EP2968407A1 - Cellules souches de carcinome du côlon individualisées, de pureté élevée, procédés et utilisation associés - Google Patents

Cellules souches de carcinome du côlon individualisées, de pureté élevée, procédés et utilisation associés

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Publication number
EP2968407A1
EP2968407A1 EP14779579.3A EP14779579A EP2968407A1 EP 2968407 A1 EP2968407 A1 EP 2968407A1 EP 14779579 A EP14779579 A EP 14779579A EP 2968407 A1 EP2968407 A1 EP 2968407A1
Authority
EP
European Patent Office
Prior art keywords
csc
cells
population
biomarkers
immunogenic composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14779579.3A
Other languages
German (de)
English (en)
Other versions
EP2968407A4 (fr
Inventor
Candace Y. HSIEH
Andrew N. CORNFORTH
Gabriel Nistor
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NeoStem Oncology LLC
Original Assignee
NeoStem Oncology LLC
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Filing date
Publication date
Priority claimed from PCT/US2013/053850 external-priority patent/WO2014028274A1/fr
Application filed by NeoStem Oncology LLC filed Critical NeoStem Oncology LLC
Publication of EP2968407A1 publication Critical patent/EP2968407A1/fr
Publication of EP2968407A4 publication Critical patent/EP2968407A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001116—Receptors for cytokines
    • A61K39/001118—Receptors for colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001122—Ephrin Receptors [Eph]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001128—CD44 not IgG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001166—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0695—Stem cells; Progenitor cells; Precursor cells
    • 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
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/50—Colon

Definitions

  • the present disclosure relates to reagents and methods for stimulating immune response against antigens of colon cancer, and against colon cancer tumors.
  • the 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.
  • the regenerative capacity of any tissue resides in the existence of a subpopulation of progenitor cells that under certain signals can restart the growth cycle and produce cells that are identical to the originating tissue.
  • a systemic determination such as hormonal control can strongly modulate the tissue specific stem cell niche.
  • Colorectal cancer is the third most common cancer diagnosed in North America and the major of patients will undergo surgical resection for treatment. The 5-year overall-survival rates of patients that undergo surgery for colon carcinoma averages around 40 percent.
  • An immunotherapy approach that using the immune system to target those cells responsible for new disease, namely tumorigenic cancer stem cells, could have profound and lasting impact on patient recurrence and survival.
  • the present disclosure provides a cell population obtained from human colon carcinoma tumors that consist of high purity cancer stem cells. These cancer stem cells are specific for the colon progenitors and have the capacity of continuous self-renewal and differentiation to a certain level.
  • the present disclosure also provides a method to produce a purified population of colon carcinoma-derived stem cells that are further used as an antigen source for autologous immune therapy for cancer, as well as the use of the high purity colon carcinoma stem cell population for genetic analysis to identify unique changes that determine the formulation of personalized medicines.
  • the high purity colon carcinoma stem cell population can be used for in vitro assays for chemical compounds, drugs, or biologic agents screening.
  • an immunogenic composition comprising dendritic cells activated ex vivo by tumor antigens derived from the population of purified colon carcinoma (CC) cancer stem cells (CSC).
  • the tumor antigens comprise cell extracts of the CC-CSC, lysates of the CC-CSC, or intact CC-CSC.
  • the intact CC-CSC are rendered non-proliferative.
  • the intact CC-CSC are rendered non-proliferative by irradiation.
  • the intact CC-CSC are rendered non-proliferative by exposure of the cells to a protein or nuclear 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.
  • the immunogenic composition comprises activated dendritic cells and CC-CSC, wherein the CC-CSC are in the form of CC-CSC spheroids, early CC-CSC, mixed CC-CSC, or EMT-CC-CSC.
  • a method of treating colon 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 in the manufacture of a medicament for the treatment of colon carcinoma.
  • a method for preparing a population of colon carcinoma (CC) cancer stem cells comprising: acquiring a sample of CC; dissociating the cells of the sample, and in vitro culturing the dissociated cells in a defined medium on a non-adherent 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 CC-CSC spheroids; wherein at least 80% of the cells in the CC-CSC spheroid population express two or more of the biomarkers CD133, Hes1 , CD44, CD24, CD166, and CD29.
  • MPK mitogen activated protein kinase
  • At least 80% of the cells in the CC-CSC spheroid population further express one or more of the biomarkers CK7, CK19, E-cadherin, CD20, ESA, ALDH, CDX1 , LGR5, and DCIkl .
  • at least 90% of the cells in the CC-CSC spheroid population express two or more of the biomarkers CD133, Hes1 , CD44, CD24, CD166, and CD29.
  • the method further comprises culturing the CC-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 CC-CSC, wherein at least 80% of the cells in the early CC- CSC population express two or more of the biomarkers Nanog, Sox2, Oct3/4, c-kit, FoxA2, and CD133.
  • At least 80% of the cells in the early CC-CSC population further express one or more of the biomarkers EpCAM, E-cadherin, Sox7, Sox17, CD9, KRAS, ESA, BMI1 , CD166, CD24, CD29, CD44, CD166, and CDCP1.
  • at least 90% of the cells in the early CC-CSC population express two or more of the biomarkers Nanog, Sox2, Oct3/4, c-kit, FoxA2, and CD133.
  • the method further comprises culturing the CC-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 mixed CC-CSC, wherein at least 80% of the cells in the mixed CC- CSC population express two or more of the biomarkers Hes1 , MSI1 , ALDH1 B1 , ALDH1A1 , EpCAM, G-CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki-67, AFP, and DCIkl .
  • At least 90% of the cells in the mixed CC-CSC population express two or more of the biomarkers Hes1 , MSI1 , ALDH1 B1 , ALDH1A1 , EpCAM, G-CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki-67, AFP, and DCIkl .
  • the method further comprises culturing the CC-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)-CC-CSC, wherein at least 80% of the cells in the EMT-CC-CSC population express two or more of the biomarkers N- cadherin, Slug/Snail, vimentin, Twist, and CD1 17.
  • EMT epithelial to mesenchymal transitioned
  • At least 80% of the cells in the EMT-CC-CSC population further express one or more of the biomarkers CD44, CD24, Y-synuclein, FMNL2, b-catenin, Nanog, CD147, 33GhT8, LGR5, CD29, CXCR4, CD133, and DCIk1.
  • at least 90% of the cells in the EMT-CC-CSC population express one or more of the biomarkers N-cadherin, Slug/Snail, vimentin, Twist, and CD1 17
  • the method further comprises culturing CC-CSC spheroids, mixed CC-CSC, or EMT-CC-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 CC-CSC, wherein at least 80% of the cells in the early CC-CSC population express two or more of the biomarkers Nanog, Sox2, Oct3/4, c-kit, FoxA2, and CD133.
  • At least 80% of the cells in the early CC-CSC population further express one or more of the biomarkers EpCAM, E-cadherin, Sox7, Sox17, CD9, KRAS, ESA, BMI1 , CD166, CD24, CD29, CD44, CD166, and CDCP1.
  • at least 90% of the cells in the early CC-CSC population express one or more of the biomarkers Nanog, Sox2, Oct3/4, c-kit, FoxA2, and CD133.
  • the method further comprises culturing CC-CSC spheroids, early CC-CSC, or EMT-CC-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 CC-CSC, wherein at least 80% of the cells in the mixed CC-CSC population express two or more of the biomarkers Hes1 , MSI1 , ALDH1 B1 , ALDH1A1 , EpCAM, G-CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki-67, AFP, and DCIkl .
  • At least 90% of the cells in the mixed CC-CSC population express two or more of the biomarkers Hes1 , MSI1 , ALDH1 B1 , ALDH1A1 , EpCAM, G- CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki-67, AFP, and DCIkl .
  • the method further comprises culturing CC-CSC spheroids, early CC-CSC, or mixed CC-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 EMT-CC-CSC, wherein at least 80% of the cells in the EMT-CC-CSC population express two or more of the biomarkers N-cadherin, Slug/Snail, vimentin, Twist, and CD1 17.
  • At least 80% of the cells in the EMT-CC-CSC population further express one or more of the biomarkers CD44, CD24, ⁇ -synuclein, FMNL2, b-catenin, Nanog, CD147, 33GhT8, LGR5, CD29, CXCR4, CD133, and DCIkl .
  • at least 90% of the cells in the EMT-CC-CSC population express one or more of the biomarkers N-cadherin, Slug/Snail, vimentin, Twist, and CD1 17.
  • the defined media is any media described in Table 2, a combination of Table 2 and Table 3, a combination of Table 2, Table 3, and Table 4, or 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 CC-CSC.
  • the medium further 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 such as superoxide dismutase, catalase, glutathione, putrescine, or ⁇ -mercaptoethanol. In another embodiment, the medium is supplemented with glutathione.
  • an antioxidant such as superoxide dismutase, catalase, glutathione, putrescine, or ⁇ -mercaptoethanol.
  • the medium is supplemented with glutathione.
  • the adherent substrate is configured to adhere to, and 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.
  • CC-CSC prepared by a method disclosed herein, wherein the purified CC-CSC are CC-CSC spheroids, early CC-CSC, mixed CC- CSC, or EMT-CC-CSC.
  • CC-CSC cell line prepared by a method disclosed herein, wherein the CC-CSC are CC-CSC spheroids, early CC-CSC, mixed CC-CSC, or EMT-CC-CSC.
  • CC-CSC cells disclosed herein or a CC-CSC cell line disclosed herein in the manufacture of a medicament for the treatment of colon carcinoma.
  • CC-CSC cells disclosed herein or a CC-CSC cell line disclosed herein for the treatment of colon carcinoma.
  • FIG. 1 is a flow chart of the process of isolation, expansion, and harvest of the colon carcinoma (CC) stem cells (CC-CSC) from an excised tumor (solid boxes and arrows) or from a small sample such as a needle biopsy (dashed boxes and arrows) into spheroids. After generation of spheroids, the pathway of producing CC-CSC subpopulations is a common pathway.
  • CC colon carcinoma
  • FIG. 2 depicts spheroid formation in ultralow adherent cell culture flasks from dissociated bulk colorectal tumor (phase contrast x10).
  • FIG. 3 depicts CC-CSC colony formation after the dissociation of spheroids and plating on regular tissue culture plastic (phase contrast 10X).
  • FIG. 3A-D depicts a patient-derived CC-CSC culture staining positive for alpha fetoprotein (AFP) and the epithelial marker CK19, with a nuclear counterstain (bisbenzimide) (epifluorescence, 20x).
  • FIG. 3B depicts a red channel image of the cells of FIG. 3A for AFP staining.
  • FIG. 3C depicts a green channel image of the cells of FIG. 3A for CK19 staining.
  • FIG. 3D depicts a blue channel image for bisbenzimide.
  • FIG. 4A-D depicts a patient-derived CC-CSC culture labeled positive for ABCG2 and the epithelial marker CK7. The cells are also stained with a nuclear counterstain, bisbenzimide (epifluorescence, 20x).
  • FIG. 4B depicts a red channel image of the cells of FIG. 4A for ABCG2 staining.
  • FIG. 4C depicts a green channel image of the cells of FIG. 4A for CK7.
  • FIG. 4D depicts a blue channel image for bisbenzimide.
  • FIG. 5A-C depicts a patient-derived CC-CSC culture with a high percentage of FoxA2 staining, a marker for a pioneer transcription factor that occurs early in gastrulation, thus demonstrating the early cancer stem cell phenotype.
  • FIG. 5B depicts a red channel image of the cells of FIG. 6A for FoxA2 staining.
  • FIG. 6C depicts a blue channel image for bisbenzimide.
  • FIG. 6A depicts a patient-derived CC-CSC culture with a high percentage of neural cell adhesion molecule (NCAM) staining, confirming the early cancer progenitor phenotype selection in the established cultures (includes nuclear counterstain, bisbenzimide) (epifluorescence, 40x).
  • FIG. 6B depicts a red channel image of the cells of FIG. 6A for NCAM staining.
  • FIG. 6C depicts a blue channel image for bisbenzimide.
  • FIG. 7A-D depicts a patient-derived CC-CSC culture labeled positive for CD44, a marker of invasiveness, specific to cancer stem cells with high metastatic potential and the Sox2 marker specific for early cancer stem cells.
  • FIG. 7B depicts a red channel image of the cells of FIG. 7A for Sox2 staining.
  • FIG. 7C depicts a green channel image of the cells of FIG. 7A for CD44 staining.
  • FIG. 7D depicts a blue channel image for bisbenzimide.
  • FIG. 8A-D depicts a patient-derived CC-CSC culture labeled for HLA1 and proliferation marker Ki67.
  • FIG. 8B depicts a red channel image of the cells of FIG. 8A demonstrating intense proliferation (positive for Ki67 marker).
  • FIG. 8C depicts a green channel image of the cells of FIG 8A that shows the up-regulation of HLA1 , a marker that is associated with poor immunogenicity and high invasiveness.
  • FIG. 8D depicts a blue channel image for bisbenzimide nuclear staining.
  • FIG. 9A-D depicts a patient-derived CC-CSC culture labeled for HLA1 and proliferation marker Ki67.
  • FIG. 8B depicts a red channel image of the cells of FIG. 8A demonstrating intense proliferation (positive for Ki67 marker).
  • FIG. 8C depicts a green channel image of the cells of FIG 8A that shows the up-regulation of HLA1 , a marker that is associated with poor immunogenicity and high invasiveness.
  • FIG. 9A depicts epithelial to mesenchymal transition markers, vimentin and Slug/Snail, in a patient-derived CC-CSC culture with a bisbenzimide nuclear counterstain.
  • FIG. 9B depicts a red channel image of the cells of FIG. 9A for Slug/Snail staining.
  • FIG. 9C depicts a green channel image of the cells of FIG. 9A for vimentin.
  • FIG. 9D depicts a blue channel image for bisbenzimide.
  • the present disclosure provides a cell population obtained from human colon carcinoma (CC) 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 colon 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 CC-derived stem cells, for further use as an antigen source for autologous immune therapy of cancer.
  • colon carcinoma or "colon cancer” include colorectal cancer and colorectal carcinoma.
  • the present disclosure uses the high purity CC 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 CC.
  • the CC 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 applications.
  • the exemplary cell line of the present disclosure is also used for manufacturing of a drug for treating CC.
  • 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 nucleic acid cross-linking agent but are still metabolically active 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.
  • 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 to the cell, as well as contact of a reagent to 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 CC comprising acquiring a biopsy of CC, 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 CC-CSC spheroids, or single cell preparations of CC-CSC.
  • 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 CC-CSC express one or more of the biomarkers ATP-binding cassette sub-family G member 2 (ABCG2; GenBank Accession Number AAG52982.1 ), alpha-fetoprotein (AFP), CD133, CD44, CD24, CD166, CD29, CD49f, CD9, CD1 17, CD147, cytokeratin 19 (CK19), cytokeratin 7 (CK7), cytokeratin 20 (CK20), epithelial specific antigen (ESA), aldehyde dehydrogenase (ALDH), homeobox protein CDX1 , leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), serine/threonine-protein kinase DCLK1 , aldehyde dehydrogenase 1 family, member A1 (ALDH1A1 ), aldehyde dehydrogenase 1 family
  • 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 CC-CSC spheroid population express two or more of the biomarkers CD133, Hes1 , CD44, CD24, CD166, and CD29. In other embodiments, at least 80% of the cells in the CC-CSC spheroid population express two or more of the biomarkers CD133, Hes1 , CD44, CD24, CD166, CD29, CK7, CK19, E-cadherin, CK20, ESA, ALDH, CDX1 , LGR5, and DCIkl . In another embodiment, at least 90% of the cells in the CC-CSC spheroid population express two or more of the biomarkers CD133, Hes1 , CD44, CD24, CD166, and CD29.
  • 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 populations are presented in Table 1.
  • CC Colon Cancer
  • CSC cancer stem cell
  • EMT epithelial to mesenchymal transition
  • any of the early CC-CSC, mixed CC-CSC, or EMT-CC-CSC populations can be obtained from CC-CSC spheroids, early CC-CSC, mixed CC-CSC, or EMT-CC-CSC by changing the media and conditions as disclosed in Table 1 .
  • the CC 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 CC-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 CC-CSC population express two or more of biomarkers Nanog, Sox 2, Oct3/4, c-kit, FoxA2, and CD133.
  • At least 80% of the cells in the early CC-CSC population express two or more of biomarkers Nanog, Sox 2, Oct3/4, c-kit, FoxA2, CD133, EpCAM, E-cadherin, Sox7, Sox17, ALDH1A1 , LGR5, Hes1 , DPPA2, CD9, KRAS, ESA, BMI 1 , CD166, CD24, CD29, CD44, CD166, and CDCP1 .
  • at least 90% of the cells in the early CC-CSC population express two or more of biomarkers Nanog, Sox 2, Oct3/4, c-kit, FoxA2, and CD 133.
  • the CC spheroids are further cultured on an adherent substrate in the presence of FGF, EGF, and a serum-containing media (expansion media) to yield colonies mixed with a monolayer wherein the cells have heterogeneous morphologies.
  • CC-CSC CC-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 CC-CSC population express two or more of biomarkers Hes1 , MSI 1 , ALDH1 B1 , ALDH1A1 , EpCAM, G-CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki-67, AFP, and DCIkl .
  • biomarkers Hes1 MSI 1 , ALDH1 B1 , ALDH1A1 , EpCAM, G-CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki-67, AFP, and DCIkl .
  • At least 90% of the cells in the early CC-CSC population express two or more of biomarkers Hes1 , MSI 1 , ALDH1 B1 , ALDH1A1 , EpCAM, G-CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki-67, AFP, and DCIkl .
  • the CC 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 CC-CSC or "EMT-CC-CSC" (epithelial to mesenchymal transitioned [EMT] cancer stem cells).
  • the spheroids have undergone a process of EMT characterized by the loss of the expression of at least one epithelial marker
  • 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 markers Slug/Snail, CD44, Twist, 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-CC-CSC population express two or more of the biomarkers N-cadherin, Slug/Snail, vimentin, Twist, and CD1 17.
  • at least 80% of the cells in the EMT-CC-CSC population express two or more of the biomarkers N- cadherin, Slug/Snail, vimentin, CD1 17, CD44, CD24, ⁇ -synuclein, FMNL2, b-catenin, Nanog, CD147, 33GhT8, LGR5, CD29, CXCR4, CD133, and DCIkl .
  • at least 90% of the cells in the EMT-CC-CSC population express two or more of the biomarkers N- cadherin, Slug/Snail, vimentin, Twist, and CD1 17.
  • 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.
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • 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 an example surgically excised tumor, so that purification of CC-CSC can begin immediately. If the sample is very small, such as needle aspirate, metastasis, or peritoneal lavage, and non-adherent culture is not feasible, an adherent culture may be used for initial expansion, followed by
  • 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 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 CC-CSC.
  • Other ligands to receptor tyrosine kinases (RTK) such as VEGF, HGF, or PDGF may have the same effect on the tumor cell expansion.
  • CC-CSC cell line that is unique to each patient obtained from the patient's primary colon 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%.
  • the present disclosure encompasses nucleic acids, gene products, polypeptides, and peptide fragments, where identity can be reasonably established by a trivial name alone.
  • 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 CC stem cells, or antigens derived therefrom, for use in therapy of colon carcinoma.
  • immunogenic compositions such as vaccine compositions, comprising dendritic cells loaded with (exposed to) the CC-CSC ex vivo.
  • the dendritic cells and tumor cells are from the same human subject although embodiments where the dendritic cells and CC cells are from different subjects are within the scope of the present disclosure.
  • Dendritic cells can be loaded with CC tumor cell antigens comprising whole cells, cell lysates, cell extracts, irradiated cells or any protein derivative of a CC tumor cell.
  • 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 CC-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 CC stem cell population, as disclosed above, as a source of antigen for autologous immune therapy, for example, where the CC stem cells 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.
  • a radiant energy e.g., gamma, UV, X
  • temperature e.g., heat or cold
  • chemical e.g., cytostatic, aldehyde, alcohol
  • the CC stem cells are used as a source of antigen for ex vivo activation of dendritic cells.
  • the present disclosure provides prepared CC cells, provides DC loaded with the prepared CC cells, and provides immunogenic compositions (or vaccines) comprising dendritic cells loaded the prepared CC cells.
  • an immunogenic composition of the present disclosure can comprise DC loaded with CC 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 CC-CSC, mixed CC-CSC, or EMT-CC- CSC.
  • the DC are loaded with tumor antigens comprising messenger RNA from CC-CSC.
  • Also disclosed herein is a population of CC-CSC that are capable of stimulating an effective immune response against a cell expressing at least one CC-specific antigen, wherein the CC-CSC population is contacted with at least one dendritic cell, wherein the CC-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 CC cells, or are not loaded with peptide derived from CC 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 CC-CSC spheroids, early CC-CSC, mixed CC-CSC, and/or EMT-CC-CSC from tumor samples according to the methods disclosed herein and/or reagents for characterizing the CC-CSC spheroids, early CC-CSC, mixed CC-CSC, and/or EMT-CC-CSC, and instructions for generating and/or characterizing the CC-CSC spheroids, early CC-CSC, mixed CC-CSC, and/or EMT-CC- CSC.
  • the kit additionally, or alternatively, includes reagents and instructions for isolating dendritic cells, for loading the dendritic cells with CC-CSC, and/or for administering the DC-CC composition to a subject.
  • the colon carcinoma (CC) 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 a tumor-containing tissue.
  • CC 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, or combinations thereof, may be used in the disclosed methods.
  • Deoxyribonuclease DNAse
  • 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.
  • 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 (FIG. 2) that contain high purity cancer stem cells.
  • the mature cells will remain isolated and nonadherent.
  • 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 colon 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. In some instance one can stimulate and sustain the stem cell proliferation with the addition or adjustment of amount of growth factors that possess a mitogenic activity, such as the FGF family and EGF.
  • 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.
  • FIG. 3 depicts a representative cell population that is attached and expanding to an ultralow adherent surface. 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 CC-CSC 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 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%.
  • 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.
  • 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.
  • the lipid mix is made by o/w emulsions using
  • a medium which is not supplemented with one or both of superoxide dismutase (SOD) or catalase.
  • 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 CC stem cells. This subpopulation is associated with more aggressive forms of CC and earlier (less differentiated) cancer stem cells.
  • Follistatin is used to block the activin A receptors and prevent spontaneous differentiation of the CC stem cells, especially when large numbers of cells that endogenously secrete activin A are present, such as fibroblasts and normal cells.
  • the use of follistatin has no effect if the cells are insensitive to activin A or in high purity CC 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 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 colon 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.
  • an antagonist of activin A such as, but not limited to, follistatin or an antibody that specifically
  • 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 CC 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 formulation described in Tables 2, 3 and 4.
  • FGF and EGF cause proliferation of CC-CSC 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 CC-CSC that are insensitive to activin A. If the CC-CSC 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 CC-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, then plating on adherent substrate containing RGD (Arg-Gly- Asp) peptide motifs (e.g., collagen, gelatin, etc).
  • RGD Arg-Gly- Asp
  • the EMT-CC-CSC subpopulation is obtained by culturing CC-CSC spheroids, early CC-CSC, or mixed CC-CSC under culture conditions as described in Table 1 and FIG. 1.
  • CC-CSC can generally refer to CC-CSC spheroids, early CC-CSC, mixed CC-CSC, or EMT-CC-CSC.
  • CC-CSC 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 core biopsy, 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 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 CC-CSC can be propagated and expanded indefinitely, as an additional characteristic of stem cells.
  • An expanding culture on an adherent substrate is represented in FIG. 4.
  • the CC-CSC can be partial or totally differentiated. If the stem cell expansion conditions are removed, the CC-CSC 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 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 CC-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
  • 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.
  • Colon 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).
  • 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.
  • Loading dendritic cells are particularly advantageous that the patient will not develop immune resistance such as the more traditional biosimilars.
  • the individual CC-CSC cell line 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 CC-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 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 TLR5
  • 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.
  • 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.
  • CC-CSC 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 CC-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 hepatocellular carcinoma tumor cells have been used in clinical trials with no evidence of proliferation.
  • whole CC-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 1x10 6 loaded DCs.
  • each dose comprises about 8x10 6 loaded DCs, about 9x10 6 loaded DCs, about 10 x10 6 loaded DCs, about 1 1x10 6 loaded DCs, or about 12x10 6 loaded DCs.
  • the loaded DCs comprise a mixture of DCs and residual CC-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.
  • a pharmaceutically acceptable carrier or excipients 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.
  • 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. In other exemplary implementations, there is no adjuvant at all.
  • 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 CC, or a control subject not having CC.
  • 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.
  • the immunogenic compositions can be administered directly to the site of a tumor or metastasis.
  • Example 1 Isolation and expansion of CC cancer stem cells
  • Colorectal carcinoma (CC) tumor samples are histologically heterogeneous, consisting in more or less differentiated cancer cells, along with normal parenchymal, stromal and vascular cells.
  • the purpose of the methods presented here are to isolate and expand a population of cancer stem cells derived from CC samples. Further, these cells are used to prepare an autologous therapy for the treatment and recurrence prevention of the CC.
  • the procedures and reagents were designed to sustain typical stem cells, and do not sustain the persistence/proliferation in vitro of differentiated epithelia, vascular endothelial cells, smooth muscle and fibroblasts.
  • Needle aspiration biopsies and tissue fragments were obtained from consented patients diagnosed with colorectal cancer tumors from the macroscopically identified pathological areas at the primary lesion or from colon metastases.
  • the biopsies were transferred immediately in a closed container in transport media and delivered to the tissue processing facility at controlled temperature (4-8°C).
  • the biopsies were than dissociated in a solution of collagenase IV (4 mg/mL) for 30 minutes.
  • the larger biopsies were transferred after dissociation in ultralow adherent cell culture flasks (Corning) in serum free media consisting of DMEM:F12 and supplemented with lineage supplement (as described in Tables 2, 3 and 4), and containing 10 ng/mL bFGF and 10 ng/mL of EGF, while the smaller samples such as needle biopsies were first expanded first on adherent substrate.
  • the spheres were gently dissociated with exposure to collagenase IV and mechanical pipetting, the enzyme removed by centrifugation, the cells resuspended in fresh media and transferred in regular, gas plasma treated tissue culture flasks.
  • the cell lines were immunocytochemically analyzed for cytokeratins CK19 (FIG. 3C) and CK7 (FIG. 4C), tumor specific markers alpha fetoprotein (FIG. 3B) and ABCG2 (FIG. 4B), adhesion molecules EpCAM and NCAM (FIG. 6A-C), CD44 (FIG. 7C), proliferation marker Ki67 (FIG. 8B), and epithelial to mesenchymal transition (EMT) markers Slug/Snail (FIG. 9B) and Twist. Table 5.
  • 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 CC- CSC.
  • normal cells such as lymphocytes, stromal cells and connective tissue are eliminated.
  • the enriched proliferating CC-CSC tumor cells, TC
  • irradiation apoptosis, which facilitates antigen exposure to antigen presenting cells
  • 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.

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Abstract

La présente invention concerne des cellules souches cancéreuses, pour leur utilisation dans la stimulation d'une réponse immunitaire contre un cancer, par exemple contre le carcinome du côlon (CC). La présente invention concerne également la préparation et la purification des cellules souches cancéreuses.
EP14779579.3A 2013-03-13 2014-03-12 Cellules souches de carcinome du côlon individualisées, de pureté élevée, procédés et utilisation associés Withdrawn EP2968407A4 (fr)

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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
PCT/US2014/024382 WO2014165101A1 (fr) 2013-03-13 2014-03-12 Cellules souches de carcinome du côlon individualisées, de pureté élevée, procédés et utilisation associés

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