WO2016081554A1 - Compositions immunogènes préparées à partir de cellules tumorales dérivées du sang périphérique et provenant d'une tumeur solide et leur utilisation - Google Patents

Compositions immunogènes préparées à partir de cellules tumorales dérivées du sang périphérique et provenant d'une tumeur solide et leur utilisation Download PDF

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WO2016081554A1
WO2016081554A1 PCT/US2015/061260 US2015061260W WO2016081554A1 WO 2016081554 A1 WO2016081554 A1 WO 2016081554A1 US 2015061260 W US2015061260 W US 2015061260W WO 2016081554 A1 WO2016081554 A1 WO 2016081554A1
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cells
cell
tumor
cancer
population
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Gabriel I. Nistor
Hans S. Keirstead
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Neostem Oncology, Llc
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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Definitions

  • the described invention generally relates to autologous cancer vaccines comprising dendritic cells contacted with at least one cancer-specific antigen.
  • Immune responses are initiated by an individual's encounter with a foreign antigenic substance/immunogen, for example, an infectious agent.
  • the individual rapidly responds with the production of antibody molecules specific for epitopes of the immunogen and with the expansion and differentiation of antigen- specific regulatory and effector T-lymphocytes.
  • the latter include cells that produce cytokines and killer T cells capable of lysing the infected cells. Generally this initial immune response is sufficient to control and eradicate the foreign substance.
  • lymphocytes which are the cells that determine the specificity of immunity, and cells that interact with lymphocytes, which play roles in the presentation of antigen and in the mediation of immunologic functions. These cells include the monocyte/macrophages, dendritic cells and closely related Langerhans' cells, natural killer (NK) cells, mast cells, basophils and other members of the myeloid lineage of cells.
  • NK natural killer
  • a series of specialized epithelial and stromal cells provide the anatomic environment in which immunity occurs, often by secreting critical factors that regulate growth and/or gene activation in cells of the immune system. Such cells also play direct roles in the induction and effector phases of the response. Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999).
  • lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens. This commitment, which exists before the first contact of the immune system with a given antigen, is
  • lymphocytes expressed by the presence on the lymphocyte's surface membrane of receptors specific for determinants (epitopes) on the antigen.
  • Each lymphocyte possesses a population of receptors, all of which have identical combining sites.
  • One set, or clone, of lymphocytes differs from another clone in the structure of the combining region of its receptors and thus differs in the epitopes that it can recognize.
  • B-cells B- lymphocytes
  • T-cells T- lymphocytes
  • B-lymphocytes are derived from hematopoietic cells of the bone marrow.
  • a mature B-cell can be activated with an antigen that expresses epitopes that are recognized by its cell surface.
  • Na ' ive B cells are distinguished in that they do not express the marker CD27, while antigen-specific B cells do express CD27
  • the activation process may be direct, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B-cell activation), or indirect, via interaction with a helper T-cell, in a process referred to as cognate help.
  • Antibody responses to most protein antigens are dependent on helper T cells.
  • receptor cross-linkage stimuli and cognate help synergize to yield more vigorous B-cell responses.
  • Cross-linkage dependent B-cell activation requires that the antigen express multiple copies of the epitope complementary to the binding site of the cell surface receptors because each B-cell expresses Ig molecules with identical variable regions.
  • Such a requirement is fulfilled by other antigens with repetitive epitopes, such as capsular polysaccharides of microorganisms or viral envelope proteins.
  • Cross-linkage-dependent B-cell activation is a major protective immune response mounted against these microbes.
  • Cross-linkage-dependent B-cell activation is a major protective immune response mounted against these microbes.
  • Cognate help allows B-cells to mount responses against antigens that cannot cross-link receptors and, at the same time, provides costimulatory signals that rescue B cells from inactivation when they are stimulated by weak cross-linkage events.
  • Cognate help is dependent on the binding of antigen by the B-cell's membrane immunoglobulin (Ig), the endocytosis of the antigen, and its fragmentation into peptides within the endosomal/lysosomal compartment of the cell. Some of the resultant peptides are loaded into a groove in a specialized set of cell surface proteins known as class II major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the resultant class ll/peptide complexes are expressed on the cell surface and act as ligands for the antigen-specific receptors of a set of T-cells designated as CD4+ T- cells.
  • the CD4+ T-cells bear receptors on their surface specific for the B-cell's class ll/peptide complex.
  • B-cell activation depends not only on the binding of the T cell through its T cell receptor (TCR), but this interaction also allows an activation ligand on the T-cell (CD40 ligand) to bind to its receptor on the B-cell (CD40) signaling B- cell activation.
  • T helper cells secrete several cytokines that regulate the growth and differentiation of the stimulated B-cell by binding to cytokine receptors on the B cell. (Paul, W. E., "Chapter 1 : The immune system: an introduction,"
  • the CD40 ligand is transiently expressed on activated CD4+ T helper cells, and it binds to CD40 on the antigen-specific B cells, thereby transducing a second costimulatory signal.
  • the latter signal is essential for B cell growth and differentiation and for the generation of memory B cells by preventing apoptosis of germinal center B cells that have encountered antigen.
  • Hyperexpression of the CD40 ligand in both B and T cells is implicated in the pathogenic autoantibody production in human SLE patients. (Desai- Mehta, A. et al., "Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production," J. Clin. Invest., 97(9): 2063-2073 (1996)).
  • T-lymphocytes derive from precursors in hematopoietic tissue, undergo differentiation in the thymus, and are then seeded to peripheral lymphoid tissue and to the recirculating pool of lymphocytes. T-lymphocytes or T cells mediate a wide range of immunologic functions. These include the capacity to help B cells develop into antibody-producing cells, the capacity to increase the microbicidal action of monocytes/macrophages, the inhibition of certain types of immune responses, direct killing of target cells, and mobilization of the inflammatory response. These effects depend on their expression of specific cell surface molecules and the secretion of cytokines. (Paul, W. E., "Chapter 1 : The immune system: an
  • T cells differ from B cells in their mechanism of antigen recognition.
  • Immunoglobulin the B cell's receptor, binds to individual epitopes on soluble molecules or on particulate surfaces.
  • B-cell receptors see epitopes expressed on the surface of native molecules.
  • Antibody and B-cell receptors evolved to bind to and to protect against microorganisms in extracellular fluids.
  • T cells recognize antigens on the surface of other cells and mediate their functions by interacting with, and altering, the behavior of these antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • Immature dendritic cells are located in tissues throughout the body, including the skin, gut, and respiratory tract. When they encounter invading microbes at these sites, they endocytose the pathogens and their products, and carry them via the lymph to local lymph nodes or gut associated lymphoid organs. The encounter with a pathogen induces the dendritic cell to mature from an antigen-capturing cell to an antigen- presenting cell (APC) that can activate T cells.
  • APC antigen- presenting cell
  • APCs display three types of protein molecules on their surface that have a role in activating a T cell to become an effector cell: (1 ) MHC proteins, which present foreign antigen to the T cell receptor; (2) costimulatory proteins which bind to complementary receptors on the T cell surface; and (3) cell-cell adhesion molecules, which enable a T cell to bind to the antigen- presenting cell (APC) for long enough to become activated.
  • (1 ) MHC proteins which present foreign antigen to the T cell receptor
  • costimulatory proteins which bind to complementary receptors on the T cell surface
  • cell-cell adhesion molecules which enable a T cell to bind to the antigen- presenting cell (APC) for long enough to become activated.
  • T-cells are subdivided into two distinct classes based on the cell surface receptors they express.
  • the majority of T cells express T cell receptors (TCR) consisting of a and ⁇ chains.
  • T cells express receptors made of Y and ⁇ chains.
  • TCR T cell receptors
  • CD4+ T cells those that express the coreceptor molecule CD4
  • CD8+ T cells those that express CD8
  • CD4+ T cells are the major regulatory cells of the immune system. Their regulatory function depends both on the expression of their cell-surface molecules, such as CD40 ligand whose expression is induced when the T cells are activated, and the wide array of cytokines they secrete when activated.
  • T cells also mediate effector functions, some of which are
  • the cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms.
  • T cells can develop into cytotoxic T-lymphocytes (CTLs) capable of efficiently lysing target cells that express antigens recognized by the CTLs.
  • CTLs cytotoxic T-lymphocytes
  • T cell receptors recognize a complex consisting of a peptide derived by proteolysis of the antigen bound to a specialized groove of a class II or class I MHC protein.
  • the CD4+ T cells recognize only peptide/class II complexes while the CD8+ T cells recognize peptide/class I complexes.
  • the TCR's ligand i.e., the peptide/MHC protein complex
  • APCs antigen-presenting cells
  • class II MHC molecules bind peptides derived from proteins that have been taken up by the APC through an endocytic process. These peptide-loaded class II molecules are then expressed on the surface of the cell, where they are available to be bound by CD4+ T cells with TCRs capable of recognizing the expressed cell surface complex.
  • CD4+ T cells are specialized to react with antigens derived from extracellular sources.
  • class I MHC molecules are mainly loaded with peptides derived from internally synthesized proteins, such as viral proteins. These peptides are produced from cytosolic proteins by proteolysis by the proteosome and are translocated into the rough endoplasmic reticulum. Such peptides, generally nine amino acids in length, are bound into the class I MHC molecules and are brought to the cell surface, where they can be recognized by CD8+ T cells expressing
  • T cell system particularly CD8+ T cells, the ability to detect cells expressing proteins that are different from, or produced in much larger amounts than, those of cells of the remainder of the organism (e.g., vial antigens) or mutant antigens (such as active oncogene products), even if these proteins in their intact form are neither expressed on the cell surface nor secreted.
  • vial antigens e.g., vial antigens
  • mutant antigens such as active oncogene products
  • T cells can also be classified based on their function as helper T cells; T cells involved in inducing cellular immunity; suppressor T cells; and cytotoxic T cells.
  • Helper T cells are T cells that stimulate B cells to make antibody responses to proteins and other T cell-dependent antigens.
  • T cell-dependent antigens are immunogens in which individual epitopes appear only once or a limited number of times such that they are unable to cross-link the membrane
  • immunoglobulin (Ig) of B cells or do so inefficiently.
  • B cells bind the antigen through their membrane Ig, and the complex undergoes endocytosis.
  • the antigen is fragmented into peptides by proteolytic enzymes and one or more of the generated peptides are loaded into class II MHC molecules, which traffic through this vesicular compartment. The resulting
  • peptide/class II MHC complex is then exported to the B-cell surface membrane. T cells with receptors specific for the peptide/class II molecular complex recognize this complex on the B-cell surface.
  • B-cell activation depends both on the binding of the T cell through its TCR and on the interaction of the T-cell CD40 ligand (CD40L) with CD40 on the B cell.
  • T cells do not constitutively express CD40L. Rather, CD40L expression is induced as a result of an interaction with an APC that expresses both a cognate antigen recognized by the TCR of the T cell and CD80 or CD86.
  • CD80/CD86 is generally expressed by activated, but not resting, B cells so that the helper interaction involving an activated B cell and a T cell can lead to efficient antibody production.
  • CD40L on T cells is dependent on their recognition of antigen on the surface of APCs that constitutively express CD80/86, such as dendritic cells.
  • Such activated helper T cells can then efficiently interact with and help B cells.
  • Cross-linkage of membrane Ig on the B cell even if inefficient, may synergize with the CD40L/CD40 interaction to yield vigorous B-cell activation.
  • the subsequent events in the B-cell response, including proliferation, Ig secretion, and class switching (of the Ig class being expressed) either depend or are enhanced by the actions of T cell-derived cytokines.
  • CD4+ T cells tend to differentiate into cells that principally secrete the cytokines IL-4, IL-5, IL-6, and IL-10 (TH2 cells) or into cells that mainly produce IL-2, IFN- ⁇ , and lymphotoxin (TH1 cells).
  • TH2 cells are very effective in helping B-cells develop into antibody-producing cells
  • TH1 cells are effective inducers of cellular immune responses, involving enhancement of microbicidal activity of monocytes and macrophages, and consequent increased efficiency in lysing microorganisms in intracellular vesicular compartments.
  • TH1 cells Although the CD4+ T cells with the phenotype of TH2 cells (i.e., IL-4, IL-5, IL-6 and IL-10) are efficient helper cells, TH1 cells also have the capacity to be helpers. (Paul, W. E., “Chapter 1 : The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
  • T cells also may act to enhance the capacity of monocytes and macrophages to destroy intracellular microorganisms.
  • interferon- gamma (IFN- ⁇ ) produced by helper T cells enhances several mechanisms through which mononuclear phagocytes destroy intracellular bacteria and parasitism including the generation of nitric oxide and induction of tumor necrosis factor (TNF) production.
  • the THi cells are effective in enhancing the microbicidal action because they produce IFN- ⁇ .
  • two of the major cytokines produced by TH2 cells IL-4 and IL-10, block these activities.
  • Treg Suppressor or Regulatory T (Treg) cells
  • a controlled balance between initiation and downregulation of the immune response is important to maintain immune homeostasis.
  • Mechanisms that contribute to the downregulation of the immune response include both apoptosis and T cell anergy (a tolerance mechanism in which the T cells are intrinsically functionally inactivated following an antigen encounter (Scwartz, R. H., "T cell anergy,” Annu. Rev. Immunol., 21 : 305-334 (2003)).
  • a third mechanism is provided by active suppression of activated T cells by suppressor or regulatory CD4+ T (Treg) cells. (Reviewed in Kronenberg, M.
  • CD4+ Tregs that constitutively express the IL-2 receptor alpha (IL-2Ra) chain are a naturally occurring T cell subset that are anergic and suppressive.
  • IL-2Ra IL-2 receptor alpha
  • CD4+ CD25+ are a naturally occurring T cell subset that are anergic and suppressive.
  • Depletion of CD4+CD25+ Tregs results in systemic autoimmune disease in mice; transfer of these Tregs prevents development of autoimmune disease.
  • Human CD4+CD25+ Tregs similar to their murine counterpart, are generated in the thymus and are characterized by the ability to suppress proliferation of responder T cells through a cell-cell contact-dependent mechanism, the inability to produce IL-2, and the anergic phenotype in vitro.
  • Human CD4+CD25+ T cells can be split into suppressive (CD25high) and nonsuppressive (CD25low) cells, according to the level of CD25 expression.
  • a member of the forkhead family of transcription factors, FOXP3 has been shown to be expressed in murine and human CD4+CD25+ Tregs and appears to be a master gene controlling CD4+CD25+ Treg development. (Battaglia, M. et al., "Rapamycin promotes expansion of functional CD4+CD25+Foxp3+ regulator T cells of both healthy subjects and type 1 diabetic patients," J. Immunol., 177: 8338-8347 (200)).
  • CTL Cytotoxic T Lymphocytes
  • the CD8+ T cells that recognize peptides from proteins produced within the target cell have cytotoxic properties in that they lead to lysis of the target cells.
  • the mechanism of CTL-induced lysis involves the production by the CTL of perforin, a molecule that can insert into the membrane of target cells and promote the lysis of that cell.
  • Perforin-mediated lysis is enhanced by a series of enzymes produced by activated CTLs, referred to as granzymes.
  • Many active CTLs also express large amounts of Fas ligand on their surface. The interaction of Fas ligand on the surface of CTL with Fas on the surface of the target cell initiates apoptosis in the target cell, leading to the death of these cells.
  • CTL-mediated lysis appears to be a major mechanism for the destruction of virally infected cells.
  • the elements of the immune system include cellular immunity, humoral immunity, and the complement system.
  • NK cells Natural Killer (NK cells)
  • NK cells belong to the lymphoid lineage. Although 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 constant region of antibodies is mediated by the 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 (see, e.g., Caligiuri (2008) Blood 1 12:461 -469). NK cells have been reported to mediate cytotoxicity against cancer stem cells (see, e.g., Jewett and Tseng (201 1 ) J. Cancer. 2:443-457).
  • Antigen presenting cells are cells of the immune system used for presenting antigen to T cells.
  • APCs include dendritic cells, monocytes, macrophages, marginal zone Kupffer cells, microglia, Langerhans cells, T cells, and B cells (see, e.g., Rodriguez-Pinto and Moreno (2005) Eur. J. Immunol. 35:1097- 1 105).
  • Antigen-presenting cells display three types of protein molecules on their surface that have a role in activating a T cell to become an effector cell: (1 ) MHC proteins, which present foreign antigen to the T-cell receptor; (2) costimulatory proteins, which bind to complementary receptors on the T cell surface; and (3) cell- cell adhesion molecules, which enable a T cell to bind to the antigen-presenting cell for long enough to become activated. Alberts, B. et al., Molecular Biology of the Cell, 4 th Ed., Garland Science, NY (2002), p. 1394. The function of class I and class II MHC molecules is to bind and present antigen-derived peptides to T cells whose receptors can recognize the peptide/MHC complex that is generated. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)), p. 15.
  • Dendritic cells are discrete leukocyte population(s) of antigen presenting cells that initiate specific T-lymphocyte activation and proliferation. Their key properties include (1 ) the ability to take up, process, and present antigen; (2) the ability to migrate selectively through tissues; and (3) the ability to interact with, stimulate and direct T-lymphocyte responses. Hart, DNJ, Blood 90(9): 3245-87, 3245 (1997). The encounter with an antigen induces the dendritic cell to mature from an antigen-capturing cell to an antigen-presenting cell that can activate T cells.
  • DCs exhibit several features necessary for the generation of T-cell- mediated antitumor immunity (Dermime S, et al., British Medical Bulletin (2002) 62: 149-162; Cella M, et al., Curr. Opin. Immunol. (1997) 9: 10-16).
  • a DC acquires 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.
  • dendritic cells presenting antigen to a T-cell can "activate" that T-cell, the activated T-cell might not be capable of mounting an effective immune response.
  • Effective immune response by the CD8+ T-cell for example, often requires prior stimulation of the DC by one or more of a number of interactions.
  • These interactions include direct contact of a CD4+ T-cell to the DC (by way of contact of the CD4+ T-cell's CD40 ligand to the DCs CD40 receptor), or direct contact of a toll-like receptor (TLR) agonist to one of the dendritic cell's toll-like receptors (TLRs).
  • TLR toll-like receptor
  • the described invention provides agents that, alone or in combination with a dendritic cell vaccine, can stimulate an immune response against tumor antigens; agents that can deplete T regulatory cells (Tregs); agents that can enhance activity of CD8+ T cells or CD4+ T cells, and other agents that modulate the immune system.
  • T regulatory cells T regulatory cells
  • TLR tolllike receptor
  • TLR9 CpG-oligonucleotide
  • TLR7 imiquimod
  • TLR3 poly(l:C)
  • TLR4 glucopyranosyl lipid A
  • TLR2 murein
  • TLR5 flagellin
  • CD40 agonists e.g., CD40-ligand
  • cytokine interferon-gamma
  • prostaglandin E2 and the like.
  • TLR toll-like receptor
  • the imidazoquinollines imiquimod and resiquimod are TLR7 or TLR8 agonists. Stimulation of TLR7 of plasmacytoid dendritic cells results in expression of IFN-alpha.
  • CpG ODNs are TLR9 agonists.
  • CpG ODNs occur in three classes, CpG-A, CpG-B, and CpG-C.
  • CpG-A stimulates NK cells, owing to its IFN-alpha producing effect on plasmacytoid DCs.
  • CpG-B induces IFN-alpha, and upregulates co-stimulatory markers on pDCs and B cells.
  • TLR3 agonists include polyriboinosinic-polyribocytidylic acid (poly l:C), which is an analogue of viral double stranded RNA (dsRNA).
  • TLR4 agonists include monophosphoryl lipid A (MPL), which is a derivative of Salmonella minnesota lipopolysaccharide, and which is used as part of a vaccine against human papillomavirus. Attenuated bacteria are used in anti-cancer therapy.
  • Mycobacterium bovis stimulates TLR2, and TLR4, and TLR9.
  • Listeria monocytogenes stimulates various TLRs (US 2007/0207171 of Dubensky et al, which is incorporated herein by reference, in its entirety). See also, Galluzzi et al (2012) Oncolmmunology. 1 :699- 716; Adams (2009) Immunotherapy. 1 :949-964.
  • Alpha-galactosylceramide (alpha-GalCer) is also provided (Schwaab and Ernstoff (201 1 ) Therapy. 4:369-377).
  • Alpha-GalCer provokes the activation of NKT cells that express certain T cell receptors (Lopez-Sagaseta et al (2012) PLoS Biol. 10:e1001412 (1 1 pages)).
  • NOD nucleotide- binding and oligomerization domain
  • NOD receptors include NOD1 and NOD2, which recognize distinct motifs of peptidoglycan (PGN).
  • NOD agonists include N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyldipeptide (MDP)), which binds to NOD 2.
  • MDP muramyldipeptide
  • NOD agonists include gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), which binds to NOD1 .
  • NOD agonists include desmuramylpeptides (DMP), which binds to NOD1 . See, e.g., Uehara et al (2006) J. Immunol. 177:1796-1804).
  • NOD agonists are derived from fragments of peptidoglycan.
  • Regs T regulatory cells
  • These agents include cyclophosphamide, gemcitabine, and antibodies that deplete Tregs (see, e.g., Le and Jaffee (2012) Cancer Res. 72:3439-3444; Klages et al (2010) Cancer Res. 70:7788-7799).
  • Gemcitabine reduces the immunosuppressive activity of myeloid-derived suppressor cells (MDSCs) (Le et al (2009) Int.
  • Anti-CD25 antibodies can deplete Tregs, and have been used in the treatment of cancer (Klages et al (2010) Cancer Res. 70:7788- 7799).
  • Daclizumab is an anti-CD25 antibody, which blocks binding of interleukin-2 (IL-2) to CD25, thereby blocking a signal needed for Treg maintenance.
  • Some Tregs express high levels of CTLA4 (CD45RA+ Tregs).
  • Anti-CTLA4 antibodies such as ipilimumab, target CTLA4. Ipilimumab is used for treating melanoma (Rech et al (2012) Immunotherapy. 4:1 103-1 105).
  • Lymphocyte activation gene-3 (LAG-3) blocking agents such as anti-LAG-3 antibodies or soluble LAG-3 (e.g., LAG-3 Ig), can enhance immune response to cancers or infections.
  • Anti-LAG-3 antibodies reduce the activity of Tregs (see, e.g., Huang, et al. (2004) Immunity 21 :503-513; Triebel (2003) Trends Immunol. 24:619-622).
  • Antibodies are available that directly target CD8+ T cells or CD4+ T cells. These antibodies include those that target costimulatory receptors (4-1 BB, OX40 and GITR) or the blockade of co-inhibitory receptors (CTLA-4, PD-1 and PD- L1 ).
  • GITR stands for glucocorticoid-induced TNFR-related protein. See, e.g., Schaer et al (2010) Curr. Opin. Investig. Drugs. 1 1 : 1378-1386). Anti-GITR can stop the suppressive function of Tregs, thus explaining anti-GITR's efficacy in anti-cancer therapy (Coe et al (2010) Cancer Immunol. Immunother. 59:1367-1377). DTA-1 is an anti-GITR antibody.
  • DCs express high levels of co-stimulatory molecules.
  • the trafficking of immature DCs to sites of inflammation and or mature DCs to the T- cell area of secondary lymphoid organs is regulated by the expression of different chemokines and chemokine receptors (Rubio M T, et al., International Immunology (2005) 17(12): 1561 -1572; Sallusto F and Lanzavecchia A, Immunol. Rev. (2000) 177: 134-140; Val, M, et al., J. Ex. Med. (1998) 188: 373-386; Sallusto F, et al., Eur. J. Immunol. (1999) 29: 1617-1625).
  • Immature DCs express inflammatory
  • chemokines monocyte chemoattractant protein-1 (CCL2/MCP-1 ), macrophage inflammatory protein-1 a (CCL3/MIP-1 a), macrophage inflammatory protein-1 ⁇ (CCL4/MIP-1 ⁇ ), regulated on activation, normal T-cell expressed and secreted (CCL5/RANTES), macrophage inflammatory protein-3a (CCL20/MIP-3a)) and chemokine receptors that bind to inflammatory chemokines (chemokine (C-C motif) receptor 1 (CCR1 ), chemokine (C-C motif) receptor 2 (CCR2), chemokine (C-C motif) receptor 5 (CCR5), chemokine (C-C motif) receptor 6 (CCR6) and chemokine (C-X-C motif) receptor 1 (CXCR1 )).
  • DCs down-regulate the inflammatory chemokines and their receptors and up-regulate constitutive chemokines such as interferon-y-inducible protein 10 (CXCL10/IP-10), thymus and activation regulated chemokine (CCL17/TARC), pulmonary and activation regulated chemokine
  • constitutive chemokines such as interferon-y-inducible protein 10 (CXCL10/IP-10), thymus and activation regulated chemokine (CCL17/TARC), pulmonary and activation regulated chemokine
  • CCL18/PARC macrophage inflammatory protein-3 (0 ⁇ _19/ ⁇ -3 ⁇ )
  • macrophage derived chemokine CCL22/MDC
  • C-C motif chemokine receptor chemokine 7
  • CCL21 /SLC secondary lymphoid-tissue chemokine
  • the co-stimulatory chemokines expressed by mature DCs function to attract T-cells and to interact with receptors on T-cells to provide a second signal required to optimally activate antigen-specific T-cells (Dermime S, et al., British Medical Bulletin (2002) 62: 149-162; June C, et al., Immunol. Today (1994) 15: 321 - 331 ).
  • DCs have been shown to attract T helper 1 (Th1 ) and T helper 2 (Th2) cells, as well as na ' ive T-cells and memory T-cells.
  • T helper cells express CCR4, a G-coupled protein that is the receptor for the chemokines MCP-1 , MIP-1 , RANTES,
  • CD17/TARC macrophage-derived chemokine and thymus and activation regulated chemokine
  • DCs are known to express high levels of CCL17/TARC, which acts on the CCR4 receptor, and thus attracts and activates downstream immune cells (Vissers J L, et al., J. Leukoc. Biol. (2001 ) 69: 785-793).
  • a particular advantage of attracting or "recruiting" CD4+ T-cells is that they provide "help" for cross-priming naive T-cells by expression of CD40L.
  • CD40L permits DCs to activate CD8+ cytotoxic T-lymphocytes (Schoenberger S P, et al., Nature (1998) 393: 480-483). DCs also produce heterodimer IL-12p70, which is the major determinant of their ability to promote Th1 differentiation (Rubio M T, et al., International Immunology (2005) 17(12): 1561 -1572; Hilkens C M, et al., Blood (1997) 90: 1920-1926).
  • DCs also express the CD45 antigen, including the CD45 RA, CD45RO and CD45 RC isoforms. Hart, DN, Blood 90(9): 3245-87 (1997), at 3250.
  • Dendritic cells occur in at least two lineages.
  • the first lineage encompasses pre-DC1 , myeloid DC1 , and mature DC1 .
  • the second lineage encompasses CD34+ +CD45RA- early progenitor multipotent cells,
  • CD34++CD45RA+ cells CD34++CD45RA++ CD4+ IL-3Ralpha++ pro-DC2 cells
  • CD4 * CD1 1 c- plasmacytoid pre-DC2 cells lymphoid human DC2 plasmacytoid- derived DC2s, and mature DC2s (see, e.g., Gilliet and Liu (2002) J. Exp. Med.
  • Dendritic cells receive maturation signals in the form of cytokines and toll-like receptor ligands such as CD40 ligand, interferon-gamma and
  • LPS lipopolysaccride
  • maturation which induces the expression of maturation markers such as CD83 and the induction of T-cell activating cytokines such as interleukin-12 (IL-12) and
  • Dendritic cells differ functionally and phenotypically depending on their stage of activation. Hart, D.N.J. "Dendritic cells: unique leukocyte populations which control the primary immune response," Blood 90: 3245-87 (1997). Immature DCs are efficient at capturing and processing antigens, O'Doherty, U.
  • Mature DCs express large amounts of MHC class II molecules and lack lineage markers, such as CD14 (monocytes/macrophages;) CD3 (T-cells); CD19, CD20 and CD24 (B-cells); CD56 (NK cells); and CD66b (granulocytes).
  • CD1 a is preferentially expressed on human immature myeloid DC, whereas CD83 is found on mature DCs which also express high levels of costimulatory molecules (e.g., CD80, CD86, CD40) and adhesion molecules, including CD1 1 a, CD1 1 c, CD50, CD54, CD58 and CD102 Brossart, P. et al., "Dendritic cells in cancer vaccines," Exp. Hematol. 29: 1247-55 (2001 ).
  • costimulatory molecules e.g., CD80, CD86, CD40
  • adhesion molecules including CD1 1 a, CD1 1 c, CD50, CD54, CD58 and CD102 Brossart, P. et al., "Dendritic cells in cancer vaccines," Exp. Hematol. 29: 1247-55 (2001 ).
  • Cancer has been described as a disorder of the immune system, based on the fact that the immune system fails, at least in certain segments of the afflicted human population, to respond optimally to cancerous cells.
  • cancer cells consist mainly of self-antigens, in striking contrast to the situation with infectious organisms. Some antigens that are classified as cancer antigens are actually normal antigens that are overexpressed, or normal antigens that have a mutation in only one or two amino acids in the polypeptide chain.
  • cancer cells down-regulate Major Histocompatibility Complex (MHC), and thus do not much present tumor cell-derived peptides by way of MHC.
  • MHC Major Histocompatibility Complex
  • cancer cells and associated tumor-associated macrophages, express cytokines that dampen the immune response (see, e.g., Yu et al (2007) Nature Rev. Immunol. 7:41 -51 ). This dampening is caused, for example, by the secretion of interleukin-10 (IL-10) by the cancer cells or by the associated macrophages.
  • IL-10 interleukin-10
  • cancer cells do not provide any immune adjuvant.
  • Pathogens express a variety of naturally-occurring immune adjuvants, which take the form of toll-like receptor (TLR) agonists and NOD agonists (see, e.g., Kleinnijenhuis et al (201 1 ) Clin. Dev. Immunol.
  • TLRs toll-like receptors
  • DCs generated ex vivo by culturing hematopoietic progenitor cells or monocytes with cytokine combinations have been tested as therapeutic vaccines in cancer patients for more than a decade (Ueno H, et al., Immunol. Rev. (2010) 234: 199-212).
  • sipuleucel-T also known as APC 8015
  • APC 8015 is a cellular product based on enriched blood APCs that are briefly cultured with a fusion protein of prostatic acid phosphatase (PAP) and granulocyte macrophage colony-stimulating factor (GM-CSF)
  • PAP prostatic acid phosphatase
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • DCs can be provided with tumor-specific antigens by culturing DCs ex vivo with an adjuvant and a tumor-specific antigen, and then injecting these cells back into the patient.
  • Tumor cells obtained from an excised tumor, needle biopsy, core biopsy, vacuum-assisted biopsy or peritoneal lavage have been used to generate
  • immunogenic compositions comprising tumor-specific-antigen presenting dendritic cells.
  • CTCs Cancer stem cells circulating within the blood stream are known as circulating tumor cells (CTCs).
  • CTCs express the markers of cancer stem cells (CSCs) that have adopted an epithelial to mesenchymal transition (EMT) for increased mobility and migration.
  • CTCs express biomarkers including, for example, N-cadherin, vimentin, CD44 and fibronectin (Liu et al., Stem Cell Reports, 2014; 2: 78-91 ); Mani et al., Cell 2008; 4: 704-715).
  • Circulating tumor cells are shed by a primary tumor and ultimately cause distant metastases.
  • CTC sampling is minimally invasive, repeatable, prognostic/predictive; and provides information about tumor biology and drug effects. Circulating tumor cells captured from peripheral blood have been shown to predict disease outcome and therapy response in cancer patients.
  • CTCs have been isolated from blood by methods that rely on immunomagnetic binding of cell surface epithelial cell adhesion molecules. This method is limited to EpCAM-expressing tumors. See, e.g., CELLSEARCH® CTC Kit, Janssen Diagnostics, LLC, Cristofanill, M et al, N. Engl. J. Med. 351 : 781 -91 (2004).
  • Zheng et al captured CTCs on a parylene-C pore microfilter, which traps CTCs based on their size differential from other blood cells, but relied on fixation, staining and visual enumeration of captured cells.
  • telomerase activity-a recognized cancer marker- was reliably detected by quantitative PCR from as few as 25 cancer cells added into 7.5 mL of whole blood and captured on the microfilter. Significant telomerase activity elevation was also measured from captured live cells processed from patients' blood samples for up to 24 hours from the time of blood draw and from single cancer cells lifted off of the microfilter.
  • Rho associated coiled-coil kinase is a key regulator of actin organization and thus a regulator of cell migration.
  • Rho kinase inhibitor Y-27632 in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro (Liu, Xuefeng et al. ROCK
  • ROCK inhibits the de-polymerization of actin filaments indirectly by phosphorylating and activating LIM kinase, which in turn phosphorylates ADF/cofilin, thereby inactivating its actin-de- polymerization activity. This results in the stabilization of actin filaments and an increase in their numbers. The increase in stable actin filaments and the loss of actin monomers contribute to a reduction of cell migration and cell shape definition.
  • ROCK inhibitors by preventing tumor spreading and blocking cell migration into neighboring tissue, may be useful for treating cancer disease (Hahmann C, Schroeter T (2010). "Rho-kinase inhibitors as therapeutics: from pan-inhibition to isoform selectivity”. Cell Mol Life Sci 67 (2): 171 - 7).
  • ROCK inhibitors may be effective in reducing adherent tumor cell behavior, other research indicates that these inhibitors could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy (Bhandary L, Whipple RA, Vitolo Ml, et al. ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells. Oncotarget. 2015; 6(8):6251 - 6266).
  • Phosphatase and tensin homolog functions as a tumor suppressor. It acts as a dual-specificity protein phosphatase, dephosphorylating tyrosine-, serine- and threonine-phosphorylated proteins, and as a lipid phosphatase, removing the phosphate in the D3 position of the inositol ring from
  • ROCK which is a well-known effector of RhoA, upregulates the activity of PTEN.
  • Activated PTEN downregulates Akt activity, which is essential for cell proliferation.
  • PTEN regulates the phosphatidylinositol 3,4, 5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival (Sun H et al.
  • RhoA-ROCK-PTEN pathway acts as a molecular switch to control cell proliferation and determine anchorage dependence. In cells that are poorly attached to
  • the described invention provides CTCs isolated from blood for expansion and production of autologous circulating tumor cancer stem cell lines for producing autologous dendritic cell-circulating tumor cell (DC-CTC) vaccines for the treatment of solid tumors.
  • DC-CTC dendritic cell-circulating tumor cell
  • the described invention provides an immunopotent tumor-specific cancer cell product comprising: an immunogenic composition comprising: an immunostimulatory amount of an activated population of dendritic cells contacted ex vivo with a cancer stem cell population expressing at least one cancer-specific antigen, the cancer stem cell population derived from a tumor cell, tumor cell cluster, tumor cell aggregate or a combination thereof derived from peripheral blood and originating from a solid tumor; an adjuvant; and a pharmaceutically acceptable carrier, wherein the immunopotent tumor-specific cancer cell product stimulates an effective immune response against one or more cancer- specific antigens.
  • the described invention provides a method for preparing the tumor-specific cancer cell product according to claim 1 , comprising: obtaining a blood sample from a patient comprising a tumor cell, tumor cell cluster, tumor cell aggregate or a combination thereof isolated from peripheral blood and originating from a tumor; expanding the tumor cell, tumor cell cluster, tumor cell aggregate or a combination thereof by: transferring the tumor cell, tumor cell cluster, tumor cell aggregate or a combination thereof to an adherent substrate for about 2-4 weeks, wherein the adherent substrate comprises one or more of an Arginine-Glycine-Aspartic Acid (RGD)-rich compound and a serum-free medium; and feeding the cell population comprising the tumor cell, tumor cell cluster, tumor cell aggregate or a combination thereof every 2 days with fresh media, wherein the fresh media consists of a basal stem cell formulation supplemented with a protein/growth factor mixture, wherein the growth factor is a fibroblast growth factor (FGF), and epidermal growth factor (EGF), or a combination
  • FGF fibroblast growth
  • PBMCs peripheral blood mononuclear cells
  • step b. incubating the purified PBMCs of step b. with GM-CSF and IL-4 for 6 days to generate the population of dendritic cells; thawing the irradiated cancer stem cells; contacting the population of dendritic cells with the thawed irradiated cancer stem cells for 18-24 hours to form a stimulated dendritic cell-tumor-derived cell antigen composition; and collecting and storing the dendritic cell-tumor-derived cell composition in vapor phase liquid nitrogen.
  • the described invention provides a method for treating a cancer patient comprising: preparing for the cancer patient a patient-specific immunopotent tumor-specific cancer cell product comprising an immunostimulatory amount of an isolated population of dendritic cells contacted ex vivo with a population of cancer-specific cancer stem cells expressing a cancer- specific antigen by the method according to claim 1 ; administering the immunopotent tumor-specific cancer cell product to the cancer patient, wherein the immunogenic composition is effective to generate an immune response comprising activation and proliferation of CD4+ T cells, CD8+ T cells, B cells or a combination thereof; wherein the effective immune response is effective to improve a clinical parameter selected from progression-free survival, disease-free survival, time to progression, time to distant metastasis, overall survival of the subject when compared to a control.
  • the adjuvant is granulocyte- macrophage colony stimulating factor (GM-CSF).
  • GM-CSF granulocyte- macrophage colony stimulating factor
  • the cancer stem cell population expresses at least one cancer-specific antigen with mesenchymal characteristics. According to some embodiments, the cancer stem cell population expresses at least one cancer-specific antigen with embryonic stem cell characteristics. According to some embodiments, the cancer stem cell population expresses at least one cancer- specific antigen with mixed stem cell characteristics.
  • the solid tumor is selected from the group consisting of a colon carcinoma, a renal carcinoma, a glioblastoma multiforme, a hepatocellular carcinoma, an ovarian carcinoma, a breast carcinoma and a prostate carcinoma.
  • the cancer stem cell population expressing at least one cancer-specific antigen is inactivated by irradiation.
  • the immunostimulatory amount comprises at least 1 X 10 3 cancer cell-specific antigen-stimulated dendritic cells.
  • the effective immune response comprises an effective T cell response, an effective B cell response, or a combination thereof.
  • the effective immune response comprises activation and proliferation of CD4+ T cells, CD8+ T cells, B cells, or a combination thereof.
  • the described invention provides a composition
  • a composition comprising (a) an inhibitor selected from the group consisting of a ROCK inhibitor, a PTEN inhibitor and a combination thereof; (b) a growth factor selected from the group consisting of FGF, EGF and a combination thereof; and (c) N-acetyl cysteine, wherein the composition is effective for isolating or purifying circulating tumor cells (CTCs) from a blood sample obtained from a cancer patient.
  • CTCs circulating tumor cells
  • the ROCK inhibitor is selected from the group consisting of Y-27632, Thiazovivin, Slx-21 19, WF-536 [(+)-(R)-4-(1 - aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride] , RK1 -1447, 5-(1 ,4- Diazepane-1 -sulfonyl)isoquinoline (Fasudil®), GSK429286A, 3-(4-Pyridyl)-1 H-indole (Rockout®), SR 3677 dihydrochloride, SB 772077B, AS 1892802, H 1 152
  • the PTEN inhibitor is selected from the group consisting of potassium bisperoxo(1 , 10-phenanthroline)oxovanadate (V) (bpV), SF 1670 and VO-OHpic.
  • the concentration of the ROCK inhibitor in the composition ranges from about 5 ⁇ to about 20 ⁇ , i.e., the concentration of the Rock inhibitor is about 5 ⁇ , 6 ⁇ . 7 ⁇ , 8 ⁇ . 9 ⁇ , 10 ⁇ , 1 1 ⁇ , 12 ⁇ , 13 ⁇ , 14 ⁇ , 15 ⁇ , 16 ⁇ , 17 ⁇ , 18 ⁇ , 19 ⁇ , or 20 ⁇ .
  • the concentration of the ROCK inhibitor in the composition is about 5 ⁇ .
  • concentration of the PTEN inhibitor ranges from about 0.01 ⁇ to about 10 ⁇ , i. .., about 0.01 ⁇ , 0.02 ⁇ , 0.03 ⁇ , 0.04 ⁇ , 0.05 ⁇ , 0.06 ⁇ , 0.07 ⁇ , 0.08 ⁇ , 0.09 ⁇ , 1 .0 ⁇ , 1 .1 ⁇ , 1 .2 ⁇ , 1 .3 ⁇ , 1 .4 ⁇ , 1 .5 ⁇ , 1 .6 ⁇ , 1 .7 ⁇ , 1 .8 ⁇ , 1 .9 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , or 10 ⁇ .
  • the concentration of the PTEN inhibitor in the composition ranges from about 0.5 ⁇ to about 2 ⁇ , i.e., the concentration of the PTEN inhibitor is about 0.5 ⁇ , 0.6 ⁇ , 0.7 ⁇ , 0.8 ⁇ , 0.9 ⁇ , 1 ⁇ , 1 .1 ⁇ , 1 .2 ⁇ , 1 .3 ⁇ , 1 .4 ⁇ , 1 .5 ⁇ , 1 .6 ⁇ , 1 .7 ⁇ , 1 .8 ⁇ , 1 .9 ⁇ or 2 ⁇ .
  • the concentration of the PTEN inhibitor is 1 ⁇ .
  • the growth factor is epidermal growth factor (EGF) or fibroblast growth factor (FGF). According to some embodiments, the growth factor is epidermal growth factor (EGF) or fibroblast growth factor (FGF). According to some embodiments, the growth factor is epidermal growth factor (EGF) or fibroblast growth factor (FGF). According to some embodiments, the growth factor is epidermal growth factor (EGF) or fibroblast growth factor (FGF). According to some embodiments, the growth factor is epidermal growth factor (EGF) or fibroblast growth factor (FGF). According to some embodiments, the growth factor is epidermal growth factor (EGF) or fibroblast growth factor (FGF). According to some embodiments, the growth factor is epidermal growth factor (EGF) or fibroblast growth factor (FGF). According to some embodiments, the growth factor is epidermal growth factor (EGF) or fibroblast growth factor (FGF). According to some embodiments, the growth factor is epidermal growth factor (EGF) or fibroblast growth factor (FGF
  • the concentration of the growth factor in the composition is 10 ng/mL.
  • the concentration of N-acetyl cysteine in the composition is 1 mM.
  • the composition is formulated for deposition in a blood collection tube.
  • the described invention provides a method for isolating/purifying circulating tumor cells (CTCs) from a blood sample comprising: (a) obtaining a blood sample from a patient comprising a tumor cell, tumor cell cluster, tumor cell aggregate or a combination thereof originating from a tumor; (b). exposing the blood sample to the composition; (c). removing mononuclear cells from the blood sample, leaving the tumor cell, tumor cell cluster, tumor cell aggregate or a combination thereof;; (d).
  • CTCs circulating tumor cells
  • exposing step (b) is performed immediately after obtaining the blood sample.
  • removing step (c) is performed by a method selected from the group consisting of density gradient and tangential centrifugation.
  • FIG. 1 shows a schematic of therapeutic targeting of cancer stem cells (CSCs).
  • Figure 2 shows a schematic of static electroporation.
  • Figure 3 shows the precision and consistency of flow
  • Figure 4 shows overall survival for 54 metastatic melanoma patients treated with patient-specific vaccine of dendritic cells loaded with autologous proliferating tumor cells.
  • Figure 5 shows the overall survival for metastatic melanoma patients treated with autologous dendritic cell-tumor cell (DC-TC) vaccine and autologous tumor cell (TC) vaccine.
  • DC-TC dendritic cell-tumor cell
  • TC tumor cell
  • Figure 6 shows survival differences between metastatic melanoma patients treated with DC-TC or TC who had no evidence of disease (NED) at the time of treatment.
  • Figure 7 shows survival differences between metastatic melanoma patients treated with DC-TC or TC who had detectable metastatic disease (Mets) at the time of treatment.
  • Figure 8 shows a non-adherent culture of melanoma cancer stem cell spheroids isolated using media formulated to select for cancer stem cells.
  • Figure ⁇ shows expression of CD148/CD271 during the production of melanoma tumor ceils for use in an autologous DC-TC vaccine.
  • Figure 10 shows immunohistochemicaS staining for expression of vimentin and CD44 in melanoma cancer stem cell lines established from bulk tumor samples.
  • Figure 11 shows a microscopic image of a dendritic cell
  • Figure 12 is a diagram summarizing the spike and retrieval experiment of Example 1 .
  • Figure 13 is a schematic diagram for establishing the
  • Figure 14 is a schematic diagram representing the Spike/Retrieval (S/R) procedure using FACS analysis described in Example 1 .
  • Figure 15 is a schematic diagram representing the survival factor cocktail (SFC) optimization procedure described in Examples 2 and 3.
  • Figure 16 shows a Parylene-C slotted membrane filter used to filter circulating tumor cells (CTCs) by size from other blood cells.
  • Figure 17 shows clonally expanding CTCs captured on the
  • Figure 18 shows that cells grown in serum containing media resulted in the expansion of monocyte/macrophage and dendritic cells on the Parylene-C slotted membrane filter.
  • Figure 19 shows that in serum free conditions, the myeloid lineage cleared, progressively leaving behind colonies of expanding tumor cells.
  • Figure 20 shows tumor cells transferred to regular cell culture flasks separating from contaminant macrophages.
  • Figure 21 shows the phenotypic change of cultured CTCs. Cells losing adherence (round cells) float, allowing for their removal by media exchange.
  • Figure 22 is a schematic depicting the analysis of biological processes affected by exomic mutations in cultured tumor cells. The analysis revealed perturbations in o-glycosylation and in plasma membrane cell adhesion molecules.
  • Figure 23 shows cells exposed to ROCK inhibitor after passage (A) and cells not exposed to ROCK inhibitor after passage (B). Cells exposed to ROCK inhibitor (A) displayed better adherence compared to cells that were not exposed to ROCK inhibitor (B).
  • Figure 24 shows an established cell line from circulating tumor cells (CTCs).
  • Figure 25 shows immunocytochemical characterization of expanded cell populations.
  • Panels a,b vimentin, Panel c- vimentin nuclear stain;
  • Panels d,e Magel ,
  • Panel f Magel nuclear counterstain.
  • Figure 26 shows immunocytochemical characterization of expanded cell populations.
  • Panel a EpCAM/NCAM
  • Panel b EpCAM
  • Panel c NCAM
  • Panel d nuclear stain
  • Panel e Vimentin/Snail
  • Panel f Vimentin
  • Panel g Snail
  • Panel h nuclear counterstain.
  • lymphocyte activation refers to stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells resulting in synthesis of RNA, protein and DNA and production of lymphokines; it is followed by proliferation and differentiation of various effector and memory cells.
  • a mature B cell can be activated by an encounter with an antigen that expresses epitopes that are recognized by its cell surface immunoglobulin Ig).
  • the activation process may be a direct one, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B cell activation) or an indirect one, occurring most efficiently in the context of an intimate interaction with a helper T cell ("cognate help process").
  • T-cell activation is dependent on the interaction of the TCR/CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule.
  • tyrosine kinases leading to the tyrosine phosphorylation of a set of substrates that control several signaling pathways.
  • These include a set of adapter proteins that link the TCR to the RAS pathway, phospholipase Cy1 , the tyrosine phosphorylation of which increases its catalytic activity and engages the inositol phospholipid metabolic pathway, leading to elevation of intracellular free calcium concentration and activation of protein kinase C, and a series of other enzymes that control cellular growth and differentiation.
  • Full responsiveness of a T cell requires, in addition to receptor engagement, an accessory cell-delivered costimulatory activity, e.g., engagement of CD28 on the T cell by CD80 and/or CD86 on the antigen presenting cell (APC).
  • the soluble product of an activated B lymphocyte is immunoglobulins (antibodies).
  • the soluble product of an activated T lymphocyte is lymphokines.
  • 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 in vitro 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.
  • 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.
  • 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.
  • biomarkers refers to peptides, proteins, nucleic acids, antibodies, genes, metabolites, or any other substances used as indicators of a biologic state. It is a characteristic that is measured objectively and evaluated as a cellular or molecular indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.
  • indicator refers to any substance, number or ratio derived from a series of observed facts that may reveal relative changes as a function of time; or a signal, sign, mark, note or symptom that is visible or evidence of the existence or presence thereof.
  • a biomarker may be used as a surrogate for a natural endpoint, such as survival or irreversible morbidity. If a treatment alters the biomarker, and that alteration has a direct connection to improved health, the biomarker may serve as a surrogate endpoint for evaluating clinical benefit.
  • Clinical endpoints are variables that can be used to measure how patients feel, function or survive.
  • Surrogate endpoints are biomarkers that are intended to substitute for a clinical endpoint; these biomarkers are demonstrated to predict a clinical endpoint with a confidence level acceptable to regulators and the clinical community.
  • cancer refers to diseases in which abnormal cells divide without control and are able to invade other tissues. There are more than 100 different types of cancer. Most cancers are named for the organ or type of cell in which they start - for example, cancer that begins in the colon is called colon cancer; cancer that begins in melanocytes of the skin is called melanoma. Cancer types can be grouped into broader categories.
  • Carcinoma meaning a cancer that begins in the skin or in tissues that line or cover internal organs, and its subtypes, including adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma
  • Sarcoma meaning a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue
  • Leukemia meaning a cancer that starts in blood- forming tissue (e.g., bone marrow) and causes large numbers of abnormal blood cells to be produced and enter the blood
  • Lymphoma and myeloma meaning cancers that begin in the cells of the immune system
  • Central nervous system cancers meaning cancers that begin in the tissues of the brain and spinal cord).
  • Myelodysplastic syndrome refers to a type of cancer in which the bone marrow does not make enough healthy blood cells (white blood cells, red blood cells, and platelets) and there are abnormal cells in the blood and/or bone marrow. Myelodysplastic syndrome may become acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • cancer stem cell CSC
  • TIC tumor initiating cell
  • TPC tumor propagating cell
  • chemokine refers to a class of
  • chemotactic cytokines that signal leukocytes to move in a specific direction.
  • the terms “chemotaxis” or “chemotactic” refer to the directed motion of a motile cell or part along a chemical concentration gradient towards environmental conditions it deems attractive and/or away from surroundings it finds repellent.
  • cross-presentation refers to a process whereby APCs acquire proteins from other tissue cells through endocytic mechanisms, especially phagocytosis or macropinocytosis.
  • the internalized antigen can then be processed through at least two different mechanisms. In one pathway, the antigen is transferred from the
  • phagosome into the cytosol where it is hydrolyzed by proteasomes into oligopeptides that then are transported by the transporter associated with antigen processing to MHC class I molecules in the endoplasmic reticulum or phagosomes.
  • the antigen is cleaved into peptides by endosomal proteases, particularly cathepsin S, and bound by class I molecules probably in the endocytic compartment itself.
  • endosomal proteases particularly cathepsin S
  • class I molecules probably in the endocytic compartment itself.
  • one or both of these pathways can contribute to cross-presentation in vivo.
  • the outcome of cross-presentation can be either tolerance or immunity. Id.
  • cytokine refers to small soluble protein substances secreted by cells, which have a variety of effects on other cells.
  • Cytokines mediate many important physiological functions, including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Generally, cytokines act locally.
  • type I cytokines which encompass many of the interleukins including interleukin 2 (IL-2), as well as several hematopoietic growth factors; type II cytokines, including the interferons and interleukin-10; tumor necrosis factor (“TNF”)-related molecules, including TNFa and lymphotoxin; immunoglobulin super-family members, including interleukin 1 ("IL-1 "); and the chemokines, a family of molecules that play a critical role in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on a cell depending on the state of the cell. Cytokines often regulate the expression of, and trigger cascades of, other cytokines.
  • TNF tumor necrosis factor
  • IL-1 immunoglobulin super-family members
  • contact refers to the state or condition of touching or of being in immediate or local proximity. Contacting a composition to a target destination, such as, but not limited to, an organ, a tissue, a cell, or a tumor, may occur by any means of administration known to the skilled artisan.
  • DC-TC dendritic cell-tumor cell product
  • peptides derived from one or more cancer cells encompasses the following.
  • 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 providing dendritic cells with 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.
  • detectable response refers to any signal or response that may be detected in an assay, which may be performed with or without a detection reagent. Detectable responses include, but are not limited to, radioactive decay and energy (e.g., fluorescent, ultraviolet, infrared, visible) emission, absorption, polarization, fluorescence, phosphorescence, transmission, reflection or resonance transfer. Detectable responses also include chromatographic mobility, turbidity, electrophoretic mobility, mass spectrum, ultraviolet spectrum, infrared spectrum, nuclear magnetic resonance spectrum and x-ray diffraction. Alternatively, a radioactive decay and energy (e.g., fluorescent, ultraviolet, infrared, visible) emission, absorption, polarization, fluorescence, phosphorescence, transmission, reflection or resonance transfer. Detectable responses also include chromatographic mobility, turbidity, electrophoretic mobility, mass spectrum, ultraviolet spectrum, infrared spectrum, nuclear magnetic resonance spectrum and x-ray diffraction. Alternatively, a radioactive decay and energy (e.g.,
  • detectable response may be the result of an assay to measure one or more properties of a biologic material, such as melting point, density, conductivity, surface acoustic waves, catalytic activity or elemental composition.
  • a "detection reagent” is any molecule that generates a detectable response indicative of the presence or absence of a substance of interest. Detection reagents include any of a variety of molecules, such as antibodies, nucleic acid sequences and enzymes. To facilitate detection, a detection reagent may comprise a marker.
  • differentiation refers to a property of cells to exhibit tissue-specific differentiated properties in culture.
  • dose refers to the quantity of a
  • biosynthesis of mRNA polypeptide biosynthesis, polypeptide activation, e.g., by post- translational modification, or an activation of expression by changing the subcellular location or by recruitment to chromatin.
  • increased expression encompasses increased biosynthesis, or increased activity that is caused by phosphorylation, or an increased activity that is caused by migration from the cytosol to the nucleus.
  • fragment or "peptide fragment” as used herein refers to a small part derived, cut off, or broken from a larger peptide, polypeptide or protein, which retains the desired biological activity of the larger peptide, polypeptide or protein.
  • growth refers to a process of becoming larger, longer or more numerous, or an increase in size, number, or volume.
  • immune response and “immune-mediated” are used interchangeably herein to refer to any functional expression of a subject's immune system, against either foreign or self antigens, whether the consequences of these reactions are beneficial or harmful to the subject.
  • immunomodulatory cell(s) refer(s) to cell(s) that are capable of augmenting or diminishing immune responses by expressing chemokines, cytokines and other mediators of immune responses.
  • immunopotent refers to the ability to activate and guide a na ' ive immune system to mount a response toward a foreign protein.
  • the term "immunostimulatory amount" of the disclosed compositions refers to an amount of an immunogenic composition that is effective to stimulate an immune response, for example, as measured by ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay) and major histocompatibility complex (MHC) tetramer assay to detect and quantify antigen-specific T cells, quantifying the blood population of antigen-specific CD4+ T cells, or quantifying the blood population of antigen specific CD8+ T cells by a measurable amount, or where the increase is by at least 10%, etc. when compared to a suitable control (e.g., a control composition where dendritic cells are not loaded with tumor-specific cells, or not loaded with peptide derived from tumor-specific cells).
  • ELISPOT assay cellular immune response
  • ICS intracellular cytokine staining assay
  • MHC major histocompatibility complex
  • inflammatory cytokines or "inflammatory mediators” as used herein refers to the molecular mediators of the inflammatory process, which may modulate the inflammation process being either pro- or anti-inflammatory in their effect. These soluble, diffusible molecules act both locally at the site of tissue damage and infection and at more distant sites. Some inflammatory mediators are activated by the inflammatory process, while others are synthesized and/or released from cellular sources in response to acute inflammation or by other soluble mediators.
  • inflammatory mediators include, but are not limited to, plasma proteases, complement, kinins, clotting and fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, platelet- activating factor (PAF), peptides and amines, including, but not limited to, histamine, serotonin, and neuropeptides, and pro-inflammatory cytokines, including, but not limited to, interleukin-1 -beta (IL-1 ⁇ ), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-a), interferon-gamma (IF- ⁇ ), and interleukin-12 (IL-12).
  • IL-1 ⁇ interleukin-1 -beta
  • IL-4 interleukin-4
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • TNF-a tumor necrosis factor-alpha
  • IL interleukin
  • interleukins include interleukin-1 (IL-1 ), interleukin 2 (IL-2), interleukin-1 ⁇ (IL-1 ⁇ ), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-12 (IL-12).
  • IL-1 interleukin-1
  • IL-2 interleukin 2
  • IL-1 ⁇ interleukin-1 ⁇
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • IL-12 interleukin-12
  • interleukin-12 refers to a cytokine that regulates the differentiation of naive T-cells into Th1 cells. It stimulates the growth and function of T-cells and alters the normal cycle of apoptotic cell death.
  • IL-12 is one of a large group of cytokines that folds into a bundle of four alpha- helices. It is a heterodimer of 70kDa that is composed of two disulfide-linked subunits, of mass 35kDa and 40kDa.
  • inhibitor and its various grammatical forms, including, but not limited to, “inhibiting” or “inhibition”, are used herein to refer to reducing the amount or rate of a process, to stopping the process entirely, or to decreasing, limiting, or blocking the action or function thereof.
  • Inhibition can include a reduction or decrease of the amount, rate, action function, or process of a substance by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.
  • isolated is used herein to refer to material, such as, but not limited to, a nucleic acid, peptide, polypeptide, protein or cell, which is
  • An "isolated molecule” is a molecule that is substantially pure and is free of other substances with which it is ordinarily found in nature or in vivo systems to an extent practical and appropriate for its intended use.
  • the term "loaded with antigen(s)" as used herein refers to the ability of an antigen-presenting cell to capture whole cells, including live cells, necrotic cells, and dead cells, a cell lysate, acid elution cell extract, partially purified antigens, isolated antigens, partially purified peptides, purified peptides, isolated peptides, synthetic peptides, or any combination and then administered to a subject, including by cross-presentation.
  • MHC major histocompatibility complex
  • MHC II MHC class II
  • MHC III MHC class III
  • MHC II molecules are found only on specialized, antigen-presenting cell types such as macrophages, dendritic cells, activated T-cells and B-cells.
  • MHC II presents peptides derived from extracellular proteins that are internalized by the cell from its environment, digested by lysosomes and bound by MHC II before its migration to the plasma membrane.
  • MHC II interacts with helper (CD4+) T-cells to trigger an appropriate immune response.
  • MHC III molecules include several secreted proteins comprising components of the
  • complement system e.g., C2, C and B factor
  • cytokines e.g., TNF-a, LTA and LTB
  • HSP heat shock proteins
  • markers are used herein to refer to a receptor, or a combination of receptors, found on the surface of a cell. These markers allow a cell type to be distinguishable from other kinds of cells. Specialized protein receptors (markers) that are capable of selectively binding or adhering to other signaling molecules coat the surface of every cell in the body. Cells use these receptors and the molecules that bind to them as a way of communicating with other cells and to carry out their proper function in the body.
  • 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 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.
  • Expression of biomarkers by a single cell or by a population of cells 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.
  • Kaplan Meier plot or "Kaplan Meier survival curve” as used herein refers to the plot of probability of clinical study subjects surviving in a given length of time while considering time in many small intervals.
  • the Kaplan Meier plot assumes that: (i) at any time subjects who are censored (i.e., lost) have the same survival prospects as subjects who continue to be followed; (ii) the survival probabilities are the same for subjects recruited early and late in the study; and (iii) the event (e.g., death) happens at the time specified. Probabilities of occurrence of events are computed at a certain point of time with successive probabilities multiplied by any earlier computed probabilities to get a final estimate.
  • the survival probability at any particular time is calculated as the number of subjects surviving divided by the number of subjects at risk. Subjects who have died, dropped out, or have been censored from the study are not counted as at risk.
  • lymphocyte refers to a small white blood cell formed in lymphatic tissue throughout the body and in normal adults making up about 22-28% of the total number of leukocytes in the circulating blood that plays a large role in defending the body against disease.
  • modify means to change, vary, adjust, temper, alter, affect or regulate to a certain measure or proportion in one or more particulars.
  • module means to regulate, alter, adapt, or adjust to a certain measure or proportion.
  • peptide is used herein to refer to two or more amino acids joined by a peptide bond.
  • polypeptide and protein are used herein in their broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. The subunits generally are linked by peptide bonds.
  • the polypeptides described herein may be isolated, chemically synthesized or recombinantly expressed.
  • peptidomimetic refers to a small protein-like chain designed to mimic a peptide. A peptidomimetic typically arises from modification of an existing peptide in order to alter the molecule's properties.
  • protein is used herein to refer to a large complex molecule or polypeptide composed of amino acids. The sequence of the amino acids in the protein is determined by the sequence of the bases in the nucleic acid sequence that encodes it.
  • peripheral blood mononuclear cells or "PBMCs” are used interchangeably herein to refer to blood cells having a single round nucleus such as, for example, a lymphocyte or a monocyte.
  • composition refers to a composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition, syndrome, disorder or disease.
  • pharmaceutically acceptable carrier refers to any substantially non-toxic carrier conventionally useable for administration of pharmaceuticals in which the isolated polypeptide of the present invention will remain stable and bioavailable.
  • the pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for
  • the pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition.
  • salts refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.
  • Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic.
  • such salts may be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • pharmaceutically acceptable salt is meant those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well-known in the art. For example, P. H. Stahl, et al. describe
  • salts in detail in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use” (Wiley VCH, Zurich, Switzerland: 2002).
  • the salts may be prepared in situ during the final isolation and purification of the compounds described within the present invention or separately by reacting a free base function with a suitable organic acid.
  • Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate(isethionate), lactate, maleate,
  • methanesulfonate nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and
  • the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates
  • long chain halides such as decyl
  • Basic addition salts may be prepared in situ during the final isolation and purification of compounds described within the invention by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine.
  • Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like.
  • Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.
  • salts also may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal for example, sodium, potassium or lithium
  • alkaline earth metal for example calcium or magnesium
  • polypeptide is used herein in its broadest sense to refer to a sequence of subunit amino acids, amino acid analogs or peptidomimetics, wherein the subunits are linked by peptide bonds.
  • potency refers to efficacy, effectiveness, strength or, typically, the dissociation constant, which indicates the concentration needed to inhibit binding.
  • prevent refers to effectual stoppage of action or progress.
  • primary refers to the process of increasing sensitivity to. When used in an immunological sense it refers to a process whereby a specific antigen is presented to na ' ive lymphocytes causing them to differentiate.
  • unprimed cells also referred to as virgin, na ' ive, or inexperienced cells
  • TCR antigen receptor
  • BCR BCR for B cells
  • primary refers to the process whereby T cells and B cell precursors encounter the antigen for which they are specific.
  • helper T cells and B cells can interact to produce specific antibody
  • the antigen-specific T cell precursors must be primed. Priming involves several steps: antigen uptake, processing, and cell surface expression bound to class II MHC molecules by an antigen presenting cell, recirculation and antigen-specific trapping of helper T cell precursors in lymphoid tissue, and T cell proliferation and differentiation. Janeway, CA, Jr., "The priming of helper T cells, Semin. Immunol. 1 (1 ): 13-20 (1989). Helper T cells express CD4, but not all CD4 T cells are helper cells. Id. The signals required for clonal expansion of helper T cells differ from those required by other CD4 T cells.
  • the critical antigen- presenting cell for helper T cell priming in vivo appears to be a macrophage; and the critical second signal for helper T cell growth in vivo is the macrophage product interleukin 1 (IL-1 ). Id. If the primed T cells and/or B cells receive a second, co- stimulatory signal, they become activated T cells or B cells.
  • IL-1 interleukin 1
  • PTEN inhibitor refers to any molecule that inhibits a PTEN protein.
  • purify and its other grammatical forms as used herein refers to the process of isolating or freeing from foreign, extraneous, or objectionable elements.
  • ROCK inhibitor refers to any molecule that inhibits a ROCK protein.
  • 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.
  • stimulate in any of its grammatical forms as used herein refers to inducing activation or increasing activity.
  • subject or “individual” or “patient” are used interchangeably to refer to a member of an animal species of mammalian origin, including humans.
  • subject in need thereof refers to a patient that (i) will be administered an immunogenic composition according to the described invention, (ii) is receiving an immunogenic composition according to the described invention; or (iii) has received an immunogenic composition according to the described invention, unless the context and usage of the phrase indicates otherwise.
  • substrate refers to a surface upon which cells can grow.
  • exemplary substrates include, without limitation, adherent substrates and non-adherent substrates.
  • An 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 also can be grafted on polymeric backbones such as polystyrene, hyaluronan, polylactic acid or combination thereof.
  • the backbone can further carry proteoglycans.
  • the proteoglycans can carry growth factors, e.g., FGF, epidermal growth factor (EGF), activin A or follistatin.
  • a non-adherent substrate can cause fast and efficient enrichment of the cultures with cancer stem cells and may be used when a large enough sample is provided, so that purification of tumor-CSC can begin immediately. If the sample is small (needle aspirate, peritoneal lavage) and non-adherent culture is not feasible, an adherent culture may be used for initial expansion, followed by a purification step on a non-adherent substrate, then followed by another expansion under adherent conditions.
  • target refers to a biological entity, such as, for example, but not limited to, a protein, cell, organ, or nucleic acid, whose activity can be modified by an external stimulus. Depending upon the nature of the stimulus, there may be no direct change in the target, or a conformational change in the target may be induced.
  • therapeutic agent refers to a drug, molecule, nucleic acid, protein, metabolite, composition or other substance that provides a therapeutic effect.
  • active refers to the ingredient, component or constituent of the compositions of the described invention responsible for the intended therapeutic effect.
  • therapeutic agent and “active agent” are used interchangeably herein.
  • therapeutic component refers to a therapeutically effective dosage (i.e., dose and frequency of administration) that eliminates, reduces, or prevents the progression of a particular disease manifestation in a percentage of a population.
  • An example of a commonly used therapeutic component is the ED50 which describes the dose in a particular dosage that is therapeutically effective for a particular disease manifestation in 50% of a population.
  • terapéuticaally effective amount refers to an amount that is sufficient to provide the intended benefit of treatment.
  • dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Additionally, the terms “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the described invention.
  • compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment.
  • dose and “dosage” are used interchangeably herein.
  • a therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation.
  • a therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.
  • the therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models.
  • a therapeutically effective dose may also be determined from human data.
  • the applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.
  • Drug products are considered to be pharmaceutical equivalents if they contain the same active ingredients and are identical in strength or
  • Two pharmaceutically equivalent drug products are considered to be bioequivalent when the rates and extents of bioavailability of the active ingredient in the two products are not significantly different under suitable test conditions.
  • the term "therapeutic window” refers to a concentration range that provides therapeutic efficacy without unacceptable toxicity. Following administration of a dose of a drug, its effects usually show a characteristic temporal pattern. A lag period is present before the drug concentration exceeds the minimum effective concentration (“MEC") for the desired effect. Following onset of the response, the intensity of the effect increases as the drug continues to be absorbed and distributed. This reaches a peak, after which drug elimination results in a decline in the effect's intensity that disappears when the drug concentration falls back below the MEC. Accordingly, the duration of a drug's action is determined by the time period over which concentrations exceed the MEC. The therapeutic goal is to obtain and maintain concentrations within the therapeutic window for the desired response with a minimum of toxicity.
  • Drug response below the MEC for the desired effect will be subtherapeutic, whereas for an adverse effect, the probability of toxicity will increase above the MEC.
  • Increasing or decreasing drug dosage shifts the response curve up or down the intensity scale and is used to modulate the drug's effect.
  • Increasing the dose also prolongs a drug's duration of action but at the risk of increasing the likelihood of adverse effects. Accordingly, unless the drug is nontoxic, increasing the dose is not a useful strategy for extending a drug's duration of action.
  • concentration that produces about half of the greatest possible therapeutic effect and the upper limit of the therapeutic range is such that no more than about 5% to about 10% of patients will experience a toxic effect.
  • a target level strategy is reasonable, wherein a desired target steady-state concentration of the drug (usually in plasma) associated with efficacy and minimal toxicity is chosen, and a dosage is computed that is expected to achieve this value. Drug concentrations subsequently are measured and dosage is adjusted if necessary to approximate the target more closely.
  • transplantation refers to removal and transfer of cells, a tissue or an organ from one part or individual to another.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).
  • variant of a cancer-specific cancer stem cell antigen is used herein to refer to a protein, peptide or polypeptide sequence in which at least one amino acid residue has been modified by deletion of an amino acid, insertion of an amino acid, or substitution of a second amino acid for a first amino acid at a specific position on the polypeptide.
  • a skilled artisan can produce polypeptide variants having single or multiple amino acid substitutions, deletions, additions or replacements.
  • variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid includes a substituent group; (d) variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions; and (d) variants in which a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, such as, for example, an epitope for an antibody.
  • a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, such as, for example, an epitope for an antibody.
  • mutation refers to a change of the DNA sequence within a gene or chromosome of an organism resulting in the creation of a new character or trait not found in the parental type, or the process by which such a change occurs in a chromosome, either through an alteration in the nucleotide sequence of the DNA coding for a gene or through a change in the physical arrangement of a chromosome.
  • Three mechanisms of mutation include substitution (exchange of one base pair for another), addition (the insertion of one or more bases into a sequence), and deletion (loss of one or more base pairs).
  • the described invention provides an immunopotent tumor-specific cancer cell product.
  • the tumor-specific cancer cell product is an immunogenic composition.
  • the immunogenic composition comprises a therapeutic amount of an isolated population of dendritic cells activated in vitro with a tumor-specific antigen derived from a population of purified cultivated tumor cells derived from circulating tumor cells (CTCs) obtained from a patient.
  • CTCs circulating tumor cells
  • the therapeutic amount is effective to stimulate an immune response to one or more tumor specific antigens.
  • the composition comprises a pharmaceutically acceptable carrier.
  • the immunogenic composition further comprises an adjuvant.
  • the adjuvant is GM-CSF.
  • a source of the cancer cell expressing a cancer cell-specific antigen is a population of live tumor cell derived cancer stem cells found in peripheral blood.
  • Antigenic specificity can arise from the amino acid sequence of the antigen, from the degree of expression of that antigen by the tumor cell, from post- translational modification of the antigen, and the like.
  • Specificity to a certain type of cancer cell can also arise from a particular fingerprint of a plurality of tumor antigens. Specificity can also arise, from the fact that a particular antigen, while expressed by a wide variety of tumor cells, has particular use in immunotherapy against a smaller number of tumor types. Specificity can also arise from the fact that a particular collection of MHC class I presentable and MHC class II presentable epitopes exist, on a particular polypeptide or polypeptide fragment. Also, specificity in an administered antigen can arise by omitting one or more peptides that can provoke immunotolerance. The skilled artisan can locate the relevant nucleic acid and polypeptide sequences, e.g., on the U.S. government's web site, at ncbi.nlm.nih.
  • Exemplary glioblastoma-specific antigens include, but are not limited to, PTPRZ1 ; EGFR; SEC61 G; TNC; HER2; TRP-2; gp100; MAGE-1 ; IL13Ralpha2; AIM-2 (Phuphanich et al (2013) Cancer Immunol. Immunother. 62:125-135; Neidert et al (2012) J. Neurooncol.).
  • Exemplary colorectal cancer-specific antigens include, but are not limited to, SPARC, CEA, Cep55/c10orf3 (Inoue et al (2010) Int. J. Cancer. 127:1393- 1403, Parkhurst et al (201 1 ) Mol. Ther. 19:620-626; Inoda et al (201 1 ) Exp. Mol. Pathol. 90:55-60).
  • Exemplary renal cancer-specific antigens include, but are not limited to, carbonic anhydrase IX (CA-IX), MUC-1 , and NYESO-1 , and 5T4 (Tykodi et al (2012) J. Immunother. 35:523-533).
  • Exemplary ovarian cancer-specific antigens include, but are not limited to, WT1 , mesothelin, NY-ESO-1 , p53, p53 carrying specific mutations, HER- 2/neu, folate receptor-alpha, IGFBP; MUC1 , MUC4, MUC16, EpCAM; CTA (Dohi et al (201 1 ) Anticancer Res. 31 :2441 -2445; Vermeij et al (2012) Curr. Pharm. Des. 18:3804-381 1 ; Preston et al (201 1 ) Immunother. 3:539-556).
  • Exemplary melanoma-specific antigens include, but are not limited to, tyrosinase; gp75 (tyrosinase related protein-1 (TRP-1 )); gp100 (Pmel17); Melan A/MART-1 ; TRP-2; MAGE family; BAGE family; GAGE family; NY-ESO-1 ; CDK4; ⁇ - catenin; mutated introns; N-acetylglucosaminyltransferase V gene product; MUM-1 ; p15; gangliosides (e.g., GM2, GD2, GM3, GD3); high molecular weight chondroitin sulfate proteoglycan; p97 melanotransferrin; and SEREX antigens (e.g., D-1 , SSX-2) (Hodi FS, Clin Cancer Res, February 1 , 2006; 12: 673-678).
  • TRP-1 tyrosina
  • the population of live tumor cell derived cancer stem cells expressing a tumor-specific antigen is incapable of cell division but metabolically active.
  • the population of live tumor cell derived cancer stem cells expressing a tumor-specific antigen is incapable of cell division after exposure to radiant energy, temperature (e.g., heat or cold), chemical methods (e.g., cytostatic, aldehyde, alcohol), or a combination thereof.
  • radiant energy e.g., heat or cold
  • chemical methods e.g., cytostatic, aldehyde, alcohol
  • the population of live tumor cell derived cancer stem cells expressing a tumor-specific antigen is an irradiated cancer cell.
  • the term "irradiated” refers to a process by which a subject is exposed to radiation, meaning the process in which energy is emitted by one body, transmitted through an intervening medium or space, and absorbed by another body.
  • the radiation is selected from gamma-radiation, X-ray radiation, electron beam radiation, neutron beam radiation, proton beam radiation, electromagnetic radiation, visible light radiation, ultraviolet light radiation and the like.
  • irradiation functions to prevent cell division of the cancer stem cells.
  • irradiation prevents cell division, but also denatures cellular proteins.
  • the population of live tumor cell derived cancer stem cells expressing a tumor-specific antigen is a physically disrupted cancer cell.
  • methods for physical disruption include, without limitation, sonication, cavitation, dehydration, ion depletion, or by toxicity from exposure to one or more salts.
  • the population of live tumor cell derived cancer stem cells expressing a tumor-specific antigen is a cancer cell in which nucleic acids are cross-linked with a nucleic acid cross-linking agent.
  • nucleic acid targeting agents include, without limitation, beta-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester; psoralens in combination with ultraviolet (UVA) irradiation; 4'-(4-amino-2-oxa)butyl-4,5',8- trimethylpsoralen ("S-59”); and 150 micromolar of psoralen S-59 and 3 J/cm2 UVA light (FX 1019 irradiation device, Baxter Fenwal, Round Lake, IL). See, U.S. Pat. Nos. 7,833,775 and 7,691 ,393.
  • the population of live tumor cell- derived cancer stem cells is derived from cells of a solid tumor.
  • solid tumors include, without limitation, a melanoma, a tissue of endodermal, mesodermal, or ectodermal origin (e.g., melanoma of neural crest origin, colon cancer of endoderm origin, renal cancer of mesoderm origin, glioblastoma of ectoderm origin, ovarian cancer of mixed mesoderm plus extraembryonic origin) a hepatocellular carcinoma, colon carcinoma, ovarian carcinoma, glioblastoma multiforme, and tumor-cell derived vasculogenic-like cells, the source of which, for example is uveal melanoma, cutaneous melanoma, breast cancer, ovarian cancer, prostate cancer, and glioblastoma multiforme.
  • the cells of the solid tumor are a form of whole cells, including live cells, necrotic cells, dead cells, a cancer cell extract, a cancer cell lysate, or an acid eluate.
  • the cells of the solid tumor comprise purified cancer cell peptides or cancer cell synthetic peptides.
  • the live cancer stem cell is a cancer progenitor cell characterized by continuous self-renewal and differentiation.
  • the live cells of the solid tumor are derived from peripheral blood of the subject.
  • the live cells of the solid tumor in the peripheral blood of the subject are in cell clusters.
  • the cell clusters are aggregates of cells.
  • the aggregates of cells originate from the tumor and enter the vasculature.
  • the population of live tumor cell- derived cancer stem cells comprises a population of cancer stem cells expressing at least one tumor-specific antigen with mesenchymal characteristics.
  • the population of hepatocellular cancer stem cells expressing at least one tumor-specific antigen with mesenchymal characteristics includes, but is not limited to, a loss of expression of at least one of the epithelial markers CK7, CK19, EpCAM and E-cadherin; and an increase in expression of at least one of the mesenchymal markers Slug/Snail, Twist, CD44, NCAM, N-cadherin, and vimentin by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, of the cells in the population.
  • the tumor antigen expressed by a population of vascular mimicry stem cells expressing at least one tumor-specific antigen with mesenchymal characteristics includes two or more of the biomarkers NCAM, Slug/Snail, CD24, and Twist by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, of the cells in the population.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population express two or more of the biomarkers NCAM, Slug/Snail, CD24, Twist, N-cadherin, CD44, vimentin, CD133, Nanog and CD1 17.
  • at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population express two or more of the biomarkers NCAM, Slug/Snail, CD24, and Twist.
  • the population of ovarian cancer stem cells expressing at least one tumor-specific antigen with mesenchymal characteristics includes, but is not limited to, loss of expression of at least one of epithelial biomarkers CK8, CK18 and EpCAM, wherein loss of expression of a biomarker refers to undetectable expression or expression in 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1 % or less, 0.5% or less, 0.1 % or less of the cells, and an increase in expression of at least one of the mesenchymal biomarkers Slug/Snail, Twist, CD44, NCAM, N- cadherin, and vimentin in at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%
  • the population of colon cancer stem cells expressing at least one tumor-specific antigen with mesenchymal characteristics includes, but is not limited to, an increase in the expression of at least one of the mesenchymal markers Slug/Snail, CD44, Twist, N-cadherin, and vimentin in at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population express two or more of the biomarkers N-cadherin, Slug/Snail, vimentin, Twist and CD1 17.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population express two or more of the biomarkers N-cadherin, Slug/Snail vimentin, CD1 17, CD44, CD24, ⁇ -synuclein, FMNL2, b-catenin, Nanog, CD147, 3GhT8, LGR5, CD29, CXCFr, CD133, and DCIkl .
  • at least 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population express two or more of the biomarkers N-cadherin, Slug/Snail, vimentin, Twist and CD1 17.
  • the population of glioblastoma multiforme stem cells expressing at least one tumor-specific antigen with
  • mesenchymal characteristics includes, but is not limited to, expression of two or more of the biomarkers Slug/Snail, Sox10, Twist, vimentin and N-cadherin by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population. According to some embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population express two or more of the biomarkers Slug/Snail, Sox10, Twist, vimentin, N-cadherin, nestin, S100, P53 and Ki-67.
  • At least 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population express two or more of the biomarkers Slug/Snail, Sox10, Twist, vimentin and N-cadherin.
  • the population of live tumor cell- derived cancer stem cells comprises a population of "early” cancer stem cells with characteristics of embryonic stem cells.
  • at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" hepatocellular cancer stem cells express two or more of biomarkers EpCAM, E-cadherin, Nanog, Sox2, Sox7, Sox17, Oct3/4, Fox2A, Ov1 , OV6, c-kit, CD133, and CD90.
  • At least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" hepatocellular cancer stem cells express two or more of biomarkers EpCAM, E-cadherin, Nanog, Sox2, Sox7, Sox17, Oct3/4, Fox2A, Ov1 , OV6, c-kit, CD133 and CD90.
  • at least 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early” hepatocellular cancer stem cells express two or more of biomarkers Nanog, Sox2, Oct3/4, and c-kit.
  • At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" vascular mimicry stem cells express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, and Ki-67.
  • at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 of the cells in the population of "early” vascular mimicry stem cells express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, Ki-67, TGF R, and CD24.
  • At least 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" vascular mimicry stem cells express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, and Ki- 67.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" ovarian cancer stem cells express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17 and Ki-67.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" ovarian cancer stem cells express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, Ki-67, CA-125, MUC-1 , TGF R, and CD24.
  • at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early” ovarian cancer stem cells express two or more of biomarkers EPCAM; CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, and Ki-67.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" colon cancer stem cells express two or more of biomarkers Nanog, Sox2, Oct3/4, c-kit, FoxA2 and CD133.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" colon cancer stem cells express two or more of biomarkers Nanog, Sox2, Oct3/4, c-kit, FoxA2, CD133, EpCAM, E-cadherin, Sox7, Sox17, ALDH1 A1 , LGR5, Hes1 , DPPA2, CD9, KRAS, ESA, BMI1 , CD166, CD24, CD29, CD44, CD166 and CDCP1 .
  • At least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" colon cancer stem cells express two or more of biomarkers Nanog, Sox2, Oct3/4, c-kit, FoxA2 and CD133.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" glioblastoma multiforme cancer stem cells express two or more of biomarkers CD133, Oct3/4, Sox2, CD271 , nestin, Nanog, and CD15.
  • the population of live tumor cell- derived cancer stem cells comprises a population of cancer stem cells with mixed stem cell characteristics having a mixed differentiation profile.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of hepatocellular cancer stem cells with mixed stem cell characteristics express two or more of biomarkers AFP, CK7, CK19, EpCAM, E-cadherin, Nanog, FoxA2, HNF4a, and ABCG2.
  • At least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of hepatocellular cancer stem cells with mixed stem cell characteristics express two or more of biomarkers AFP, CK7, CK19, EpCAM, E- cadherin, Nanog, FoxA2, HNF4a and ABCG2.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of vascular mimicry cancer stem cells with mixed stem cell characteristics express two or more of biomarkers ABCG2, CD133, CD24, CD44, CD34, CD1 17, CK8, EpCAM, Ki-67, Nanog, N-cadherin, NCAM, Oct3/4, Slug/Snail, Twist, vimentin, ALDH, TGF R, Sox2, EGFR, nestin, TP53, VEGF-R1 , VEGF-R2, VE-Cadherin, VEGF-A, vWF, PECAM, and UEA-1 .
  • At least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of vascular mimicry cancer stem cells with mixed stem cell characteristics express two or more of biomarkers ABCG2, CD133, CD24, CD44, CD34, CD1 17, CK8, EpCAM, Ki-67, Nanog, N-cadherin, NCAM, Oct3/4, Slug/Snail, Twist, vimentin, ALDH, TGF R, Sox2, EGFR, nestin, TP53, VEGF-R1 , VEGF-R2, VE-cadherin, VEGF-A, vWF, PECAM and UEA-1 .
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of ovarian cancer stem cells with mixed characteristics express two or more of biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18 and Ki-67.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of ovarian cancer stem cells with mixed characteristics express two or more of biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18, Ki-67, CA 19-9, Her2/neu, NCAM, ganglioside CD2, esterogen receptor alpha, testosterone TGF R, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin and TP53.
  • At least about 90%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of ovarian cancer stem cells with mixed stem cell characteristics express two or more of biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18 and Ki-67.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of colon cancer stem cells with mixed characteristics express two or more of biomarkers Hes1 , MSI1 , ALDH1 B1 , ALDH1 A1 , EpCAM, -CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki67, AFP and DCIkl .
  • biomarkers Hes1 , MSI1 , ALDH1 B1 , ALDH1 A1 , EpCAM, -CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki67, AFP and DCIkl According to some embodiments, MSI1 , ALDH1 B1 , ALDH1 A1 , EpCAM, -CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG
  • At least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of colon cancer stem cells with mixed characteristics express two or more of biomarkers HES1 , MSI1 , ALDH1 B1 , ALDH1 A1 , EpCAM, G-CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki-67, AFP and DCIkl .
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of glioblastoma multiforme cancer stem cells with mixed characteristics express two or more of biomarkers CD133, nestin, beta-tubulin III, GFAP, 01 , EphA2, ABCG2, EGFRvlll, survivin, vimentin, S100, PDGF-Ra, NSE, nestin, MAGE1 , CD271 , TRP2, NG2, CD44, ALDH and P53.
  • the isolated population of dendritic cells comprises at least one tumor-specific cancer cell antigen.
  • CD83 is expressed by at least some of the isolated population of dendritic cells comprising at least one tumor-specific cancer cell antigen.
  • the therapeutic amount of the isolated population of dendritic cells comprises 1 x 10 3 or more dendritic cells contacted and activated ex vivo with a cancer cell-specific antigen derived from a population of patient-specific isolated cancer cells sourced from circulating tumor cells (CTCs).
  • CTCs circulating tumor cells
  • the dendritic cells are immune adjuvant-activated dendritic cells.
  • the immune adjuvant for activating dendritic cells comprises a TLR agonist or an adjuvant.
  • the TLR agonist is selected from CpG-oligonucleotide (TLR9), imiquimod (TLR7), poly(l:C) (TLR3), glucopyranosyl lipid A (TLR4), murein (TLR2), flagellin (TLR5).
  • the adjuvant is a CD40 agonist.
  • the CD40 agonist is selected from a CD40-ligand, interferon-gamma, or prostaglandin E2.
  • the stimulated effective immune response comprises an effective T cell response, an effective B cell response, or both.
  • the therapeutic amount is immunostimulatory and is effective (1 ) to stimulate an effective immune response; and (2) to improve a clinical parameter selected from progression-free survival, disease-free survival, time to progression, time to distant metastasis, overall survival of the subject when compared to dendritic cells not displaying the tumor-specific stem cell antigen or a suitable control.
  • a stimulated effective immune response comprises a population of cancer-antigen stimulated dendritic cells interacting with a T cell population, resulting in activation and proliferation of antigen- specific T cells.
  • the T cells are CD4+ T cells, CD8+ T cells, or a combination thereof.
  • a stimulated effective immune response comprises a population of cancer-antigen stimulated dendritic cells interacting with a B cell population, resulting in activation and proliferation of antigen- specific B cells.
  • an effective B cell response comprises production of antibodies that specifically recognize a cancer stem cell antigen that mediates antibody dependent cell cytotoxicity (ADCC).
  • ADCC antibody dependent cell cytotoxicity
  • the antibody-dependent cell cytotoxicity is mediated by NK cells.
  • the described invention provides a method for preparing an immunopotent tumor-specific cancer stem cell product comprising an immunopotent immunogenic composition comprising a therapeutic amount of an isolated population of dendritic cells activated in vitro with a tumor- specific antigen derived from a population of purified circulating tumor cells (CTCs) derived from a patient.
  • an immunopotent immunogenic composition comprising a therapeutic amount of an isolated population of dendritic cells activated in vitro with a tumor- specific antigen derived from a population of purified circulating tumor cells (CTCs) derived from a patient.
  • CTCs purified circulating tumor cells
  • Part A Preparing a tumor-specific population of cancer stem cells expressing a tumor-specific antigen.
  • STEP 1 Obtaining a tumor sample comprising a tumor cell expressing a tumor specific antigen.
  • the tumor sample expressing a tumor specific antigen is derived from a solid tumor.
  • Examples of solid tumors include, without limitation, a melanoma, a tissue of endodermal, mesodermal, or ectodermal origin (e.g., melanoma of neural crest origin, colon cancer of endoderm origin, renal cancer of mesoderm origin, glioblastoma of ectoderm origin, ovarian cancer of mixed mesoderm plus extraembryonic origin) a hepatocellular carcinoma, colon carcinoma, ovarian carcinoma, glioblastoma multiforme, and tumor-cell derived vasculogenic-like cells, the source of which, for example is uveal melanoma, cutaneous melanoma, breast cancer, ovarian cancer, prostate cancer, and glioblastoma multiforme.
  • a melanoma a tissue of endodermal, mesodermal, or ectodermal origin
  • a melanoma of neural crest origin e.g., colon cancer of endoderm
  • the tumor sample expressing a tumor specific antigen derived from a solid tumor expressing a tumor specific antigen is a tumor cell circulating in peripheral blood.
  • the live tumor cells in the peripheral blood of the subject are in cell clusters.
  • the cell clusters are aggregates of cells.
  • the aggregates of cells originate from the tumor and enter the vasculature.
  • the method comprises expanding the number of tumor cells expressing a tumor specific antigen in vitro by
  • step (d) Feeding cultures containing the adherent cells of step (c) every 2 days with fresh medium;
  • the adherent substrate contains one or more Arginine-Glycine-Aspartic Acid (RGD)-rich compound and a serum-free medium.
  • the fresh medium consists of a basal stem cell formulation supplemented with a protein/growth factor mixture.
  • the growth factor is a fibroblast growth factor (FGF), epidermal growth factor (EGF), or both.
  • spheroids of cancer stem cells are characterized by expression of biomarkers.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the hepatocellular cancer stem cell spheroid population express two or more of biomarkers AFP, CK7, CK19, EpCAM, E-cadherin, Ov1 and OV6.
  • at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the hepatocellular cancer stem cell spheroid population express two or more of biomarkers AFP, EpCAM, Ov1 and Ov6.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in a purified the hepatocellular cancer stem cell spheroid population express one or more of biomarkers ABCG2, AFP, CD133, CD44, CD90, CK19, CK7; c-KIT, E-cadherin, EpCAM, FoxA2, HNF4a, Ki67, Nanog, N- cadherin, NCAM (CD56), Oct3/4, Ov1 , OV6, Slug/Snail, Sox17, Sox2, Sox7, Twist and vimentin.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the vascular mimicry cancer stem cell spheroid population express two or more biomarkers EpCAM, CD1 17, ALDH, CD133, CD24, Ki-67, NCAM, vimentin, CK8, TGF R, EGFR, CD44, ABCG2, Slug/Snail, nestin and TP53.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the vascular mimicry cancer stem cell spheroid population express two or more of biomarkers EpCAM, CD1 17, ALDH, CD133, CD24 and Ki-67. According to some embodiments, at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the vascular mimicry spheroid population express two or more of biomarkers EpCAM, CD1 17, ALDH, CD133, CD24, and Ki-67.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the colon cancer stem cell spheroid population express two or more biomarkers CD133, Hes1 , CD44, CD24, CD166 and CD29.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the colon cancer stem cell spheroid population express two or more of biomarkers CD133, Hes1 , CD44, CD24, CD166, CD29, CK7, CK19, E-cadherin, CK20, ESA, ALDH, CDX1 , LGR5, and DCIkl .
  • at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the colon cancer stem cell spheroid population express two or more of biomarkers CD133, Hes1 , CD44, CD24, CD166 and CD29.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the ovarian cancer stem cell spheroid population express two or more biomarkers EpCAM, CA-125, MUC-1 , CD1 17, He-4, ALDH, CD133, CD24, Ki-67, CA19-9, HER/neu, NCAM, galglioside CD2, estrogen receptor alpha, vimentin, CK8, CK18, AFP, testosterone, TGF R, EGFR, TAG-72, CD46, CD44, ABCG2, Slug/Snail, nestin and TP53.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the ovarian cancer stem cell spheroid population express two or more of biomarkers AFP, CK7, CK19, EpCAM, E-cadherin, Ov1 and OV6. According to some embodiments, at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the colon cancer stem cell spheroid population express two or more of biomarkers EpCAM, CA-125, MUC-1 , CD1 17, He-4, ALDH, CD133, CD24 and Ki-67.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the glioblastoma multiforme (GBM) cancer stem cell spheroid population express two or more biomarkers CD133, nestin, Sox2 and CD271 .
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the GBM cancer stem cell spheroid population express two or more of biomarkers CD133, nestin, Sox2, CD271 , EphA2, EGFRvlll, Her2, L1 CAM, beta tubulin III, GFAP, and 04.
  • at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the colon cancer stem cell spheroid population express two or more of biomarkers CD133, nestin, Sox2 and CD271 .
  • step (h) the spheroids are gently dissociated by exposure to collagenase IV and mechanical pipetting.
  • step (h) the spheroids are gently dissociated by exposure to dispase 2U/ml_ for 24-48 hours in the culture media.
  • step (e) for those CSCs sensitive to physical and enzymatic manipulation during passaging, the method comprises:
  • STEP 3 Differentiating cancer stem cells
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" hepatocellular cancer stem cells express two or more of biomarkers EpCAM, E-cadherin, Nanog, Sox2, Sox7, Sox17, Oct3/4, Fox2A, Ov1 , OV6, c-kit, CD133, and CD90.
  • At least at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" hepatocellular cancer stem cells express two or more of biomarkers EpCAM, E-cadherin, Nanog, Sox2, Sox7, Sox17, Oct3/4, Fox2A, Ov1 , OV6, c-kit, CD133 and CD90.
  • at least at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early” hepatocellular cancer stem cells express two or more of biomarkers Nanog, Sox2, Oct3/4, and c-kit.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" vascular mimicry stem cells express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, and Ki-67.
  • at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early” vascular mimicry cells express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, Ki-67, TGF R, and CD24.
  • At least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" vascular mimicry cells express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, and Ki-67.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" ovarian cancer stem cells express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17 and Ki-67.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" ovarian cancer stem cells express two or more of biomarkers EpCAM, CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, Ki-67, CA-125, MUC-1 , TGF R, and CD24.
  • at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early” ovarian cancer stem cells express two or more of biomarkers EPCAM; CD133, CD44, Nanog, Sox2, Oct3/4, CD1 17, and Ki-67.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" colon cancer stem cells express two or more of biomarkers Nanog, Sox2, Oct3/4, c-kit, FoxA2 and CD133.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" colon cancer stem cells express two or more of biomarkers Nanog, Sox2, Oct3/4, c-kit, FoxA2, CD133, EpCAM, E-cadherin, Sox7, Sox17, ALDH1 A1 , LGR5, Hes1 , DPPA2, CD9, KRAS, ESA, BMI1 , CD166, CD24, CD29, CD44, CD166 and CDCP1 .
  • At least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" colon cancer stem cells express two or more of biomarkers Nanog, Sox2, Oct3/4, c-kit, FoxA2 and CD133.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early" glioblastoma multiforme cancer stem cells express two or more of biomarkers CD133, Oct3/4, Sox2, CD271 , nestin, Nanog, and CD15.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of "early” of the cells in the population of "early” GBM cancer stem cells express two or more of biomarkers CD133, Oct3/4, Sox2, CD271 , nestin, Nanog, CD15, Sox3, EGFR, vimentin, S100 and CD44.
  • at least about 90%95%, 96%, 97%, 98%, 99% of the cells in the population of "early” GBM cancer stem cells express two or more of biomarkers CD133, Oct3/4, Sox2, CD271 , nestin, Nanog and CD15.
  • about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mixed hepatocellular cancer stem cells express two or more of biomarkers AFP, CK7, CK19, EpCAM, E-cadherin, Nanog, FoxA2, HNF4a, and ABCG2.
  • at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mixed hepatocellular cancer stem cells express two or more of biomarkers AFP, CK7, CK19, EpCAM, E-cadherin, Nanog, FoxA2, HNF4a and ABCG2.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mixed vascular mimicry cancer stem cells express two or more of biomarkers ABCG2, CD133, CD24, CD44, CD34, CD1 17, CK8, EpCAM, Ki-67, Nanog, N-cadherin, NCAM, Oct3/4, Slug/Snail, Twist, vimentin, ALDH, TGF R, Sox2, EGFR, nestin, TP53, VEGF-R1 , VEGF-R2, VE-Cadherin, VEGF-A, vWF, PECAM, and UEA-1 .
  • At least 1 at least 1 .
  • at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mixed vascular mimicry cancer stem cells express two or more of biomarkers ABCG2, CD133, CD24, CD44, CD34, CD1 17, CK8, EpCAM, Ki-67, Nanog, N-cadherin, NCAM, Oct3/4, Slug/Snail, Twist, vimentin, ALDH, TGF R, Sox2, EGFR, nestin, TP53, VEGF-R1 , VEGF-R2, VE-cadherin, VEGF-A, vWF, PECAM and UEA-1 .
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mixed ovarian cancer stem cells express two or more of biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18 and Ki-67.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mixed ovarian cancer stem cells express two or more of biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18, Ki-67, CA 19-9, Her2/neu, NCAM, ganglioside CD2, esterogen receptor alpha, testosterone TGF R, EGFR, TAG-72, CD46, He-4, ALDH, CD133, CD44, ABCG2, nestin and TP53.
  • At least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of in the mixed ovarian cancer stem cell population express two or more of biomarkers EpCAM, CA-125, MUC-1 , CD1 17, CK8, CK18 and Ki-67.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mixed colon cancer stem cells express two or more of biomarkers Hes1 , MSI1 , ALDH1 B1 , ALDH1 A1 , EpCAM, -CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki67, AFP and DCIkl .
  • At least at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mixed colon cancer stem cells express two or more of biomarkers HES1 , MSI1 , ALDH1 B1 , ALDH1 A1 , EpCAM, G-CSF, Hiwi, CD44, CD49f, ESA, EphBR, ABCG2, NCAM, Ki- 67, AFP and DCIkl .
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mixed glioblastoma multiforme cancer stem cells express two or more of biomarkers CD133, nestin, beta-tubulin III, GFAP, O1 , EphA2, ABCG2, EGFRvlll, survivin, vimentin, S100, PDGF-Ra, NSE, nestin, MAGE1 , CD271 , TRP2, NG2, CD44, ALDH and P53.
  • At least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mixed GBM cancer stem cells express two or more of CD133, nestin, beta-tubulin I II, GFAP, O1 , EphA2, ABCG2, EGFRvlll, survivin, vimentin, S100, PDGF-Ra, NSE, nestin, MAGE1 , CD271 , TRP2, NG2, CD44, ALDH, and P53.
  • EMT epithelial to mesenchymal transition
  • the term "mesenchymal-like tumor-specific CSC” or ⁇ -HCC-CSC (epithelial to mesenchymal transitioned (EMT) cancer stem cells)” is used to refer to a population in which the spheroids have undergone a process of EMT characterized by the loss of the expression of at least one epithelial marker, e.g., CK7, CK19, EpCAM and E-cadherin, and an increase in the expression of at least one mesenchymal markers, e.g., Slug/Snail (Slg/Snl), Twist, CD44, NCAM, N-cadherin, and vimentin.
  • epithelial marker e.g., CK7, CK19, EpCAM and E-cadherin
  • mesenchymal markers e.g., Slug/Snail (Slg/Snl), Twist, CD44, NCAM, N-cadherin, and vi
  • the population of hepatocellular cancer stem cells expressing at least one tumor-specific antigen with mesenchymal characteristics includes, but is not limited to, a loss of the expression of at least one of the epithelial markers CK7, CK19, EpCAM and E-cadherin; and an increase in the expression of at least one of the mesenchymal markers Slug/Snail, Twist, CD44, NCAM, N-cadherin, and vimentin to at least 30% of the cells in the population.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal vascular mimicry cancer stem cells express two or more of the biomarkers NCAM, Slug/Snail, CD24, and Twist. According to some embodiments,
  • At least about at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal vascular mimicry cancer stem cells express two or more of the biomarkers NCAM, Slug/Snail, CD24, Twist, N- cadherin, CD44, vimentin, CD133, Nanog and CD1 17. According to some
  • At least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal vascular mimicry cancer stem cells express two or more of the biomarkers NCAM, Slug/Snail, CD24, and Twist.
  • 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal ovarian cancer stem cells are characterized by a loss of expression of at least one of epithelial biomarkers CK8, CK18 and EpCAM, where loss of expresion of a biomarker refers to undetectable expression or expression in 40% or less of the cells, and an increase in expression of at least one of mesenchymal biomarkers Slug/Snail, Twist, CD44, NCAM, N- cadherin, and vimentin.
  • At least about 30%. 35%. 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal colon cancer stem cells are
  • the mesenchymal markers Slug/Snail, CD44, Twist, N-cadherin, and vimentin at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal colon cancer stem cells express two or more of the biomarkers N-cadherin, Slug/Snail, vimentin, Twist and CD1 17.
  • at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal colon cancer stem cells express two or more of the biomarkers N-cadherin,
  • at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal colon cancer stem cells express two or more of the biomarkers N-cadherin, Slug/Snail, vimentin, Twist and CD1 17.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal glioblastoma multiforme cancer stem cells express two or more of the biomarkers Slug/Snail, Sox10, Twist, vimentin and N-cadherin.
  • At least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal GBM cancer stem cells express two or more of the biomarkers Slug/Snail, Sox10, Twist, vimentin, N- cadherin, nestin, S100, P53 and Ki-67.
  • at least about 90%, 95%, 96%, 97%, 98%, 99% of the cells in the population of mesenchymal GBM cancer stem cells express two or more of the biomarkers Slug/Snail, Sox10, Twist, vimentin and N-cadherin.
  • STEP 4 Passaging and expanding the number of tumor-derived cancer stem cells in the cultures.
  • STEP 5 Inactivating the tumor-derived population of cancer stem cells expressing a tumor-specific antigen.
  • the inactivated population of live tumor cell derived cancer stem cells expressing a tumor-specific antigen is incapable of cell division but metabolically active.
  • the inactivated population of live tumor cell derived cancer stem cells expressing a tumor-specific antigen is incapable of cell division after exposure to radiant energy, temperature (e.g., heat or cold), chemical methods (e.g., cytostatic, aldehyde, alcohol), or a combination thereof.
  • radiant energy e.g., heat or cold
  • chemical methods e.g., cytostatic, aldehyde, alcohol
  • the inactivated population of live tumor cell derived cancer stem cells expressing a tumor-specific antigen is an irradiated cancer cell.
  • the term "irradiated” refers to a process by which a subject is exposed to radiation, meaning the process in which energy is emitted by one body, transmitted through an intervening medium or space, and absorbed by another body.
  • the radiation is selected from gamma-radiation, X-ray radiation, electron beam radiation, neutron beam radiation, proton beam radiation, electromagnetic radiation, visible light radiation, ultraviolet light radiation and the like.
  • irradiation functions to prevent cell division of the cancer stem cells.
  • irradiation prevents cell division, but also denatures cellular proteins.
  • the population of live tumor cell derived cancer stem cells expressing a tumor-specific antigen is a physically disrupted cancer cell.
  • methods for physical disruption include, without limitation, sonication, cavitation, dehydration, ion depletion, or by toxicity from exposure to one or more salts.
  • the inactivated population of live tumor cell derived cancer stem cells expressing a tumor-specific antigen is a cancer cell in which nucleic acids are cross-linked with a nucleic acid cross-linking agent.
  • nucleic acid targeting agents include, without limitation, beta-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester; psoralens in combination with ultraviolet (UVA) irradiation; 4'-(4-amino-2-oxa)butyl-4,5',8- trimethylpsoralen ("S-59”); and 150 micromolar of psoralen S-59 and 3 J/cm2 UVA light (FX 1019 irradiation device, Baxter Fenwal, Round Lake, IL). See, U.S. Pat. Nos. 7,833,775 and 7,691 ,393.
  • STEP 6 cryostoring the irradiated tumor-derived cells in a vapor phase liquid nitrogen storage at -86 Q C.
  • PART B preparing a population of immunopotent dendritic cells activated in vitro with a tumor-specific antigen derived from the population of purified cultivated tumor cells derived from the subject.
  • a method to prepare a population of immunopotent dendritic cells activated in vitro with a tumor-specific antigen derived from the population of purified cultivated tumor cells derived from the subject comprises:
  • PBMCs peripheral blood mononuclear cells
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs Peripheral blood mononuclear cells
  • DCs are purified from PBMCs by counter flow density centrifugation (elutriation), meaning a process by which monocytes are purified from other lymphocytes in order to enrich for cells that can be turned into APCs or DCs).
  • low density DCs can be prepared by gradient separation over a density gradient to obtain PBMCs, T-lymphocyte depletion
  • low density DCs can be prepared by gradient separation over a density gradient to obtain
  • PBMCs T-lymphocyte depletion (optional), overnight incubation for 16 hours in autologous serum/medium ( ⁇ cytokines), metrizamide gradient separation, and positive selection (e.g., flow cytometry, immunomagnetic beads) to obtain a lineage- negative, CD83+ cell population.
  • ⁇ cytokines autologous serum/medium
  • metrizamide gradient separation e.g., flow cytometry, immunomagnetic beads
  • purified PBMCs are incubated with GM-CSF and IL-4 for 6 days to generate DCs.
  • the method for preparing the immunopotent immunogenic composition comprises:
  • Step 1 Thawing the purified tumor cell product of Step 6;
  • Step 2 activating the population of dendritic cells in vitro by contacting the population of dendritic cells with a form of the population of tumor-specific cancer stem cells expressing a tumor-specific antigen by: Contacting the thawed irradiated tumor-derived cell antigen product with the activated dendritic cells for 18-24 hours to form an activated dendritic cell/tumor-derived cell antigen composition; and collecting and storing the dendritic cell/tumor-derived cell composition in vapor phase liquid nitrogen.
  • the dendritic cell presents a processed tumor-specific peptide within the dendritic cell.
  • the dendritic cell presents a tumor-specific processed peptide on a dendritic cell surface.
  • the tumor antigen derives from a tumor cell, tumor cell cluster, tumor cell aggregate, or a combination thereof derived from peripheral blood.
  • the source of the tumor cell, tumor cell cluster, tumor cell aggregate or combination thereof derived from peripheral blood is a solid tumor.
  • the immunopotent immunogenic composition can be prepared by transfection.
  • exemplary transfection methods include, but are not limited to, chemical-based methods and non-chemical based methods.
  • Chemical-based methods include, without limitation, calcium phosphate, highly branched organic compounds (e.g., dendrimers), liposomes, and cationic polymers (e.g., DEAD-destran, polyethylenimine).
  • Non-chemical based methods include, but are not limited to, cell squeezing, sonoporation, optical transfection, impalefection, hydrodynamic delivery, magnetofection, viral transduction,
  • MaxCyte® transfection technology (MaxCyte, Inc., Gaithersburg, MD) is based on the general principles of electroporation, which involves the application of an electric field to a cell suspension, causing the cell membrane to become transiently permeable and encouraging external material to enter the cell.
  • MaxCyte® electroporation enables transfection of a wide range of cells with loading efficiencies exceeding 90%.
  • the technology is highly scalable from, for example, 5x10 5 cells in seconds using small-scale, static electroporation to 1 x10 10 cells in less than 30 minutes using flow electroporation. MaxCyte® scalable electroporation has been successfully applied in ex vivo cell therapy, protein production and drug discovery pipelines where reproducibility, efficiency and the need for increased cell numbers are critical.
  • the MaxCyte® system can perform small and large-scale electroporation using a single instrument and a single electroporation protocol.
  • the term "static electroporation” refers to small-scale electroporation in which cells and the material(s) to be transfected are mixed and placed into a small processing chamber that is then loaded into the instrument for electroporation ( Figure 2).
  • flow electroporation refers to large-scale electroporation in which cells and the material(s) to be transfected are mixed and placed into the source bag of a large-scale processing assembly.
  • the sterile processing assembly is then loaded onto the MaxCyte® instrument. For example, when cell processing begins, 3 ml_ fractions flow in succession through the electroporation chamber and continue into the cell collection bag. Up to 1 x10 10 cells can be transfected in less than 30 minutes using flow electroporation. Transfected cells can be used immediately following a 20 minute recovery period or cryopreserved for later use.
  • the form of the population of tumor-specific cancer stem cells expressing a tumor-specific antigen is as a population of whole cells, a population of live cells, a population of dead cells, a population of necrotic cells, a cell lysate, a cell extract, a purified tumor-specific antigen, fragment or variant thereof
  • the described invention provides a method for treating a subject suffering from a cancer, comprising:
  • a patient-specific immunogenic composition comprising a therapeutic amount of an isolated population of dendritic cells contacted ex vivo with a population of cancer stem cells expressing a cancer- specific antigen derived from a tumor cell, a tumor cell cluster, a tumor cell aggregate or a combination thereof derived from peripheral blood
  • the effective immune response is effective to improve a clinical parameter selected from progression-free survival (PFS), disease-free survival, time to progression, time to distant metastasis and overall survival (OS) of the subject when compared to a control.
  • PFS progression-free survival
  • OS overall survival
  • the tumor sample expressing a tumor specific antigen is derived from a tumor cell, a tumor cell cluster, a tumor cell aggregate or a combination thereof derived from peripheral blood.
  • the live tumor cells in the peripheral blood of the subject are in cell clusters.
  • the cell clusters are aggregates of cells.
  • the aggregates of cells originate from the tumor and enter the vasculature.
  • the described invention provides a composition for isolating/purifying CTCs from a blood sample.
  • the composition comprises a ROCK inhibitor.
  • ROCK inhibitors include, but are not limited to, Y-27632 2HCI (R&D Systems Inc., Minneapolis, MN ), Thiazovivin (StemRD, Burlingame, CA), Slx- 21 19 (MedChem Express, Namiki Shoji Cop., LTD), WF-536 [(+)-(R)-4-(1 -aminoethyl)- N-(4-pyridyl) benzamide monohydrochloride] (Mitsubishi Pharma Corporation, Osaka, Japan), RK1 -1447 (University of South Florida, Tampa, FL, and Moffitt Cancer Center, Tampa, FL; Pireddu R, et al.
  • concentration of the ROCK inhibitor in the composition ranges from about 1 ⁇ to about 20 ⁇ . According to some embodiments, concentration of the ROCK inhibitor ranges from about 5 ⁇ to about 10 ⁇ . According to some embodiments, the concentration of the ROCK inhibitor is 1 ⁇ . According to some embodiments, the concentration of the ROCK inhibitor is 2 ⁇ . According to some embodiments, the concentration of the ROCK inhibitor is 3 ⁇ . According to some embodiments, the concentration of the ROCK inhibitor is 4 ⁇ .
  • the concentration of the ROCK inhibitor is 5 ⁇ .
  • the concentration of the ROCK inhibitor is 6 ⁇ .
  • the concentration of the ROCK inhibitor is 7 ⁇ .
  • the concentration of the ROCK inhibitor is 8 ⁇ .
  • the concentration of the ROCK inhibitor is 9 ⁇ .
  • the concentration of the ROCK inhibitor is 10 ⁇
  • the concentration of the ROCK inhibitor is 1 1 ⁇
  • the concentration of the ROCK inhibitor is 12 ⁇
  • the concentration of the ROCK inhibitor is 13 ⁇
  • the concentration of the ROCK inhibitor is 14 ⁇
  • the concentration of the ROCK inhibitor is 15 ⁇
  • the concentration of the ROCK inhibitor is 16 ⁇
  • the concentration of the ROCK inhibitor is 17 ⁇
  • the concentration of the ROCK inhibitor is 18 ⁇
  • the concentration of the ROCK inhibitor is 19 ⁇
  • the concentration of the ROCK inhibitor is 20 ⁇
  • the composition comprises a PTEN inhibitor.
  • PTEN inhibitors include, but are not limited to, potassium bisperoxo(1 ,10-phenanthroline)oxovanadate (V) (bpV) (Sigma-Aldrich), SF 1670 (Tocris/R&D Systems), VO-OHpic (Tocris/R&D Systems) and the like.
  • concentration of the PTEN inhibitor ranges from about 0.01 ⁇ to about 10 ⁇ . According to some embodiments, the concentration of PTEN inhibitor ranges from about 0.5 ⁇ to about 2 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 0.01 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 0.025 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 0.05 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 0.075 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 0.1 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 0.25 ⁇ .
  • the concentration of the PTEN inhibitor is 0 5 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 0 75 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 1 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 2 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 3 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 4 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 5 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 6 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 7 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 8 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 9 ⁇ . According to some embodiments, the concentration of the PTEN inhibitor is 10 ⁇
  • the tumor includes, but is not limited to, a melanoma, a tissue of endodermal, mesodermal, or ectodermal origin (e.g., melanoma of neural crest origin, colon cancer of endoderm origin, renal cancer of mesoderm origin, glioblastoma of ectoderm origin, ovarian cancer of mixed mesoderm plus extra-embryonic origin) a hepatocellular carcinoma, colon carcinoma, ovarian carcinoma, glioblastoma multiforme, and tumor-cell derived vasculogenic-like cells, the source of which, for example is uveal melanoma, cutaneous melanoma, breast cancer, ovarian cancer, prostate cancer, and glioblastoma multiforme.
  • a tissue of endodermal e.g., melanoma of neural crest origin, colon cancer of endoderm origin, renal cancer of mesoderm origin, glioblastoma of ecto
  • Dendritic cells were generated by a plastic adherence method as described by Choi and Lucas (Choi (1998) Clin. Cancer Res. 4:2709-2716; Heil (1998) Exp. Hematol. 26:489-500). Briefly, patients underwent leukapheresis (i.e., the filtering of blood to collect peripheral blood mononuclear cells (PBMCs) (about 6 liters). The autologous leukapheresis product was subjected to ficoll-hypaque (GE Healthcare, Buckinghamshire, United Kingdom) density gradient separation.
  • leukapheresis i.e., the filtering of blood to collect peripheral blood mononuclear cells (PBMCs) (about 6 liters).
  • PBMCs peripheral blood mononuclear cells
  • the autologous leukapheresis product was subjected to ficoll-hypaque (GE Healthcare, Buckinghamshire, United Kingdom) density gradient separation.
  • the resulting PBMCs were placed in antibiotic-free AIMV medium (Invitrogen, Grand Island, NY) supplemented with 1 ,000 lU/mL each of IL-4 (CellGenix, Freisberg, Germany) and GM-CSF (Berlex, Seattle, WA) (DC medium) at 15 x 10 8 cells/mL in cell cultivation flasks (Corning-Costar, Corning, NY). After one hour incubation, the non-adherent population was discarded and fresh DC medium was added to the flasks. The following morning, the non-adherent cells were discarded, the flasks were washed once with ambient temperature PBS, and fresh DC medium was added. The flasks were then cultivated for 6 days at which time flow cytometry evaluation was performed to determine the percentage and phenotype of DC generated by this approach (pre-load DC).
  • pre-load DC pre-load DC
  • Phenotypic characterization of the dendritic cell populations was performed using monoclonal antibodies against the following surface markers obtained from BD Pharmingen San Diego, CA: anti-MHC class II conjugated to PerCp, anti CD1 1 c conjugated to APC, anti-CD80, anti-CD83, anti-CD86 conjugated to PE. Isotype controls were used to determine percent positive cells.
  • CTCs can be isolated by targeting their physical properties, such as size, density, electric charges, deformability, and their biological properties, such as cell surface protein expression, and viability (Alix-Panabieres and Pantel, 2013).
  • a Parylene-C slot microfilter can be used to capture and enrich live CTCs from whole blood rapidly and efficiently based on differential size and deformability (Xu et al, 2010). Captured cells can be immunofluorescently stained for cancer-specific protein markers and imaged on the filter using a fully automated, multi-spectral scanning and imaging platform (Zeiss) specially configured for this purpose.
  • captured cells can be lysed for qPCR analysis, or picked cell-by-cell using a motorized micropipette apparatus (Eppendorf) specially configured for this application.
  • Eppendorf motorized micropipette apparatus
  • a LiquidBiopsy EpCAM-based microfluidic CTC capture platform (Cynvenio, Inc., Westlake Village, CA), a ClearCell FX size-based microfluidic CTC platform
  • Autologous CTCs can be characterized by the expression of cytokeratins, tumor specific markers, adhesion molecules, proliferation markers and/or EMT markers and can be detected by flow cytometry or
  • Autologous CTCs can be exposed to 100 Gy radiation in a cobalt 60 source irradiator. Irradiation efficiency can be confirmed by nonproliferative assay. Irradiated CTCs can either be used immediately to create autologous DC-TC vaccine or placed in vapor phase liquid nitrogen storage prior to use.
  • DC-CTC Dendritic Cell-Circulating Tumor Cell
  • Patient-specific vaccine compositions are prepared by co-incubating autologous circulating tumor cells with IFN- ⁇ . After incubation with IFN- ⁇ , the autologous circulating tumor cells are co-incubated with the activated autologous dendritic cells for 18 to 24 hrs.
  • patient-specific vaccine compositions are prepared by electroporation of autologous dendritic cells with a cell lysate prepared from cultures of circulating tumor cells incubated with IFN- ⁇ or from immunogenic proteins isolated from proliferating tumor cells or circulating tumor cells incubated with IFN- ⁇ .
  • the DC-CTC mixtures are collected and cryopreserved in vapor phase liquid nitrogen to retain viability of the DC-CTC population.
  • the DC-CTC mixtures are tested for activating dendritic cells using one or more immune adjuvants, for example, toll-like receptor (TLR) agonist (e.g., CpG-oligonucleotide (TLR9), imiquimod (TLR7), pol l :C (TLR3), glucopyranosyl lipid A (TLR4), murein (TLR2), flagellin (TLR5)), CD40 agonists (e.g., CD40-ligand), interferon-gamma, prostaglandin E1 , etc. (US Patent Nos. 7,993,659; 7,993,648, 7,935,804).
  • Dendritic activation is determined, for example, by ELISPOT assay, ICS assay and/or tetramer assay which determine an immune stimulatory amount of the composition compared to control.
  • Example 1 Use of Established Tumor Cell Lines to Determine the Viability and Proliferative Capacity of Circulating Tumor Cells (CTCs) in Blood
  • an established tumor cell line can be used as a proxy for CTCs in order to determine conditions that will make whole blood more conducive to CTC survival.
  • a spike/retrieval (S/R) system can be used to (i) introduce a pre-determined number of live tumor cells to whole blood collected in K3EDTA vacutainer tubes (BD Diagnostics, Franklin Lakes, NJ) or to Hanks
  • HBSS Balanced Salt Solution
  • a tumor cell line representative of a highly metastatic cancer for example, melanoma (Sk-Mel-28), breast (BT20) or non-small lung cancer (NCI-H460) can be obtained.
  • the acquired line can be expanded and then frozen in a small working bank containing approximate 50 vials of 1 x10 6 cells/vial.
  • the viability of the cells in the frozen bank can be tested by thawing one random vial and performing a standard viability test (e.g., trypan blue exclusion).
  • Whole blood can be freshly obtained from consented healthy donors.
  • the blood samples can be stored refrigerated during transportation (4°C) and used immediately (within 2-3 hours from collection).
  • vacutubes can be mixed with various numbers of cells from a cryopreserved stock. Two days prior to mixing, the tumor cells can be thawed and maintained in culture conditions to restore metabolic and proliferative activity. The culture can be harvested by enzymatic dissociation and the cell count and viability can be recorded. The cell pellet can be re-suspended in a minimum amount of saline and used for five dilutions in 1 mL of blood each (Table 2 and Figure 13). Another set of mixtures with identical concentrations of tumor cells can be made using Hanks Balanced Salt Solution (HBSS) instead of blood. The mixtures can be analyzed immediately for tumor cell retrieval.
  • HBSS Hanks Balanced Salt Solution
  • Table 2 Exemplary experimental set up for spiking 1 ml_ of anticoagulated whole blood or HBSS with various concentrations of tumor cells
  • the laced blood and HBSS mixtures can be subjected to a flow- cytometric analysis. Identical procedures of preparation and labeling can be performed on each sample.
  • the blood sample can be washed with saline solution and the red blood cells can be lysed with FACS Lysing Solution (BD Biosciences,
  • the mixture can be labeled for CD3 (T cell specific antigen) and
  • CD45 leucocyte common antigen
  • 7-AAD 7-Aminoactinomycin D
  • Ficoll density gradient GE Healthcare Bio-Sciences, Pittsburgh, PA
  • the buffy coat can be subjected to FACS analysis ( Figure 14).
  • the viable tumor cells are CD3/CD45/7ADD negative.
  • the test can be repeated 3 times as in Table 2 and the results from blood and HBSS can be statistically analyzed.
  • the accuracy of the retrieval system can be validated and correction factors can be introduced if necessary.
  • this experiment can be used to establish a reliable threshold of detection, by setting an acceptable variance between determinations. Using power analyses, the minimum number of cells necessary for a reliable detection can be established. The detection threshold is necessary to establish a spiking concentration for the subsequent experiments that minimizes the detection system noise.
  • Dynabeads® enable isolation or depletion of CD45+ leukocytes. The remaining cells unbound to the beads can be collected from supernatant and analyzed for total number and viability. In addition, this method can be used to analyze the proliferative and spherogenic properties of the retrieved circulating tumor cells (Figure 15).
  • a baseline survival curve of the tumor cells can be established anticipating standard transportation conditions
  • a minimum volume of spiked blood sample and a reliable concentration of tumor cells can be used.
  • the samples can be exposed for 1 hour to room temperature, simulating blood collection and preparation for
  • the results of the retrieval can be included in a table and the survival plotted on a Kaplan-Meier (K-M) graph. This experiment can be used to establish a baseline survival of tumor cells exposed to blood.
  • K-M Kaplan-Meier
  • Blood is a composite of erythrocytes (red blood cells), leukocytes (white blood cells), platelets, protein rich plasma (e.g., albumin, coagulation proteins), nutrients/metabolites (glucose) and dissolved gases (i.e., oxygen and carbon dioxide).
  • protein rich plasma e.g., albumin, coagulation proteins
  • nutrients/metabolites glucose
  • dissolved gases i.e., oxygen and carbon dioxide
  • RTK FGF EGF
  • PDGF PDGF
  • NGF, HGF, IGF1 NGF, HGF, IGF1
  • Coagulation is a major concern for CTC viability in whole blood. Blood contains platelets and serum proteins (i.e., coagulation factors) that induce clotting as a mechanism to prevent blood loss. If drawn blood is not properly treated with an anti-coagulant, the blood will clot and trap/compromise the viability of the CTCs. Coagulation is a complex cascade process with many steps and layers of regulation (reviewed Furie et al., Cell 1988, 53:505-18). Despite this complexity, calcium (Ca 2+ ) is absolutely required for coagulation.
  • anti-coagulation agents e.g., heparin, sodium citrate, EDTA
  • K3 EDTA can be incorporated into these experiments using the recommended concentration of 1 .5 mg/mL of blood (NCCLS, 1996). It is unknown whether K3EDTA is cytotoxic to CTCs. Therefore, initial experiments can be performed to test the in vitro toxicity of K3EDTA.
  • a 96 well plate containing 10,000 cells/well in off-the-clot human serum (Valley Biomedical, Winchester, VA) or culture media can be subjected to various K3EDTA concentrations (0.0; 0.5, 1 .0; 1 .5 mg/mL) and analyzed for morphology and survival at 12, 24, and 48 hours at 37°C (Table 4).
  • K3EDTA concentrations 0.0; 0.5, 1 .0; 1 .5 mg/mL
  • Metabolism One milliliter (1 ml_) of blood contains 4 - 8 million erythrocytes, which accounts for 40-45% of the blood volume.
  • An erythrocyte uses glucose and glycolysis as its sole source of energy. Pyruvate is the end product of glycolysis.
  • pyruvate In cells with mitochondria and oxidative metabolism, pyruvate is converted completely into CO 2 and H 2 O, termed aerobic glycolysis (Feig et al., Journal of clinical Investigation 1972, 51 :1547-54). In erythrocytes, which lack mitochondria and oxidative metabolism, pyruvate is reduced to lactic acid (the product of anaerobic glycolysis). As peripheral blood is collected from a venous source, the oxygen load is already diminished. It is expected that blood kept at body temperature will rapidly consume the glucose from the serum, decrease O 2 levels, and increase the concentration of CO 2 and lactic acid. As a consequence, hydrogen ions accumulate and can decrease the pH of the blood (acidosis).
  • Oxygenation of the blood sample is not practical after collection in vacutainer tubes. Measures that reduce the basal metabolism and consequently the oxygen and energetic substrate consumption should be in place for any transportation system. A "hibernation" status could be accomplished by simple reduction in the storage temperature during transportation and manipulations. The effect of temperature induced hibernation can be tested by subjecting whole blood samples spiked with tumor cells to ambient temperature (18°C) and 4 °C (Table 4).
  • supplementing whole blood with cell culture medium can improve survival by increasing metabolic substrate (e.g., glucose, L-glutamine) levels, adding metabolic substrate (e.g., glucose, L-glutamine) to the metabolic substrate (e.g., glucose, L-glutamine) levels, adding metabolic substrate (e.g., glucose, L-glutamine) to the metabolic substrate (e.g., glucose, L-glutamine) levels, adding metabolic substrate (e.g., glucose, L-glutamine) levels, adding metabolic substrate
  • metabolic substrate e.g., glucose, L-glutamine
  • the addition of cell culture medium can also produce a dilution of the whole blood that may be beneficial for survival by reducing cell density.
  • the culture medium can contain: a basal formulation such as DMEM:F12 supplemented with insulin, glutathione, putrescine, and HEPES buffer.
  • whole blood can be diluted with cell culture medium or a specialized commercial hibernation media (e.g., HypoThermosol®, Sigma-Aldrich, St. Louis, MO; or Prime-XV®, Irvine Scientific, Santa Ana, CA) and tested with the lacing/retrieval system (Table 4).
  • Hemolysis of erythrocytes during storage and shipment could reduce O 2 levels in the blood due to released hemoglobin.
  • the addition of the hemoglobin-binding glycoprotein heptaglobin can be used to scavenge free hemoglobin and prevent oxygen reduction (Ascenzi et al., IUBMB Life 2005, 57:749-59).
  • the inclusion of heptaglobin has the added benefit of acting as an anti-inflammatory agent when complexed with hemoglobin. More specifically, during inflammation, macrophages express the cell surface receptor CD163 which binds heptaglobin-hemoglobin complexes and mediates its endocytosis.
  • heptaglobin 1 -1 Three different phenotypic variants of the heptaglobin gene exist in human: heptaglobin 1 -1 , 2-1 and 2-2 (Langlois et al., Clinical Chemistry 1996, 42:1589-600). These heptaglobin polymorphisms have different abilities to interact with CD163 when bound to hemoglobin. For example, heptaglobin 1 -1 interacts weakly with CD163, heptaglobin 2-1 moderately binds to CD163 and heptaglobin strongly interacts with CD163.
  • the cytoprotective effect of heptaglobin 2-2 on CTCs viability at concentrations of 10 ⁇ g/mL and 100 ⁇ g/mL can be determined (Table 4).
  • Nitric Oxide and free radicals The effect of the NO scavenger carboxy-PTIO at concentrations of 10 and 100 ⁇ on CTC viability in whole blood can be tested (Table 4). Cells in the blood rapidly consume dissolved oxygen and release nitric oxide (NO) as a specific response to hypoxia. High levels of NO may be cytostatic or cytotoxic for tumor cells, whereas low level activity of NO can have the opposite effect and promote tumor growth. Therefore, one challenge in keeping CTCs viable in blood up to 96 hours is the potential cytotoxic effect of NO.
  • NO nitric oxide
  • white blood cells neutrils, basophils, eosinophils, monocytes, and lymphocytes. Commonly referred to as leukocytes, white blood cells are principally responsible for both the innate and adaptive immune systems.
  • Biochemical and physiological perturbations during whole blood storage can decrease the pH and alter metabolite levels triggering immune responses
  • a cocktail of cytokines that enhance the survival pathways of the cells can be used to extend the viability of CTCs.
  • cytokines include, but are not limited to, ligands for receptor tyrosine kinases (e.g., FGF, EGF, PDGF, NGF and HGF), TGF beta proteins, Rho kinase (ROCK) inhibitors and PTEN inhibitors.
  • FGF receptor tyrosine kinases
  • EGF EGF
  • PDGF EGF
  • NGF and HGF TGF beta proteins
  • Rho kinase (ROCK) inhibitors Rho kinase
  • PTEN inhibitors e.g., PTEN inhibitors.
  • CTCs enter the blood stream via an epithelial-to-mesenchymal (EMT) transition.
  • EMT epithelial-to-mesenchymal
  • EMT driven by the interplay between growth factor signaling activities, induces epithelial cells to lose their polarity and adhesion properties and adopt a migratory mesenchymal fate.
  • EMT supports metastasis through tumor-initiating cell dissociation from the primary tumor and spread to other sites in the body.
  • EMT epidermal growth factor
  • WNT WNT signaling pathway
  • TGF transforming growth factor beta
  • Notch Notch pathway
  • Hedgehog Hedgehog pathway
  • integral signaling either alone or in combination
  • Rho-associated protein kinases mediate various cellular functions such as cell shape, motility, secretion, proliferation, and gene expression.
  • ROCK expression is increased in metastatic tumors and is believed to contribute to invasive properties (Liu et al., Cancer Research 2009, 69:8742-51 ). In other tumors, increased ROCK expression causes tumor inhibition (Castro et al. Stem Cells 2013, 31 :12-22).
  • ROCK inhibitors e.g., Y27632
  • Y27632 in embryonic stem cell culture systems permits survival of isolated cells at clonal dilutions. Therefore, CTC survival in the presence of blood and Y27632 can be tested (Table 4).
  • PI3K phosphoinositide 3-kinase
  • ESC embryonic stem cells
  • Sox2 transcription factors involved in self-renewal of ESCs.
  • PI3K pathway is known to be negatively regulated by phosphatase and tensin homolog deleted on chromosome ten (PTEN), a well-known tumor suppressor that is deleted or mutated in various types of cancer. Inhibition of PTEN tumor suppressor promotes the generation of induced pluripotent stem cells (Liao et al., Molecular Therapy 2013, 21 :1242-50).
  • Tumor cells can be seeded at a density of 10,000 cells/well on cell culture treated plastic in a media that contains RPMI (Sigma-Aldrich, St. Louis, MO) and 5% FBS
  • 96-well plates offer the advantage that multiple growth factor combinations can be tested in parallel and provide a convenient platform for cell proliferation assays (e.g., MTT, EMD Millipore, Billerica, MA).
  • cell proliferation assays e.g., MTT, EMD Millipore, Billerica, MA.
  • a plate containing a group of factors in 3 replicates, and one control line with no factors added can be prepared.
  • the blood will be removed by aspiration, cells rinsed with HBSS and cell culture media added.
  • a second control line on each plate can be seeded containing a serial dilution of increasing cell densities from 1000 to 50,000 cells per well in culture media.
  • the plate can be incubated for 24 hours in a 37°C/CO 2 incubator. After incubation, an MTT assay can be performed to assess cell viability. The replicates can be averaged and the readings can be normalized to the serial diluted line to determine the viable cell number. Each test factor condition can be compared to a control (no factor condition). These experiments can be used to determine the factors that are significant for cell survival by analyzing the survival curves at 12; 24; 36; 48; 60; 72; 84; 96; 108 hours.
  • experiments can be mixed with blood collected on K3EDTA laced with tumor cells.
  • the mixture can be incubated at the optimal storage temperature in parallel with a control mixture not containing survival factors.
  • the mixture can be analyzed with the validated S/R method and compared to baseline survival curve. This experiment can be used to confirm that the composition of the survival factor cocktail (SFC), when added to whole blood, contributes to the extended viability of CTCs.
  • SFC survival factor cocktail
  • Cells retrieved by Ficoll gradient and Dynabead separation can be used to demonstrate the proliferative and spherogenic ability of tumor cells in collected blood.
  • Retrieved cells can be plated in a cell culture system consisting of polystyrene cell culture vessels (flasks, 6-well plate) and media that is formulated for cell expansion.
  • cells not exposed to blood samples can be plated at the same density and used as a control.
  • the expansion rate can be compared to the control cells, and phenotypical analyses with specific markers (e.g., CD146, CD271 ) for the cell line can be used to confirm the preservation of phenotype and proliferative capability.
  • a spherogenic assay can be initiated by dissociating the adherent cultures and transferring the cells in ultra-low adherent cell culture vessels (Corning, Tewksbury, MA) in serum-free media.
  • the spherogenic assay has been routinely used with cancer stem cells to definitively demonstrate the intrinsic properties of 'stem cells' of self-renewal in an isolated sub-population of tumor cells (Yuan et al, Oncogene 2004, 23: 9392-9400).
  • SFC Survival Factors Cocktail
  • the survival factor cocktail can be tested on a second solid carcinoma type tumor (e.g., melanoma, hepatocellular carcinoma, ovarian carcinoma, colon carcinoma, GBM, breast or non-small lung).
  • a second solid carcinoma type tumor e.g., melanoma, hepatocellular carcinoma, ovarian carcinoma, colon carcinoma, GBM, breast or non-small lung.
  • Recovered cells after Ficoll and CD45-Dynabeads separation can be subjected to cell proliferation assays and spherogenic assays. It has been observed that the majority of carcinomas that are successfully cultivated towards isolation of tumor initiating cells are spherogenic in appropriate conditions. In some cases, tumor initiating cell phenotype can be demonstrated by the presence of markers such as CD133 in ovarian cancer side population or CD20 in some cases of non-spherogenic melanoma cell lines. The proliferative potential can be assessed by cultivation for 3 passages and establishment of growth curves in comparison to equivalent starting cell numbers that were not exposed to the blood
  • the results can be used to demonstrate that (i) the SFC can be applied to other tumor types; (ii) these tumor cells maintain viability greater than 50%; and (iii) the tumor cells retain their proliferative and spherogenic/phenotypic characteristics.
  • CTCs Cancer stem cells circulating within the blood stream are known as circulating tumor cells (CTCs).
  • CTCs express the markers of cancer stem cells (CSCs) that have adopted an epithelial to mesenchymal transition (EMT) for increased motility and migration.
  • CTCs express biomarkers including, for example, N-cadherin, vimentin, CD44, and fibronectin (Liu et al., (2014) Stem Cell Reports 2:78-91 ; Mani et al., Cell 2008, 4:704-715).
  • CTCs isolated from blood for expansion and production of autologous circulating tumor cancer stem cell lines can be used to produce autologous dendritic cell-circulating tumor cell (DC-CTC) vaccines for the treatment of solid tumors.
  • DC-CTC autologous dendritic cell-circulating tumor cell
  • a double-blind study can be performed in which study patients can be randomized in a 2:1 ratio to receive either DC-CTC (autologous dendritic cells loaded with irradiated autologous circulating tumor cells in GM-CSF) or control (MC) (autologous PBMC in GM-CSF).
  • Patients eligible for treatment can be those (1 ) who have undergone debulking surgery, (2) for whom a cell line has been established, (3) who have undergone leukapheresis, (4) are scheduled for primary adjuvant chemotherapy, and (5) who have an ECOG performance grade of 0 or 1 .
  • the primary endpoint of this trial can be death from any cause with the primary endpoint of overall survival (OS) from the date of randomization.
  • OS overall survival
  • Progression-free survival can be a secondary endpoint and can be calculated as the time from the date of randomization for treatment to subjective tumor progression or death. Progression can be subjectively defined by the treating physician, and can be based on tumor marker levels (e.g. CA-125) and/or imaging. Secondarily, PFS and OS can also be defined from the date of debulking surgery.
  • tumor marker levels e.g. CA-125
  • OS can also be defined from the date of debulking surgery.
  • Patients can be stratified into two groups: (1 ) platinum-resistant, based on progression during adjuvant chemotherapy or detectable disease at the conclusion of adjuvant therapy or (2) platinum-sensitive, with no evidence of disease (NED) at the conclusion of adjuvant therapy (per elevated blood CA-125 and/or tumor markers and/or detection of disease by physical examination or imaging) as summarized in the Table 5.
  • NED no evidence of disease
  • Optimal tumor debulking is defined as a debulking procedure that leaves patients with residual tumor nodules less than or equal to 1 cm in diameter.
  • DC-CTC Autologous dendritic cells loaded with antigens from irradiated autologous circulating tumor cells and suspended in GM-CSF (treatment arm).
  • MC Autologous peripheral blood mononuclear cells suspended in GM-CSF will serve as the control arm.
  • Patient Population Female subjects 18 years or older with newly diagnosed stage III or IV ovarian cancer, who are candidates for receipt of
  • Performance status must be an ECOG score of 0 or 1 at the time of enrollment for treatment.
  • Pre-Treatment Phase Tissue Procurement and Establishment of Tumor Cell Line
  • Each patient must be aware of the neoplastic nature of her disease process and must willingly consent to the manipulation of tumor tissue for efforts to establish a tumor cell line. Patients will sign an Informed Consent to allow their blood/tissue to be given to NeoStem and for efforts to establish an autologous circulating tumor cell line
  • NeoStem Successful establishment of an autologous epithelial ovarian, fallopian tube, or primary peritoneal cancer cell line by NeoStem.
  • Patients must previously have been staged as having stage III (intraperitoneal) or Stage IV (distant metastatic) ovarian, fallopian tube, or primary peritoneal cancer and have initiated or completed standard adjuvant chemotherapy, which may include intravenous and/or intraperitoneal chemotherapy using standard regimens. Patients will be characterized as being in complete remission per physical exam, CT scans, and CA-125, or not in complete remission.
  • Standard adjuvant chemotherapy which may include intravenous and/or intraperitoneal chemotherapy using standard regimens.
  • Patients will be characterized as being in complete remission per physical exam, CT scans, and CA-125, or not in complete remission.
  • Medical fitness to undergo a leukapheresis including peripheral venous access or access by central vein if necessary.
  • Active infection or other active medical condition that could be eminently life-threatening, including active blood clotting or bleeding diathesis.
  • Active central nervous system metastases at the time of treatment.
  • Treatment Duration The study product will be administered subcutaneously weekly for three consecutive weeks and then approximately every three to four weeks (at the time subsequent maintenance or secondary therapy is given), for up to a total of eight doses of treatment given over four to six months.
  • Each dose of DC-CTC or MC contains approximately 5-10 million cells. Each dose is suspended in 500 ⁇ g GM-CSF prior to administration.
  • PFS Progression Free Survival
  • OS and PFS for the subset of patients who are platinum-sensitive after adjuvant therapy.
  • OS and PFS for the subset of patients who are platinum-resistant after/during primary adjuvant chemotherapy.
  • OS and PFS from dates of initial diagnosis and/or debulking surgery.
  • CTCs autologous circulating tumor cells
  • HIPAA Health Insurance Portability and Accountability Act
  • Kaplan-Meier curves will display survival times for each treatment group.
  • the log rank test will be used to analyze OS to test the null hypothesis of no treatment difference.
  • the Cox regression model and the Wald test will be used to estimate the hazard ratio associated with treatment and to identify the significance of potential prognostic factors and their impact, if any on the treatment differences.
  • Analyses based on the intention-to-treat population will be considered as the primary analysis
  • analyses based on the Per-Protocol population will be considered as a sensitivity analysis.
  • Subgroup analyses of the OS endpoint will mimic the plan laid out for the primary endpoint for platinum-resistant patients and platinum- sensitive patients separately.
  • the media was supplemented with growth factors (EGF 10 ng/mL, and FGF 10 ng/mL).
  • growth factors EGF 10 ng/mL, and FGF 10 ng/mL.
  • the media was supplemented with N-Acetyl-Cysteine 1 mM (163.19 ⁇ g/mL); 1 ⁇ PTEN inhibitor bpV(phen) (Potassium Bisperoxo(1 ,10-phenanthroline)oxovanadate(V)trihydrate, Sigma-Aldrich) and with 5 ⁇ Rho-Associated-Kinase (ROCK) inhibitor (Y-27632, Stem Cell Technologies).
  • the media was partially replaced Monday-Wednesday-Friday and supplemented with FGF/EGF combination, 10 ng/mL of each growth factor.
  • ROCK inhibitors may be effective in reducing adherent tumor cell behavior, these inhibitors could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy (Bhandary L, Whipple RA, Vitolo Ml, et al. ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells. Oncotarget. 2015; 6(8):6251 - 6266). Therefore, the ability of ROCK inhibitor Y-27632 to reverse the loss of adherence by tumor cells was investigated. 5 ⁇ of ROCK inhibitor Y-27632 was added to culture media of non-adherent tumor cells. The tumor cells exposed to 5 ⁇ of ROCK inhibitor re-gained adherence after overnight exposure, thus indicating that the loss of adherence by tumor cells can be reversed by the addition of ROCK inhibitor Y-27632 into the culture media ( Figure 23).
  • non-adherent CTCs were exposed to ROCK inhibitor Y-27632 as described in Example 6 in order to restore adherence and facilitate isolation.
  • Adherent tumor cells were separated from adherent macrophages by exposing the cell cultures to a gentle enzymatic digestion using TrypLE (Life Technologies). This gentle enzymatic digestion lifts only tumor cells from the culture surface; leaving macrophages attached.
  • PTEN inhibitors activates the phosphoinositide 3-kinase (PI3K) pathway, which is important for self-renewal, proliferation, and maintenance of self-renewing cells.
  • PI3K phosphoinositide 3-kinase
  • the use of a PTEN inhibitor alone or combination with a ROCK inhibitor can influence survival and proliferation pathways, thereby establishing a tumor cell line from isolated CTCs.
  • Non-adherent cells were exposed to 1 ⁇ PTEN inhibitor potassium bisperoxo(1 ,10- phenanthroline)oxovanadate (V) (Sigma-Aldrich) and 5 ⁇ ROCK inhibitor Y-27632.

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Abstract

La présente invention décrit un produit cellulaire cancéreux spécifique d'une tumeur, puissant au plan immunologique, contenant une composition immunogène contenant une quantité immunostimulante d'une population isolée de cellules dendritiques mises en contact ex vivo avec une cellule souche cancéreuse spécifique du cancer dérivée d'une cellule tumorale, un amas de cellules tumorales, un agrégat de cellules tumorales ou une combinaison de ces dernières exprimant au moins un antigène spécifique du cancer, la cellule tumorale, l'amas de cellules tumorales, l'agrégat de cellules tumorales ou leur combinaison étant dérivé du sang périphérique ; et un adjuvant ; et un support pharmaceutiquement acceptable ; ainsi que des procédés pour les préparer et les utiliser.
PCT/US2015/061260 2014-11-18 2015-11-18 Compositions immunogènes préparées à partir de cellules tumorales dérivées du sang périphérique et provenant d'une tumeur solide et leur utilisation WO2016081554A1 (fr)

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WO2019152767A1 (fr) * 2018-02-02 2019-08-08 Wake Forest University Health Sciences Organoïdes associés à une immunothérapie et leurs procédés de préparation et d'utilisation
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US11364264B2 (en) 2016-09-09 2022-06-21 The General Hospital Corporation Ex vivo antigen-presenting cells or activated CD-positive T cells for treatment of cancer
CN114921411A (zh) * 2022-03-08 2022-08-19 闫书印 一种nk细胞的培育方法及应用
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