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WO2016048872A1 - Compositions, procédés et kits utilisés pour déterminer la puissance de cellules dendritiques dans l'immunothérapie du cancer - Google Patents

Compositions, procédés et kits utilisés pour déterminer la puissance de cellules dendritiques dans l'immunothérapie du cancer Download PDF

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WO2016048872A1
WO2016048872A1 PCT/US2015/051174 US2015051174W WO2016048872A1 WO 2016048872 A1 WO2016048872 A1 WO 2016048872A1 US 2015051174 W US2015051174 W US 2015051174W WO 2016048872 A1 WO2016048872 A1 WO 2016048872A1
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cells
cell
dendritic cells
dendritic
tarc
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Andrew CORNFORTH
Monica SIEGENTHALER
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NeoStem Oncology LLC
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NeoStem Oncology LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/05—Adjuvants
    • C12N2501/052—Lipopolysaccharides [LPS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/52—CD40, CD40-ligand (CD154)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G01N15/147—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the described invention generally relates to compositions, methods and kits for determining the potency of dendritic cells (DC) used in cancer immunotherapy.
  • DC dendritic cells
  • the immune system encompasses cellular immunity, humoral immunity, and complement response.
  • Cellular immunity includes a network of cells and events involving dendritic cells (DC), CD8 + T-cells (a.k.a., cytotoxic T-cells or cytotoxic lymphocytes), and CD4 + T-cells (a.k.a., helper T-cells).
  • DC dendritic cells
  • CD8 + T-cells a.k.a., cytotoxic T-cells or cytotoxic lymphocytes
  • CD4 + T-cells a.k.a., helper T-cells
  • T-cells recognize antigens on the cell surface of target T-cells as small peptides presented by class I or class II of the major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • CD8 + cytotoxic T-cells recognize short peptides derived from intracellular cytoplasmic proteins and presented at the cell surface by class I MHC molecules that are expressed by virtually all nucleated cells (Dermime S, et al., British Medical Bulletin (2002) 62: 149-162; Grey H M, et al.. Cancer Ser. (1995) 22: 3749).
  • CD4 + helper T-cells recognize longer peptides derived from engulfed extracellular proteins and presented at the cell surface by class II MHC molecules by antigen presenting cells (APCs) (Dermime S, et al., British Medical Bulletin (2002) 62: 149-162; Pieters J, Adv. Immunol. (2000) 75: 159-208).
  • APCs are central to the priming of T-cells by specific antigens, and dendritic cells (DCs) are the most potent stimulatory APCs (Dermime S, et al., British Medical Bulletin (2002) 62: 149-162).
  • DCs Dendritic cells
  • 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.
  • a DC bearing a MHC class I/peptide complex contacts a CD8 + T-cell, the result is activation and proliferation of the CD8 + T-cell.
  • 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
  • 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-la (CCL3/MlP-la), macrophage inflammatory protein-ip (CCL4/MIP-lf3), regulated on activation, normal T- cell expressed and secreted (CCL5/RANTES) and macrophage inflammatory protein-3a (CCL20/MIP-3a)) and chemokine receptors that bind to inflammatory chemokines (chemokine (C-C motif) receptor 1 (CCRl), 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 (CXCRl )).
  • CCL2/MCP- 1 CCL2/MCP- 1
  • macrophage inflammatory protein-la CL3/Ml
  • DCs down-regulate the inflammatory chemokines and their receptors and up-regulate constitutive chemokines such as interferon-y-inducible protein 10 (CXCLl O/IP-10), thymus and activation regulated chemokine (CCL 17/TARC), pulmonary and activation regulated chemokine (CCL 18/PARC), macrophage inflammatory protein-3p (CCL 19/MIP-3P), macrophage derived chemokine (CCL22/MDC), the chemokine receptor chemokine (C-C motif) receptor 7 (CCR7) and secondary lymphoid-tissue chemokine
  • constitutive chemokines such as interferon-y-inducible protein 10 (CXCLl O/IP-10), thymus and activation regulated chemokine (CCL 17/TARC), pulmonary and activation regulated chemokine (CCL 18/PARC), macrophage inflammatory protein-3p (CCL 19/MIP-3P), macrophag
  • 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: 149162; June C, et al, Immunol. Today (1994) 15: 321 -331).
  • DCs have been shown to attract T helper 1 (Thl) and T helper 2 (Th2) cells, as well as naive 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, macrophage-derived chemokine and thymus and activation regulated chemokine (CCL 17/TARC).
  • DCs are known to express high levels of CCL 17/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 Thl differentiation (Rubio M T, et al., International Immunology (2005) 17(12): 1561-1572; Hilkens C M, et al., Blood (1997) 90: 1920-1926).
  • Thymus and activation regulated chemokine is a chemokine secreted from monocyte-derived DCs and endothelial cells, and is responsible for selective recruitment and migration of activated Th2 lymphocytes to affected tissue (Dallos T, et al.. Arthritis & Rheumatism (2010) 62(1 1): 3496-3503).
  • CCR4 and CCR8 both of its receptors (CCR4 and CCR8) is transiently up-regulated on activated T-cells and preferentially on cells of the Th2 subset, and conversely, production of CCL 17/TARC by DCs is stimulated by Th2 cytokines (Dallos T, et al., Arthritis & Rheumatism (2010) 62(1 1): 3496- 3503; DAmbrosio D, et al., J. Immunol. (1998) 161 : 51 1 1 -51 15; De Lavareille A, et al., Eur. J. Immunol. (2001) 31 : 1037-1046).
  • CCL17/TARC production by DCs in affected tissue may be up-regulated by Th2 cytokines and thus provide additional chemoattraction for Th2 cells (Dallos T, et al.. Arthritis &
  • CCL 17/TARC is expressed in CD 1 lc + DCs in murine lung tissue and plays a crucial role in Th2-mediated experimental allergen-induced asthma in mice, and development of murine asthma could be inhibited by a monoclonal antibody against CCL 17/TARC (Dallos T, et al., Arthritis & Rheumatism (2010) 62(1 1): 3496-3503; Lieberman I and Forster I, J. Immunol. (1999) 29: 2684-2694; Kawasaki S, et al., J.
  • CCL 17/TARC's role in Th2-mediated allergen-induced asthma is also supported by the finding of production of high levels of CCL 17/TARC by bronchial epithelial cells of asthma patients (Dallos T, et al., Arthritis & Rheumatism (2010) 62(1 1): 3496- 3503; Sekiya T, et al, J. Immunol. (2000) 165: 2205-2213).
  • DCs that are 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).
  • the therapeutic use of DC cancer vaccines has recently been revived owing to a series of clinical trials that have yielded encouraging clinical outcomes.
  • sipuleucel-T also known as APC 801
  • APC 801 sipuleucel-T
  • PAP prostatic acid phosphatase
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • the described invention provides compositions, methods and kits useful for the reliable and reproducible detection of potent DCs generated for use as a cancer vaccine.
  • the potency of DCs can be determined by measuring (i) the ability of these cells to respond to maturation signals such as cytokines and toll-like receptor (TLR) ligands; (ii) changes in the expression of CD80, CD83, MHC 11 and/or CD54 on the surface of these cells; (iii) secretion of immune stimulating factors such as IL-12 and/or CCL 17/TARC; or (iv) a combination thereof.
  • maturation signals such as cytokines and toll-like receptor (TLR) ligands
  • TLR toll-like receptor
  • the described invention provides compositions, methods and kits for determining potency of dendritic cells used in cancer immunotherapy.
  • the described invention provides a method for preparing a population of immunopotent dendritic cells activated in vitro with a tumor-specific antigen derived from a population of purified cultivated tumor cells derived from a patient comprising: (a) obtaining peripheral blood mononuclear cells (PBMCs) by leukapheresis from the patient from whom the tumor cells were derived; (b) optionally shipping the collected PBMCs from (a) to a manufacturing facility; (c) purifying the PBMCs from (a)
  • PBMCs peripheral blood mononuclear cells
  • the immunopotency of the dendritic cells formed in (e) is measured by an amount of a biomarker produced by the dendritic cells formed in (e), and wherein the dendritic cells formed in (e) are effective to generate an effective immune response against the tumor-specific antigen comprising activation and proliferation of CD4+ T-cells, CD8+ T-cells, B-cells or a combination thereof.
  • the amount of the biomarker is measured by an immunoassay.
  • the immunoassay is selected from the group consisting of Western blot, ELISA and flow cytometry.
  • the biomarker is CCL 17/TARC.
  • the amount of the biomarker is at least 274.3 pg/mL/day.
  • the described invention provides a method for treating a subject suffering from a cancer comprising: (a) preparing for a cancer patient a patient- specific immunogenic composition comprising an immunopotent amount of an isolated population of dendritic cells contacted ex vivo with a cancer cell expressing a cancer-specific antigen by the method according to claim 1 ; (b) administering the immunogenic composition to the cancer patient; and (c) generating an effective immune response against the cancer- specific antigen 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 the group consisting of progression-free survival, disease- free survival, time to progression, time to distant metastasis and overall survival of the subject when compared to a control.
  • Figure 1 depicts a CCL17/TARC standard curve for Sample # 1955 and Sample # 1 893.
  • Figure 2 depicts calculated concentrations of Sample # 1955 and Sample # 1893 from diluted concentrations within an acceptable percent linearity. Data is expressed as mean ⁇ standard error.
  • Figure 3 depicts calculated concentrations of Sample # 1955 and 1893 from all diluted concentrations tested. Data is expressed as mean ⁇ standard error.
  • Figure 4 depicts a CCL17/TARC standard curve for Sample #2353.
  • Figure 5 depicts a bar graph representing CCL17/TARC concentrations for Sample # 2353 calculated from a 1 :8 dilution.
  • Figure 6 depicts an IL-12 standard curve for Sample # 1955 and Sample # 1 893.
  • Figure 7 depicts a bar graph representing IL-12 concentrations for Sample # 1955 and 1893 calculated from 1 :2 and 1 :20 dilutions.
  • Figure 8 depicts an IL-12 standard curve for Sample #2353.
  • Figure 9 depicts a bar graph representing IL-12 concentrations for Sample #2353 calculated from undiluted, 1 :2 and 1 :4 dilutions.
  • Figure 10 depicts cytometry data for various dendritic cell media conditions. Representative flow cytometry of DC-TC incubated under control conditions (DC media plus 5% FBS only) or under dendritic cell maturation conditions (DC media plus 5% FBS plus 0.5
  • the extracellular/intracellular flow cytometry optimization consisted of applying extracellular antigen antibodies (CD40 and CD83) prior to fixation and permeabilization (Pre-fix) or after fixation and permeabilization (Post-fix) followed by staining with intracellular antigen antibodies (CCL 17 and IL12).
  • Figure 11 depicts AIM-V media conditions. Representative flow cytometry of DC- TC incubated under control conditions (AIMV media plus 5% FBS only) or under dendritic cell maturation conditions (AIMV media plus 5% FBS plus 0.5
  • control conditions AIMV media plus 5% FBS only
  • dendritic cell maturation conditions AIMV media plus 5% FBS plus 0.5
  • CD40/CCL 17 double positive cells for each of the eight conditions tested. Each bar represents a single experiment.
  • AIM AIMV plus 5%FBS.
  • DC Dendritic cell media plus 5%FBS.
  • FIG. 13 depicts a bar graph representing flow cytometry results for Sample # 1986 for CD83/IL- 12 double positive cells for each of the eight conditions tested. Each bar represents a single experiment.
  • AIM AIMV plus 5%FBS.
  • DC Dendritic cell media plus 5%FBS.
  • Figure 14 depicts a bar graph representing flow cytometry results for Sample # 1955 for
  • CD40/CCL 17 double positive cells for each of the eight conditions tested. Each bar represents a single experiment.
  • AIM AIMV plus 5%FBS.
  • DC Dendritic cell media plus 5%FBS.
  • Figure 15 depicts a bar graph representing flow cytometry results for Sample # 1955 for CD83/IL- 12 double positive cells for each of the eight conditions tested. Each bar represents a single experiment.
  • AIM AIMV plus 5%FBS.
  • DC Dendritic cell media plus 5%FBS.
  • Figure 16 depicts a bar graph representing flow cytometry results for Sample #1718 for
  • CD40/CCL 17 double positive cells for each of the eight conditions tested. Each bar represents a single experiment.
  • AIM AIMV plus 5%FBS.
  • DC Dendritic cell media plus 5%FBS.
  • FIG. 17 depicts a bar graph representing flow cytometry results for Sample #1718 for CD83/IL-12 double positive cells for each of the eight conditions tested. Each bar represents a single experiment.
  • AIM AIMV plus 5%FBS.
  • DC Dendritic cell media plus 5%FBS.
  • Figure 18 depicts flow cytometry results for CD40, CD80, MHC-II and CD54. Incubation of normal donor dendritic cells generated in Cell Genix or AIMV media followed by standard maturation cytokine cocktail or toll-like receptor ligand with CD40L/IFNgamma overnight in 5%FBS/AIMV. CTL is equal to 5% FBS/AIMV only.
  • CK1 is equal to 5% FBS/AIMV plus 0.5 Lj,g/mL CD40L, 1000 lU/mL IFNgamma, IOng/mL TNFalpha, lOng/mL ILl-beta, and 15ng/mL IL6 and CK2 is equal to 5% FBS/AIMV plus 0.5
  • FIG. 19 depicts flow cytometry results for CD83 and IL- 12.
  • CTL is equal to 5% FBS/AIMV only.
  • C 1 is equal to 5% FBS/AIMV plus 0.5
  • Figure 20 depicts flow cytometry results for CD54, CD40, CD83 and MHC-class II. Incubation of normal donor dendritic cells generated in AIM-V dendritic cell media followed by sequential treatment with toll-like receptor ligand and maturation cytokines.
  • FIG. 141 j Figure 22 depicts flow cytometry results of for IL-12.
  • Antigen- loaded dendritic cells were exposed to 1
  • Control consisted of AIMV plus 5% FBS only. Three separate antigen- loaded dendritic cells were assayed, only a single sample is shown.
  • Figure 23 depicts a bar graph representing expression of CD83 and CD40 in monocytes, unloaded dendritic cells (DC only) and antigen-loaded dendritic cells (DC-TC) after incubation for approximately 24 hours in either unstimulated (media only),
  • Figure 25 depicts CCL 17/TARC ELISA results normalized on a per day basis.
  • N 1 1.
  • Figure 26 depicts a Kaplan Meier survival curve correlating cancer patient outcome with
  • 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/CDS complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule.
  • the molecular events set in motion by receptor engagement are complex. Among the earliest steps appears to be the activation of tyrosine kinases leading to the tyrosine phosphorylation of a set of substrates that control several signaling pathways.
  • T-cell 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).
  • APC antigen presenting cell
  • the soluble product of an activated B lymphocyte is immmunoglobulins (antibodies).
  • the soluble product of an activated T lymphocyte is lymphokines.
  • CCL2/MCP-1 refers to monocyte chemoattractant protein- 1 , which is a member of the C-C chemokine family, and a potent chemotactic factor for monocytes.
  • CCL2/MCP-1 is produced by many cell types, including dendritic, endothelial, fibroblasts, epithelial, smooth muscle, mesangial, astrocytic, monocytic and microglial cells.
  • CCL2/MCP-1 regulates the migration and infiltration of monocytes, memory T-cells and natural killer (NK) cells.
  • CCL3/MIP-la refers to macrophage inflammatory protein-la, which is a member of the C-C family of chemokines.
  • CCL3/MIP-la expression can be induced in a variety of cell types, including dendritic cells, Langerhans cells, fibroblasts and T-cells.
  • CCL3/MIP-la induces migration of monocytes and T-cells. It is primarily chemotactic for B-cells and activated CD8 + T-cells.
  • CCL4/MIP-1 P refers to macrophage inflammatory protein-ip, which is a member of the C-C family of chemokines.
  • CCL4/MIP-1P is secreted by a variety of cell types, including dendritic cells and is the principal regulator of macrophage migration.
  • CCL4/MIP- I P binds to and signals through the CCR5 receptor.
  • CCL5/RANTES refers to Regulated on Activation Normal T-cell Expressed and Secreted, which is a member of the C-C family of chemokines.
  • CCL5/RANTES is secreted by many hematopoietic and non-hematopoietic cells, including dendritic cells.
  • CCL5/RANTES plays an important role in homing and migration of effector and memory T- cells by binding to and signaling through CCR5 receptor.
  • CCL20/MIP-3a refers to macrophage inflammatory protein-3a, which is a member of the C-C family of chemokines. CCL20/MIP-3a is predominantly expressed in
  • extralymphoid tissue is known to direct migration of dendritic cell precursors and memory
  • lymphocytes to sites of antigen invasion.
  • CCR1 refers to chemokine (C-C motif) receptor 1.
  • CCR1 is a member of the beta chemokine receptor family, predicted to be a seven transmembrane protein.
  • Ligands for CCR1 include CCL3/MIP-la, CCL5/RANTES, monocyte chemoattractant protein 3 (MCP-3) and myeloid progenitor inhibitory factor-1 (MPIF-1 ).
  • CCR1 -mediated signal transduction is critical for recruitment of effector immune cells to the site of inflammation and plays a role in host protection from inflammatory response and susceptibility to virus and parasite.
  • CCR2 refers to chemokine (C-C motif) receptor 2.
  • CCR2 is a receptor for monocyte chemoattractant protein- 1 (CCL2/MCP-1), a chemokine involved in monocyte infiltration in inflammatory diseases.
  • CCR2 mediates agonist-dependent calcium mobilization and inhibition of adenylyl cyclase.
  • CCR5 refers to chemokine (C-C motif) receptor 5.
  • CCR5 is a member of the beta chemokine receptor family, predicted to be a seven transmembrane protein.
  • Ligands for CCR1 include CCL3/MlP-la, CCL5/RANTES, monocyte chemoattractant protein 3 (MCP-3) and myeloid progenitor inhibitory factor- 1 (MPIF-1 ).
  • CCRl -mediated signal transduction is critical for recruitment of effector immune cells to the site of inflammation and plays a role in host protection from inflammatory response and susceptibility to virus and parasite.
  • CCR6 refers to chemokine (C-C motif) receptor 6.
  • CCR6 is a member of the beta chemokine receptor family, predicted to be a seven transmembrane protein.
  • the ligand for CCR6 is macrophage inflammatory protein 3 alpha (MI -3a).
  • CCR6 has been shown to be important for B-Iineage maturation and antigen-driven B-cell differentiation, and it may regulate the migration and recruitment of dendritic and T cells during inflammatory and immunological responses.
  • CD54 refers to cluster of differentiation 54.
  • CD54 is a type I transmembrane protein present on leukocytes and endothelial cells and inducible on lymphocytes, dendritic cells, keratinocytes, chondrocytes, fibroblasts and epithelial cells. CD54 acts as a ligand for CD 1 1 and CD 18 and aids in intercellular adhesion.
  • CD80 refers to cluster of differentiation 80.
  • CD80 is a membrane receptor activated by the binding of CD28 or CTLA-4. Activated CD80 induces T-cell proliferation and cytokine production.
  • CD83 refers to cluster of differentiation 83.
  • CD83 is a single-pass type I membrane protein thought to be involved in the regulation of antigen presentation. A soluble form of this protein can bind to dendritic cells and inhibit their maturation.
  • CXCR1 refers to chemokine (C-X-C motif) receptor 1.
  • CXCR1 is a member of the G-protein-coupled receptor family. This protein is a receptor for interleukin 8 (IL-8). It binds to IL-8 with high affinity, and transduces the signal through a G- protein activated second messenger system. Knockout studies in mice suggested that this protein inhibits embryonic oligodendrocyte precursor migration in developing spinal cord.
  • T lymphocyte-mediated immunity refers to T lymphocyte-mediated immunity.
  • T lymphocytes or T-cells, are known to directly kill cells, to provide "help” for such killers, to activate other immune system cells (e.g., macrophages), to help B-cells make an antibody response, to down-modulate the activities of various immune system cells, and to secrete cytokines, chemokines, and other mediators.
  • T- cells are divided into two (2) major classes: T helper cells (Th) and regulatory T-cells (T r eg). T helper cells are further subdivided into T helper 1 (Thl) cells and T helper 2 (Th2) cells.
  • T-helper 1 (Thl) cells which are implicated in the stimulation of inflammation, produce IFN-gamma, GM-CSF, TNF-beta, and TNF alpha.
  • T helper 2 (Th- 2) cells produce IL-4, IL-5, IL-10, and iL-13, and provide help for B-cells in their activation and differentiation leading to the humoral immune response, (de Waal Malefyt, Immunity 31 : 700-702 (2009)).
  • Regulatory T-cells either natural, induced, or Trl cells, produce IL-10 and TGFp, suppress the activation of effector T-cells, and provide a counter-balance against uncontrolled and harmful T-cell responses.
  • Th9 cells may provide additional help for mast-cells through the production of IL-9. Id.
  • Thl 7 an additional T-cell subset, produces IL-17A, 17- 17F, IL-22 and CCL20, which act on stromal and epithelial cells to induce a number of secondary effector molecules, such as G-CSF, which stimulates the production and mobilization of neutrophils, acute phase proteins, chemokines, and antimicrobial peptides.
  • G-CSF secondary effector molecules
  • Naive T- cells can differentiate into any of the distinct T-cell subsets when activated in the presence of appropriate signals and cytokines. The induction of a maturation process in dendritic cells is a crucial step for efficient priming of naive T-cells. There is an extensive cross-regulation between subsets to ensure that the appropriate T-cell subset is activated. Id.
  • chemokine refers to a class of chemotactic cytokines that signal leukocytes to move in a specific direction.
  • 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.
  • 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 T-cells. Generally, cytokines act locally.
  • type I cytokines which encompass many of the interleukins, as well as several hematopoietic growth factors
  • type II cytokines including the interferons and interleukin- 10
  • TNF tumor necrosis factor
  • IL- 1 immunoglobulin super-family members
  • 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.
  • DC dendritic cell
  • the term "dendritic cell” or “DC” as used herein refers to bone marrow -derived cells that are seeded in all tissues. DCs exhibit several features which are necessary for the generation of T-cell-mediated antitumor immunity. That is, they efficiently capture and take up antigens in peripheral tissues and transport these antigens to the primary and secondary lymphoid organs where they express high levels of MHC class I and II molecules that present the processed peptides to T-cells for the priming of antigen-specific responses.
  • enzyme activity refers to the action of an enzyme (meaning a protein that catalyzes a specific chemical reaction) on its target. It is quantified as the amount of substrate consumed (or product formed) in a given time under given conditions.
  • turnover number refers to the number of molecules of substrate that can be converted into product per catalytic site of a given enzyme.
  • immunomodulatory cell(s) refer(s) to cell(s) that are capable of augmenting or diminishing immune responses by expressing chemokines, cytokines and other
  • immunopotent refers to the ability to activate and guide a naive immune system to mount a response toward a foreign protein.
  • inflammatory cytokines or "inflammatory mediators” as used herein refers to the molecular mediators of the inflammatory process, which may modulate being either pro- or antiinflammatory 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 inflammatory mediators.
  • inflammatory mediators of the inflammatory response 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, pro-inflammatory cytokines, including, but not limited to, interleukin-l-beta (IL-ip), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-a), interferon-gamma (IF-y), and interleukin- 12 (IL- 12).
  • IL-ip interleukin-l-beta
  • IL-4 interleukin-4
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • TNF-a tumor necrosis factor-al
  • interleukin refers to a cytokine secreted by, and acting on, leukocytes. Interleukins regulate cell growth, differentiation, and motility, and stimulates immune responses, such as inflammation. Examples of interleukins include interleukin- 1 (IL- 1 ), interleukin 2 (IL- 2), interleukin- 1 (3 (IL- 1 (3), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin- 12 (IL- 12).
  • IL- 1 interleukin- 1
  • IL- 2 interleukin 2
  • interleukin- 1 3
  • 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 Thl 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.
  • 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 event 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.
  • 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 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.
  • 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. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their functions.
  • B-cells which are precursors of antibody-secreting cells
  • 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.
  • 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.
  • cognate help In many physiological situations, 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. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4 lh Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).
  • Cognate help allows B-cells to mount responses against antigens that cannot crosslink receptors and, at the same time, provides co-stimulatory 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
  • MHC histocompatibility complex
  • CD4 + T-cells The resultant class II/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 Il/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.
  • cytokine receptors on the B-cell.
  • the CD40 ligand is transiently expressed on activated CD4 + T helper cells, and it binds to CD40 on the antigen-specific B- cells, thereby tranducing a second co- stimulatory 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.
  • 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
  • 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) co-stimulatory 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.
  • MHC proteins which present foreign antigen to the T-cell receptor
  • co-stimulatory 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 p chains.
  • TCR T-cell receptors
  • a small group of T- cells express receptors made of y and 5 chains.
  • CD4 + T-cells those that express the coreceptor molecule CD4
  • CDS CD8 + T-cells
  • 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 important effector functions, some of which are determined by the patterns of cytokines they secrete.
  • 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 IMHC 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 appropriate receptors.
  • 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
  • B-cells bind the antigen through their membrane Ig, and the complex undergoes endocytosis. Within the endosomal and lysosomal compartments, 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. (Paul, W. E., "Chapter 1 : The immune system: an introduction,"
  • 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 I L- l 0 (Th2 cells) or into cells that mainly produce IL-2, IFN-y, and lymphotoxin (Thl cells).
  • the Th2 cells are very effective in helping B-cells develop into antibody-producing cells, whereas the THl 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.
  • THl cells Although the CD4 + T-cells with the phenotype of TH 2 cells (i.e., IL-4, IL-5, IL-6 and IL-l 0) are efficient helper cells, THl cells also have the capacity to be helpers. (Paul, W. E., “Chapter 1 : The immune system: an introduction,” Fundamental Immunology, 4 th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).-
  • T-cells also may act to enhance the capacity of monocytes and macrophages to destroy intracellular microorganisms.
  • interferon-gamma IFN-y
  • 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 Thl cells are effective in enhancing the microbicidal action because they produce IFN-y.
  • two of the major cytokines produced by Th2 cells IL-4 and IL- 10, block these activities.
  • CD4 + constitutively express the IL-2 receptor alpha (IL-2Ra) chain CD4 + CD25 +
  • IL-2Ra IL-2 receptor alpha chain
  • Tregs results in systemic autoimmune disease in mice. Furthermore, transfer of these
  • Human CD4 + CD25 + Tc g 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 1L-2, and the anergic phenotype in vitro.
  • Human CD4 + CD25 + T- cells can be split into suppressive (CD25 hlsh ) and nonsuppressive (CD25 low ) cells, according to the level of CD25 expression.
  • CTL Cytotoxic TLymphocytes
  • 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 target 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.
  • 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) and heat shock proteins (hsp).
  • unprimed cells also referred to as virgin, naive, 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.
  • helper T-cells differ from those required by other CD4 + T-cells.
  • the critical antigen-presenting cell for helper T-cell priming appears to be a macrophage; and the critical second signal for helper T-cell growth 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
  • modulate refers to the regulation, alteration, adaptation or adjustment to a certain measure or proportion.
  • 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.
  • PBMCs peripheral blood mononuclear cells
  • potency refers to the relationship between a therapeutic effect of a therapeutic substance and the dose necessary to achieve that effect; the amount of a substance required to produce a given percentage of its maximal effect, irrespective of the size of maximal effect; or the relative pharmacologic activity of a substance.
  • TARC chemokine (C-C motif) 17 (also known as thymus and activation regulated chemokine).
  • CCL17/TARC is a chemokine that is secreted from monocyte-derived dendritic cells and endothelial cells and is responsible for selective recruitment and migration of activated Th2 lymphocytes to affected tissue.
  • the described invention provides methods, compositions and kits useful in determining the potency of dendritic cells used in cancer immunotherapy.
  • the described invention provides a dendritic cell (DC) that presents a processed peptide within the dendritic cell.
  • DC dendritic cell
  • the described invention provides a dendritic cell that presents a processed peptide on the dendritic cell surface.
  • the processed peptide is a tumor antigen.
  • the tumor antigen is from a tumor that 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 ovarian carcinoma, glioblastoma multiforme and the like.
  • the described invention provides methods for determining the potency of dendritic cells used in cancer immunotherapy. These methods include, but are not limited to, immunoassays. Exemplary immunoassays include Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry and the like. Flow cytometry includes, but is not limited to, fluorescence-activated cell sorting (FACS®), magnetic-activated cell sorting (MACS®), high-dimensional flow cytometry and cytometric bead array.
  • the enzyme-linked immunosorbent assay employs highly-purified capture antibodies that are non-covalently adsorbed ("coated") onto plastic microwell plates. After washings, the immobilized antibodies capture specifically soluble proteins (e.g., chemokine) present in samples applied to the plate. After washing away unbound material, the captured proteins are detected by biotin-conjugated detection antibodies followed by an enzyme-labeled avidin or streptavidin reporter. Following addition of a chromogenic (color- developing) substrate-containing solution, the level of colored product generated by the bound, enzyme-linked, detection reagents can be measured spectrophotometrically using an ELISA- plate reader at an appropriate optical density.
  • soluble proteins e.g., chemokine
  • Flow cytometry a technique that may be used for counting and examining cells, allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of each individual cell. Briefly, a beam of light (usually laser light) of a single wavelength is directed onto a laser beam.
  • a number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter (FSC)), several perpendicular to it (SSC)), and one or more fluorescence detectors.
  • FSC Forward Scatter
  • SSC Segment Scatter
  • _im Each suspended cell (from 0.2 y.m to 150
  • the FSC correlates with the cell volume; SSC depends upon the inner complexity of the cell (i.e., shape of the nucleus, type of cytoplasmic granules, etc.).
  • the data generated by flow cytometers may be plotted as a histogram. The regions on these plots can be separated sequentially based on fluorescence intensity by creating a series of subset extractions ("gates"). Specific gating protocols have been developed for diagnostic and clinical purposes.
  • Flow cytometers may use either light scattering in combination with fluorescence or light scattering only for analysis.
  • Flow cytometers are available from a variety of commercial sources, including BD Biosciences (San Jose, CA), EMD Millipore (Billerica, MA), Life Technologies (Carlsbad, CA), Agilent (Santa Clara, CA), Miltenyi Biotec (Cambridge, MA) and the like.
  • Fluorescence activated cell sorting provides a method of sorting a heterogeneous mixture of cells into two or more containers, a single cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. Briefly, the cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid and the flow is arranged such that there is a large separation between cells relative to their diameter. The stream of individual cells passes through a fluorescence detector, and an electrical charge is assigned to each cell (based on the cell's fluorescence) just as the stream is broken into individual drops (usually via vibration) such that there is a low probability of more than one cell per droplet. Each charged droplet (containing an individual cell) may be sorted, via electrostatic deflection, into separate containers.
  • FACS Fluorescence activated cell sorting
  • MACS provides a cell separation technique in which cells that express a specific surface antigen may be isolated from a heterogeneous mixture of cells using magnetic particles coated with a binding agent (e.g., antibody) that recognizes the specific surface antigen.
  • a binding agent e.g., antibody
  • cells expressing the specific surface antigen bind to the magnetic particles.
  • the heterogeneous mixture of cells is transferred to a column placed in a magnetic field. The magnetic field captures the magnetic particles (including magnetic particles bound to cells expressing the specific surface antigen) while cells not expressing the specific surface antigen (i.e., not bound to magnetic particles) may be eluted as flow through.
  • Negative selection involves the isolation and removal of undesired cells expressing a specific surface antigen from a heterogeneous mixture of cells by binding the cells expressing the specific surface antigen to magnetic particles coated with a binding agent (e.g., antibody) that recognizes the specific surface antigen.
  • a magnetic field captures the magnetic particles (including magnetic particles bound to undesired cells expressing the specific surface antigen) while cells not expressing the specific surface antigen (i.e., not bound to magnetic particles) may be eluted and collected.
  • MACS products are commercially available. These products include, but are not limited to, MACS microbeads (Miltenyi Biotec, Cambridge, MA), autoMACS® columns (Miltenyi Biotec, Cambridge, MA), autoMACS Pro Separator Instrument (Miltenyi Biotec, Cambridge, MA), and the like.
  • High-dimensional flow cytometry provides a method of sorting a heterogeneous mixture of cells into two or more containers, a single cell at a time, using 6- 12 fluorescent colors (i.e., fluorophores).
  • 6- 12 fluorescent colors i.e., fluorophores
  • the following protocol may be used to perform FACS to detect antigen-specific B lymphocytes.
  • Cryopreserved peripheral blood mononuclear cell (PBMC) samples may be thawed and washed in deficient RPMI media supplemented with 4% FCS.
  • PBMC peripheral blood mononuclear cell
  • Biotin-coupled antigen (DBY-2 or DBX-2) may be added to the cells and 20 minutes later, a "cocktail" of fluorochrome conjugated monoclonal antibodies detecting CD19, CD2r, CD43, CD5, CD23, IgM and IgG, CD27 and dead cells may be added. Following 20 minute incubation, cells may be spun and washed and incubated for 20 min with fluorochrome-conjugated streptavidin. Data may be collected for 1 -5 x 10 6 cells on a LSRII flow cytometer (BDBiosciences.com). The data may be analyzed using FlowJo (TreeStar.com) and further analyzed with Excel and Prism (GraphPad software, Inc).
  • Cytometric bead array (BD Biosciences, San Jose, CA) is a flow cytometry application that allows users to quantify multiple proteins simultaneously.
  • the system employs the broad dynamic range of fluorescence detection offered by flow cytometry and antibody-coated beads to efficiently capture analytes.
  • Each bead in the array has a unique fluorescence intensity so that beads can be mixed and run
  • the data generated by flow cytometers may be plotted in a single dimension to produce a histogram or in two-dimensional or three-dimensional plots.
  • the regions on these plots may be sequentially separated, for example, based on fluorescence intensity, by creating a series of subset extractions termed "gates."
  • gates One skilled in the art recognizes that specific gating protocols exist for diagnostic and clinical purposes, including, but not limited to, classification of immune system cells.
  • a light scattering gate to include only B lymphocytes by placing upper and lower limits on the forward and side scatter distributions.
  • the surfaces of all cells in the body are coated with specialized protein receptors that selectively can bind or adhere to other signaling molecules. These receptors and the molecules that bind to them are used for communicating with other cells and for carrying out proper cell functions in the body.
  • Each cell type has a certain combination of receptors (or surface markers) on its surface that makes it distinguishable from other kinds of cells.
  • Cells may be fluorescently labeled, i.e., a reactive derivative of a fluorophore may be covalently attached to a cell.
  • the most commonly used labeled molecules are antibodies; their specificity towards certain surface markers on a cell surface allows for more precise detection and monitoring of particular cells.
  • fluorescent labels used may include, but are not limited to, fluorescein isothiocyanate (FITC), Alexa Fluor® 488, green fluorescent protein (GFP), carboxyfluorescein (CFSE), carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), DyLight® 488 (Dyomics), phycoerythrin (PE), propidium iodide (PI), peridinin chlorophyll protein (PerCP), PerCP-CyTM5.5, PE-AlexaFluor 700, PE- CyTM5; PE-CyTM5.5, PE- AlexaFluor® 750 and PE-CyTM7; when a red diode laser (635 run) is used, fluorescent labels used may include, but are not limited to, allophycocyanin (FITC), Alexa Fluor® 488, green fluorescent protein (GFP), carboxyfluorescein (CFSE), carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), DyL
  • Conjugation of a label to a binding agent may be accomplished by covalent or non-covalent (including hydrophobic) bonding, or by adsorption. Techniques for conjugation are well-known in the art and may be readily adapted for the particular reagents employed.
  • PBMCs Peripheral blood mononuclear cells
  • a tumor sample e.g., a needle biopsy, a lavage of a tumor-containing tissue, or a bulk tumor
  • the collected PBMCs are shipped to a manufacturing facility.
  • the collected PBMCs are purified from other lymphocytes.
  • 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 (optional), overnight incubation for 16 hours in autologous serum/medium ( ⁇ cytokines), gradient separation, and positive selection (e.g., flow cytometry, immunomagnetic beads).
  • positive selection is for activation antigens, e.g., CD80, CD83, CD86, and CMRF-44 (Feamley, DB et al. Blood 89; 3708 (1997); Caux, C. et al.., J. Exp. Med. 180 (5); 1841 -47 (1994)),CD80. Hart, DNJ, Blood 90(9); 3245-87 (1997); Hock, B.D. et al, Immunol. 83; 573-81 (1994).
  • activation antigens e.g., CD80, CD83, CD86, and CMRF-44
  • 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.
  • PBMCs PBMCs
  • T-lymphocyte depletion optionallymphocyte depletion
  • metrizamide gradient separation e.g., flow cytometry, immunomagnetic beads
  • IL-12 human Interleukin 12
  • IL-12 Quantikine ELISA Kit R&D Systems, catalog # D1200, Minneapolis, MN
  • reagents, standard dilutions and samples were prepared as directed in the product insert. Excess microplate strips were removed from the plate frame and returned to the foil pouch containing the desiccant pack and the pouch was resealed.
  • 50 [iL of Assay Diluent was added to each well followed by 200
  • the plate was covered with a plate sealer and incubated at room temperature for 2 hours. Following incubation, each well was aspirated and washed for a total of 3 washes. Next, 200
  • a microtiter plate reader Molecular Devices, Sunnyvale, CA
  • 1128 J Prepare all bead suspensions immediately prior to use. 1129] Label two tubes, Tube A and Tube B. Tube A for PMT adjustment and Tube B for fluorescence compensation and sensitivity testing. Note: Do not dilute PerCP-Cy5.5 beads in sheath fluid.
  • J 311 Dispense approximately 1.5 mL sheath fluid into Tube B (fill to about one inch from the bottom).
  • Tube B add 1 drop unlabeled beads + 1 drop allophycocyanin (APC)-labeled bead + 1 drop fluorescein isothiocyanate (FITC) labeled bead + 1 drop phycoerythrin (PE)- labeled bead + 1 drop peridinin chlorophyll protein (PerCP)-labeled bead.
  • API drop allophycocyanin
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • PerCP peridinin chlorophyll protein
  • 1135J Keep prepared bead suspensions on ice or at 2° to 8°C and protect from direct light until ready to use.
  • J 1361 CaliBRITE® bead suspensions prepared in FACSFlow® sheath fluid are stable for 8 hours at 4°C, or if PerCP-labeled beads are included, then for 1 hour at 4°C.
  • the fluorescence sensitivity is determined by the amount of channel separation between the unlabeled and labeled bead populations.
  • the light scatter sensitivity is determined by the amount of channel separation between the mixed bead population and instrument background signal.
  • 146I Resuspend cells to a concentration of at least 2.5xl0 5 cells/100)jL in 10% HSA in PBS.
  • a DC sample requires testing for CD45/14-FITC/PE, 7AAD-PerCP and CDl Ic-APC.
  • 7AAD is a nucleic acid dye for non-viable cells and requires a fresh/live cell sample.
  • 1150J Prepare isotype controls and conjugated antibodies. Refer to Table 1 - Dilutions of Antibodies for required antibodies and dilutions. Record antibody and isotype volume, lot number, part number, and expiration date in Attachment 1 - FACS Staining and Counting for an Autologous (e.g. Melanoma) Sample Worksheet, Section 5 Preparation of Antibodies. Place antibodies on chilled beads (4°C) during preparation.
  • Autologous e.g. Melanoma
  • [ 164] Resuspend cells to a concentration of at least 2.5x10 s cells/lOOpL in 10% HSA in PBS.
  • a DC- TC sample requires testing for CD45/14-FITC/PE, 7AAD-PerCP and CDl 1 c- APC.
  • CD40-FITC CD80-PE
  • MHClI-PerCP MHClI-PerCP
  • CD54-APC If testing for 7AAD, note that it is a nucleic acid dye for non-viable cells and requires a fresh/live cell sample.
  • Dendritic cells receive maturation signals in the form of cytokines and toll-like receptor ligands such as CD40 ligand, interferon-gamma and lipopolysaccride (LPS), respectively.
  • cytokines such as CD40 ligand, interferon-gamma and lipopolysaccride (LPS)
  • LPS lipopolysaccride
  • IL-12 interleukin-12
  • CCL 17/TARC Vissers J L, et al., J. Leukoc. Biol. (2001) 69: 785-793; Ping J, et al., J. Trans. Med. (2010) 8: 1 -15; Butterfield L H, et al, J. Immunother. (2008) 3 1 : 89-100).
  • DC- TC supernatants (Sample # 1955 and 1893) were serially diluted according to the manufacturer's protocol to produce the following sample dilutions: 1 :2, 1 :4, 1 :8, 1 : 16, 1 :32, 1 :64 and 1 : 128. All standards and samples were assayed in triplicate.
  • Monocytes, unloaded dendritic cells and loaded dendritic cells (DC-TC) from a patient derived sample (Sample # 2353) were cultured overnight in AIM-V media (Invitrogen, Grand Island, NY), AIM-V media containing CD40L/interferon-gamma(gIFN) and AIM-V media containing CD40L/gIFN/lipopolysaccharide (LPS). Supernatant was collected, diluted 1 :2, 1 :4, 1 :8, 1 : 16, 1 :32 and 1 :64 and assessed for CCL17/TARC (R&D Systems, catalog # DDN00,
  • the corrected optical density for each sample was then calculated for a diluted concentration by using the equation of the line.
  • the diluted concentrations were then used to calculate the linearity between dilutions.
  • %Linearity (Observed Concentration / (Previous observed value in the dilution series / Dilution Factor))* 100.
  • the acceptable criteria for mean percent linearity for each interval was 80-120%.
  • concentrations of the samples were calculated using diluted concentration values that were found to be within acceptable linearity, and multiplying by the dilution factor (e.g., 2, 4, 8, 16, 32, 64, or 128).
  • TARC concentrations for Sample # 1955 and 1893 cultured in the various conditions were calculated from the diluted concentrations that were within acceptable percent linearity. The calculated TARC concentrations are shown in Figure 2.
  • Figure 3 shows the calculated TARC concentrations for each sample from all diluted concentrations tested.
  • Sample # 2353 was first tested in the IL-12 ELISA at dilutions 1 :2, 1 :4, 1 :8, and 1 : 16.
  • Sample#2353 was re-tested in the IL-12 ELISA undiluted, 1 :2, 1 :4.
  • the standard curves from the IL-12 ELISA Immunoassays for determining the IL-12 concentration of sample 2353 are shown in Figure 8.
  • Table 5 lists the calculated concentrations of IL-12 for Sample # 2353 undiluted, diluted 1 :2 and diluted 1 :4; despite the absence of an acceptable percent linearity.
  • Figure 9 shows the calculated
  • DC-TC Three autologous tumor cell antigen-loaded dendritic cells (DC-TC) were thawed in AIM-V, counted and re-suspended at 1 x 10 6 cells per mL in media consisting of AIM-V or dendritic cell media (AIM-V plus 1000 lU/mL GM-CSF or 400 lU/mL IL-4) with 5% FBS.
  • the DC-TCs were incubated in the presence or absence of maturation conditions consisting 0.5lj,g/mL CD40L/1000 IU gammalFN/1 pg/mL LPS in their respective basal media conditions in mesh top FACS tube for 24 hours.
  • Golgi-Stop (brefeldin-A; BD Biosciences, catalog # 554724, Franklin Lakes, NJ) was not added or was added either continuously or during the final 4 hours of incubation. At the end of the incubation period, the cells were centrifuged, supernatant collected for future assaying and the cell pellets transferred to 96-well plates for processing for flow cytometry. Two types of antibody
  • CD83/IL12 positive cells CD83/IL12 positive cells.
  • CD83/IL12 was detectable optimally only in the post-fixation, 24-hour Golgi- Stop conditions in only two out of three of the samples tested.
  • CD40/CCL17 was not induced by cytokine maturation and was only marginally detectable in the pre-fixation conditions, which was comparable in either media formulation. There was slightly diminished detection rate in the cells treated for 24-hours with Golgi-Stop versus either 4-hours or without Golgi-Stop. Detecting double positive CD40/CCL17 and CD83/IL- 12 dendritic cells required completely separate incubation and fixation conditions for detection. Without being bound by theory, it may be preferential to measure cytokine maturation response for IL-12 and CCL 17/TARC by ELISA while measuring CD83 and CD40 by flow cytometry in the
  • the sequence of maturation steps (e.g., adding toll-like receptor ligands first, followed by the addition of cytokines) also was tested.
  • Cell culture supernatants were collected and the expression of IL-12 and CCL17/TARC by ELISA was determined.
  • a panel of IL-12 antibodies was used to determine which antibody is best suited for detecting IL-12 by flow cytometry.
  • dendritic cells were incubated in CellGenix DC Media (Portsmouth, NH) or AIM-V dendritic cell media containing 1000 lU/mL GM-CSF and 400 lU/mL IL-4 followed by combinatorial maturation conditions using either a standard cytokine cocktail of TNF alpha, IL-l-beta, IL-6 with CD40L/gIFN (CKl) or Poly IC with CD40L/gIFN (CK2) or AIM-V plus 5%FBS (CT) followed by 4 hours incubation with Golgi-stop (brefeldin-A).
  • CellGenix DC Media Portsmouth, NH
  • AIM-V dendritic cell media containing 1000 lU/mL GM-CSF and 400 lU/mL IL-4 followed by combinatorial maturation conditions using either a standard cytokine cocktail of TNF alpha, IL-l-beta, IL-6 with CD40L/gIFN (CKl)
  • CD40L/gIFN 1 1.1 1 19. 14 913.87 34.07
  • CD40L/gIFN 3.5 2.58 680.67 1 12.52
  • IL-12 was consistently detectable by ELISA in conditions were dendritic cells were incubated with LPS and CD40L/gIFN. It was noted that 1940 failed to induce any IL- 12 under any condition (Table 14).
  • Samples were thawed, washed in AIM-V and the cells were counted by hemocytometer (data not shown). Immediately following thawing and cell counting, a sample was stained for CD 14/CD45/7-AAD/CD 1 lc to determine the viability and identity of the prepared samples. Samples sets included paired samples in which three types of samples were available from each patient;
  • DC-TC tumor antigen-loaded dendritic cells
  • Figure 25 shows the range of CCL 17/TARC secretion from samples normalized to time.
  • a DC-TC product must contain at least 25% CD1 l c positive dendritic cells (data not shown).
  • the lowest threshold of CCL 17/TARC secretion was calculated based on the lowest CCL 17/TARC value extrapolated to 25% CD 1 l c positive dendritic cells.
  • the lowest value for CCL 17/TARC secretion per day was 783.9 pg/mL from a sample containing 71.5% CD1 l c positive dendritic cells. Therefore, a final product containing 25% CD1 l c positive cells would secrete 274.3 pg/mL/day.
  • CCL 17/TARC is a suitable marker for potency considering that increased levels of CCL17/TARC were correlated with antigen loading and maturation status.
  • a lower limit of acceptable CCL 17/TARC secretion by a potent, antigen-loaded DC (DC- TC) product can be set at 274 pg/mL/day.

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Abstract

La présente invention concerne des compositions, des procédés et des kits pour déterminer la puissance de cellules dendritiques (DC) utilisées dans l'immunothérapie du cancer.
PCT/US2015/051174 2014-09-23 2015-09-21 Compositions, procédés et kits utilisés pour déterminer la puissance de cellules dendritiques dans l'immunothérapie du cancer Ceased WO2016048872A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017048875A1 (fr) * 2015-09-15 2017-03-23 Northwest Biotherapeutics, Inc Procédés relatifs à des compositions de cellules dendritiques activées et traitements immunothérapeutiques pour des sujets atteints de cancers avancés
WO2020154135A1 (fr) * 2019-01-21 2020-07-30 Essen Instruments, Inc. D/B/A Essen Bioscience, Inc. Cytométrie en flux comprenant une analyse de données destinée à une dilution optimisée d'échantillons de fluide pour une étude de cytométrie en flux
US11709116B2 (en) 2020-02-04 2023-07-25 Sartorius Bioanalytical Instruments, Inc. Liquid flourescent dye concentrate for flow cytometry evaluation of virus-size particles and related products and methods
US12196652B2 (en) 2018-08-01 2025-01-14 Sartorius Bioanalytical Instruments, Inc. Methods, kits and stain compositions for flow cytometry evaluation of unassociated virus-size particles using multiple fluorogenic dyes
US12455287B2 (en) 2019-03-26 2025-10-28 Sartorious BioAnalytical Instruments, Inc. Flow cytometry evaluation for unassociated non-enveloped viral particles

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103599528A (zh) * 2013-12-04 2014-02-26 深圳市合一康生物科技有限公司 人树突状细胞疫苗的制备方法
WO2014138455A1 (fr) * 2013-03-07 2014-09-12 California Stem Cell, Inc. Cellules souches de carcinome hépatocellulaire individualisées et de haute pureté, procédés et utilisation de celles-ci

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014138455A1 (fr) * 2013-03-07 2014-09-12 California Stem Cell, Inc. Cellules souches de carcinome hépatocellulaire individualisées et de haute pureté, procédés et utilisation de celles-ci
CN103599528A (zh) * 2013-12-04 2014-02-26 深圳市合一康生物科技有限公司 人树突状细胞疫苗的制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CORNFORTH, ANDREW N. ET AL.: "Increases in serum TARC/CCL17 levels are associated with progression-free survival in advanced melanoma patients in response to dendritic cell -based immunotherapy", JOURNAL OF CLINICAL IMMUNOLOGY, vol. 29, no. 5, 7 May 2009 (2009-05-07), pages 657 - 664, XP019731105, DOI: doi:10.1007/s10875-009-9299-3 *
JIN, PING ET AL.: "Molecular signatures of maturing dendritic cells: implications for testing the quality of dendritic cell therapies", JOURNAL OF TRANSLATIONAL MEDICINE, vol. 8, no. 4, 15 January 2010 (2010-01-15), pages 1 - 15 *
SABADO, RACHEL LUBONG ET AL.: "Preparation of tumor antigen-loaded mature dendritic cells for immunotherapy", JOURNAL OF VISUALIZED EXPERIMENTS, vol. 78, 1 August 2013 (2013-08-01), pages e50085 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017048875A1 (fr) * 2015-09-15 2017-03-23 Northwest Biotherapeutics, Inc Procédés relatifs à des compositions de cellules dendritiques activées et traitements immunothérapeutiques pour des sujets atteints de cancers avancés
US12196652B2 (en) 2018-08-01 2025-01-14 Sartorius Bioanalytical Instruments, Inc. Methods, kits and stain compositions for flow cytometry evaluation of unassociated virus-size particles using multiple fluorogenic dyes
WO2020154135A1 (fr) * 2019-01-21 2020-07-30 Essen Instruments, Inc. D/B/A Essen Bioscience, Inc. Cytométrie en flux comprenant une analyse de données destinée à une dilution optimisée d'échantillons de fluide pour une étude de cytométrie en flux
CN113167716A (zh) * 2019-01-21 2021-07-23 以埃森生物科学股份有限公司的名义经营的埃森仪器股份有限公司 具有用于流式细胞术研究的流体样本的优化稀释的数据分析的流式细胞术
US11137337B2 (en) 2019-01-21 2021-10-05 Essen Instruments, Inc. Flow cytometry with data analysis for optimized dilution of fluid samples for flow cytometry investigation
US12455287B2 (en) 2019-03-26 2025-10-28 Sartorious BioAnalytical Instruments, Inc. Flow cytometry evaluation for unassociated non-enveloped viral particles
US11709116B2 (en) 2020-02-04 2023-07-25 Sartorius Bioanalytical Instruments, Inc. Liquid flourescent dye concentrate for flow cytometry evaluation of virus-size particles and related products and methods

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