 5 years from date of randomization or until death, whichever occurs first.
  • Kaplan-Meier curves will display survival times for each treatment group.
  • the log rank test will be used to analyze OS to test the null hypothesis of no treatment difference.
  • the Cox regression model and the Wald test will be used to estimate the hazard ratio associated with treatment and to identify the significance of potential prognostic factors and their impact, if any on the treatment differences.
  • Analyses based on the intention to treat population will be considered as the primary analysis, analyses based on the Per-Protocol population will be considered as a sensitivity analysis.
  • Subgroup analyses of the OS endpoint will mimic the plan laid out for the primary endpoint for platinum-resistant patients and platinum-sensitive patients separately.

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Abstract

La présente invention concerne des cellules souches cancéreuses destinées à être utilisées pour stimuler la réponse immunitaire contre un cancer, tel qu'un carcinome ovarien. L'invention porte également sur des procédés de préparation et de purification des cellules souches cancéreuses.
PCT/US2014/022494 2012-08-15 2014-03-10 Cellules souches cancéreuses ovariennes de haute pureté pour immunothérapie autologue active Ceased WO2014164464A1 (fr)

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EP14778635.4A EP2968531A4 (fr) 2013-03-12 2014-03-10 Cellules souches cancéreuses ovariennes de haute pureté pour immunothérapie autologue active
CA2905288A CA2905288A1 (fr) 2013-03-12 2014-03-10 Cellules souches cancereuses ovariennes de haute purete pour immunotherapie autologue active
JP2016500982A JP2016512423A (ja) 2013-03-12 2014-03-10 能動的自家免疫療法のための高純度卵巣癌幹細胞
KR1020157028644A KR20150139529A (ko) 2013-03-12 2014-03-10 활성 자가조직 면역 요법을 위한 고순도 난소암 줄기세포
US14/775,313 US20160058855A1 (en) 2012-08-15 2014-03-10 High purity ovarian cancer stem cells for active autologous immune therapy
HK16108669.5A HK1220616A1 (zh) 2013-03-12 2014-03-10 用於活性自体免疫疗法的高纯度卵巢癌干细胞
CN201480027224.XA CN105339000A (zh) 2013-03-12 2014-03-10 用于活性自体免疫疗法的高纯度卵巢癌干细胞
AU2014249346A AU2014249346A1 (en) 2013-03-12 2014-03-10 High purity ovarian cancer stem cells for active autologous immune therapy

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PCT/US2013/053850 WO2014028274A1 (fr) 2012-08-15 2013-08-06 Procédé de production rapide de cellules souches cancéreuses de pureté élevée et d'une population de cellules souches cancéreuses de pureté élevée

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WO2016081554A1 (fr) * 2014-11-18 2016-05-26 Neostem Oncology, Llc Compositions immunogènes préparées à partir de cellules tumorales dérivées du sang périphérique et provenant d'une tumeur solide et leur utilisation
WO2016112172A1 (fr) * 2015-01-07 2016-07-14 Dana-Farber Cancer Institute, Inc. Culture cellulaire microfluidique de sphéroïdes de cellules tumorales dérivées de patient
WO2017207737A1 (fr) * 2016-06-02 2017-12-07 Stemtek Therapeutics Sl Procédé de production de sphéroïdes de cellules souches cancéreuses
US11098369B2 (en) 2017-03-31 2021-08-24 Dana-Farber Cancer Institute, Inc. Methods for evaluating tumor cell spheroids using 3D microfluidic cell culture device
US11464838B2 (en) * 2015-04-01 2022-10-11 Colorado State University Research Foundation Optimized cancer stem cell vaccines

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CN110623982B (zh) * 2019-09-23 2023-04-25 四川大学华西医院 卵巢表面上皮细胞的3d-emt免疫活性制剂及制备与应用
CN112618712A (zh) * 2021-01-21 2021-04-09 武汉轻工大学 一种含波形蛋白的佐剂及应用

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016081554A1 (fr) * 2014-11-18 2016-05-26 Neostem Oncology, Llc Compositions immunogènes préparées à partir de cellules tumorales dérivées du sang périphérique et provenant d'une tumeur solide et leur utilisation
WO2016112172A1 (fr) * 2015-01-07 2016-07-14 Dana-Farber Cancer Institute, Inc. Culture cellulaire microfluidique de sphéroïdes de cellules tumorales dérivées de patient
US10472599B2 (en) 2015-01-07 2019-11-12 Dana-Farber Cancer Institute, Inc. Microfluidic cell culture of patient-derived tumor cell spheroids
US11464838B2 (en) * 2015-04-01 2022-10-11 Colorado State University Research Foundation Optimized cancer stem cell vaccines
WO2017207737A1 (fr) * 2016-06-02 2017-12-07 Stemtek Therapeutics Sl Procédé de production de sphéroïdes de cellules souches cancéreuses
US11098369B2 (en) 2017-03-31 2021-08-24 Dana-Farber Cancer Institute, Inc. Methods for evaluating tumor cell spheroids using 3D microfluidic cell culture device
US11572590B2 (en) 2017-03-31 2023-02-07 Dana-Farber Cancer Institute, Inc. Methods for evaluating tumor cell spheroids using 3D microfluidic cell culture device
US12116636B2 (en) 2017-03-31 2024-10-15 Dana-Farber Cancer Institute, Inc. Methods for evaluating tumor cell spheroids using 3D microfluidic cell culture device

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AU2014249346A8 (en) 2015-10-15
JP2016512423A (ja) 2016-04-28
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CA2905288A1 (fr) 2014-10-09
AU2014249346A1 (en) 2015-10-01

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