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WO2011063336A2 - Site secondaire de stimulation d'antigène pour vaccination thérapeutique - Google Patents

Site secondaire de stimulation d'antigène pour vaccination thérapeutique Download PDF

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Publication number
WO2011063336A2
WO2011063336A2 PCT/US2010/057630 US2010057630W WO2011063336A2 WO 2011063336 A2 WO2011063336 A2 WO 2011063336A2 US 2010057630 W US2010057630 W US 2010057630W WO 2011063336 A2 WO2011063336 A2 WO 2011063336A2
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WIPO (PCT)
Prior art keywords
antigen
tumor
cells
days
plg
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PCT/US2010/057630
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English (en)
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WO2011063336A3 (fr
Inventor
Omar Abdel-Rahman Ali
Dwaine Emerich
Glenn Dranoff
David J. Mooney
Original Assignee
President And Fellows Of Harvard College
Dana-Farber Cancer Institute, Inc.
Incytu, Inc.
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Application filed by President And Fellows Of Harvard College, Dana-Farber Cancer Institute, Inc., Incytu, Inc. filed Critical President And Fellows Of Harvard College
Publication of WO2011063336A2 publication Critical patent/WO2011063336A2/fr
Publication of WO2011063336A3 publication Critical patent/WO2011063336A3/fr
Priority to US13/741,271 priority Critical patent/US9370558B2/en
Priority to US15/135,216 priority patent/US9821045B2/en
Priority to US15/818,509 priority patent/US10568949B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma

Definitions

  • This invention relates generally to the field of immunology.
  • Vaccines are largely ineffective for patients with established disease, as advanced pathology requires potent and sustained activation of CD8(+), cytotoxic T lymphocyte (CTL) mediated responses.
  • Established diseases such as cancer, normally suppress responses by the immune system leading to immune tolerance to foreign antigen and the disease. As such, there is a pressing need for new strategies to activate the immune system in patients with established disease.
  • a method for eliciting and sustaining an anti-tumor immune response is carried out by administering a device containing an amount of antigen sufficient to stimulate an innate immune response and an adaptive immune response.
  • a method for eliciting and sustaining an anti-tumor immune response is carried out by administering to a first anatomical site a first antigen-loaded acellular biomaterial device (polyactide-co-glycolide (PLG) vaccine) at a first time point to elicit an innate immune response and administering to a second anatomical site at a second time point a second antigen loaded acellular biomaterial device to sustain an adaptive immune response.
  • PLG polyactide-co-glycolide
  • the antigen comprises a tumor cell lysate, granulocyte-macrophage colony- stimulating factor (GM-CSF), and/or cytosine-guanosine (CpG)-oligodeoxynucleotides (ODN).
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • CpG cytosine-guanosine-oligodeoxynucleotides
  • Suitable tumor cells include those from, e.g., central nervous system (CNS) cancers, lung cancer, breast cancer, Leukemia, Multiple Myeloma, Renal Cancer, Malignant Glioma, MeduUoblastoma, and Melanoma.
  • purified tumor antigen e.g. , those listed below is used instead of or together with tumor lysate as the antigen.
  • the methods described herein are suitable for any mammal in need of treatment to elicit or boost an immune response.
  • the mammal can be, e.g., any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, or a pig.
  • the mammal is a human.
  • Each implant stimulates both innate and adaptive immune responses.
  • the device After implantation, the device first recruits antigen presenting cells (dendritic cells (DCs) and macrophages), and then at a later time (at the same implant) cells of the adaptive immune system (T cells) are stimulated. Stimulation of innate immunity followed by adaptive immunity occurs after administration of a single device, provided that antigen is present in the device longer than the onset of innate immunity, i.e., antigen is not depleted from the device during the first 3 days after implantation.
  • DCs dendritic cells
  • T cells adaptive immune system
  • an improved device contains enough antigen to be make the antigen available for continued presentation to endogenous cells for at least 3 days, 7 days, 12 days, 16 days, 30 days, 45 days, 90 days, 120 days, or more to prolong the adaptive immune response.
  • a second (and subsequent administrations/implantations of the device) are made to prolong the stimulation of the immune response to generate adaptive immune cells. Multiple rounds of vaccinations increase and extend an anti-tumor response.
  • Vaccination requires stimulation for at least 3 days, e.g., at least 4 days, 5 days, 6 days, or 7 days) to induce the onset of innate immunity (i.e., DC responses) and effector cytotoxic T lymphocyte (CTL) responses are maintained until antigen is depleted from the vaccine site.
  • innate immunity i.e., DC responses
  • CTL cytotoxic T lymphocyte
  • the innate immune response which is initially induced by the first device during the first time period (days following the first time point) is characterized by an infiltration of dendritic cells and macrophages, i.e., antigen-presenting cells.
  • the innate immune response is characterized by an increase in plasmacytoid dendritic cells (pDCs).
  • the vaccine induces persistent interleukin-12 (IL-12) and interferon- ⁇ (IFN- ⁇ ) production consistent with DC and T cell infiltration into the vaccine site.
  • the adaptive immune response is characterized by cytotoxic T cells (CTLs).
  • CTLs cytotoxic T cells
  • the second time point occurs after onset of the innate immune response and before depletion of antigen in the biomaterial device.
  • a plurality of devices is administered to elicit and sustain an anti-tumor immune response for an extended period of time.
  • the first and second time points are sequential or simultaneous (or closely spaced, e.g., within minutes or hours of one another).
  • the second time point is between 1-120 days, e.g., 1-5 days, 1-10 days, 1-15 days, 1-20 days, 1-30 days, 1-60 days, or 3-38 days, after the first time point.
  • An exemplary vaccination protocol is characterized by a first vaccination followed by a second vaccination that is 10 days after said first time point (first vaccination).
  • implantation of a plurality of devices occurs at or about the same time.
  • the secondary antigen site is provided using a single device containing an ample amount of antigen to span the time during which both innate and adaptive immunity is developed or by a plurality of devices that are temporally spaced and/or geographically spaced such that multiple devices provide a continuing source of antigen to sustain the adaptive immune response to reduce and eliminate tumors.
  • the vaccine devices and methods described herein are useful to reduce tumor burden, e.g., tumor mass of solid tumors, and eradicate such tumors.
  • Types of cancers to be treated include central nervous system (CNS) cancers, CNS germ cell tumors, lung cancer, breast cancer, prostate cancer, liver cancer, Leukemia, Multiple Myeloma, renal cancer, Malignant Glioma, Medulloblastoma, and Melanoma.
  • CNS central nervous system
  • the methods and devices are also useful to treat infections, e.g., at discrete anatomical sites caused by microbial pathogens such as bacteria, viruses, fungi.
  • a primary antigen (device) site is at or near a target tumor site, and a secondary antigen site or sites (device(s)) are implanted at, near, or at a distant site relative to the location of the tumor.
  • device(s) are implanted at a distance of 1, 3, 5, 10, 15, 20, 25, 40 mm from a tumor site or site of excision, e.g., 16-21 mm away from a tumor mass.
  • the second or subsequent implant(s) are optionally placed anywhere in the body. For microbial infections, a similar device location strategy is employed.
  • An adjuvant is optionally included in the secondary antigen site to boost adaptive responses.
  • exemplary adjuvants are listed as follows: mineral salts, e.g., aluminium hydroxide and aluminium or calcium phosphate gels; oil emulsions and surfactant based formulations, e.g., MF59 (microfiuidised detergent stabilised oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion + MPL + QS-21), Montanide ISA- 51 and IS A-720 (stabilised water-in-oil emulsion); particulate adjuvants, e.g.
  • virosomes unilamellar liposomal vehicles incorporating influenza haemagglutinin
  • AS04 [SBAS4] Al salt with MPL
  • ISCOMS structured complex of saponins and lipids
  • PLG polylactide co- glycolide
  • microbial derivatives naturally and synthetic, e.g., monophosphoryl lipid A (MPL), Detox (MPL + M.
  • Phlei cell wall skeleton Phlei cell wall skeleton
  • AGP RC-529
  • DC Chol lipoidal immunostimulators able to self organize into liposomes
  • OM-174 lipid A derivative
  • modified LT and CT genetically modified bacterial toxins to provide non-toxic adjuvant effects
  • immunomodulators e.g., hGM-CSF or hIL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array); inert vehicles, such as gold particles.
  • hGM-CSF or hIL-12 cytokines that can be administered either as protein or plasmid encoded
  • Immudaptin C3d tandem array
  • inert vehicles such as gold particles.
  • the invention includes a method for progressively reducing tumor burden by successively administering an antigen-loaded acellular biomaterial device.
  • functionalized biomaterials incorporating various combinations of an inflammatory cytokine, immune danger signal, and tumor lysates are administered to control the activation and localization of host dendritic populations in situ.
  • the methods include the steps of identifying a subject with an established disease (at a defined location) and implementing a successive administration schedule until the disease, e.g. , tumor or infection, is substantially reduced or eradicated.
  • the schedule comprises a first implantation and a second implantation of an antigen- loaded acellular biomaterial device ⁇ e.g., PLG vaccine device) and a second implantation occurring at least 24 hours prior to antigen depletion of first device.
  • This schedule, first and second implantations, are repeated successively for as many cycles as required to progressively reduce (or eliminate) tumor burden.
  • the invention therefore features methods for treating established pathologies using vaccines that contain secondary immunostimulatory sites of sustained antigen presentation.
  • Effective vaccination against established tumors was dramatically enhanced with sustained presentation of tumor associated antigens at a secondary site during the clearance of the disease.
  • Secondary sites include immunostimulatory signals to amplify immune responses.
  • Vaccination is enhanced by sustained antigen stimulation to immune cells that are maintained longer than the onset of innate immunity (i.e., infiltration of macrophages, DCs - typically 24 hours).
  • Multiple vaccinations (at least two rounds of administration) are better than one vaccination to sustain antigen presentation. Appropriate timing between multiple vaccinations.
  • vaccinations is between 3-28 days to effectively sustain antigen presentation, i.e., vaccines are administered between days 3-28 after administration of the previous vaccine.
  • the device recruit cells in vivo, modifies these cells, and then promotes their migration to another site in the body.
  • This approach is exemplified herein in the context of immune cells and cancer vaccine development, but is also useful to other vaccines such as those against microbial pathogens as well as cell therapies in general.
  • Figure 1 is a series of panels illustrating that granulocyte-macrophage colony- stimulating factor (GM-CSF) delivery from polyactide-co-glycolide (PLG) matrices promotes CD1 lb+ DC recruitment and activation.
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • PLG polyactide-co-glycolide
  • A H&E staining of sectioned PLG scaffolds explanted from subcutaneous pockets in the backs of C57BL/6J mice after 14 days: Blank scaffolds (BLANK), and GM-CSF (3000 ng) loaded scaffolds (GM-CSF).
  • B The number of CD1 lc(+) DCs isolated from PLG scaffolds at day 14 after implantation in response to doses of 0, 1000, 3000 and 7000 ng of GM-CSF.
  • C Fluorescence-activated cell sorting (FACS) plots of cells isolated from explanted scaffolds and stained for CD1 lc and CD1 lb. Cells were isolated from PLG matrices incorporating 3,000 ng of GM-CSF at day 10 post- implantation. Numbers in FACS plots indicate the percentage of the cell population positive for CD1 lc and CD1 lb, or for both markers.
  • D The number of CD1 lc(+)CD86(+),
  • Figure 2 is a series of panels demonstrating that cytosine-guanosine (CpG)- oligodeoxynucleotides (ODN) and GM-CSF delivery from PLG matrices promotes plasmacytoid DC generation and the production of anti-tumor cytokines.
  • CpG cytosine-guanosine
  • ODN oligodeoxynucleotides
  • GM-CSF delivery from PLG matrices promotes plasmacytoid DC generation and the production of anti-tumor cytokines.
  • A FACS plots of cells isolated from explanted scaffolds and stained for the plasmacytoid DC markers, CDl lc and PDCA-1. Cells were isolated from PLG matrices incorporating 0, 1, 10, and 100 ⁇ g of CpG-ODN at day 10 post-implantation. Numbers in FACS plots indicate the percentage of the cell population positive for CDl lc only or for both markers.
  • GM-CSF in combination with 10 (10+GM), or 100 ⁇ g (100+GM) of CpG-ODN.
  • Figure 3 is a series of panels showing that tumor lysate, CpG-ODN, and GM-CSF co- delivery from PLG matrices stimulates CD8+ DC generation and IL-12 production.
  • A FACS density plots of CDl lc and CD8 staining of cells infiltrating Blank PLG matrices (blank) or matrices loaded with 3000 ng GM-CSF and 100 ⁇ g CpG-ODN without
  • CpG+GM or with tumor lysates (CpG+GM+Ant) at day 10. Numbers in FACS plots indicate the percentage of the cell population positive for CDl lc and CD8 or for both markers.
  • B-D The number of (B) CD 11 c(+)CD8(+) cDCs, (C) plasmacytoid DCs, and (D) CDl lc(+)CDl lb(+)cDCs at day 10 after implantation in blank matrices (Blank) and in response to 3000ng GMCSF (GM) or 100 ⁇ g CpG-ODN (CpG) alone or in combination (CpG+GM) or co-presented with tumor lysates (GM+Ant, CpG+Ant and CpG+GM+Ant).
  • E-G The in vivo concentration of (E) IL-12, (F) IFN-a, and (G) IFN- ⁇ at day 10 after implantation in blank matrices (Blank) and in response to doses of 3000 ng GM-CSF (GM) or 100 ⁇ g CpG-ODN (CpG) alone or in combination (CpG+GM) or co-presented with tumor lysates (GM+Ant, CpG+Ant and CpG+GM+Ant).
  • Figure 4 is a series of panels illustrating that tumor lysate, CpG-ODN, and GM-CSF co-delivery in PLG matrices stimulates potent local and systemic CD8+ cytoxic T cells.
  • A FACS plots of cells isolated from explanted matrices and stained for the cytotoxic T cell markers, CD3 and CD8a. Cells were isolated from PLG matrices with 3000 ng GM-CSF, 100 ⁇ g CpG-ODN and tumor lysates at days 1, 5, 12 and 21 after implantation.
  • C The number of CD8 T cells at day 12 after implantation in blank scaffolds (Blank) or in response to lysate alone (Lys) or in combination with CpG-ODN (CpG+Lys) or GM-CSF (GM+Lys) or both factors (GM+Lys+CpG).
  • D FACS plots of splenocytes of naive mice and mice vaccinated with PLG vaccines containing 3000 ng GM- CSF, 100 ⁇ gCpG-ODN, and tumor lysates at day 16 post-implantation. Cells were stained with anti-CD8-PE Ab, and Kb/TRP2 pentamers.
  • the gates represent the TRP2-specific, CD8(+) T cells and numbers provide the percentage of gated cells.
  • Figure 5 is a series of graphs demonstrating that tumor protection stimulated by engineered PLG matrices is correlated with DC subsets and IL-12 production. Survival times of mice vaccinated with PLG vaccines 14 days prior to B16-F10 melanoma tumor challenge (10 5 cells).
  • (A) A comparison of survival times in mice treated with blank PLG matrices or with PLG matrices loaded with tumor lysates and 1, 10, 50 or 100 ⁇ g of CpG-ODN.
  • B A comparison of survival times in mice vaccinated with PLG matrices loaded with tumor lysates, 3000 ng GM-CSF and either 1, 10, 50 or 100 ⁇ g of CpG-ODN.
  • CD1 lc(+) DC population consisting of CD1 lb(+) cDCs (white bar), PDCA-1(+) pDCs (black bar), and CD8(+) cDCs (striped bar) generated at the PLG vaccine site at day 10.
  • Vaccines were loaded with either 3000 ng GM-CSF, or 100 ⁇ g of CpG-ODN alone or in combination. Survival percentages recorded at day 100 after tumor challenge.
  • Figure 6 is a series of charts showing that engineered PLG matrices attenuate FoxP3+ Tregs and immunosuppressive cytokines.
  • A The total number of CD3(+)CD4(+) T cells isolated from PLG matrices loaded with GMCSF, CpG-ODN and tumor lysates as a function of time after implantation.
  • B The number of CD4 T cells at day 12 after implantation in blank scaffolds (Blank) or in response to lysate alone (Lys) or in combination with CpG- ODN (CpG+Lys) or GM-CSF (GM+Lys) or both factors (GM+Lys+CpG).
  • Cells were isolated from PLG matrices incorporating GM-CSF and lysates without (GM+Lys) or with GM-CSF, lysates and CpGODN (GM+Lys+CpG) at day 12 after implantation. Numbers in FACS plots indicate the percentage of the cell population positive for both markers.
  • F The number of FoxP3(+) Tregs at day 12 post-implantation in blank scaffolds (Blank) or in response to lysate alone (Lys) or in combination with CpG-ODN (CpG+Lys) or GM-CSF (GM+Lys) or both factors (GM+Lys +CpG).
  • Figure 7 is a series of graphs demonstrating that Engineered PLG matrices stimulate the regression of established melanomas
  • mice were also treated once (Vax, lx; at day 9) or twice (Vax, 2x; at days 9 and 19) with PLG matrices incorporating GM-CSF, CpG-ODN and tumor lysates (Vax). Mice were also vaccinated with 5 10 5 irradiated, GM-CSF transduced B 16-F 10 cells.
  • C The individual tumor growth curves for each mouse surviving tumor challenge (5 ⁇ 10 5 cells) after a two-time treatment with PLG vaccines at days 9 and 19.
  • Figure 8 is a series of FACS plots of cells isolated from explanted scaffolds and stained for the DC marker, CDl lc, and for activation markers MHCII and CCR7.
  • Cells were isolated from blank PLG matrices or matrices incorporating either 3 or 7 ⁇ g of GM-CSF at day 28 post-implantation. Numbers in FACS plots indicate the percentage of the cell population positive for CDl lc only or positive for both CDl lc and either MHCII or CCR7.
  • Figure 9 shows the effect of PLG vaccine duration on efficacy in B16 melanoma tumor models.
  • A Photomicrograph of surface of macroporous, PLG-based vaccine loaded with GM-CSF, tumor lysates and CpG-ODN.
  • B SEM micrograph of cross section of macroporous, PLG vaccine. Scale bar - 200 ⁇ .
  • C Schematic of PLG vaccine regimen for both prophylactic and therapeutic B16 models in mice. [Prophylactic] Vaccine duration is varied from 0, 1, 3, 7, 12, and 16 days.
  • D A comparison of the survival time and
  • E the survival percentage at day 100 in mice treated with blank PLG scaffolds, and PLG vaccines for a duration of 1, 3, 7, 12, 16 days or indefinitely (>16).
  • mice were challenged with 10 4 B16-F10 melanoma tumor cells at day 14 after the start of vaccination.
  • F Effect of vaccine duration on therapeutic efficacy [Therapeutic]. Tumor growth curves for mice vaccinated at day 9 after tumor challenge (10 5 B16 melanoma cells), with PLG vaccines for a duration of 1, 3, 7, 12, 16 days or indefinitely (>16). The total GM-CSF dose was 3000 ng, and CpG-ODN dose was 100 ⁇ g. Values in E represent mean and standard error.
  • Figure 10 demonstrates the kinetics of DC and T cell infiltration into PLG vaccine site.
  • A Hematoxylin & Eosin staining of sectioned PLG vaccines explanted from
  • Figure 11 illustrates the kinetics of IL-12 and IFN- ⁇ production at vaccine site.
  • A The concentrations of IL-12 at the implant site of PLG vaccines as a function of time post- implantation.
  • B The in vivo concentration of IL-12 at the implant site of blank matrices (Blanks), PLG vaccines (GM+CpG+LYS) or matrices loaded with either Lysate alone (Lys) or Lysate with 3000 ng of GM-CSF (GM+Lys) or Lysate withlOO ⁇ g PEI-CpG-ODN (CpG+Lys) or the combination of GM-CSF and PEI-CpG-ODN (GM+CpG) at Day 12 after implantation into the backs of C57BL/6J mice.
  • (C) The concentration of IFN- ⁇ at the implant site of PLG vaccines as a function of time post-implantation.
  • D The in vivo concentration of IFN- ⁇ at the implant site of blank matrices (Blanks), PLG vaccines (GM+CpG+LYS) or matrices loaded with either Lysate alone (Lys) or Lysate with 3000 ng of GM-CSF
  • GM+Lys Lysate withlOO ⁇ g PEI-CpG-ODN (CpG+Lys) or the combination of GM-CSF and PEI-CpG-ODN (GM+CpG) at Day 12 after implantation into the backs of C57BL/6J mice.
  • Figure 12 demonstrates the effects of PLG vaccination on cells in draining lymph nodes (A & B) The number of phagocytic cells: CDl lb(+)CDl lc(-) macrophages, CDl lc(+) DCs, and PDCA-1(+)CD1 lc(+) plasmacytoid DCs, in the draining inguinal lymph nodes of mice at (A) day 3 and at (B) day 10 after implantation of blank matrices (Blank) or PLG matrices containing GM-CSF and Lysate (GMCSF+LYS) only or PLG vaccines
  • T cells CD3(+) T cells, CD3(+)CD8(+) T cells, and CD3(+)CD4(+) T cells, in the draining inguinal lymph nodes of mice at day 10 after implantation of blank matrices (Blank) or PLG matrices containing GM-CSF and Lysate (GMCSF+LYS) only or with CpG-ODN (GMCSF+Lys+CpG).
  • Figure 13 shows the sustained tumor antigen presentation by PLG vaccines.
  • the invention described herein is based on the discovery that recruitment and activation of multiple dendritic cell (DC) and T cell subsets provide therapeutic vaccination against established tumors.
  • DC dendritic cell
  • the methods described herein are utilized to treat established pathologies using vaccines containing secondary and immunostimulatory sites of antigen presentation during immune clearance of disease.
  • secondary and stimulatory sites of antigen presentation are required to maintain effector T cell responses until the primary site of established disease has cleared.
  • one implant single administration of vaccine
  • the vaccine is sufficient to both stimulate innate immunity and provide the secondary site of antigen stimulation required to further stimulate an adaptive response.
  • the vaccine is fabricated to persist for longer than the onset of innate immunity, it also provides a secondary site of antigen presentation.
  • the methods described herein are suitable for any mammal in need of treatment.
  • the mammal can be, e.g., any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, or a pig.
  • the mammal is a human.
  • DCs Dendritic cells
  • DCs Dendritic cells
  • PAMPs unique to invading pathogens
  • PAMPs including lipopolysacharides and cytosine-guanosine (CpG) sequences in bacterial deoxyribonucleic acid (DNA), trigger a particular toll-like receptor (TLR), that allows DCs to classify the pathogen and induce their expression of T cell-activating molecules
  • TLR toll-like receptor
  • Activated DCs then migrate to draining lymph nodes (LNs) where they prime the appropriate T cell response, via the presentation of antigen, costimulatory molecules and the appropriate cytokines.
  • T regulatory (Treg) cells allows solid tumors to develop by dysregulating DC activity and the cytotoxic T lymphocyte (CTL) responses required to kill tumor cells
  • CTL cytotoxic T lymphocyte
  • Cancer vaccines are frequently developed with easily accessible, patient- derived blood monocytes that are transformed into DCs ex vivo with cytokine mixtures and pulsed with tumor antigens to promote antigen presentation (R. M.
  • TGF-b transforming growth factor-b
  • IL-10 interleukin-10
  • Hematopoietic precursor cells of both the myeloid and lymphoid lineage have the capacity to differentiate into two main categories of DCs: conventional DCs (cDCs) and plasmacytoid DCs (pDCs) (S. H. Naik, P. Sathe, H. Y. Park, D. Metcalf, A. I. Proietto, A. Dakic, S. Carotta, M. O'Keeffe, M. Bahlo, A. Papenfuss, J. Y. Kwak, L. Wu, K. Shortman, Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. Nat. Immunol. 8, 1217-1226 (2007); A. O'Garra, G.
  • cDCs include CD1 lc+CDl lb+ and CD1 lc+CD8+ cells and exhibit classic DC morphology, protruding dendrites that make these cells especially adept at antigen processing and antigen presentation to T cells (S. H. Naik, P. Sathe, H. Y. Park, D. Metcalf, A. I. Proietto, A. Dakic, S. Carotta, M. O'Keeffe, M. Bahlo, A. Papenfuss, J. Y. Kwak, L. Wu, K. Shortman,
  • Plasmacytoid DCs exhibit a spherical morphology and can produce large amounts of type 1 interferons (IFNs) in response to "danger signals," such as unmethylated CpG dinucleotide sequences found in bacterial or viral DNA (A. M. Krieg, Development of TLR9 agonists for cancer therapy. J. Clin. Invest.
  • IFNs type 1 interferons
  • Plasmacytoid DC-derived type 1 IFNs link innate and adaptive immunity to viral infection by directly inducing naive T cell differentiation to TH1 cells (J. J. O'Shea, R.
  • biomaterials are utilized to extend the duration of immunostimulation, and act as adjuvants and to enable controlled presentation of antigens to the immune system (Hubbell JA, Thomas SN, Swartz MA. Materials engineering for immunomodulation. Nature. 462, 449-60. (2009); Uchida M, Natsume H, Kishino T, Seki T, Ogihara M, Juni K, Kimura M, Morimoto Y. Immunization by particle bombardment of antigen-loaded poly-(DL- lactide-co-glycolide) microspheres in mice. Vaccine. 12, 2120-30. (2006)).
  • Material-based particulate systems control antigen localizaton in tissue or to target host DCs specifically via antibody or ligand conjugation (Reddy, S. T. et al. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nature Biotechnol. 25, 1159-1164 (2007); Kwon YJ, et al. In vivo targeting of dendritic cells for activation of cellular immunity using vaccine carriers based on pH-responsive microparticles, Proc. Natl. Acad. Sci. U. S. A. 102 18264-18268. (2005)).
  • biomaterial-based vaccines prior to the invention described herein could enhance antigen loading to DCs, leading to antigen specific T cell activation, they lacked concurrent regulation of effector T cell activity.
  • therapeutic vaccination against an established disease including solid tumors, requires methods that stimulate not only innate immunity and DCs, but also persistent cytotoxic T lymphocyte (CTL) responses, which kill tumor cells, until the disease has cleared. Therefore, the natural kinetics of innate and adaptive immune responses and the coordination of these responses by vaccines impact vaccine design and application.
  • CTL cytotoxic T lymphocyte
  • Described herein is the in situ generation of a heterogeneous DC network capable of CTL induction to activate robust CD8+ T cell effector responses to established tumors, by providing a secondary immunostimulatory site of tumor antigen presentation.
  • the results presented herein describe the kinetics of innate DC responses and adaptive T cell responses and illustrate their fundamental relationship to potent tumor rejection when implanted subcutaneously in a mouse B16 model of late-stage melanoma.
  • Also described in detail below is the relationship between vaccine duration and efficacy in melanoma models and its correlation to local kinetics of DC and T-cell responses to infection-mimicking polymers.
  • Implantable, synthetic polylactide-co-glycolide (PLG) matrices that spatially and temporally control the in vivo presentation of cytokines, tumor antigens and TLR-activating, danger signals have been described (Ali OA, Huebsch N, Cao L, Dranoff G, Mooney DJ. Infection-mimicking materials to program dendritic cells in situ. Nat Mater 2, 151-158 (2009); Ali OA, Emerich D, Dranoff G, and Mooney DJ. In situ Regulation of DC Subsets and T Cells Mediates Tumor Regression in Mice Sci Transl Med. 1, 8-19. (2009)).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • CpG-ODN CpG-rich oligonucleotides
  • antigen tumor lysates
  • the vaccination systems described herein cause immune-mediated eradication of distant and established B16 melanoma tumors in mice. As described below, the duration of vaccination was varied to determine the impact on vaccine efficacy. Also described herein is the kinetics of innate (DCs) and adaptive cellular responses (cytotoxic T cells) to these vaccines, and the inflection point when innate responses transition to effective anti-tumor immune responses after vaccination. Because the cytokines interleukin (IL)-12 and interferon (IFN)-y are important mediators of cytotoxic T cell responses to viruses and tumors (O'Shea JJ, and Visconti R. Type 1 IFNs and regulation of TH1 responses: enigmas both resolved and emerge.
  • IL interleukin
  • IFN interferon
  • the vaccine's induction of systemic cellular responses were monitored at the inguinal LN draining the site of vaccination, as these are points where naive T cells interact with antigen presenting cells (APCs; DCs and macrophages) and are important locations where CD8+ CTL cells are primed.
  • APCs antigen presenting cells
  • vaccines Prior to the invention described herein, vaccines were unable to monitor and trigger the T effector profile described here, but instead induced at least partially dysfunctional T cells that are more likely to undergo exhaustion within the immunosuppressive
  • PLG Antigen-laden, macroporous poly (lactide-co-glycolide) (PLG) scaffolds induce potent dendritic cell (DC) and cytotoxic T-lymphocyte (CTL) responses that maintain a "danger" microenvironment via the controlled signaling of inflammatory cytokines and tolllike receptor agonists (PLG vaccines).
  • DC dendritic cell
  • CTL cytotoxic T-lymphocyte
  • Scaffolds for cell transplantation are generally described in US 2008-0044900 Al and PCT Publication WO 2009/102465, both of which are incorporated herein by reference in their entirety.
  • Components of the scaffolds are organized in a variety of geometric shapes (e.g., beads, pellets), niches, planar layers (e.g., thin sheets).
  • multicomponent scaffolds are constructed in concentric layers each of which is characterized by different physical qualities (% polymer, % crosslinking of polymer, chemical composition of scaffold, pore size, porosity, and pore architecture, stiffness, toughness, ductility, viscoelasticity, and or composition of bioactive substances such as growth factors, homing/migration factors, differentiation factors.
  • Each niche has a specific effect on a cell population, e.g., promoting or inhibiting a specific cellular function, proliferation, differentiation, elaboration of secreted factors or enzymes, or migration.
  • Cells incubated in the scaffold are educated and induced to migrate out of the scaffold to directly affect a target tissue, e.g., and injured tissue site.
  • stromal vascular cells and smooth muscle cells are useful in sheetlike structures are used for repair of vessel-like structures such as blood vessels or layers of the body cavity.
  • vessel-like structures such as blood vessels or layers of the body cavity.
  • such structures are used to repair abdominal wall injuries or defects such as gastroschisis.
  • sheetlike scaffolds seeded with dermal stem cells and/or keratinocytes are used in bandages or wound dressings for regeneration of dermal tissue.
  • the device is placed or transplanted on or next to a target tissue, in a protected location in the body, next to blood vessels, or outside the body as in the case of an external wound dressing.
  • Devices are introduced into or onto a bodily tissue using a variety of known methods and tools, e.g., spoon, tweezers or graspers, hypodermic needle, endoscopic manipulator, endo- or trans-vascular- catheter, stereotaxic needle, snake device, organ-surface-crawling robot (United States Patent Application
  • a scaffold or scaffold device is the physical structure upon which or into which cells associate or attach
  • a scaffold composition is the material from which the structure is made.
  • scaffold compositions include biodegradable or permanent materials such as those listed below.
  • the mechanical characteristics of the scaffold vary according to the application or tissue type for which regeneration is sought. It is biodegradable ⁇ e.g., collagen, alginates, polysaccharides, polyethylene glycol (PEG), poly(glycolide) (PGA), poly(L-lactide) (PLA), or poly(lactide-co-glycolide) (PLGA) or permanent ⁇ e.g., silk).
  • the composition is degraded by physical or chemical action, e.g., level of hydration, heat or ion exchange or by cellular action, e.g., elaboration of enzyme, peptides, or other compounds by nearby or resident cells.
  • the consistency varies from a soft/pliable ⁇ e.g., a gel) to glassy, rubbery, brittle, tough, elastic, stiff.
  • the structures contain pores, which are nanoporous, microporous, or macroporous, and the pattern of the pores is optionally homogeneous, heterogenous, aligned, repeating, or random.
  • Alginates are versatile polysaccharide based polymers that may be formulated for specific applications by controlling the molecular weight, rate of degradation and method of scaffold formation. Coupling reactions can be used to covalently attach bioactive epitopes, such as the cell adhesion sequence RGD to the polymer backbone.
  • Alginate polymers are formed into a variety of scaffold types. Injectable hydrogels can be formed from low MW alginate solutions upon addition of a cross-linking agents, such as calcium ions, while macroporous scaffolds are formed by lyophilization of high MW alginate discs. Differences in scaffold formulation control the kinetics of scaffold degradation.
  • Release rates of morphogens or other bioactive substances from alginate scaffolds is controlled by scaffold formulation to present morphogens in a spatially and temporally controlled manner. This controlled release not only eliminates systemic side effects and the need for multiple injections, but can be used to create a microenvironment that activates host cells at the implant site and transplanted cells seeded onto a scaffold.
  • the scaffold comprises a biocompatible polymer matrix that is optionally
  • a hydrogel is one example of a suitable polymer matrix material.
  • materials which can form hydrogels include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids such as polypeptides particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon.- caprolactone, polyanhydrides; polyphosphazines, poly( vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymers of the above, including graft copolymers.
  • the scaffolds are fabricated from a variety of synthetic polymers and naturally- occurring polymers such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels.
  • One preferred material for the hydrogel is alginate or modified alginate material.
  • Alginate molecules are comprised of (l-4)-linked ⁇ -D-mannuronic acid (M units) and a L-guluronic acid (G units) monomers, which can vary in proportion and sequential distribution along the polymer chain.
  • Alginate polysaccharides are polyelectrolyte systems which have a strong affinity for divalent cations (e.g. Ca +2 , Mg +2 , Ba +2 ) and form stable hydrogels when exposed to these molecules.
  • calcium cross-linked alginate hydrogels are useful for dental applications, wound dressings chondrocyte transplantation and as a matrix for other cell types.
  • An exemplary device utilizes an alginate or other polysaccharide of a relatively low molecular weight, preferably of size which, after dissolution, is at the renal threshold for clearance by humans, e.g., the alginate or polysaccharide is reduced to a molecular weight of 1000 to 80,000 daltons. Prefereably, the molecular mass is 1000 to 60,000 daltons, particularly preferably 1000 to 50,000 daltons. It is also useful to use an alginate material of high guluronate content since the guluronate units, as opposed to the mannuronate units, provide sites for ionic crosslinking through divalent cations to gel the polymer.
  • U.S. Patent Number 6,642,363, incorporated herein by reference discloses methods for making and using polymers containing polysachharides such as alginates or modified alginates that are particularly useful for cell transplantation and tissue engineering applications.
  • Useful polysaccharides other than alginates include agarose and microbial polysaccharides such as those listed in the table below.
  • Xanthan gum (A) 1,4- ⁇ .-D-Glucan with D-mannose;
  • Curdlan (N) 1.3- ⁇ .-D-Glucan (with branching)
  • the scaffolds of the invention are porous or non-porous.
  • the scaffolds are nanoporous having a diameter of less than about 10 nm; microporous wherein the diameter of the pores are preferably in the range of about 100 nm-20 ⁇ ; or macroporous wherein the diameter of the pores are greater than about 20 ⁇ , more preferably greater than about 100 ⁇ and even more preferably greater than about 400 ⁇ .
  • the scaffold is macroporous with aligned pores of about 400-500 ⁇ in diameter.
  • the preparation of polymer matrices having the desired pore sizes and pore alignments are described in the Examples. Other methods of preparing porous hydrogel products are known in the art. (U.S. Pat. No. 6,511 ,650 incorporated herein by reference).
  • the device includes one or more bioactive compositions.
  • Bioactive compositions are purified naturally-occurring, synthetically produced, or recombinant compounds, e.g., polypeptides, nucleic acids, small molecules, or other agents.
  • the compositions described herein are purified. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%), and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • Exemplary bioactive composition includes tumor antigens, tumor cell lysates, or tumor cells.
  • the bioactive composition includes tumor cell lysate, GM-CSF, and CpG-ODN.
  • the biocompatible scaffolds are useful as delivery vehicles for cancer vaccines.
  • the cancer vaccine stimulates an endogenous immune response against cancer cells.
  • vaccines Prior to the invention described herein, vaccines predominantly activated the humoral immune system (i.e., the antibody dependent immune response).
  • Other vaccines focused on activating the cell-mediated immune system including cytotoxic T lymphocytes which are capable of killing tumor cells.
  • Cancer vaccines generally enhance the presentation of cancer antigens to both antigen presenting cells (e.g., macrophages and dendritic cells) and/or to other immune cells such as T cells, B cells, and NK cells.
  • cancer vaccines may take one of several forms, their purpose is to deliver cancer antigens and/or cancer associated antigens to antigen presenting cells (APC) in order to facilitate the endogenous processing of such antigens by APC and the ultimate presentation of antigen presentation on the cell surface in the context of MHC class I molecules.
  • APC antigen presenting cells
  • One form of cancer vaccine is a whole cell vaccine which is a preparation of cancer cells which have been removed from a subject, treated ex vivo and then reintroduced as whole cells in the subject. These treatments optionally involve cytokine exposure to activate the cells, genetic manipulation to overexpress cytokines from the cells, or priming with tumor specific antigens or cocktails of antigens, and expansion in culture.
  • Dendritic cell vaccines activate antigen presenting cells directly, and their proliferation, activation and migration to lymph nodes is regulated by scaffold compositions to enhance their ability to elicit an immune response.
  • Types of cancers to be treated include central nervous system (CNS) cancers, CNS Germ Cell tumor, lung cancer, Leukemia, Multiple Myeloma, Renal Cancer, Malignant Glioma, Medulloblastoma, and Melanoma.
  • CNS central nervous system
  • a scaffold device is implanted into a mammal.
  • the device is tailored to activate immune cells and prime the cells with a specific antigen thereby enhancing immune defenses and destruction of undesired tissues and targeted microorganisms such as bacterial or viral pathogens.
  • the device attracts appropriate immune cells, such as macrophages, T cells, B cells, NK cells, and dendritic cells, by containing and/or releasing signaling substances such as GM-CSF.
  • signaling substances are incorporated in the scaffold composition in such a way as to control their release spatially and temporally using the same techniques used to integrate other bioactive compounds in the scaffold composition.
  • the device programs the immune cells to attack or cause other aspects of the immune system to attack undesired tissues (e.g., cancer, adipose deposits, or virus-infected or otherwise diseased cells) or microorganisms.
  • Immune cell activation is accomplished by exposing the resident immune cells to preparations of target-specific compositions, e.g., ligands found on the surface of the undesired tissues or organisms, such as cancer cell surface markers, viral proteins, oligonucleatides, peptide sequences or other specific antigens.
  • target-specific compositions e.g., ligands found on the surface of the undesired tissues or organisms, such as cancer cell surface markers, viral proteins, oligonucleatides, peptide sequences or other specific antigens.
  • useful cancer cell-specific antigens and other tissue or organism-specific proteins are listed in the table below.
  • the device optionally contains multiple ligands or antigens in order to create a multivalent vaccine.
  • the compositions are embedded in or coated on the surface of one or more compartments of the scaffold composition such that immune cells migrating through the device are exposed to the compositions in their traverse through the device. Antigens or other immune stimulatory molecules are exposed or become exposed to the cells as the scaffold composition degrades.
  • the device may also contain vaccine adjuvants that program the immune cells to recognize ligands and enhance antigen presentation.
  • Exemplary vaccine adjuvants include chemokines/cytokines, CpG rich oligonucleotides, or antibodies that are exposed concurrently with target cell-specific antigens or ligands.
  • the device attracts immune cells to migrate into a scaffold where they are educated in an antigen-specific manner and activated.
  • the programmed immune cells are then induced to egress towards lymph nodes in a number of ways.
  • the recruitment composition and deployment signal/composition e.g., a lymph node migration inducing substance, is released in one or more bursts, programmed by the method of incorporation and/or release from the scaffold material, or controlled by the sequential degradation of scaffold compartments which contain the attractant. When a burst dissipates, the cells migrate away. Compartments containing repulsive substances are designed to degrade and release the repulsive substance in one or more bursts or steadily over time.
  • Relative concentration of the repulsive substances cause the immune cells to migrate out of the device.
  • cells which have been placed in or have migrated into the device are programmed to release repulsive substances or to change their own behavior.
  • localized gene therapy is carried out by cell exposure to plasmid DNA attached to the scaffold.
  • Useful repulsive substances include chemokines and cytokines.
  • the device may cause immune cells to egress by degrading and releasing them.
  • LPS Lipopolysacharides
  • telomerase ferment hTRT
  • MUCIN 1 TUMOR-ASSOCIATED MUCIN
  • CARCINOMA-ASSOCIATED MUCIN POLYMORPHIC EPITHELIAL MUCIN
  • PEM EPITHELIAL MEMBRANE ANTIGEN
  • EEMBRANE ANTIGEN EPITHELIAL MEMBRANE ANTIGEN
  • EMA EMB
  • H23 AG H23 AG
  • PEANUT-REACTIVE URINARY MUCIN PUM
  • PUM BREAST CARCINOMA- ASSOCIATED ANTIGEN DF3
  • MAGE-C1 (cancer/testis antigen CT7)
  • MAGE-B1 ANTIGEN MAGE-XP ANTIGEN
  • Adenocarcinoma antigen ART1 28.
  • Paraneoplastic associated brain-testis-cancer antigen onconeuronal antigen MA2; paraneoplastic neuronal antigen
  • Chromogranin A parathyroid secretory protein 1
  • CEA Carcinoembryonic antigen
  • Vaccines are largely ineffective for patients with established cancer, as advanced disease requires potent and sustained activation of CD8 + cytotoxic T lymphocytes (CTLs) to kill tumor cells and clear the disease.
  • CTLs cytotoxic T lymphocytes
  • DCs dendritic cells
  • cytokines which regulate both CTLs and T regulatory (Treg) cells that shut down effector T cell responses.
  • Treg T regulatory
  • pDCs plasmacytoid DCs
  • CD8 + DCs enhances host immunity in mice.
  • Functionalized biomaterials incorporating various combinations of an inflammatory cytokine, immune danger signal, and tumor lysates were used to control the activation and localization of host DC populations in situ.
  • GM- CSF cytokine granulocyte-macrophage colony-stimulating factor
  • Implantable synthetic polymer matrices (antigen-loaded acellular biomaterial device) that spatially and temporally control the in vivo presentation of cytokines, tumor antigens, and danger signals were utilized herein.
  • GM-CSF is released from these polylactide-co- glycolide (PLG) [a Food and Drug Administration (FDA)-approved biomaterial] matrices into the surrounding tissue to recruit DC precursors and DCs.
  • PLG polylactide-co- glycolide
  • FDA Food and Drug Administration
  • CpG-rich oligonucleotides are immobilized on the matrices as danger signals, and antigen (tumor lysates) is released to matrix -resident DCs to program DC development and maturation.
  • These matrices quantitatively regulate DC activation and trafficking in situ and induce prophylactic immunity against inoculations of murine B16-F10 melanoma cells (P. Schnorrer, G. M.
  • the biopsies of B16-F10 tumors that had grown subcutaneous ly in the backs of C57BL/6J mice were digested in collagenase (250 U/ml) (Worthington) and suspended at a concentration equivalent to 10 7 cells per milliliter after filtration through 40- ⁇ cell strainers.
  • the tumor cell suspension was subjected to four cycles of rapid freeze in liquid nitrogen and thaw (37°C) and then centrifuged at 400 rpm for 10 min.
  • microspheres to make PLG cancer vaccines are microspheres to make PLG cancer vaccines.
  • Blank PLG matrices and matrices containing 3000 ng of GM-CSF alone or in combination with either 1, 10, 50, or 100 ⁇ g of CpG-ODN (studies were also performed with tumor lysates copresented with either 3000 ng of GM-CSF or 100 ⁇ g of CpG-ODN alone or in combination) were implanted into subcutaneous pockets on the back of 7- to 9-week-old male C57BL/6J mice.
  • scaffolds were excised and fixed in Z- fix solution (Anatech), embedded in paraffin, and stained with hematoxylin and eosin (H&E).
  • H&E hematoxylin and eosin
  • APC- conjugated CD1 lc, fluorescein isothiocyanate (FITC)-conjugated CCR7, and PE-conjugated MHCII stains were conducted for DC programming analysis.
  • FITC fluorescein isothiocyanate
  • MHCII PE-conjugated MHCII stains
  • cells were stained with APC-conjugated CD1 lc and PE- conjugated PDCA-1 (pDC marker), APC-conjugated CD1 lc and PE-conjugated CD8 (CD8 DCs), or APC-conjugated CD1 lc and FITC-conjugated CD1 lb (CD1 lb DCs).
  • PE-Cy7-conjugated CD3 stains were performed in conjunction with APC- conjugated CD8a (CD8 T cells), FITC-conjugated CD4 (CD4 T cells), and PE-conjugated FoxP3 (Treg) and analyzed with flow cytometry.
  • Cells were gated according to positive FITC, APC, and PE with isotype controls, and the percentage of cells staining positive for each surface antigen was recorded.
  • PLG scaffolds with melanoma tumor lysates and various dosages of GM-CSF and/or various quantities of PEI-CpG-ODN condensates were implanted subcutaneously into the lower left flank of C57BL/6J mice.
  • animals were challenged 14 days later with a subcutaneous injection of 10 5 B16-F10 melanoma cells [American Type Culture Collection (ATCC)] in the back of the neck. Animals were monitored for the onset of tumor growth ( ⁇ 1 mm ) and killed for humane reasons when tumors grew to 20 to 25 mm (longest diameter).
  • C57BL/6J mice were challenged with a subcutaneous injection of 5 x 10 5 B16-F10 melanoma cells (ATCC) in the back of the neck.
  • ATC B16-F10 melanoma cells
  • PLG vaccines loaded with 3000 ng of GM-CSF, 100 ⁇ g of CpG-ODN, and tumor lysates were implanted
  • mice subcutaneously into the lower left flank of C57BL/6J mice. A subset of mice was vaccinated again at 10 days after the initial vaccination (days 19 and 23).
  • IFN-a, IFN- ⁇ , and TGF- ⁇ concentrations at the matrix implant site the adjacent tissue was excised and digested with tissue protein extraction reagent (Pierce). After centrifugation, the concentrations of IL-12, IFN- a, IFN- ⁇ , and TGF- ⁇ in the supernatant were then analyzed with enzyme-linked immunosorbent assay (R&D Systems) according to the manufacturer's instructions.
  • single-cell suspensions were prepared from the spleens of mice immunized with PLG vaccines (lysate + 3000 ng of GM- CSF + 100 ⁇ g of CpG) at various time points. These cells were initially stained with APC-H- 2Kb-TRP2 pentamers (Proimmune) and subsequently stained with PE-conjugated
  • Macroporous PLG matrices were fabricated for GM-CSF release to recruit DCs and with an interconnected porous structure facilitates cell infiltration. Matrices were loaded with 0, 3000, and 7000 ng of GM-CSF and implanted into the subcutaneous pockets of C57BL/6J mice. Histological analysis at day 14 after implantation of PLG matrices loaded with 3000 ng of GM-CSF revealed enhanced cellular infiltration when compared to blank controls ( Figure 1A). Fluorescence-activated cell sorting (FACS) analysis for CD1 lc DCs showed that GM-CSF delivery recruited significantly more DCs (a factor of ⁇ 8 increase) than blank PLG matrices ( Figure IB).
  • FACS Fluorescence-activated cell sorting
  • the matrix -resident DCs were almost exclusively CD1 lb + (-87%), in accordance with other studies of GM-CSF effects on DC recruitment in vivo (N. Mach, S. Gillessen, S. B. Wilson, C. Sheehan, M. Mihm, G. Dranoff, Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage
  • GM-CSF delivery promoted greater cellular penetration into and association with the PLG material, as indicated by histological analysis ( Figure 8) and measurement of DC numbers ( Figure 1, B and D), allowing for the subsequent programming of resident DC precursors and DCs.
  • In situ delivery of CpG-oligodeoxynucleotide promotes pDC recruitment and IFN production
  • CpG-ODN The dose of CpG-ODN presented in combination with 3000 ng of GM-CSF was altered to regulate the numbers of resident pDCs, resulting in 190,000, 520,000, and 1,200,000 cells at doses of 0, 10, and 100 ⁇ g of CpG-ODN, respectively ( Figure 2B). Copresentation of CpG-ODN had little effect on the ability of GM-CSF to enhance CD1 lc + -CDl lb + cDCs ( Figure 2C). High doses of CpG-ODN promoted the local production of IFN-a (-1010 pg/ml) and IFN- ⁇ (-600 pg/ml)
  • Tumor lysate co-delivery with CpG-ODN and GM-CSF stimulates CD8+ generation and IL-12 production
  • necrotic tumor cells may be particularly immunostimulatory, as they release a variety of endogenous mediators (for example, heat shock proteins and damaged nucleic acids) that trigger innate immune recognition (C.
  • CD8 + CD1 lc + cDCs are especially efficient at cross-presenting exogenous antigen on MHCII molecules (J. D. Farrar, H. Asnagli, K. M. Murphy, T helper subset development: Roles of instruction, selection, and transcription. J. Clin. Invest. 109, 431-435 (2002); D. Skokos, M. C. Nussenzweig, CD8 DCs induce IL-12-independent Thl
  • tumor lysate in combination with GM-CSF and CpG enhanced the numbers of recruited pDCs at day 10 after implantation by a factor of 2 over matrices without lysate and by a factor of 10 over blank controls (Figure 3C).
  • No significant difference in pDC numbers was observed with tumor lysate in combination with only GM-CSF or CpG signaling.
  • the CD1 lc + CDl lb + DC population at the vaccine site depended on GM-CSF alone ( Figure 3D), as tumor lysate or CpG signaling alone or in combination had no significant effect on the recruitment and expansion of these DCs (Figure 3D).
  • TRP2 MHCII-tyrosinase-related protein 2
  • This system is capable of generating prophylactic immunity against poorly
  • mice unvaccinated mice were killed by day 23 due to tumor burden.
  • GM-CSF-mediated DC recruitment was combined with lysate and CpG-ODN delivery, the mice showed significant protection from tumor-induced lethality.
  • CpG-ODN doses of 10, 50, and 100 ⁇ g resulted in 50%, 60%, and 90%> survival rates, respectively (Figure 5B).
  • melanoma tumors were established for 13 days and then mice were vaccinated.
  • Onetime (day 13) and two-time (days 13 and 23) vaccination decreased tumor progression (Figure 7D).
  • engineered PLG vaccines evoke a coordinated response of multiple DC subtypes, which together trigger sustained and potent antitumor CD8 + CTLs while inhibiting immunoregulatory pathways.
  • the combination of tumor cell lysates, GM- CSF, and CpG-ODN in the vaccine matrix was required for optimal tumor protection, which was strongly associated with the recruitment of pDCs and CD8 + DCs and the local production of IL-12.
  • the accumulation of CD8 + DCs at the vaccine site is a notable feature of this vaccination strategy because this DC subset is typically localized to secondary lymphoid structures. Plasmacytoid DC numbers were closely linked with the generation of type I IFNs (H. Kanzler, F. J. Barrat, E. M. Hessel, R. L.
  • the findings suggest that a minimum number of DCs are required to induce high concentrations of protective immunity.
  • Vaccines that generated about 1,200,000 pDCs and 600,000 CD8 + DCs (-43% of total DCs) in a total population of -4.2 million DCs resulted in 90% survival in a subsequent tumor challenge.
  • the engineered matrices appear to program T cell responses efficiently by providing a site of sustained immunostimulatory tumor antigen presentation, which evokes robust CTLs, both locally and systemically, and attenuates immune regulation mediated through TGF- ⁇ , IL-10, and FoxP3 + Treg cells.
  • the vaccine system described herein is useful to modulate DC and CTL responses for the control of other solid cancers and perhaps chronic infections.
  • the approach also facilitates the study of DC subset development and the mechanisms through which these subsets are coordinated in vivo for the eradication of established diseases. It is striking that tumor regression induced by these PLG vaccines outperformed gene-modified tumor cell vaccines in direct comparison and outperformed ex vivo DC vaccines reported in literature.
  • This acellular biomaterial system was designed with components that either are FDA approved (PLG and GM-CSF) or have been utilized clinically (CpG-ODN) and do not require the maintenance and modification of live cell cultures.
  • PLG system has considerable advantages in terms of clinical application as compared to other approaches. Scaling to humans does not require significant modification of the size or structure of the material but simply require utilizing effective human analogs (for example, human GM-CSF and CpG-ODN sequences) that evoke human DC and CTL responses.
  • Cancer vaccines are typically formulated for bolus injection and often produce shortlived immunostimulation resulting in poor temporal control over immune cell activation, and weak oncolytic activity.
  • One means of overcoming these limitations utilizes
  • Antigen-laden, macroporous PLG scaffolds induce potent dendritic cell (DC) and cytotoxic T-lymphocyte (CTL) responses via the controlled signaling of inflammatory cytokines, antigen and toll-like receptor agonists.
  • DC dendritic cell
  • CTL cytotoxic T-lymphocyte
  • a seamless relationship was observed between the production of controlled CTL responses, tumor growth and long- term survival in both prophylactic and therapeutic models. Scaffolds must be implanted for >7 days to augment CTL responses via the prolonged presentation of tumor antigen, and the benefits included a notable regression of established tumors.
  • PLG microspheres encapsulating GM-CSF were first made using a standard double emulsion process (Cohen S., Yoshioka T., Lucarelli, M., Hwang L.H., and Langer R. Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres. Pharm. Res. 8,713-720 (1991)).
  • PLG microspheres were then mixed with 150 mg of the porogen, sucrose (sieved to a particle size between 250 ⁇ and 425 ⁇ ), and compression molded. The resulting disc was allowed to equilibrate within a high-pressure C0 2 environment, and a rapid reduction in pressure causes the polymer particles to expand and fuse into an interconnected structure. The sucrose was leached from the scaffolds by immersion in water yielding scaffolds that were 90% porous.
  • biopsies of B16-F10 tumors that had grown subcutaneously in the backs of C57BL/6J mice (Jackson Laboratory, Bar Harbor Maine), were digested in collagenase (250 U/ml) (Worthington,
  • CpG-ODN 1826 5 '-tec atg acg ttc ctg acg tt-3', (Oligofactory, Holliston, MA) was first condensed with
  • PEI poly(ethylenimine)
  • Mi -60,000 Sigma Aldrich
  • Sigma Aldrich poly(ethylenimine)
  • the charge ratio between PEI and CpG-ODN (NH3+:P04-) was kept constant at 7 during condensation.
  • PEI-CpG-ODN condensate solutions were then vortexed with 60 ⁇ of 50% (wt/vol) sucrose solution, lyophilized and mixed with dry sucrose to a final weight of 150 mg.
  • the sucrose containing PEI-CpG-ODN condensate was then mixed with blank, GM-CSF and/or tumor lysate loaded PLG microspheres to make PLG cancer vaccines.
  • scaffolds were excised at various time-points and the ingrown tissue was digested into single cell suspensions using a collagenase solution (Worthingtion, 250 U/ml) that was agitated at 37°C for 45 minutes. The cell suspensions were then poured through a 40 ⁇ cell strainer to isolate cells from scaffold particles and the cells were pelleted and washed with cold PBS and counted using a Z2 coulter counter (Beckman Coulter). To assess DC infiltration, subsets of the total cell population isolated from PLG matrices were then stained with primary antibodies (BD Pharmingen, San Diego, CA) conjugated to fluorescent markers to allow for analysis by flow cytometry.
  • primary antibodies BD Pharmingen, San Diego, CA
  • APC- conjugated CD1 lc (dendritic cell marker) stains were conducted for DC recruitment analysis.
  • PE-Cy7 conjugated CD3 stains were performed in conjunction with APC-conjugated CD8a (CD8 T cells), FITC-conjugated CD4 (CD4 T cells) and analyzed with flow cytometry.
  • Cells were gated according to positive FITC, APC and PE using isotype controls, and the percentage of cells staining positive for each surface antigen was recorded. Tumor growth assays, and in situ cytokine concentrations
  • PLG-based vaccines were developed utilizing matrices incorporating melanoma tumor lysates, 3,000ng GM-CSF and 100 ⁇ g PEI-CpG-ODN condensates and were implanted subcutaneously into the lower left flank of C57BL/6J mice.
  • animals were challenged 14 days later with a subcutaneous injection of 10 5 B16-F10 melanoma cells (ATCC, Manassas, NJ) in the back of the neck.
  • B16-F10 melanoma cells ATCC, Manassas, NJ
  • PLG vaccines were explanted at days, 1, 3, 7, 12, and 16 days after implantation. Animals were monitored for the onset of tumor growth (approximately 1mm ) and sacrificed for humane reasons when tumors grew to 20 - 25 mm (longest diameter).
  • IFN- ⁇ concentrations at the matrix implant site excised tissue was digested with tissue protein extraction reagent (Pierce). After centrifugation, the concentration of GM-CSF, IL- 12p70, and IFN- ⁇ in the supernatant was then analyzed with ELISA (R&D systems), according to the manufacturers instructions.
  • Macroporous PLG scaffolds were fabricated to control the presentation of GM-CSF, tumor lysate (B16 melanoma), and CpG-ODN to serve as vaccines (PLG vaccines) (Figure 9 A &B).
  • the duration of vaccination using PLG vaccines was controlled by explanting the material system at various timepoints. The relationship between vaccine duration and efficacy was examined in prophylactic and therapeutic B16-F10 melanoma models.
  • PLG vaccines were implanted subcutaneously into the backs of C57BL/6J mice and removed at days 1, 3, 7, 12 and 16 after implantation and these mice were challenged at day 14 with an otherwise lethal dose of B16-F10 cells (Figure 9C).
  • the engineered PLG matrices were designed to release a pulse of GM-CSF to recruit host DCs. Histological analysis at day 14 post-implantation of PLG matrices loaded with 3000 ng of GM-CSF revealed enhanced cellular infiltration and penetration into the void volume of the material, relative to cell infiltration at 3 days after implantation ( Figure 10A).
  • the T cell response to PLG vaccines is predominantly comprised of CD8(+) cytotoxic T cells (Figure 10D).
  • Local cytotoxic T cell responses persisted at significant levels between days 7 and 28 after implantation.
  • CD8(+) cytotoxic T-lymphocytes (CTLs) were detectable at day 5, peaked at day 12 and subsided at day 28.
  • CTLs manifested potent effector function, as vaccination resulted in a prototypical activation phase that gradually plateaus, followed by a contraction phase as antigen is cleared.
  • the data indicates that the CTL response also effectively terminated the innate response by day 12 as indicated by the sharp decline of antigen presenting DCs, likely via effector, CTL killing.
  • IL-12 which is a T cell growth and stimulating factor and an activating factor for DCs, is produced by DCs and macrophages in response to intercellular pathogens and tumor cells.
  • Local IL-12 concentrations peaked at 800 pg/ml after one day of vaccination, and then subsided to approximately 300 pg/ml between days 5-16 of vaccination (Figure 11A). All three components of the vaccine, GM-CSF, CpG-ODN and tumor lysates were required to promote and maintain high IL-12 concentrations, as blank controls and all other combinations of the vaccine's bioactive factors produced significantly lower IL-12 levels (Figure 1 IB).
  • PLG vaccines sustained the induction of IL-12 and IFN- ⁇ from infiltrating immune cells for 16 days, which is likely to be important for Thl polarization and prolonged CD8(+) CTL responses to the tumor antigens embedded within the vaccine's matrix.
  • PLG vaccines To determine the systemic effects of PLG vaccines, cellular populations in the draining LNs were monitored over time. Blank matrices and PLG matrices containing GM- CSF, lysate and CpG-ODN (PLG vaccines) were implanted subcutaneously into the backs of mice at a distance of approximately 9 mm from the inguinal LN. PLG vaccines resulted in a rapid enhancement in the numbers of phagocytic cells in the proximal inguinal LN, as 4 fold increases in resident DCs and plasmacytoid DCs numbers were observed at day 3 ( Figure 12A).
  • the T cell expansion induced by PLG vaccines was mostly due to the expansion of the CD8(+) CTL subset, as their numbers in LNs increased similarly in scale from 2.8x10 5 CTLs (blank matrices) to 8.2xl0 5 CTLs (PLG vaccines) at day 10 of vaccination (Figure 12C).
  • Provision of persistent CpG-ODN signaling alongside antigenic signals (tumor lysate) was required of the vaccines ability to induce polarization of LN T cells toward CD8(+) CTL expansion (Figure 12C), and to down-regulate FoxP3(+) T regulatory cell activity in LNs ( Figure 12D and E). FoxP3 T cells may suppress the cytotoxicity of CD8(+) T cells and extinguish vaccine activity.
  • Vaccination utilizing melanoma vaccines fabricated into PLG matrices resulted in two distinct phases of immune responses in situ.
  • innate responses and DC activation began at day 3, peaked at day 5 and subsided at day 12 post- vaccination ( Figure 10A & B).
  • the second phase consisted of adaptive T cell responses consisting mostly of CD8(+) cytotoxic T- lymphocytes (CTLs) (Figure IOC), which began at day 5, peaked at day 12 and subsided at day 28 ( Figure 10A & B).
  • CTL responses to the PLG matrix manifested potent effector function, with vaccination resulting in a prototypical activation phase that gradually plateaued, and was followed by a contraction phase as the antigen was cleared.
  • the time lag associated with the onset of the effector response is likely the time required to induce sufficient DC recruitment and priming.
  • the vaccine also induced persistent IL-12 and IFN- ⁇ production consistent with DC and T cell infiltration into the vaccine site; this is a hallmark of Thl and cytotoxic T cell responses against tumors.
  • PLG vaccines induce CTLs expressing TCRs specific for the melanoma antigen, Trp-2, and the kinetics of cytotoxic T cell homing to the vaccine site is likely in response to prolonged antigen presentation (tumor lysates) by the PLG matrix.
  • the data described herein demonstrates that the CTL response terminated the local innate responses as the significant drop in local DC numbers after day 7 was likely due to CD8(+)T- cell cytotoxicity against these antigen-presenting cells.

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Abstract

La présente invention concerne un procédé qui permet de provoquer et de conserver une réponse immunitaire anti-tumorale et qui est mis en œuvre par l'administration d'un dispositif contenant une quantité d'antigènes suffisante pour stimuler une réponse immunitaire innée et une réponse immunitaire adaptative. Un procédé permettant de provoquer et de conserver une réponse immunitaire anti-tumorale est également mis en œuvre par l'administration d'une pluralité de dispositifs, par exemple un premier dispositif de biomatériau acellulaire chargé d'antigènes (vaccin PLG) à un premier instant, pour stimuler une réponse immunitaire innée et une réponse immunitaire adaptative, et l'administration d'un second dispositif sur un second site anatomique à un second instant pour prolonger la réponse immunitaire adaptative.
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