US20140065173A1 - Tumor lysate loaded particles - Google Patents

Tumor lysate loaded particles Download PDF

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Publication number
US20140065173A1
US20140065173A1 US14/019,007 US201314019007A US2014065173A1 US 20140065173 A1 US20140065173 A1 US 20140065173A1 US 201314019007 A US201314019007 A US 201314019007A US 2014065173 A1 US2014065173 A1 US 2014065173A1
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Prior art keywords
particle
tumor lysate
tumor
cell
dendritic cell
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US14/019,007
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Thomas E. Wagner
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Orbis Health Solutions LLC
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Orbis Health Solutions LLC
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Priority to US14/019,007 priority Critical patent/US20140065173A1/en
Priority to CN201380057518.2A priority patent/CN104936612B/zh
Priority to ES13835508T priority patent/ES2777207T3/es
Priority to EP20156351.7A priority patent/EP3708183A1/en
Priority to EP13835508.6A priority patent/EP2892551B1/en
Priority to CN202011473846.9A priority patent/CN112891522A/zh
Priority to CA2883949A priority patent/CA2883949C/en
Priority to JP2015531351A priority patent/JP6494511B2/ja
Priority to PCT/US2013/063091 priority patent/WO2014040089A1/en
Priority to AU2013312135A priority patent/AU2013312135B2/en
Publication of US20140065173A1 publication Critical patent/US20140065173A1/en
Assigned to ORBIS HEALTH SOLUTIONS LLC reassignment ORBIS HEALTH SOLUTIONS LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WAGNER, THOMAS E.
Priority to AU2018203586A priority patent/AU2018203586B2/en
Priority to JP2018211024A priority patent/JP6956701B2/ja
Priority to AU2020203845A priority patent/AU2020203845C1/en
Abandoned legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung

Definitions

  • a tumor cell exists in part because it has selected for one or more mutations that allows it to partially or completely escape immune surveillance in vivo.
  • dendritic cells which are professional antigen-presenting cells, to present tumor antigens to the immune system.
  • dendritic cells pulsed with peptide or tumor lysate have been used to vaccinate melanoma patients.
  • MHC Major Histocompatibility Complex
  • presenting tumor antigens via the MHC class I pathway contributes to a more robust anti-tumor immunity by activating CD8+ T cells.
  • Previous researchers have attempted to present tumor antigens through the MHC class I pathway by using gene transfer methods. However, these methods have disadvantages, including (1) a limited ability to identify all of the important tumor-specific antigens, (2) a limited ability to map the genes of specific tumor antigens, (3) only one or a small number of known tumor antigen genes can be introduced into a dendritic cell and (4) the methods are time-consuming and cumbersome.
  • an isolated dendritic cell comprising a phagocytosed component consisting essentially of (i) a particle and (ii) a tumor lysate loaded within the particle.
  • the tumor lysate is present in an amount from about 200 ⁇ g to about 500 ⁇ g. In specific embodiments, the tumor lysate is present in an amount of about 200 ⁇ g.
  • the tumor lysate is a lysate selected from a cancer selected from the group consisting of breast cancer, small cell lung cancer, non-small cell lung cancer, glioma, medulloblastoma, neuroblastoma, Wilms tumors, rhabdomyosarcoma, osteosarcoma, liver cancer, pancreatic cancer, melanoma, prostate cancer and ocular melanoma.
  • the particle is a yeast cell wall particle.
  • the dendritic cell is an immature cell that has been isolated for no more than 8 days.
  • Another embodiment relates to a vaccine comprising the foregoing isolated dendritic cell.
  • Some embodiments relate to a method for producing an isolated dendritic cell containing a tumor lysate loaded particle comprising: (i) loading the tumor lysate into the particle to produce the tumor lysate loaded particle; (ii) freeze-drying the tumor lysate loaded particle; and (iii) incubating the tumor lysate loaded particle with a dendritic cell, wherein the incubating causes the dendritic cell to phagocytose the tumor lysate loaded particle.
  • the foregoing method further comprises (a) resuspending the tumor lysate loaded particle in solution and (b) freeze-drying the resuspended solution before step (iii).
  • the tumor lysate is produced by freezing and thawing the tumor.
  • the foregoing method further comprises repeating the freezing and thawing steps.
  • the foregoing method further comprises cryopreserving the tumor lysate at ⁇ 150° C.
  • step (iii) comprises: (a) adding tumor lysate into a yeast cell wall particle, (b) incubating the yeast cell wall particle, (c) freeze-drying the yeast cell wall particle and (d) washing the yeast cell wall, wherein steps (b)-(c) are repeated at least once with a step of adding water into the yeast cell particle before step (b) is repeated.
  • step (iii) comprises: (a) contacting the tumor lysate loaded particle and the dendritic cell at a ratio of about 100:1, (b) incubating the tumor lysate loaded particle with the dendritic cell for 1 to 2 hours and (c) collecting the dendritic cell and washing the cell.
  • Some embodiments relate to a method for treating cancer, comprising administering a vaccine comprising the foregoing isolated dendritic cell.
  • the cancer is selected from the group consisting of breast cancer, small cell lung cancer, non-small cell lung cancer, glioma, medulloblastoma, neuroblastoma, Wilms tumors, rhabdomyosarcoma, osteosarcoma, liver cancer, pancreatic cancer, melanoma, prostate cancer and ocular melanoma.
  • FIG. 1 depicts a process for producing dendritic cells.
  • FIG. 2 depicts a process for producing tumor lysate.
  • FIG. 3 depicts a process for producing yeast cell wall particles.
  • FIG. 4 depicts a process for loading tumor lysate into yeast cell wall particles.
  • FIG. 5 depicts a process for producing tumor lysate particle loaded dendritic cells.
  • FIG. 6 is a graph comparing the effect of tumor lysate particle loaded dendritic cells versus antigen pulsing of dendritic cells on B3Z cells.
  • FIG. 7A shows the lungs of control mice afflicted with B16F0 murine melanoma.
  • FIG. 7B shows the lungs of mice afflicted with Bl6F0 murine melanoma but treated with tumor lysate particle loaded dendritic cells.
  • subject or “patient” denotes any animal in need of treatment with a vaccine.
  • a subject may be suffering from or at risk of developing a condition that can be treated or prevented with a vaccine.
  • subject or “patient” includes humans.
  • therapeutically effective amount and “therapeutic level” mean that vaccine dosage or plasma concentration in a subject, respectively, that provides the specific response for which the vaccine is administered in a subject in need of such treatment.
  • exemplary dosages, vaccine delivery amounts, therapeutically effective amounts and therapeutic levels are provided below with reference to adult human subject. Those skilled in the art can adjust such amounts in accordance with standard practices as needed to treat a specific subject and/or condition/disease.
  • tumor lysate refers to a tumor that has been lysed. Tumor lysis can occur under a number of conditions, including repeated freezing and thawing of the tumor, physical breakage of the tumor by homogenizing, contact with a hyper- or hypo-tonic solution and contact with one or more non-ionic detergents. The tumor lysate is not cross-linked during the lysing process.
  • the tumor lysate is produced by mincing, grinding or mashing the tumor, or otherwise pulverizing the tumor using any known technique in the art.
  • the tumor lysate is produced by mincing, grinding, mashing or pulverizing the tumor in the presence of phosphate buffer solution (PBS), such as 1 ⁇ PBS.
  • PBS phosphate buffer solution
  • the tumor lysate is produced from a solid tumor weighing a minimum of 200 to 500 ⁇ g.
  • the tumor lysate is produced by mincing, grinding, mashing or pulverizing the tumor followed by repeated freezing and thawing.
  • the minced tumor is frozen and thawed multiple times.
  • the minced tumor is frozen and thawed at least 1, 2, 3 or 4 times.
  • the freezing is performed in liquid nitrogen, and can be performed for 20 minutes.
  • the thawing is performed at room temperature.
  • the tumor lysate is stored at a temperature of about ⁇ 135° C. or below after the freezing and thawing process.
  • the tumor lysate is store at a temperature of ⁇ 150° C. or below after the freezing and thawing process.
  • the tumor lysate can be prepared from any solid tumor including, but not limited to carcinomas, and sarcomas.
  • the solid tumors are from tumors relating to breast cancer, small cell lung cancer, non-small cell lung cancer, glioma, medulloblastoma, neuroblastoma, Wilms tumors, rhabdomyosarcoma, osteosarcoma, liver cancer, pancreatic cancer, melanoma, prostate cancer and ocular melanoma.
  • the size of the particle refers to any hollow and porous structure that can contain tumor lysate therein and also allow the lysate to exit the structure.
  • the size of the particle is about 0.5 to about 5 ⁇ m, which approximates the size of bacterium to allow the particle to be ingested by monocytes, such as dendritic cells.
  • the size of the particle is about 0.5 to about 1 ⁇ m.
  • the size of the particle is about 0.5 to about 2.5 ⁇ m.
  • the particle can be any particle with a glycan network, so long as the particle is about 0.5 to about 5 ⁇ m in size.
  • the particle is a bead vector.
  • the bead vector is not limited by shape or material, but can be any shape, size or material that allows the bead vector to be phagocytosed by monocytes, including dendritic cells.
  • the particle is a yeast cell wall particle (“YCWP”).
  • the YCWP is prepared from yeast cell wall such that the particle is porous and can contain lysate therein.
  • the YCWP is prepared from Saccharomyces cerevisiae .
  • the YCWP is a zymosan particle.
  • the YCWP approximates the size of microbial structures that cells of the mononuclear phagocyte system and other phagocytic cells typically ingests. In specific embodiments, the YCWP is about 1-5 ⁇ m.
  • the YCWP is prepared by (a) suspending yeast to produce a suspension, (b) incubating the suspension, (c) centrifuging the suspension and removing the supernatant and (d) recovering the resulting YCWP. In another embodiment, steps (a)-(d) are repeated at least 1, 2, 3 or 4 times.
  • the YCWP is prepared by (a) suspending yeast in a solution to produce a first suspension, (b) incubating the first suspension, (c) centrifuging the first suspension and removing the supernatant, (d) suspending the resulting pellet to produce a second suspension, (e) incubating the second suspension, (f) centrifuging the second suspension and removing the supernatant and (g) washing the resulting pellet to recover the YCWP.
  • the YCWP is sterilized.
  • the yeast is suspended in NaOH, including 1M NaOH.
  • the first suspension is incubated at about 80° C. for about 1 hour or for 1 hour.
  • the centrifuging is performed at about 2000 times gravity for about 10 minutes, or at 2000 times gravity for 10 minutes.
  • the pellet is suspended in water, including water at about pH 4.5 or at pH 4.5.
  • the second suspension is incubated at about 55° C. for about 1 hour or at 55° C. for 1 hour.
  • the pellet is washed in water at least 1, 2, 3 or 4 times. In specific embodiments, the pellet is washed once.
  • the YCWP is sterilized using isopropanol and/or acetone following washing of the pellet. In specific embodiments, other known alcohols are appropriate.
  • the YCWP is allowed to fully dry after sterilization.
  • the YCWP is resuspended after being allowed to dry.
  • the YCWP is resuspended in PBS, such as 1 ⁇ PBS.
  • the YCWP is allowed to dry and then frozen before the tumor lysate is loaded into the YCWP, in order to place it in storage before use.
  • the YCWP is freeze dried and store at about 4° C. or lower. In specific embodiments, the YCWP is freeze dried and store at 4° C.
  • the particle is loaded with tumor lysate.
  • the tumor lysate is loaded into the particle by incubating the lysate and a suspension of particles together and allowing the lysate to penetrate into the hollow insides of the particles.
  • the combination is freeze-dried to create an anhydrous tumor lysate within the particle.
  • the lysate is trapped within the particle and ready to be phagocytosed by a monocyte, such as a dendritic cell.
  • the freeze-drying is the only mechanism used to trap the lysate within the particle.
  • the entrapment is not caused by a separate component blocking the lysate from exiting the particle, for example, by physical entrapment, hydrophobic binding, any other binding.
  • the entrapment is not caused by crosslinking or otherwise attaching the lysate to the particle outside of any attachment that may occur upon freeze-drying.
  • the particle is resuspended in solution after the freeze-drying.
  • the solution is water.
  • the particle is resuspended to allow additional tumor lysate to penetrate the particle and then the combination is freeze-dried again.
  • the combination is subjected to multiple freeze-drying and resuspensions.
  • the tumor lysate loaded particle is sterilized in ethanol after the freeze-drying and before use.
  • the tumor lysate is loaded into the particle by (a) incubating the lysate and a suspension of the particles, allowing the lysate to penetrate into the hollow insides of the particles and freeze-drying the suspension of particles loaded with lysate and (b) optionally resuspending the particles, incubating the resuspended particles and freeze drying the resuspended particles and any tumor lysate not already in the particle.
  • the number of YCWPs is about 1 ⁇ 10 9 and the volume of tumor lysate is about 50 ⁇ L (generated from about 200 ⁇ g of tumor tissue). In specific embodiments, the number of YCWPs is 1 ⁇ 10 9 and the volume of tumor lysate is 50 ⁇ L (from about 200 ⁇ g of tumor tissue).
  • the incubation in step (a) is for about 2 hours at about 4° C. In specific embodiments, the incubation in step (a) is for 2 hours at 4° C. In some embodiments, the foregoing suspension is freeze dried in step (b) over a period of about 2 hours or over a period of 2 hours.
  • the YCWPs in step (c) are resuspended in water, including about 50 ⁇ L of water or 50 ⁇ L of water. In some embodiments, the resuspended YCWPs are incubated in step (d) for about 2 hours at about 4° C. or for 2 hours at 4° C.
  • dendritic cell refers to a cell generated from a peripheral blood mononuclear cell (“PBMC”).
  • PBMC peripheral blood mononuclear cell
  • a dendritic cell is prepared by (a) collecting blood, (b) diluting the blood, (c) performing a density gradient separation of PBMCs, (d) lysing red blood cells and washing the PBMCs, (e) incubating the PBMCs, (f) removing nonadherent cells and (g) culturing adherent cells in media.
  • the dendritic cell is an immature dendritic cell that has been cultured for no more than 5 days. In other embodiments, the dendritic cell has been cultured for 6-8 days.
  • the blood is heparinized.
  • the density gradient separation at step (c) comprises placing the blood in a Lymphocyte Separation Medium and then centrifuging the blood.
  • the centrifuging is performed at about 1000 times gravity for about 20 minutes or at 1000 times gravity for 20 minutes.
  • a second centrifuging is performed before step (d) and is performed at about 500 g for about 5 minutes or is performed at 500 g for 5 minutes.
  • a third centrifuging is performed before step (d) and is performed at about 500 g for about 10 minutes or is performed at 500 g for 10 minutes.
  • the lysing is performed using an ACK lysing solution, followed by incubation, preferably at room temperature for about 5 minutes, and followed by a subsequent centrifugation.
  • the PBMCs are washed in RPMI-1640 medium.
  • the PBMCs are incubated at step (e) in flasks at about 37° C. for about 1-2 hours or at 37° C. for 1-2 hours.
  • serum-free DC media is added to the flask.
  • one or more cytokines is present in the culture media, including, but not limited to, granulocyte macrophage colony stimulating factor (800 units/ml) and IL-4 (500 units/ml).
  • the tumor lysate loaded particle is phagocytosed within a monocyte, preferably a dendrite cell.
  • the tumor lysate loaded particle is incubated with a dendritic cell such that the cell phagocytoses the tumor lysate loaded particle.
  • the particle is incubated with the dendritic cell at a ratio of about 100:1 or at a ratio of 100:1.
  • the incubation can be performed for in about 1 hour, 1 hour or preferably less than 1 hour.
  • the incubated dendritic cell containing the tumor lysate particle is collected and washed, for example, at least 1, 2, 3 or 4 times.
  • the dendritic cells are incubated after washing and resuspended in freezing medium.
  • the resuspension produces a concentration of about 10 ⁇ 10 6 cells per ml or 10 ⁇ 10 6 cells per ml.
  • the resuspension is frozen for storage before use.
  • the dendritic cell containing a tumor lysate loaded particle is used as a vaccine to prevent and/or treat a disease, including cancer.
  • the disease to be treated is not particularly limiting, but depends on the particular tumor lysate loaded into the particle.
  • a vaccine using tumor lysate from a breast cancer tumor is used to treat breast cancer.
  • a patient's own tumor cells are used to create the vaccine.
  • the vaccine can be produced using tumor lysate from a tumor associated with breast cancer and then administered to the breast cancer patient from which the tumor was extracted.
  • about 200 ⁇ L of a 10 ⁇ 10 6 concentration of dendritic cells containing tumor lysate loaded particles forms one dose of the vaccine.
  • the dose is administered by diluting the 200 ⁇ L aliquot to a final volume of 1 ml before administering the dose to a patient.
  • the aliquot is diluted with sterile saline containing 5% human serum albumin.
  • the 200 ⁇ L aliquot will need to be thawed before dilution. In such a scenario, the length of time between thawing and administration of the dose to a patient will be no longer than 2 hours.
  • the diluted aliquot is administered in a 3 cc syringe. In some embodiments, a syringe needle no smaller than 23 gauge is used.
  • a patient is administered at least 1, 2, 3 or 4 doses. In specific embodiments, a patient is re-vaccinated once every 4 weeks. In specific embodiments, about 1-2 million dendritic cells containing tumor lysate loaded particles will be administered at each vaccination. In another embodiment, the dendritic cells containing tumor lysate loaded particles are administered to a patient by injection. In specific embodiments, the tumor lysate loaded particles are injected in a patient at or near (1) a tumor or (2) a lymph node.
  • the vaccine is not administered with any other immunosuppressive treatment, such as steroids or chemotherapy.
  • the vaccine can be administered using any technique, including intravenous injection and inhalation.
  • the vaccine can also contain biological adjuvants, including but not limited to nucleic acids such as GpC oligonucleotides, proteins or peptide epitopes such as the tetanus toxoid MHC class II-binding p30 peptide.
  • nucleic acids such as GpC oligonucleotides, proteins or peptide epitopes such as the tetanus toxoid MHC class II-binding p30 peptide.
  • Dendritic cells were generated from a patient's PBMCs.
  • PBMCs were collected from the patient by a blood draw of 200 ml following standard operating procedures. The blood was then transferred to 250 ml centrifuge tubes and diluted 1:1 with 1 ⁇ PBS. Then, 35 ml of the diluted blood was layered over 15 ml of room temperature Lymphocyte Separation Medium (LSM; Mediatech) in 50 ml tubes and centrifuged at 1000 g for 20 minutes at room temperature. The PBMC layers were removed by pipetting from the LSM gradients and placed into clean 50 ml centrifuge tubes. Four volumes of 1 ⁇ PBS were added and the tubes were inverted to mix the contents.
  • LSM Lymphocyte Separation Medium
  • the PBMCs were then centrifuged at 500 g at room temperature for 5 minutes. Ten ml of 1 ⁇ PBS were added into each tube and the cells were resuspended and pooled into 1 tube. The PBMCs were again centrifuged at 500 g at room temperature for 10 minutes, resuspended in 20 to 40 ml of ACK lysing solution (Cambrex) and incubated at room temperature for 5 minutes. The cells were then centrifuged again at 1500 rpm for 5 minutes. The PBMCs were resuspended in 30 ml RPMI-1640 medium (Mediatech). The cells were then transferred into 2-4 T75 flasks. The flasks were incubated at 37° C.
  • FIG. 1 provides an overview of the generation of dendritic cells.
  • a tumor sample was obtained from a patient. After separating fat and necrotic tissue away from the tumor tissue, the tissue was weighed and 1 ⁇ PBS added (50 ⁇ L of PBS per 200 ⁇ g of tissue) and the tumor was minced thoroughly with scalpels in 1 ⁇ PBS. The tumor cells were then subjected to 4 cycles of freeze and thaw. The freezing was performed in liquid nitrogen for 20 minutes and the thawing was performed at room temperature. Prepared tumor lysate was quantified by a spectrophotometer. An aliquot was taken for quality control testing. The remainder was stored at ⁇ 135° C. in preparation for vaccine preparation.
  • FIG. 2 provides an overview of the tumor cell lysate processing.
  • YCWPs were prepared from Fleishmans Baker's Yeast. Briefly, 10 g of Fleishmans Baker's yeast was suspended in 100 ml of 1 M NaOH and heated to 80° C. for one hour. The undissolved yeast cell walls were recovered by centrifugation at 2000 ⁇ g for 10 minutes. The recovered yeast cell walls were then resuspended in 100 ml of water with the pH adjusted to 4.5 with HCl and incubated at 55° C. for an additional hour, and subsequently recovered by centrifugation. The recovered YCWPs were then washed with water once, isopropanol 4 times and finally acetone 2 times. Once the YCWPs were fully dried they were resuspended in PBS, counted, aliquoted into groups of 1 ⁇ 10 9 particles and freeze dried for use in manufacturing the vaccine.
  • FIG. 3 provides an overview of the yeast cell wall particles processing.
  • a suspension of fully anhydrous YCWPs (1 ⁇ 10 9 ) was placed in contact with 50 ⁇ L of tumor lysate in PBS (from 200 ⁇ gs of tumor tissue) over a period of 2 hours at 4° C., allowing the lysate to penetrate into the hollow insides of the YCWPs to produce loaded YCWPs.
  • the suspension was then freeze dried for 2 hours. After freeze drying, 50 ⁇ L of water was added to the loaded YCWPs, incubated for another 2 hours at 4° C. and again freeze dried to yield YCWPs with dry tumor lysate within their hollow insides.
  • the loaded YCWPs were then sterilized by washing in ethanol and maintained in ethanol.
  • FIG. 4 provides an overview of the YCWPs loading procedure.
  • dendritic cells were collected, washed twice with 1 ⁇ PBS, pelleted by centrifugation and resuspended in CryoStorTM CS-10 freezing medium at a final concentration of 10 ⁇ 10 6 cells/ml. The cell suspension was aliquoted into several 200 ⁇ l aliquots, frozen, and stored at ⁇ 135° C.
  • FIG. 5 provides an overview of the vaccine formulation and filling procedure.
  • B3Z cells are a T cell hybridoma expressing a T-cell receptor that specifically recognizes the OVA (257-264) (SIINFEKL) epitope in the context of H-2K b and carry a beta-galactosidase (lacZ) construct driven by a nuclear factor of activated T cells elements from the interleukin 2 promoter (X).
  • SIINFEKL OVA (257-264)
  • lacZ beta-galactosidase
  • X interleukin 2 promoter
  • the B3Z cells Upon activation by interaction with MHC class I molecules on dendritic cells presenting ovalbumin epitopes, the B3Z cells were engineered to respond by expressing ⁇ -galactosidase.
  • ⁇ -Galactosidase catalyzes a breakdown of X-gal (5-bromo-4-chloro-indolyl- ⁇ -D-galactopyranoside) to yield 5-bromo-4-chloro-3-hydroxyindole, a blue colored product.
  • Spectroscopic measurement of this blue color gives a measure of the effective MHC class I presentation of the ovalbumin epitope.
  • the results of this experiment shown in FIG. 6 , demonstrate that loading dendritic cells by ovalbumin loaded YCWPs gave more than a 100 fold increase in CD8+ T cell response over dendritic cells pulsed with ovalbumin.
  • Dendritic cells were prepared from cells obtained from the bone marrow of the femur and tibia of both hind legs of a female C57BL/6J mouse.
  • B16F0 murine melanoma cells were obtained (ATCC(CRL-6322)) and cultured using standard tissue culture techniques.
  • the dendritic cells were loaded with YCWPs containing B16F0 tumor lysate (around 2 ⁇ 10 ⁇ 15 g/YCWP) at a ratio of 100:1 particles:DC by adding the particles at day 7 of a dendritic cell culture for a period of 2 hours.
  • mice Three days prior to the preparation of the dendritic cells containing tumor lysate loaded particles, female C57BL/6J mice were challenged with 0.75 ⁇ 10 6 B16F0 melanoma cells in 0.4 ml 1 ⁇ PBS by intravenous injection. Once the dendritic cells containing tumor lysate loaded particles were prepared, each mouse in the treatment group was injected intravenously with 2 ⁇ 10 6 of dendritic cells containing tumor lysate loaded particles and this vaccination was repeated for three weekly doses. The mice were monitored up to four weeks for pulmonary metastasis.
  • FIG. 7A shows the lungs of three of the control mice (one mouse died prior to the end of the experiment and the lungs were not able to be photographed) in this experiment and
  • FIG. 7B shows the lungs of the four treated mice.
  • a dose of 1.25 million dendritic cells containing tumor lysate loaded particles can be cryopreserved in 0.2 mL of a serum-free, 10% dimethyl sulfoxide freezing medium (CryoStorTM CS-10, BioLife Solutinos, Inc.). Before injection, the dendritic cells can be thawed and diluted to a 1 mL with sterile saline for injection containing 5% human serum albumin (Albuminar-25, Aventis Behring). The dilution can then be transferred to a 3.0 cc syringe for injection and using a needle no smaller than 23 gauge, which should be administered within 2 hours of the thawing. The injection can be administered subcutaneously into an area of lymph nodes.
  • a serum-free, 10% dimethyl sulfoxide freezing medium (CryoStorTM CS-10, BioLife Solutinos, Inc.).
  • the dendritic cells can be thawed and diluted to

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