Antigen presenting cancer vaccine

Abstract

The disclosure provides reagents, methods, and kits, for treating melanoma. The reagent encompasses interferon-gamma (IFN-gamma) responsive melanoma cells, where the cells are autophagic and non-apoptotic melanoma cells, and where the cells express MHC Class II. In another aspect, the reagent encompassed dendritic cells loaded with the IFN-gamma responsive, non-apoptotic, MHC Class II-expressing melanoma cells.

Claims

1. A melanoma vaccine comprising: a population of melanoma cells comprising melanoma peptides from a subject with melanoma, contacted, in vitro, with an antigen presenting cell (APC) from the same subject, wherein the population of melanoma cells (a) is selected for being at least 60% autophagic, non-apoptotic, and MHC class II expressing, by one or more of flow cytometry, affinity chromatography, immunomagnetic separation, or adherence to a tissue culture surface; (b) is not treated with IFN-gamma; (c) is metabolically active; (d) is treated, in vitro, with an inhibitor of apoptosis, and wherein the contact between the melanoma cells and the APCs results in APCs comprising the melanoma peptides that are partially or substantially processed.

2. The composition of claim 1, wherein the APC is a dendritic cell.

3. The composition of claim 1, wherein the population of melanoma cells comprise melanoma-specific peptides that are acquired by the APCs and are partially or substantially processed in the APCs.

4. The composition of claim 1, wherein the APCs are loaded with melanoma specific peptides derived from the population of melanoma cells.

Description

FIGURES

(1) FIG. 1 reveals a graphic of cultured tumor cells before treatment with IFN-gamma (left) and after treating with IFN-gamma for 72 hours (right). After treatment, the cultured tumor cells are either floating, non-autophagic, and apoptotic, or adherent, autophagic, and non-apoptotic. The floating cells are shown expressing the apoptotic marker, phosphatidyl serine. The floating cells are shown with relatively few expressed MHC class II, while the adherent cells are shown with over-expressed MHC class II.

(2) FIGS. 2A-D show characterization of IFN-gamma treated autologous tumor cells used for loading dendritic cells. Autologous melanoma tumor cells were treated with or without 1000 IU/mL IFN-gamma for 72 hours in 15% FBS/ECS in RPMI, harvested and irradiated with 100Gy and cryopreserved. Cells were then thawed in AIMV and a sample taken for flow cytometry and for preparation of cell lysates for immunoblotting prior to antigen loading of DCs. An example of four separate autologous melanoma cell lines is shown (FIG. 2A, FIG. 2B and FIG. 2C). Induction major histocompatibility complexes by IFN-gamma treatment of autologous tumor cells (FIG. 2D). Tumor cells were harvested after being treated with or without 1000 IU/mL IFN-gamma for 72 hours and then assayed for MHC class I and class II. Control isotype antibodies were used to identify positive populations. Dark data points indicate median mean fluorescent plus/minus 95% confidence interval. N=65. After irradiation, melanoma cells are checked by assays to ensure that there is not any mitosis.

(3) In one aspect, the disclosure excludes non-autologous tumor cells for loading dendritic cells, and excludes methods of using non-autologous tumor cells for loading dendritic cells.

(4) FIGS. 3A and 3B describe phenotype of dendritic cells loaded with autologous melanoma cell lines treated with or without interferon-gamma. A set of four autologous melanoma cell lines were treated with or without 1000 IU/mL of IFN-gamma for 72 hours, irradiated and cryopreserved. The cells were then thawed in AIMV and combined with autologous dendritic cells for approximately 24 hours prior to harvest and assaying by flow cytometry for the expression of CD80, CD83, CD86 and MHC class II (FIG. 3A). The data is summarized in FIG. 3B. AveragesSD are shown, n=4.

(5) FIGS. 4A and 4B show phenotype of dendritic cells used for dose preparation. Samples of DC prior to loading (Pre-ATC Load DC, N=53) and after loading (Post-ATC Load DC, N=65) with IFN-gamma treated, irradiated autologous tumor cells were accessed by flow cytometry for the expression of CD80, CD83, CD86 and MHC class II. FACS Caliber beads were used to set the initial flow cytometer instrument settings which were then held constant throughout the collection of data (FIG. 4A). Values of percent expression and mean fluorescence intensity (MFI)SD are compared in FIG. 4B for Pre-ATC and Post-ATC loading. *p=0.019 and **p=0.0009.

(6) FIGS. 5A to 5C show interferon-gamma treated melanoma cells undergo autophagy. A selection of commercially available melanoma cell lines were incubated with 1000 IU/mL IFN-gamma for 72 hours in 5% FBS/RMPI. Phase-contrast photomicrographs of SK-5-Mel cell cultures were taken at the end of the incubation period (FIG. 5A) showing enlarged cells with vacuoles reminiscent of autophagosomes. Confirmation of the formation of autophagosomes was demonstrated by transfection with GFP-LC3B constructs prior to treatment with IFN-gamma (FIG. 5B). Autophagy induction after IFN-gamma treated was confirmed by western blotting using an antibody for LC3B (FIG. 5C) which identifies a faster migrating form of LC3 that has been shown to be associated with autophagic vessel formation.

(7) FIGS. 6A and 6B reveal apoptosis and autophagy induced in response to interferon-gamma. SK-5-Mel cells were incubated with 1000 IU/mL of IFN-gamma for 72 hours after which non-adherent and adherent populations were collected and assayed for apoptosis and autophagy by flow cytometry using 7-MD and Annexin-V (FIG. 6A). Enzo Cyto-ID Autophagy Detection Dye was used to measure autophagy by flow cytometry by measuring the mean intensity peak shift of dye provided by the manufacturer (FIG. 6B). Fold changes in the peak shift in comparison to 5% FBS/RPMI are shown in FIG. 6C with serum-free as positive control for the induction of autophagy.

(8) FIG. 7 discloses autophagy induction after blocking of caspase activity did not affect the induction of autophagy in response to IFN-gamma in melanoma cells. SK-5-Mel cells were treated with 1000 IU/mL of IFN-gamma in the presence of 20 uM of the pan-caspase inhibitor z-VAD or its control compound, z-FA for 72 hours. The cells were harvested and assayed for autophagy by flow cytometry as in FIG. 6C.

(9) FIG. 8 shows SK-5-Mel cells which were incubated with 1000 IU/mL of IFN-gamma in the presence of 10 uM of the autophagy inhibitor 3-methyladenine (3-MA) for 72 hours. The cells were then harvested and assayed for apoptosis and MHC class II (HLA-DR) expression by flow cytometry.

(10) FIG. 9 shows IFN-gamma treated cells from tumor cell lines generated from patient tumor specimens (N=36) were assayed for changes in MHC class II or apoptosis. The data shown are averages of mean fluorescent intensitySE.

(11) FIG. 10 shows IFN-gamma treated cells that were assayed for MHC class II or apoptosis by flow cytometry from samples used for loading dendritic cells for a patient-specific vaccine immunotherapy (N=54). Fold changes in MHC class II mean fluorescence intensity and percent apoptotic cells (Annexin-V positive) are shown.

(12) FIG. 11 and FIG. 12 show a correlation between induction of MHC class II and the absence of apoptosis (Interferon-gamma resistant) is associated with better progression-free survival (FIG. 11) and overall survival (FIG. 12) in patients received dendritic cells loaded with autophagic, non-apoptotic interferon-gamma treated tumor cells.

(13) FIG. 13 shows survival curves from three trials. The plot (Kaplan-Meier plot) is a stepwise curve showing the percent of study subjects surviving during the course of clinical trials. The groups are designated DC-54 (solid circle); TC-74 (solid square); TC-24 (solid triangles); and DC-18 (line). Poorest survival occurred with TC-24. The next poorest survival was with TC-74. TC-24 refers to a vaccine of tumor cells in a study involving 24 subjects.

(14) FIG. 14 shows survival curves from three trials. The trials are the same clinical trials as those disclosed in FIG. 13, but with additional data acquired from later time points.

FURTHER DESCRIPTION

(15) Autologous Dendritic Cell Generation

(16) Dendritic cells were generated by plastic adherence method of ficoled apheresis products (Choi, et al. (1998) Clin. Cancer Res. 4:2709-2716; Luft, et al. (1998) Exp. Hematol. 26:489-500; Cornforth, et al. (2011) Cancer Immunol. Immunother. 60:123-131), in antibiotic-free AIM-V medium (Invitrogen, Grand Island, N.Y.) supplemented with 1,000 IU/mL each of IL-4 (CellGenix, Freisberg, Germany) and GM-CSF (Berlex, Seattle, Wash.) (DC medium). The flasks were then cultivated for 6 days prior to loading with IFN-gamma treated, irradiated autologous tumor cells.

(17) IFN-Gamma Autologous Tumor Cell Line Generation and Preparation of Pharmaceutical

(18) Pure tumor cells were generated according to Cornforth, et al. (Cornforth, et al. (2011) Cancer Immunol. Immunother. 60:123-131; Dillman, et al. (1993) J. Immunother. Emphasis Tumor Immunol. 14:65-69; Dillman, et al. (2000) Cancer Biother. Radiopharm. 15:161-168). The tumor cells were then incubated with 1,000 U/mL of interferon-gamma (InterMune, Brisbane, Calif.) for 72 h, irradiated with 100Gy from a cesium source and cryopreserved (Selvan, et al. (2007) Int. J. Cancer 122:1374-1383; Selvan, et al. (2010) Melanoma Res. 20:280-292). The IFN-gamma treated and irradiated tumor cells were recovered from cryopreservation, washed with phosphate buffered saline (PBS), and then added to the cultivated dendritic cells (DCs) and then incubated for about 24 h. The antigen-loaded DCs were harvested by gentle scraping with a rubber policeman and cryopreserved. Aliquots of IFN-gamma treated or untreated tumor cells and loaded DCs were obtained for flow cytometry evaluation and trypan-blue exclusion assay.

(19) Staging of Cutaneous Melanoma

(20) The pharmaceutical or reagent of the disclosure can be administered to melanoma patients, where melanoma is diagnosed at Stage I, Stage II, Stage III, or Stage IV (Mohr, et al (2009) Ann. Oncology (Suppl. 6) vi14-vi21). Stage I, for example, refers to patients with primary melanomas without evidence of regional or distant metastasis. Stage II includes patients without evidence of lymphatic disease or distant metastases, where the patients are further characterized, e.g., by lesions greater than 1 mm and less than or equal to 2 mm thick with ulceration of the overlying epithelium, or by lesions greater than 2 mm and less than or equal to 4 mm thick with epithelial ulceration. Stage III melanoma includes lesions with pathologically documented involvement of regional lymph nodes or in-transit or satellite metastases, where patients may have, e.g., one, two, three, or four or more affected lymph nodes. Stage IV melanoma is defined by the presence of distant metastases, where the metastasis is located only in distant skin, subcutaneous tissues, or lymph nodes, where the metastasis involves lung metastases, or where the metastasis involves all other visceral sites.

(21) The disclosure encompasses methods for administration that are preventative, that is, for use with subjects not yet or never diagnosed with a melanoma. What is encompassed are methods for administration where a subject had earlier been diagnosed with a melanoma, and had earlier been successfully treated to eradicate the melanma (or had experienced a spontaneous complete remission), and where following eradication the administration is used preventatively.

(22) Tumor Antigens

(23) Without implying any limitation, melanoma cells of the disclosure express one or more of Mage, Mart-1, Mel-5, HMB45, S100, or tyrosinase (Dillman, et al. (2011) Cancer Biotherapy Radiopharmaceuticals 26:407-415). In one aspect, detection of tumor antigen uses cells that were not exposed to IFN-gamma while, in another aspect, detection of tumor antigen is conducted on cells that were treated with IFN-gamma (see, e.g., Comforth, et al. (2011) Cancer Biotherapy Radiopharmaceuticals 26:345-351). What is encompassed are melanoma cells expressing one or more melanoma antigens, or compositions comprising one or more isolated melanoma antigens, as disclosed by US2007/0207171 of Dubensky, et al, which is incorporated herein by reference in its entirety.

(24) Measuring Apoptosis

(25) Apoptosis can be detected or measured with a number of reagents, e.g., fluorochrome-labeled annexin, by staining with dyes such as propidium iodide and 7-aminoactinomycin D (7-AAD), by determining loss of mitochondrial inner membrane potential, by measuring activation or cleavage of caspases. See, e.g., George, et al. (2004) Cytometry Part A. 59A:237-245. An early event in apoptosis is exposure of phosphatidyl serine on the outer surface of the plasma membrane, which can be detected by fluorochrome-labeled annexin. The available methods can distinguish between live cells, necrotic cells, early apoptotic cells, and late apoptotic cells. The disclosure uses melanoma cells that are not apoptotic by 7-ADD assay, not apoptotic by annexin V assay, not apoptotic by an assay for apoptosis after IFN-gamma treatment (Dillman, et al. (2011) Cancer Biotherapy Radiopharmaceuticals 26:407-415), or not apoptotic by one or more of the biomarkers BcI-2, caspase-3, P53, or survivin (Karam, et al. (2007) Lancet Oncol. 8:128-136). The pharmaceutical compositions, reagents, and related methods, of the disclosure exclude IFN-gamma-treated melanoma cells that are apoptotic, where apoptosis is determined, e.g., according to U.S. Pat. No. 7,544,465 issued to Herlyn, et al; U.S. Pat. No. 7,714,109 issued to Thorpe, et al, which are incorporated herein by reference.

(26) Measuring Autophagy

(27) Autophagy is a naturally occurring process that is used for the degradation of many proteins and some organelles. Autophagy mediates protein and organelle turnover, starvation response, cell differentiation, cell death, and so on. Microtubule-associated protein light chain 3 (LC3) is to monitor autophagy. In one approach, autophagy can be detected by measuring the conversion of LC3, which involves conversion of LC3-I to LC3-II. The amount of LC3-II is correlated with the number of autophagosomes. In detail, LC3 is cytosolic and soluble, while LC3-II is present on membranes. LC3-II has a greater molecular weight because it is conjugated with a lipid. LC3 processing can be measured, e.g., by western blots, while autophagy, autophagic vesicles, and autophagosomes, can be measured by microscopy. Autophagy can be quantitated, e.g., by detecting processed LC3-II, by the ratio between early to late autophagic compartments, or by autophagic volume. See, (Mizushima and Yoshimori (2007) Autophagy 3:542-546:634-641; Tanida, et al. (2008) Methods Mol. Biol. 445:77-88; Eng, et al. (2010) Autophagy 6:634-641). In one aspect, the present disclosure uses autophagy as a screening tool, for selecting appropriate autophagic cancer cells, where the cells can be selected according to occurrence of autophagy in one or more particular stages. These autophagy stages include: (1) sequestering of cytosolic compartments by the autophagosome, (2) fusion of the autophagosome with the lysosome to form the autolysosome, and (3) degradation of the autophagosomal contents by proteases within the lysosome. In another aspect, the present disclosure includes mainly cells displaying the first stage, mainly the second stage, mainly the third stage, mainly the first and second stage, mainly the second and third stage, or mainly cells displaying all three stages. In yet another aspect, the disclosure comprises cells displaying the first stage, the second stage, the third stage, the first and second, the second and third stage, or cells displaying all three stages.

(28) Interferon-Gamma (IFN-Gamma) Signaling

(29) IFN-gamma (type II interferon) signaling depends on expression of a number of genes, e.g., IFN-gamma receptor, STAT1, STAT2, STAT1 homodimers, STAT1/STAT2 heterodimers, IRF-1, GAS, and IRF-E. Studies have shown that IFN-gamma signaling is dependent on IFN-gamma receptor (IFNGR1 chain; IFNGR2 chain). Low expression of IFNGR on the cell surface can block some aspects of IFN-gamma signaling (Schroder, et al. (2004) J. Leukocyte boil. 75:163-189). In one aspect, the present disclosure excludes using cancer cells that show low surface expression of IFNGR. In another aspect, the present disclosure screens cancer cells for those that express the STAT1 homodimer, uses these cells, and substantially excludes cells that do not express STAT1 homodimer. In yet another aspect, the disclosure contemplates screening cells for those with STAT1 phosphorylation (serine-727). What is also contemplated, is excluding cancer cells from patients having loss of function mutations in the STAT1 gene (see, e.g., Dupuis, et al. (2001) Science 293:300-303; Schroder, et al. (2004) J. Leukoc. Biol. 75:163-189). The following concerns the IRF gene family. IRF-1, IRF-2, and IRF-9, all participate in IFN-gamma signaling. The disclosure embraces using cancer cells that express one or more of these IRF gene family genes, or excluding cancer cells that do not express one or more of these genes.

(30) IFN-Gamma Responsive Genes

(31) The present disclosure embraces biologic material, compositions, reagents, and methods that require using a melanoma cell, or pre-neoplastic melanoma cell, that responds to IFN-gamma. The melanoma cell can be identified, distinguished, and selected, by an assay for the expression of one or more IFN-gamma-responsive genes. A number of IFN-gamma-responsive genes have been identified (see, e.g., Halonen, et al. (2006) J. Neuroimmunol. 175:19-30; MacMicking (2004) 11:601-609; Boehm, et al. (1997) 15:749-795). Said assay can involve removing one or more melanoma cells from the patient, culturing the cell in the presence and absence of added IFN-gamma, and determining responsiveness to IFN-gamma. In the assay, IFN-gamma induced gene expression can be detected by assays sensitive to binding of a transcription factor to the promoter of an IFN-gamma induced gene, to expression of mRNA from an IFN-gamma induced gene, to expressed polypeptide, and the like. The IFN-gamma response gene can include, e.g., a gene used for immune response, encoding a transcription factor, a transport protein, an apoptosis gene, a gene used for cell growth or maintenance, a gene used for lipid metabolism, a gene that mediates endocytosis or exocytosis, an intracellular signaling gene, a glucose metabolism gene, a cell adhesion gene, as well as genes without an established function.

(32) In one aspect, the disclosure excludes melanoma cells that, with IFN-gamma treatment, show reduced expression of MHC class II, show no detectable change in expression of MHC class II, show an increase of MHC class II expression of 10% or less, show an increase in MHC class II expression of 15% or less, show an increase in MHC class II expression of 20% or less, 25% or less, 30% or less, 40% or less, 50% or less, and the like. In one aspect, the value for percentage refers to the average expression value for the population of melanoma cells, residing in a biopsy or part of a biopsy, from a given subject or patient.

(33) Non-Limiting Lists of IFN-Gamma Inducible Genes for Use in Screening for IFN-Gamma Responsive Cancer Cells

(34) ab000677, JAB/SOCS1; m63961, IFN-gamma inducible protein (mag-1); m35590, Macrophage inflammatory protein 1-; m19681, MCP-1 (JE); y07711, zyxin; M34815, Monokine induced by IFN-gamma (MIG); m33266, Interferon inducible protein 10 (IP-10); U44731, Purine nucleotide binding protein; U88328, Sup. of cytokine signalling-3 (SOCS-3); M21065, Interferon regulatory factor 1; M63630, GTP binding protein (IRG-47); U19119, G-protein-like LRG-47; L27990, Ro protein; M31419, 204 interferon-activatable protein; af022371, Interferon-inducible protein 203; U28404, MIP-1 alpha receptor; U43085, Glucocorticoid-attenuated response 39; x56123, Talin; m31419, 204 interferon-activatable protein; U53219, GTPase IGTP; I38444, T-cell specific protein; M31418, 202 interferon-activatable protein; d38417, Arylhydrocarbon receptor; m26071, Tissue factor (mtf); D13759, Cot proto-oncogene; M18194, Fibronectin; u59463, ICH-3; M13945, pim-1 proto-oncogene; L20450, DNA-binding protein (see, Gil, et al. (2001) Proc. Natl. Acad. Sci 98:6680-6685). The disclosure encompasses use of the IFN-gamma induced gene, CIITA (see, e.g., Chan, et al. (2010) J. Leukocyte Biol. 88:303-311; Kwon, et al (2007) Mol. Immunol. 44:2841-2849).

(35) The present disclosure embraces measuring expression of one or more of the following IFN-gamma inducible genes, as a screening procedure for qualifying or selecting patients for administering a pharmaceutical. The genes include, (gene 1) FCGR1A, (gene 2) IL6R, (gene 3) CXCL9, (gene 4) CLCSF14, (gene 5) UBD, (gene 6) C/EBPalpha, and (gene 7) MHC2TA (CIITA) (see, Waddell, et al. (2010) PLoS ONE 5:e9753). Also embraced are use of specific clusters of these genes, in the qualifying procedure, such as, genes 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 1 and 3, 1 and 4, 1 and 5, 1 and 6, 1 and 7, 2 and 4, 2 and 5, 2 and 6, 2 and 7, 3 and 5, 3 and 6, 3 and 7, 4 and 6, 4 and 7, 5, and 7, and well as combinations of three genes, e.g., 1, 2, 3; or 3, 4, 5; or 4, 5, 6; or 5, 6, 7; or 1, 3, 4; or 1, 3, 5, or 1, 3, 6, or 1, 3, 7; or 1, 2, 4; or 1, 2, 5; or 1, 2, 6; or 1, 2, 7; and the like. (These gene numbers are arbitrary.)

(36) What is excluded is a population of melanoma cells that is less than 90% are autophagic, less than 80% are autophagic, less than 70% are autophagic, less than 60% are autophagic, less than 50% are autophagic, less than 40% are autophagic, and the like.

(37) What is excluded is a population of melanoma cells where, that is less than 90% are non-apoptotic, less than 80% are non-apoptotic, less than 70% are non-apoptotic, less than 60% are non-apoptotic, less than 50% are non-apoptotic, less than 40% are non-apoptotic, and the like.

(38) What is excluded is a population of melanoma cells that is less than 90% are non-adherent, less than 80% are non-adherent, less than 70% are non-adherent, less than 60% are non-adherent, less than 50% are non-adherent, less than 40% are non-adherent, and the like.

(39) Measuring Expression of MHC Class II

(40) Expression of MHC class II can be measured, for example, using antibodies or nucleic acid probes that are specific for MHC class II gene products. These MHC class II gene products include HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, as well as HLA-DM and HLA-DO (see, e.g., Apostolopoulos, et al. (2008) Human Vaccines 4:400-409).

(41) For example, the present disclosure encompasses reagents, methods of treatment, and methods of diagnosis, that require the melanoma cells to express STAT1 and STAT2, to have an active STAT1-signaling pathway, to have an active STAT2-signaling pathway, or to have active STAT1 and STAT2-signaling pathways.

(42) The disclosure provides a pharmaceutical composition or pharmaceutical reagent, related methods of administration, and methods of treatment, that result in survival data with a hazard ratio (HR) of less than 1.0, HR less than 0.9, HR less than 0.8, HR less than 0.7, HR less than 0.6, HR less than 0.5, HR less than 0.4, HR less than 0.3, and the like. The disclosure results in overall survival data, progression-free survival data, time to progression data, and so on. What is also provided is 6-month PFS of at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and so on. Moreover, what is provided is 6-month overall survival of at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and so on. Additionally, what is provided is 1-year (or 2-year) PFS of at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and so on. Moreover, what is provided is 1-year (or 2-year) overall survival of at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and so on (see, e.g., U.S. Dept. of Health and Human Services. Food and Drug Administration. Guidance for Industry. Clinical trial endpoints for the approval of cancer drugs and biologics (April 2005)).

(43) IFN-Gamma and the Induction of Autophagy

(44) Induction of autophagy after IFN-gamma treatment, as measured by increases in the expression of major histocompatibility class II complexes, can be used to determine response to systemic IFN-gamma treatment. If biopsied melanoma tumor cells, upon exposure to IFN-gamma in culture, undergo autophagy but not apoptosis, this indicates that these patients will respond favorably to systemic IFN-gamma treatment. Additionally, if successful cell lines are established from the biopsies, that patient would also benefit from cell-therapy products prepared from IFN-gamma treated purified tumor cells lines that are from autophagic but non-apoptotic adherent populations.

(45) The disclosure embraces isolating and characterizing major histocompatiability complexes isolated from autophagic, non-apoptotic cells collected from tumor cell lines treated with interferon-gamma. Major histocompatibility complexes contain antigens specific for CD4.sup.+ T cells and have been associated with antibody mediated immune responses. The complexes would represent a large repertoire of antigens would not be present in non-autophagic cells due to the action of lysosomal mediated antigen processing induced in autophagic cells.

(46) Non-apoptotic, autophagic tumor cells generated from IFN-gamma treated cell lines can be fused with dendritic cells to enhance the antigen presentation due to the high levels of major histocompatability complexes on the surface of the autophagic tumor cells. This process would yield a novel cellular product generated from the fusion of the two cell types.

(47) The process of induction of autophagy in response to IFN-gamma may be induced in a manner that does not result in apoptosis. By combining the treatment of tumor cells with caspase inhibitors and interferon gamma, the process of cell death (and ultimately the formation of tolergeneic apoptotic cells) can be blocked without inhibiting the induction of autophagy or the increase in major histocompatibility class II complexes.

(48) Procedure to Eliminate Apoptotic Cells, while Retaining Viable Autophagic Cells

(49) Studies of melanoma demonstrated a correlation between the presence of apoptotic cells and poor survival in a clinical trial (Cornforth, et al. (2011) Cancer Immunol. Immunother. 60:123-131; Dillman, et al. (2011) Cancer Biother. Radiopharmaceuticals 26:407-415). The following study investigated the induction of autophagy, apoptosis and MHC class II molecules after IFN-gamma treatment of melanoma tumor cells in vitro.

(50) The methodology of the study was as follows. Autologous and model melanoma tumor cell lines were incubated with 1000 IU/mL of IFN-gamma for 72 hours prior to assaying for autophagy, apoptosis and MHC class II expression. Autophagy was detected by immunobloting with antibodies against LC3 II and by flow cytometry with Enzo's CytoID Autophagy Detection Kit. Apoptosis and MHC class II induction were assayed by flow cytometry using 7-AAD and annexin-V staining and antibodies against MHC class II, respectively.

(51) The results from the study demonstrated that IFN-gamma induces both autophagic and apoptotic cell populations in melanoma cell lines. The apoptotic population was predominantly found in the non-adherent population while the autophagic cells remained adherent to the flask. Blocking of autophagy with the inhibitor 3-methyladenine (3-MA) inhibits the induction of MHC class II positive cells in response to IFN-gamma (39.4% IFN-gamma vs. 10.0% IFN-gamma+3-MA). Inhibition of caspase activity with the pan caspase inhibitor Z-VAD prevents apoptosis but does not perturb autophagy in IFN-gamma treated cells (2.750.15 IFN-gamma vs. 3.040.27 IFN-gamma+Z-VAD, fold change). To conclude, induction of apoptosis is associated with reduced levels of autophagy and MHC class II induction. This disclosure provides method or procedure to eliminate apoptotic cells while retaining viable autophagic cells after IFN-gamma treatment can enhance the effectiveness of this type of cell-based immunotherapy.

(52) IFN-gamma has been associated with suppression of immune response against tumors (see, e.g., Hallermalm (2008) J. Immunol. 180:3766-3774; Romieu-Mourez (2010) Cancer Res. 70:7742-7747; Lee (2005) Clinical Cancer Res. 11:107-112).

(53) A tumor can be a heterogeneous population of more or less differentiated cells. IFN-gamma treatment of melanoma cells of a tumor can act on some of the more differentiated cells, that are more susceptible to apoptosis. By eliminating these cells from the antigen source, the result can be loss of some effect on the tumor bulk following vaccination, translated by slow or no apparent regression of tumor size. Studies have shown that apoptotic cells do not activate dendritic cells (Sauter (2000) J. Exp. Med. 191:423-434).

(54) IFN-gamma may act to skew monocyte differentiation from DCs to macrophages. The amount of IFN-gamma in the preparation may influence the incomplete differentiation of DCs by skewing the phenotype to the less specialized macrophages.

(55) IFN-gamma may be used to enhance the MHC Class II molecules, and have a direct presentation to the T cells. However, the co-induction of II protein (Calprotectin) with MHC Class II molecules prevents the presentation of endogenous tumor antigens from MHC Class II molecules.

(56) Materials and Methods from First Study

(57) Autologous Dendritic Cell Generation

(58) Dendritic cells were generated by plastic adherence method as previously described (Choi (1998) Clin. Cancer Res. 4:2709-2716; Luft (1998) Exp. Hematol. 26:489-500). Briefly, autologous apheresis product was subjected to ficoll-hypaque (GE Healthcare, Buckinghamshire, United Kingdom) density gradient separation. The resulting peripheral blood mononuclear cells were placed in antibiotic-free AIM-V medium (Invitrogen, Grand Island, N.Y.) supplemented with 1,000 IU/mL each of IL-4 (CellGenix, Freisberg, Germany) and GM-CSF (Berlex, Seattle, Wash.) (DC medium) at 1510.sup.8 cells/mL in cell cultivation flasks (Corning-Costar, Corning, N.Y.). After one hour incubation, the non-adherent population was discarded and fresh DC medium was added to the flasks. The following morning, the non-adherent cells were discarded, the flasks were washed once with ambient temperature PBS, and fresh DC medium was added. The flasks were then cultivated for 6 days at which time flow cytometry evaluation is performed to determine the percentage and phenotype of DC generated by this approach (pre-load DC).

(59) Autologous Tumor Cell Line Generation

(60) Pure tumor cells generated and characterized as previously reported were expanded to 200 million cells and then incubated with 1000 IU/mL of IFN-gamma (InterMune, Brisbane, Calif.) for 72 hours in 15% FBS/ECS in RPMI (complete medium), irradiated with 100 Gy from a cesium source and cryopreserved as previously described (Choi (1998) Clin. Cancer Res. 4:2709-2716; Luft (1998) Exp. Hematol. 26:489-500; Dillman (1993) J. Immunother. Emphasis Tumor Immunol. 14:65-69). The IFN-gamma treated and irradiated tumor cells were recovered from cryopreservation, washed 3 with PBS, and then added to the in vitro cultivated DC and incubated for 24 hours. The antigen loaded DC were harvested by gentle scraping with a rubber policeman and cryopreserved at equal amounts in 9-11 aliquots. An aliquot of cells was obtained for flow cytometry evaluation which represents the post-loaded DC cells.

(61) Flow Cytometry

(62) Phenotypic characterization of the dendritic cell populations were performed using monoclonal antibodies against surface markers obtained from BD Pharmingen San Diego, Calif.: anti-MHC class II conjugated to PerCp, anti CD11c conjugated to APC, anti-CD80, anti-CD83, anti-CD86 conjugated to PE. Isotype controls were used to determine percent positive cells. Flow cytometry of tumor cells was conducted using antibodies against MHC class I and II conjugated to FITC, annexin-V-PE and 7-amino-actinomycin D (7-AAD) from BD Pharmingen. CaliBRITE flow cytometry calibration (BD Pharmingen) was used prior to each run and the same instrument settings were used throughout the collection of flow cytometric data.

(63) Immunoblot Assays

(64) Cytoplasmic cell lysates were prepared with Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, Ill.) plus protease inhibitor cocktail (Roche, Indianapolis, Ind.) at 10,000 cells/uL on ice. Approximately 25 uLs/lane of cell lysates were separated on 12.5% tris-glycine gels, transferred to PVDF membrane and probed with antibodies against the following: calreticulin (MBL, Woburn, Mass.), Hsp-60, Hsp-70, Hsp-90 (R&D Systems, Minneapolis, Minn.), HMBG-1 (Cell Signaling, Danvers, Mass.), ICAM-1 (Santa Cruz Biotech, Santa Cruz, Calif.), Mel-4, Mart-1 (Signet, Emeryville, Calif.), tyrosinase (Upstate, Lake Placid, N.Y.) and GADPH (Calbiochem, Darmstadt, Germany).

(65) Immunohistochemistry

(66) Expression of a panel antigens by melanoma lines were determined using immunocytochemical procedure. Cells were cultured in 8-chamber culture slides (Thermo Fisher, Rochester, N.Y.) in the presence or absence of 1000 IU/mL IFN-gamma. After 72 hours, the cells were washed 3 times with 1 Phosphate Buffered Saline (PBS) and fixed in cold acetone. After blocking endogenous peroxidase, cells were incubated with appropriate primary antibodies against the antigens listed. Immunohistochemistry was performed using biotinylated anitmouse or rabbit immunoglobulins, Super Sensitive enzyme-conjugated streptavidin labeling and horse radish peroxidase chromogen, and substrate kits (Biogenex, San Ramon, Calif.). The reactivity of the following anti-human polyclonal or monoclonal antibodies was investigated with isotype matched control antibody: S-100 and HMB-45 (Biogenex, San Ramon, Calif.), Mel-2, Mel-5, Mart-1 (Signet, Dedham, Mass.), Tyrosinase and Mage-1 (Thermo Scientific, Fremont, Calif.), Melan-A, HLA-class I and HLA-class II (Dako, Denmark).

(67) Statistical Analysis

(68) Student t-test of two-tailed, two samples of equal variance. Significant differences were determined by p value 0.05.

(69) Results from the First Study

(70) Cell death was differentially induced in the autologous melanoma tumor cells line in response to incubation with IFN-gamma for 72 hours in complete medium. Trypan-blue dye exclusion assay performed on cells either treated with IFN-gamma or not, revealed a significant trend toward lower viability in the IFN-gamma treated cells (89.16.8% vs. 84.99.3%, p=0.014, N=47). Analysis of a sample of four autologous melanoma cell lines by flow cytometry for apoptosis induction (FIG. 1A) revealed that melanoma cells are differentially sensitive to the effects of IFN-gamma induced apoptosis with some cells displaying more late apoptosis or dead populations (7-AAD+/Annexin-V+) while others displayed signs of early apoptosis or dying populations (7-MD/Annexin-V+). The resulting presence of apoptotic cells after IFN-gamma treatment was associated with significant decreases in progression-free and overall survival (Cornforth (2010) Cancer Immunol. Immunother. Resistance to the proapoptotic effects of interferon-gamma on melanoma cells used in patient-specific dendritic cell immunotherapy is associated with improved overall survival). A log-rank test revealed a significant association with lower viability upon IFN-gamma treatment of melanoma tumor cells and overall survival in patients under study.

(71) Lysates from cells that were incubated in the presence or absence of IFN-gamma were subjected to immunoblotting for a variety of molecules that may be important mediators of immunity (FIG. 1B). In the setting of melanoma cells treated with IFN-gamma, heat shock proteins appear to be differentially regulated but remain largely present in the cell preparations, especially in the case of hsp-70. The endoplasmic reticulum protein, calreticulin, and high-mobility group box-1 protein (HMGB-1), appear to be up-regulated in some cases upon treatment with IFN-gamma (FIG. 1B). By contrast, common melanoma antigens (mel-4, Mart-1 and tyrosinase) generally appear to be down regulated by IFN-gamma while ICAM-1, a lymphocyte adhesion molecule associated with sensitivity to lymphocyte mediated cytotoxicity (Hamai (2008) Cancer Res. 68:9854-9864), is significantly up-regulated (FIG. 1C). Indeed, IFN-gamma treated melanoma tumor cells were found to be more sensitive to cytotoxic T lymphocyte (CTL) activity. Additionally, immunohistochemistry of a panel of melanoma associated antigens revealed that IFN-gamma results in the down regulation of antigen expression in many of the antigens examined (Table I).

(72) The use of IFN-gamma results in the up-regulation of the major histocompatibility complexes, class I and class II (Bohn (1998) J. Immunol. 161:897-908). As shown in FIG. 1D, the treatment of autologous melanoma cells with IFN-gamma resulted in the near universal and significant up-regulation of MHC class I (p=2.810.sup.8) with a median fold induction of 2.911.13 (95% C.I.). Additionally, the mean fluorescence intensity of MHC class II was also significantly higher but less so (p=0.039) with a median induction of 4.232.66 (95% C.I.). The level of MHC class II molecules on the surface of the autologous melanoma cells was generally lower than that of the MHC class I molecules but in 70% of the cases the induction was greater than two fold in response to IFN-gamma treatment for the MHC class II molecules due to the low initial level of MHC class II expression. The presence of these molecules on the tumor cells during loading of antigens onto dendritic cells may provide an opportunity for cross dressing MHC complexes onto antigen presenting cells (Dolan (2006) J. Immunol. 277:6018-6024, Dolan (2006) J. Immunol. 176:1447-1455).

(73) A set of four representative autologous melanoma cell lines were incubated with IFN-gamma and loaded in equal amounts onto dendritic cells which were then assayed by flow cytometry for the expression of CD80, CD83, CD86 and MHC-class II. The results indicated that a small but appreciable increase in the percent positive population of dendritic cells expressing CD83 was seen upon the loading of the IFN-gamma treated melanoma cells (FIG. 2). Additionally, more unprocessed tumor cells are noted in the CD86 dot plot (upper left quadrant) which resulted in a discernible reduction in the percent CD86 positive population, indicating that IFN-gamma untreated tumor cells were still present. This effect is most likely due to the induction of apoptosis by IFN-gamma, as apoptotic cells are more likely to be phagocytosed by dendritic cells as previously reported.

(74) As shown in FIG. 3, a sample of pre-loaded DC showed that they expressed CD80 (39.016.2%), CD83 (7.16.9%), CD86 (73.619.5%) and were MHC class II positive with a viability of 96.25.0%. The loaded DC had a significantly higher percentage of CD83 (9.47.1%, p=0.019) with a significantly higher mean fluorescence intensity (172.979.0, p=0.0009) indicating that loading the DC with irradiated, IFN-gamma treated tumor cells induces maturation in some dendritic cells (FIG. 3B).

(75) Discussion from First Study

(76) Protocols for antigen loading, maturation, and administration, in the context of anti-tumor immunity, and guidance on dendritic cell (DC)-based immune therapy are practiced by the skilled artisan. This type of therapy encompasses use of purified autologous tumor cells as the source of antigen, and contains a patient-specific repertoire of tumor-associated antigens (Selvan (2010) Melanoma Res. 20:280-292; Dillman (2007) Cancer Biother. Radiopharm. 22:309-321). Some clinical trials are using unpurified autologous bulk tumors. This source of antigen may have contaminating fibroblasts and necrotic tissue (O'Rourke (2007) Melanoma Res. 17:316-322). Tumor stem cell associated antigens may be present in the purified cell lines (Dillman (2006) New Engl. J. Med. 355:1179-1181). IFN-gamma treatment increases expression of MHC class II molecules. MHC class II molecules are important for response to dendritic cell-based therapy. Molecules present in phagocytosed material, such as calreticulin, HMGB-1, and heat shock proteins, may contribute to a maturation signal, where this contribution may be in addition to contributions by cytokine cocktails. The present preparation of DCs shows a trend toward maturation, which can be associated with the phagocytosis of late stage apoptotic cells (Ip (2004) J. Immunol. 173:189-196). Use of apoptotic cells has been correlated with the generation of dendritic cells that were more effective at stimulating lymphocyte IFN-gamma secretion versus dendritic cells loaded with either tumor cell lysates or necrotic cells suggesting that dendritic cells loaded with apoptotic cells may be more potent in vivo. Resistance to the proapoptotic effects of IFN-gamma may be associated with a better clinical outcome (Comforth (2010) Cancer Immunol. Immunother. 60:123-131). Interleukin-12 (IL-12) secretion by mature DC can lead to robust cytotoxic lymphocyte (CTL; CD8.sup.+ cells) activity. The issue of whether ex vivo maturation leads to lasting tumor immunity, has been addressed. The risk of induction of regulatory T cells, which can suppress antigen specific CTLs, by immature DC has also been shown to occur with cytokine matured DC. A re-evaluation of the sequence of signaling events that leads to maturation is being investigated to improve DC maturation protocols. Thus, the use of irradiated whole tumor cells as the antigen source in this study, without the necessity of ex vivo cytokine maturation, may be a more preferable method of DC immunotherapy since the evidence presented here indicates that the DC have begun the process of maturation. Upon injection, these maturing DCs may complete the process of maturation by secreting chemokines which will attract licensing, antigen-specific CD40L expressing CD4.sup.+ T cells. Serum chemokines, like CCL17/TARC produced by dendritic cells in response to the adjuvant GM-CSF, have been associated with better progression-free survival rates. In some contexts, activation of lymphocytes by dendritic cells may require the expression of co-stimulatory molecules like CD80 and CD86. As a marker of maturation, CD83, is expressed on mature dendritic cells and may correspond to dendritic cells that can induce a more potent immune response (Prazma (2008) Immunol. Lett. 115:1-8). This represents a fraction of all the cells in the pharmaceutical preparation. The number of mature DCs alone, in any one pharmaceutical regiment, may or may not be correlated with a better patient response.

(77) Table from the First Study

(78) TABLE-US-00001 TABLE 1 Table I: Change in the expression level of common tumor associated antigens in response to interferon-gamma in melanoma cell lines used patient-specific cell based dendritic cell therapy. Change after No basal IFN-gamma treatment Antigens expression Basal expression None Down S-100 74.1% 25.9% 42.9% 57.1% HMB-45 18.5 81.5 54.5 45/5 Mel-2 3.7 96.3 46.2 53.8 Melan-A 11.1 88.9 29.2 70.8 Mel-5 18.5 81.5 72.7 27.3 MAGE-1 51.9 48.1 38.5 61.5 MART-1 11.1 88.9 14.8 85.2 Tyrosinase 25.9 74.1 40.0 60.0

(79) N=27 samples.

(80) Materials and Methods for the Second Study

(81) Melanoma Cell Lines

(82) The commercially available melanoma cell lines A375, SK-Mel-5 and SK-Mel-28 were purchased from American Type Culture Collection (Catalogue numbers: CRL-1619, HTB-70, and HTB-72). A375, SK-Mel-5, and SK-Mel-28 were maintained in 5% fetal bovine serum in RPMI-1640 (Invitrogen, catalogue number 11875-085). The pan-capase inhibitor, z-VAD-fmk and its control compound, z-FA-fmk, were purchased from BD Pharmingen (Catalogue numbers: 550377 and 550411). Transfections of GFP-LC3 were performed as per manufacturer instructions (InvivoGen, catalogue numbers psetz-gfplc3 and lyec-12) and photomicrograph were taken on an Olympus BX-51 microscope using a DP72 digital camera. Tumor cells lines were incubated with 1000 U/mL of IFN- (InterMune, Cat #) for 72 hours prior to assaying. Patient-specific cell lines were generated as described (Hamai (2008) Cancer Res. 68:9854-9864; Tyring (1984) J. Natl. Cancer Inst. 73:1067-1073) by enzymatic digestion of surgical tumor samples, cultivation in RPMI-1640 tissue culture media supplemented with fetal bovine and enriched calf serum (Omega Scientific, San Diego, Calif.) plus 1 mM sodium pyruvate, 1 mM glutamine and HEPES buffer. Phase contrast photomicrographs were taken on a Olympus CK-2 microscope using a Nikon DS-L1 digital microscope camera.

(83) Autologous Dendritic Cell Generation

(84) Dendritic cells were generated by plastic adherence method of ficoled apheresis products (Selvan (2007) Int. J. Cancer. 122:1374-1383; Cornforth (2010) Cancer Immunol. 60:123-131) in antibiotic-free AIM-V medium (Invitrogen, Cat#) supplemented with 1,000 IU/mL each of IL-4 (CellGenix, Cat#) and GM-CSF (Berlex, Seattle, Wash.) (DC medium). The flasks were then cultivated for 6 days prior to loading with IFN-gamma treated, irradiated autologous tumor cells.

(85) Flow Cytometry

(86) Analysis of tumor cell death and changes in major histocompatibility class II expression in response to IFN-gamma were conducted by use of antibodies directed against MHC class II, annexin-V and 7-amino-actinomycin D (7-AAD) and acquired on a Beckton-Dickenson FACS Calibur flow cytometer.

(87) Western Blotting

(88) Melanoma tumor cell lysates were resolved on 10-12.5% SDS-PAGE, transferred to nitrocellulose and probed with primary antibodies overnight prior to secondary antibody conjugation and development by Novex AP Chromogenic substrate (Invitrogen, Carlsbad, Calif.) to develop bands. Antibodies against LC3-B antibodies (Cell Signaling Technologies, Boston, Mass.) and GADPH (EMD biosciences, Germany) were used at manufacturers recommended dilutions of 1:100 and 1:10,000, respectively.

(89) Description of the Second Study

(90) What was investigated was the induction of autophagy, apoptosis and MHC class II molecules after IFN-gamma treatment of melanoma tumor cells in vitro. Autologous and model melanoma tumor cell lines were incubated with 1000 IU/mL of IFN-gamma for 72 hours prior to assaying for autophagy, apoptosis and WIC class II expression. Autophagy was detected by immunobloting with antibodies against LC3 II and by flow cytometry with Enzo's CytoID Autophagy Detection Kit. Apoptosis and MHC class II induction were assayed by flow cytometry using 7-AAD and annexin-V staining and antibodies against MHC class II, respectively.

(91) Results of the Second Study

(92) The results demonstrated that IFN-gamma induces both autophagic and apoptotic cell populations in melanoma cell lines. The apoptotic population is predominantly found in the non-adherent population while the autophagic cells remain adherent to the flask. Blocking of autophagy with the inhibitor 3-methyladenine (3-MA) inhibits the induction of MHC class II positive cells in response to IFN-gamma (39.4% IFN-gamma vs. 10.0% IFN-gamma+3-MA). Inhibition of caspase activity with the pan caspase inhibitor Z-VAD prevents apoptosis but does not perturb autophagy in IFN-gamma treated cells (2.750.15 IFN-gamma vs. 3.040.27 IFN-gamma+Z-VAD, fold change). Induction of apoptosis is associated with reduced levels of autophagy and WIC class II expression. Patients receiving autologous tumor cell loaded dendritic cells that are non-apoptotic autophagic cells derived from interferon-gamma treated purified tumor cell lines have improved progression-free and overall survival (p 0.003 and p 0.002, respectively). A procedure to eliminate apoptotic cells while retaining viable autophagic cells after IFN-gamma treatment may enhance the effectiveness of this type of cell-based immunotherapy.

(93) Pooled Analysis of Studies

(94) Autologous, proliferating, self-renewing tumor cells (putative tumor stem cells and/or early progenitor cells), are important to establishment of new depots of metastatic cancer, and may be excellent sources of antigen for vaccines. These studies addressed the impact on survival from immunizing with antigens from such cells.

(95) Methods

(96) Data was pooled from three successive phase II trials, all of which included patients with documented metastatic melanoma, who were treated in protocols that utilized antigens from cell cultures of autologous tumor cells. S.C. injections were given weekly for 3 weeks, then monthly for 5 months: 74 patients were injected with irradiated tumor cells (TC): 54 patients were injected with autologous dendritic cells (DC) that had been co-cultured with irradiated autologous tumor cells (NCI-V01-1646): in a randomized phase II trial, 24 patients were injected with TC, and 18 with DC.

(97) Results

(98) Table 2 summarizes overall survival (OS) in each trial. In the pooled analysis there were 98 TC and 72 DC patients. Characteristics were similar in terms of age (51, 52), male gender (62%, 62%), no evidence of disease at the time of treatment (46%, 47%), and presence of MU visceral disease at the time of treatment (13%, 14%). OS was longer in patients treated with DC (median 63.1 vs 20.2 months, 5-year OS 51% vs 26%, p=0.0002 Mantle-Cox log-rank test). The difference in OS in the randomized trial is also significant (p=0.007).

(99) Patient-specific DC vaccines primed with antigens from autologous proliferating, self-renewing tumor cells are associated with encouraging long-term survival rates, and are superior to patient-specific TC vaccines in populations of patients who have been diagnosed with metastatic melanoma.

(100) TABLE-US-00002 TABLE 2 # # Median 2-yr 5-yr Vaccine patients deaths OS OS OS TC 74 60 20.3 mos 45% 28% DC 54 31 58.4 mos 72% 50% (Use IFN-gamma treated melanoma cells) TC 24 16 15.9 mos 31% DC 18 5 Not Reached 72% (No IFN-gamma treatment of melanoma cells)

(101) The survival curves from the three trials of patient specific vaccines are shown in FIG. 13. Consecutive phase I and II clinical trials were conducted using autologous tumor cells, in combination with autologous dendritic cells or without the dendritic cells, were conducted. Subcutaneous injections were given weekly for three (3) weeks, then monthly for five (5) months, 74 patients were injected with irradiated tumor cells without pretreatment with IFN-gamma (TC): 54 patients were injected with autologous dendritic cells (DC) that had been co-cultured with irradiated autologous tumor cells with pretreatment with IFN-gamma: in a randomized phase II trial, 24 patients were injected with TC without pretreatment without IFN-gamma, and 18 with DC plus TC without pretreatment with IFN-gamma.

(102) FIG. 14 shows survival curves from three trials, where the trials are the same clinical trials as those disclosed in FIG. 13, but with additional data acquired from later time points, as is evident from comparing the step plots in the two figures. The melanoma cells in the clinical trials, TC-24 and TC-74, did not receive IFN-gamma. The melanoma cells in the clinical trial, DC-TC-18, did not receive IFN-gamma. The melanoma cells in the clinical trial, DC-TC-54, did get IFN-gamma.

(103) A non-limiting standard operating procedure for preparing dendritic cell vaccine includes the following (Table 3). Upon harvesting tumor cells after expansion, the following are to be made for each patient's tumor cell lot. What is needed is about 220 million cells to make the tumor cell vaccine lot. Any extra cells are to be cryopreserved as back up cells. Make stock cell suspension as 22010.sup.6 cells in 22 ml medium to distribute in the following manner (Table 3).

(104) TABLE-US-00003 TABLE 3 Operating Procedure Total cell # First Second Final Use needed action action disposition TC Vaccine 150 million 15 ml from the Cryopreserve cells after Store until needed for Doses or stock to a 50 ml irradiation in 10 small patient treatment. DC Loading conical tube, add cryovials. Cells 25 ml AIM-V, and irradiate

(105) Trial #2: DC 2000-2006 (NCI-V01-1646). Phase II Trial of Autologous Dendritic Cells Loaded with Antigens from Irradiated Autologous Tumor Cells as Patient Specific Vaccines (BB-IND 8554): Dendritic Cell (DC) Vaccine. In the production of the vaccine for this trial, autologous proliferating tumor cells were co-incubated with IFN-gamma, cryopreserved, and then subsequently co-incubated with autologous dendritic cells. Each aliquot of cells was suspended in 500 micrograms of GM-CSF for injection

(106) Trial #3: DC vs. TC 2007-2011 (NCT00436930): Randomized Phase II Trial Of Autologous Vaccines Consisting Of Adjuvant GM-CSF plus Proliferating Tumor Cells Versus GM-CSF Plus Dendritic Cells Loaded With Proliferating Tumor Cells In Patients With Metastatic Melanoma (BB-IND 8554 and BB-IND 5838): MAC VAC. The third trial was a randomized trial to determine whether there was a difference in the two approaches noted above. IFN-gamma was not used in the production of the tumor cells. As in the DC trial above, all patients were randomized to receive either TC or DC injected s.c. with 500 micrograms of GM-CSF, weekly for 3 weeks and then monthly for five months. The projected 72% 2-year survival rate for patients in the DC arm is comparable to the 71% observed 2-year survival observed in the previous 54-patient trial of DC in which the median survival was five years.

(107) Thus, while there have shown and described and pointed out fundamental novel features of the disclosure as applied to an exemplary implementation and/or aspects thereof, it will be understood that various omissions, reconfigurations and substitutions and changes in the form and details of the exemplary implementations, disclosure and aspects thereof may be made by those skilled in the art without departing from the spirit of the disclosure and/or claims. For example, it is expressly intended that all combinations of those elements and/or method steps which perform substantially the same function in substantially the same way to achieve the same results are within the scope of the disclosure. Moreover, it should be recognized that structures and/or elements and/or method steps shown and/or described in connection with any disclosed form or implementation may be incorporated in any other disclosed or described or suggested form or implementation as a general matter of design choice. It is the intention, therefore, to not limit the scope of the disclosure. All such modifications are intended to be within the scope of the claims appended hereto.

(108) All publications, patents, patent applications, references, and sequence listings, cited in this specification are herein incorporated by this reference as if fully set forth herein.

(109) The Abstract is provided to comply with 37 CFR 1.72(b) to allow the reader to quickly ascertain the nature and gist of the technical disclosure. The Abstract is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.