WO2014130823A1 - Dendritic cell enhancement of protection by human muc1-stimulated mononuclear cells against cancer - Google Patents

Abstract

The present invention includes compositions and methods for improving a cytotoxic immune response to a cancer antigen comprising obtaining peripheral blood mononuclear cells from a subject; incubating adherent peripheral blood mononuclear cells with the cancer antigen and GM-CSF in the absence of IL-4 to mature the adherent peripheral blood mononuclear cells into dendritic cells; and isolating the matured dendritic cells, wherein the matured dendritic cells stimulate cytotoxic T cells specific for the cancer antigen in vivo.

Description

DENDRITIC CELL ENHANCEMENT OF PROTECTION BY HUMAN MUC1- STIMULATED MONONUCLEAR CELLS AGAINST CANCER

Technical Field of the Invention

The present invention relates in general to the field of medical diagnosis and medical treatment, and more particularly, to dendritic cell enhancement of protection by human antigen stimulated mononuclear cells against cancer.

Background of the Invention

Without limiting the scope of the invention, its background is described in connection with cancer treatment.

Current methods of treating breast cancer such as chemotherapy and radiotherapy cause many unwanted side effects, including secondary cancers and senescence of normal cells because they are not specific to cancer cells.¹ Therefore, cancer cell-specific treatment has been one of the major goals of cancer research.² Immunotherapy is one such treatment in which the immune system is stimulated actively, by vaccination, or adoptively, by transfer of components of the immune system, e.g., antibodies or immune cells. The immune cells, which may be stimulated in culture, are generated from PBMC. Cells of the immune system, such as CTL, have been identified that recognize antigens, which are unique to the cancer cells, and kill them in preference to normal cells.³

One such method is taught in United States Patent Application No. 20100074925, filed by Carmon, Lior, and is directed to Antigen Specific Multi-Epitope Vaccines. The application is said to relate to cancer vaccines composed of the signal peptide domain of tumor associated antigens or proteins. The peptide vaccines are characterized by having multiple MHC class I and class II epitopes, which are highly abundant in the population. These vaccines are said to likely to induce a strong, comprehensive immune response against the target proteins in the majority of the vaccinated population, and thereby induce an immune reaction against tumors expressing such target proteins. Specific peptide vaccines are composed of the signal peptide domain of Mucin (MUC1), BAGE-1 or ARMET, and their use for the treatment of cancers which express Mucin (MUC1), BAGE-1 or ARMET.

Another cancer vaccine is taught in United States Patent Application No. 20090317414 filed by Pietersz, et al., and is direct to a Cancer Vaccine Comprising A Mucin 1 (Mucl) T Cell Epitope- Derived Peptide. Briefly, the application is said to teach a cancer vaccine, and a composition for the ex vivo priming of dendritic cells, which comprises a MUC1 T cell epitope-derived peptide or peptide analogue capable of provoking a cytotoxic T cell immune response. Particular MUC 1 T cell epitope-derived peptides are disclosed.

Lutz, et al, in the article entitled, "Immature dendritic cells generated with low doses of GM- CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo" 30(7) Journal of European Immunology 1813-1822, teach Dendritic cells (DC) cultured from mouse bone marrow (BM) progenitors in low concentrations of granulocyte-macrophage colony- stimulating factor (GM-CSF) (GM^lG DC) by two different protocols. In this article, the phenotype and functional properties of these GM¹" DC were compared to those of standard BM- DC cultures generated in high concentrations of GM-CSF (GM^hl DC) or in low GM-CSF plus IL-4 (GM^lo/IL-4 DC). It is said that an effect of IL-4 on maturation was observed only at low but not high doses of GM-CSF. It was also found that compared to mature DC, GM^lc DC were phenotypically immature, weak stimulators of allogeneic and peptide-specific T cell responses, but substantially more potent in presentation of native protein. Importantly, immature GM^lc DC were resistant to maturation by lipopolysaccharide, TNF-a or anti-CD40 monoclonal antibodies, as the expression of co-stimulatory molecules was not increased, and stimulatory activity in oxidative mitogenesis was not enhanced.

Seder, et al, in an article entitled, "The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4⁺ T cells from T cell receptor transgenic mice," J. Exp. Med 176(4) 1091-1098 (1992), studied the factors that determine whether CD4⁺ T cells produce interleukin 4 (IL-4) or interferon gamma (IFN-gamma) upon stimulation we used a system allowing naive T cells to be primed in vitro by specific antigen. It was found that cells primed in the absence of IL-4 produced IFN-gamma upon rechallenge but virtually no IL-4. IFN-gamma was found to diminish priming for IL-4 production when limiting amounts of IL-4 (100 U/ml) were used in the initial culture. The article argues that the dominant effect of IL-4 in determining the lymphokine-producing phenotype of primed cells was observed with dendritic cells (DC), activated B cells, and I-Ek-transfected fibroblasts as APC. However, it is said that the different APC did vary in their potency, with DC being superior to activated B cells, and that were superior to transfected fibroblasts.

Disclosure of the Invention

In one embodiment, the present invention includes a method of improving a cytotoxic T cell immune response to a cancer antigen comprising: obtaining peripheral blood mononuclear cells from a subject; incubating adherent peripheral blood mononuclear cells with the cancer antigen and GM-CSF in the absence of IL-4 to mature the adherent peripheral blood mononuclear cells into dendritic cells; and isolating the matured dendritic cells, wherein the matured dendritic cells stimulate cytotoxic T cells specific for the cancer antigen in vivo. In one aspect, the GM-CSF is added to the dendritic cells on at least one of day 0, 3, and 7. In another aspect, the dendritic cells are further incubated with at least one of tumor necrosis factor alpha or poly I:C prior to cytotoxic T cell stimulation. In another aspect, the cancer antigen is a MUC-1 peptide. In another aspect, the cancer antigen is selected from at least one of MelanA (MART -I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-family members, BAGE-family members, GAGE- family members, RAGE-family members, pl5(58), CEA, NY-ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, pl6, TAGE, PSMA, PSCA, CT7, telomerase, 43- 9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 195, CA 242, CA-50, CAM43, CD68\KP 1, CO-029, FGF-5, G250, Ga733, HTgp-175, M344, MA- 50, MG7-Ag, MOV 18, NB/70K, NY-CO- 1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, and TPS. In another aspect, a media for incubating the dendritic cells is serum free. In another aspect, the dendritic cells matured in the absence of IL-4 stimulate a stronger cytotoxic T cell immune response than dendritic cells stimulated in the presence of IL-4. In one aspect, the method further comprises the step of contacting the matured dendritic cells with peripheral blood mononuclear cells that comprise immature T cells in the presence of the cancer antigen under conditions in which the immature T cells are converted into cancer antigen-specific active cytotoxic T cells.

Yet another embodiment of the present invention includes a method of improving a cytotoxic T cell immune response to a cancer antigen comprising: obtaining dendritic cells from a subject; incubating the dendritic cells with the cancer antigen and GM-CSF in the absence of IL-4 to mature the dendritic cells; isolating the matured dendritic cells, wherein the matured dendritic cells stimulate cytotoxic T cells specific for the cancer antigen in vivo, and contacting the matured dendritic cells with peripheral blood mononuclear cells that comprise immature T cells in the presence of the cancer antigen under conditions in which the immature T cells are converted into cancer antigen-specific active cytotoxic T cells.. In one aspect, the GM-CSF is added to the dendritic cells on at least one of day 0, 3, and 7. In another aspect, the dendritic cells are further incubated with at least one of tumor necrosis factor alpha or poly I:C prior to cytotoxic T cell stimulation. In another aspect, the cancer antigen is a MUC-1 peptide. In another aspect, the cancer antigen is selected from at least one of MelanA (MART -I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-family members, BAGE-family members, GAGE- family members, RAGE-family members, pl5(58), CEA, NY-ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, pl6, TAGE, PSMA, PSCA, CT7, telomerase, 43- 9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 195, CA 242, CA-50, CAM43, CD68\KP 1, CO-029, FGF-5, G250, Ga733, HTgp-175, M344, MA- 50, MG7-Ag, MOV 18, NB/70K, NY-CO- 1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, and TPS. In another aspect, a media for incubating the dendritic cells is serum free. In another aspect, the dendritic cells matured in the absence of IL-4 stimulate a stronger cytotoxic T cell immune response than dendritic cells stimulated in the presence of IL-4.

Yet another embodiment of the present invention includes a composition for activating a cytotoxic T cell response comprising: isolated adherent peripheral blood mononuclear cells from a subject incubated with a cancer antigen and GM-CSF matured in the absence of IL-4, wherein the matured adherent peripheral blood mononuclear cells stimulate cytotoxic T cells specific for the cancer antigen in vivo. In one aspect, the GM-CSF is added to the dendritic cells on at least one of day 0, 3, and 7. In another aspect, the dendritic cells are further incubated with at least one of tumor necrosis factor alpha or poly I:C prior to cytotoxic T cell stimulation. In another aspect, the cancer antigen is a MUC-1 peptide. In another aspect, the cancer antigen is selected from at least one of MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE- family members, BAGE-family members, GAGE-family members, RAGE-family members, pl5(58), CEA, NY-ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, pl6, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta- HCG, BCA225, BTAA, CA 125, CA 15-3, CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO- 029, FGF-5, G250, Ga733, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, and TPS. In another aspect, a media for incubating the dendritic cells is serum free. In another aspect, the dendritic cells matured in the absence of IL-4 stimulate a stronger cytotoxic T cell immune response than dendritic cells stimulated in the presence of IL-4. Yet another embodiment of the present invention is an isolated dendritic cell made by the method of obtaining dendritic cells from a subject; incubating the dendritic cells with the cancer antigen and GM-CSF in the absence of IL-4 to mature the dendritic cells; and isolating the matured dendritic cells, wherein the matured dendritic cells stimulate cytotoxic T cells specific for the cancer antigen in vivo.

Description of the Drawings

For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

Figures 1A-C: Influence of IL-4 on the generation of DC for stimulation of PBMC to produce CTL. Dendritic cells (DC) were generated and maturated with or without IL-4. Percentage of specific lysis was performed on day 8 by XTT assay (MCF-7 (1A)) and alamarBlue® assays (K562 (IB), RAJI (1Q). EffectonTarget (E:T) ratios are 1.25, 2.5, 5 and 10: 1. Bars are standard error.

Figure 2: Tumor specific target, MCF-7, and nonspecific, NK target, K562, and LAK target, RAJI, specific lysis by CTL used for protection study. MUCl loaded-DC (-) IL-4 were added to PBMC at 1 : 100 ratio on days 0 and 7. Percentage of specific lysis was performed on day 8 by XTT assay (MCF-7) and alamarBlue® assays (K562, RAJI). Lysis of the cell lines was determined at 10: 1 effectontarget (E:T) ratio. Bars are standard error. Standard error bars are hidden in some of the symbols. The results are from two independent studies.

Figure 3: Cytokine production by CTL used for protection study. The quantification of cytokines (pg/mL) produced by PBMC stimulated with MUCl loaded-DC (-) IL-4, was performed with commercial ELISA kits as detailed in materials and methods. Standard error bars are hidden in some of the symbols. The results are from two independent studies.

Figure 4: Enhancement of MUCl stimulated CTL protection against MCF-7 tumor development by antigen presenting cells. Groups and treatment: Group 1, None; Group 2, MC + Unloaded DC on days 0,2,4,9, 14, 19; Group 3, MC + MlLDC(-)IL-4 on days 0,2,4,9, 14, 19; Group 4, CTL; Group 5, CTL + M1LPBMC on days 0, 14; Group 6, CTL + M1LPBMC on days 0,2,4,9, 14, 19; Group 7, CTL + MlLDC(-)IL-4 on days 0,14; Group 8, CTL + MlLDC(-)IL-4 on days 0,2,4,9, 14,19. MC (mononuclear cells, peripheral blood); CTL (cytotoxic T- lymphocytes); DC (Dendritic Cells); MlLDC(-)IL-4 (mucin 1 -loaded DC generated without IL- 4); M1LPBMC (mucin 1 -loaded peripheral blood mononuclear cells). On day 0, all NOD-SCID mice were injected with 5x 106 MCF-7 cells/mouse subcutaneously; other cell types, 5x 107 MC or CTL, with or without 5x 105 unloaded DC, MUCl loaded-DC (-) IL-4 or MUCl loaded- PBMC, were injected intraperitoneally on the days indicated. Mice were observed up to thirty- one days after the MCF-7 injection for tumor development. Comparison of all groups, except group 7, with group 8 showed a statistical difference at p < 0.05 (Fisher's Exact Test). Comparison of all groups, except group 2, with group 1 showed a statistical difference at p < 0.05 (Fisher's Exact Test). All other comparisons were not statistically significant. The arrow indicates the group to which the comparison was made. The results are from two independent studies from cells of two subjects.

Description of the Invention

While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) in stimulating peripheral blood mononuclear cells (PBMC) to generate antigen-specific cytotoxic T-lymphocytes (CTL). The objective was, first, to determine if interleukin (IL)-4 was beneficial or detrimental in generation of human DC in vitro, and subsequently, to determine if the optimized DC enhanced CTL killing of adenocarcinomas in vivo.

Mucin 1 (MUCl), a glycoprotein, found on the cell surface of adenocarcinomas, was used to load DC. MUCl loaded-DC generated without IL-4 (MUCl loaded-DC (-) IL-4) were superior to DC produced with IL-4 (MUCl loaded-DC (+) IL-4) in stimulating PBMC to kill MCF-7 in vitro. A corollary in vivo protection study was performed by injecting non-obese, diabetic - severe, combined immuno-deficient (NOD-SCID) mice subcutaneously with the human breast cancer cell line MCF-7. These same mice were injected intraperitoneally (IP) with CTL and/or PBMC, which had been loaded with MUCl, as well as MUCl loaded-DC (-) IL-4 on different schedules. The group that received CTL and MUCl loaded-DC (-) IL-4 on days 0, 2, 4, 9, 14, and 19, exhibited 100% protection against tumor development. Other combinations showed less protection. Therefore, tumor antigen-loaded DC enhanced protection in adoptive immunotherapy with CTL. This implies that the tumor antigen-loaded DC increased the killing of tumor cells by CTL. Tumor antigen-loaded DC should be considered for adoptive immunotherapy with CTL. Current methods of treating breast cancer such as chemotherapy and radiotherapy cause many unwanted side effects, including secondary cancers and senescence of normal cells because they are not specific to cancer cells.¹ Therefore, cancer cell-specific treatment has been one of the major goals of cancer research.² Immunotherapy is one such treatment in which the immune system is stimulated actively, by vaccination, or adoptively, by transfer of components of the immune system, e.g., antibodies or immune cells. The immune cells, which may be stimulated in culture, are generated from PBMC. Cells of the immune system, such as CTL, have been identified that recognize antigens, which are unique to the cancer cells, and kill them in preference to normal cells.³

Many tumor cells express tumor-associated antigens (TAA) that can be recognized by specific CTL. The defined antigenic peptide can be presented by an APC, and these cells will elicit an immune response. MUC1 is one such TAA, which has been identified as a breast cancer cell- specific epitope.^4"6

As used herein, the phrase "cancer antigens" and "tumor-associated antigens" refer to proteins and/or peptides processed from proteins and presented by antigen presenting cells to T cells that include, but are not limited to Muc-1, MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-family members, BAGE-family members, GAGE-family members, RAGE- family members, pi 5(58), CEA, NY-ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER- 2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl85erbB2, pl80erbB-3, c- met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, pl6, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha- fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 195, CA 242, CA-50, CAM43, CD68\ P1, CO-029, FGF-5, G250, Ga733, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, and the like. These cancer antigens have been associated with certain tumors such as melanoma, lung cancer, prostate cancer, breast cancer, renal cancer and others.

Mucins are polymorphic, O-linked glycosylated proteins expressed on the surface of ductal epithelial cells. MUC1 is characterized by a 20 amino acid sequence that is tandem repeated 25- 100 times in each molecule.⁵' ⁶ Adenocarcinomas are cancers of secretory organs and are typically deficient in glycosyltransferases that glycosylate mucin. As a result, adenocarcinoma cells should produce altered protein trafficking that would increase antigen processing.⁷ This would cause MUCl of adenocarcinomas to be recognized as a novel antigen and serve as a target for CTL.

Human CTL ³' ^{8 10} and B-cells ¹¹ specific for cancer-associated MUCl have been described. Previous studies showed that anti-mucin CTL cytotoxicity is MHC unrestricted.³' ^{8 10} It is hypothesized that because of its highly repetitive, multivalent structure ³' ^{8 10} mucin can bind, crosslink and thus signal the T-cell receptor in the absence of MHC presentation. Therefore, tumor-associated mucins may be effective target antigens for CTL in cancer immunotherapy. Dendritic cells, because of the use of co-stimulatory molecules, are the most potent APC of the immune system. They are the best stimulators of antigen-specific T-lymphocyte responses ¹² and, as such, could be considered ideal candidates for cancer immunotherapy in combination with T-cells. T-cells have been primed with DC in vivo,¹ with virus,¹⁴ antigens in liposomes ¹⁵ or DNA ¹⁶ in SCID mice. Addition of peptides during the maturation of DC results in an effective loading of these peptides into MHC I complexes.¹⁷ DC loaded with TAA, e.g., MUCl, can elicit specific tumor-reactive T-cells, e.g., breast cancer specific CTL. In addition, prolonged in vivo antigen stimulation enhances killing of tumor by CTL.¹⁸ This may be explained by the observation that DC restimulation maintains protective memory CTL in a viral mouse model.¹⁹

The present inventors have previously used MUCl to stimulate PBMC that showed specific killing of human breast cancer cells in vitro ³ and in vivo?⁰ The current studies were performed to determine the effects of human DC on CTL specific lysis of human breast cancer cells. The present inventors compared DC generated with or without IL-4. IL-4 is incorporated to reduce the production of macrophages.²¹' ²² The rationale for not using IL-4 is that this cytokine favors the development of type 2 immune responses, at the expense of the development of type 1 immune responses,^23"25 which is the desired cellular immune response. The reason for this is that CCL3/MIP-1 and CCL4/MIP-1 are not induced in human primary monocytes when granulocyte-macrophage colony-stimulating factor (GM-CSF) is combined with IL-4.²⁶ These chemokines are required for DC to polarize Type 1 T cells.²⁷ In addition, DC generated with GM-CSF alone have been shown to function to stimulate CTL.²⁸ The optimum method for generating DC was then used to generate the CTL for the in vivo study. The ability of MUCl loaded-DC to enhance CTL killing of human tumor cells was evaluated in a NOD-SCID mouse model.

Human cells. All human cells were obtained in accordance with the Helsinki Declaration of 1975 and the Texas Tech University Health Sciences Center Institutional Review Board from expired subjects. PBMC from two different subjects were used. PBMC were not HLA typed, as the present inventors ³' ²⁰' ³⁹ and others ⁴⁰ have found cytotoxicity by MUC1 -specific CTL may be non-major histocompatibility complex restricted. Cells used for each study were from one subject. The in vitro study and one of the in vivo studies were cells from subject 1. The other in vivo study used cells was from subject 2.

MUC1 peptide. MUCl-mtri peptide G APPAHGV APDNRPAP (SEQ ID NO.: l) (S2.12- N; T3, l 1, 16-N) ⁵ was custom synthesized by American Peptide Co., Inc..

Generation and maturation of Dendritic Cells (DC). DC were generated from PBMC, of humans with breast adenocarcinomas, obtained by apheresis,⁴¹ as published ³¹ except as modified below. PBMC were cultured in 225 cm² tissue culture flasks at 2x l0⁶ cells/mL in 75 mL of AIM-V^® serum-free lymphocyte medium (Gibco BRL, Life Technologies, Inc. catalog number 12055- 083) and incubated at 37°C and 5% CO₂ for 1 hour. After a microscopic confirmation of adherence, the non-adherent fraction was removed and 75 mL of fresh AIM-V^® serum-free lymphocyte medium was added (day 0). The non-adherent fractions were centrifuged and placed into a second set of 225 cm² tissue culture flasks for adherence. After 1-hour incubation, the non-adherent fraction in the second set of flasks was removed and 75 mL of fresh AIM-V^® serum-free lymphocyte medium was added. Both sets of flasks were incubated at 37°C and 5% CO₂. MUCl peptide (^g/mL final concentration) was added on day 0 of culture. Human recombinant GM-CSF (500 IU/mL final concentration) (Biosource International Inc., catalog number PHC2015) was added on days 0, 3, and 7. Human recombinant tumor necrosis factor- alpha (lOng/mL final concentration) (Biosource International Inc., catalog number CTP301 1), as well as polyriboinosinic polyribocytidylic acid (50μg /mL final concentration) (Sigma Chemical Co., catalog number P 1530)³⁰ was added on day 7. IL-4 (15ng/mL final concentration) (Biosource International Inc., catalog number PHC0044)³¹ was added to one set of the flasks on days 0, 3, and 7, the other set did not receive any IL-4. It has been shown that this method induced CD83 and costimulatory molecules.³⁰ On day 9, mature DC were counted and their viability was assessed by trypan blue exclusion test (Invitrogen Corporation, catalog number 15250-061).

Generation of CTL for protection studies. The CTL were generated from PBMC and grown in a gas-permeable hydrophobic bag for eight days. PBMC were cultured at 2* 10⁶ cells/mL in AIM- V^® serum-free lymphocyte medium and maintained in a 37°C humidified and 5% CO₂ atmosphere. IL-2 (Cetus, GenWayBio, Inc., catalog number GWB-76974E) was added twice per week at 100 IU/mL. PBMC were stimulated with MUCl (1 μg/mL final concentration)⁴² or MUCl loaded-DC(-)IL-4 (1 : 100 ratio) on days 0 and 7 of culture. Media was not changed, but was added on days 4 and 7, if needed, to maintain 2* 10⁶ cells/mL.³⁹ Cell and supernatant samples were collected on days 0, 3 and 8 for cytotoxicity assays and cytokine assays. PBMC stimulated under these optimized conditions⁴¹ are primarily CD+4 T-lymphocytes with the remainder being CD+8 T-lymphocytes.³ PBMC from both subjects were stimulated separately and used individually in the mouse studies.

MUC1 loading of PBMC (MUC1 loaded-PBMC). PBMC were loaded with MUC1 peptide (1 μg/mL final concentration) and maintained in a flask at 37°C humidified and 5% CO₂ atmosphere for two hours.

Cytotoxicity assays. In order to evaluate the ability of CTL to lyse specific, as well as nonspecific target cell lines in vitro, two cytotoxicity assays were used during this study: XTT assay and alamarBlue^® assay. From our previously unpublished work, it was shown that the XTT assay worked better with solid tumor adherent cell lines and the alamarBlue^® assay worked well with the hematopoietic suspension cell lines.

MCF-7 (ATCC HTB-22) breast cancer, K562 (ATCC CCL-243) erythroleukemia and RAJI (ATCC CCL-86) B-cell lymphoma cell lines were obtained from, and cultured as recommended, by the American Type Culture Collection (Manassas, VA, USA). MCF-7 expresses hypoglycosylated mucin,⁸ and was used as the target cell line in a XTT assay (Roche Diagnostics Corp., catalog number 1 1465015001). MCF-7 was cultured in Dulbecco's Modified Eagle's Medium (Gibco-BRL, Life Technologies, Inc. catalog number 12800-017) supplemented with 10% heat-inactivate fetal bovine serum (Hyclone, catalog number SH30070.02HI), 1% bovine insulin (Gibco, catalog number 128-100), and 1% L-glutamine (Gibco, catalog number 21051-024). K562, a natural killer/lymphokine-activated killer sensitive target⁴³ cell line, and RAJI, a natural killer-relatively resistant/lymphokine-activated killer- sensitive target⁴⁴ cell line were used as target cell lines in an alamarBlue^® assay (Biosource International Inc., catalog number NC9423932). K562 and RAJI were cultured in RPMI-1640 (Gibco-BRL, Life Technologies, Inc. catalog number 11875093) supplemented with 10% fetal bovine serum and 1% L-glutamine. All the cells were maintained in a 37°C humidified and 5% CO₂ atmosphere.

The cell lines were seeded into separate 96-well tissue culture plates. 5>< 10³ target cells (MCF-7, K562, or RAJI) were added to each well except the minimum wells, to which no cells were added. Washed effector CTL were added to each well in three effector cell to target cell (E:T) ratios: 10: 1, 5: 1, and 2.5: 1. The effector cells alone were seeded at the corresponding numbers per well as background. Six maximum wells were set up with the target cells only. The CTL from each subject was analyzed separately, in triplicate wells for each ratio. XTT assay. The XTT assay was used to evaluate the ability of CTL to kill the specific target cells, MCF-7, per the manufacturer's instructions. The XTT assay is a non-radioactive, colorimetric assay using XTT labeling reagent, sodium 3'-[l-(phenylamino-carbonyl)-3,4- tetrazolium] -bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate,⁴⁵ which is cleaved into formazan dye only by metabolically active cells. It is used for the determination of cellular proliferation, viability and activation in response to growth factors, cytokines, nutrients, and cytotoxicity.

The plate was set up as previously described above the day before the XTT assay was run. Fifty μL of XTT labeling mixture (5mL XTT labeling reagent with 100 μϊ_^ electron coupling reagent) were added to all wells of the plate. The plate was incubated in a 37°C humidified and 5% CO₂ atmosphere for four hours. The optical density (OD) was then measured by using a spectrophotometer (Dynatech MR 5000, Dynatech Laboratories Inc.). The plate was read at a wavelength of 450 nm with a reference wavelength of 630 nm, and background absorbance was subtracted. The maximum XTT was determined as the mean of the six wells containing only target cells and the minimum was determined as the mean of the six wells containing only medium. The nonspecific formation of formazan attributable to the presence of effector cell was determined from the wells containing effector cells alone. The percent specific lysis (%SL) was calculated as follows:⁴⁶

„, ~_T OD (target - medium) - OD (experimental wells - wells with corresponding number of effectors)

%SL 100

OD (target - medium) Alamar Blue^® assay. Alamar Blue^® assay⁴⁷ was used to evaluate the ability of CTL to kill the non-specific targets cells K562 and RAJI per the manufacturer's instructions. The internal environment of a proliferating cell is more reduced than that of non-proliferating cell ⁴⁸ AlamarBlue^®, which can be reduced by the metabolic intermediates, is useful in monitoring cell proliferation because their reduction is accompanied by a measurable shift in color. As the alamarBlue^® reacts with the cells, it accepts electrons and changes in color from the oxidized indigo blue, non-fluorescing state to the reduced fluorescing pink state. The alamarBlue^® reduction was evaluated by measuring absorbance spectrophotometrically and the calculation of the percentage of alamarBlue^® reduction is as follows according to the manufacturer's protocol:

Percentage difference in alamarBlue^® reduction:

(ε_οχ ) λ₂Αλ₁ - (ε_οχ ) λ₁Αλ₂ of test agent dilution

⁼ 7 \ 7 \ ^ 100 ε_οχ ) λ₂Α λ₁ - (ε_0Χ ) λ₁Α λ₂ of untreated positive growth control In the formula, ε_οχλι and ε_οχλ2 represent the molar extinction coefficient of alamarBlue at 570 and 630 nm, respectively, in the oxidized forms. Αλι and Αλ₂ represent absorbance of test wells at 570 and 630 nm, respectively. A and Α°λ₂ represent absorbance of untreated positive growth control wells at 570 and 630 nm, respectively. The values of percentage difference in alamarBlue^® reduction were corrected for background values of untreated positive growth controls.

The plates were set up as previously described above the same day as the alamarBlue^® assay was run. Twenty μί_^ of alamarBlue^® was added to all wells. After overnight incubation in a 37°C humidified and 5% C0₂ atmosphere, the OD was read with a spectrophotometer at wavelengths of 570 and 630 nm.

ELISA Cytokine Assay. The amount of cytokine, tumor necrosis factor-alpha (BD Pharmingen Inc., catalog number 550610), interferon-gamma (BD Pharmingen Inc., catalog number 550612), IL-10 (BD Pharmingen Inc., catalog number 550613), and GM-CSF (Biosource International Inc., catalog number 555126), present in the supernatant samples was determined by the use of enzyme linked-immunosorbent assay kits per manufacturer's instructions. The supernatant from each subject's CTL was analyzed separately, in triplicate.

In vivo protection studies. Female NOD-SCID mice (Jackson Laboratory, Bar Harbor, ME, USA), 6-12 weeks of age were injected subcutaneous ly, in the back of the neck, with O. lmL of phosphate-buffered saline: matrigel (Gibco BRL, Life Technologies, Inc. catalog number A1413202) at 1 : 1 ratio containing 5>< 10⁶ MCF-7. Washed 5x l0⁷ mononuclear cells (peripheral blood) (MC) or CTL were injected IP with or without washed 5x l0⁵ unloaded DC, MUC1 loaded-DC (-) IL-4 or MUC1 loaded-PBMC according to schedules in the figure legend for Figure 4. Control animals received phosphate-buffered saline or the above-mentioned cells individually. Each mouse was checked for tumor development three times per week for one month. Animal care was in accordance with institutional guidelines. At all times, the inventors followed the Guidelines for Ethical Conduct in the Care and Use of Animals (www.apa.org/science/anguide.html) by the APA Board of Scientific Affairs Committee on Animal Research and Ethics.

Statistical analysis. Determinations of the statistical significance of the in vitro cytotoxicity assays and cytokine assays were performed by the Mann- Whitney Rank Sum test. The Fisher's Exact Test was used to analyze the data obtained from the in vivo mouse study by testing the differences in incidence between DC groups and controls. A p value of less than 0.05 (p < 0.05) was considered statistically significant.

Generation of DC from PBMC with or without IL-4. In order to determine the optimum method for generating DC from PBMC to stimulate T lymphocytes, PBMC, with or without IL-4, were compared, in vitro. PBMC stimulated with MUCl loaded-DC (-) IL-4 showed a specific lysis of 102%, at 10: 1 E:T ratio, against MCF-7. However, when PBMC were stimulated with MUCl loaded-DC (+) IL-4, they showed a specific lysis of only 60%, at 10: 1 E:T ratio, against MCF-7 (Figure 1A). With the same day and same E:T ratio, the non-specific lysis of PBMC stimulated with MUCl loaded-DC (-) IL-4 was 72% against K562 and 56% against RAJI. PBMC stimulated with MUCl loaded-DC (+) IL-4 showed non-specific lysis of 37% against K562 (Figure IB) and 30% against RAJI (Figure 1C). These data imply that PBMC stimulated with MUCl loaded-DC (-) IL-4 had a higher specific lysis rate than those PBMC stimulated with MUCl loaded-DC (+) IL-4 against MCF-7, K562, and RAJI. Therefore, MUCl loaded-DC (-) IL-4 were used to generate CTL for the in vivo study.

Generation of CTL for the in vivo study. CTL generated from PBMC stimulated with MUCl loaded-DC (-) IL-4 used for the mouse studies exhibited higher specific lysis of MCF-7 on day 8 (47%) vs. day 0 (28%, p < 0.05), while there was minimum killing of both K562 (8.5%) and RAJI (4.7%) on day 8 (Figure 2). This implies that the killing was specific and not due to nonspecific natural killer or lymphokine-activated killer cells.

There was increased type 1 cytokines production from MUCl loaded-DC (-) IL-4-stimulated PBMC used for the in vivo protection studies. Tumor necrosis factor-alpha went from 0 pg/mL on day 0 to 19.7 on day 3, and 146.9 on day 8; Interferon-gamma went from 8.4 pg/mL on day 0 to 117 on day 3, and 406 on day 8; GM-CSF went from 0 pg/mL on day 0 to 107.8 on day 3, and 159.4 on day 8. There was no production from day 0 through day 8 of IL-10, which may be a type 1 or type 2 cytokine and is produced by inducible regulatory T lymphocytes (Figure 3).²⁹

Animal studies. Animal studies were performed in order to determine if DC, administered in vivo, enhanced tumor cell killing (Figure 4). Loaded PBMC or DC were used since CTL, alone in vivo, may not survive, or may become non responsive in five days without stimulation.¹⁸ MCF-7 was injected into all groups on day 0. Group 1 received MCF-7 alone. Unloaded DC or MUCl loaded-DC(-)IL-4 were injected IP on days 0, 2, 4, 9, 14 and 19 to ascertain if in vivo stimulation of PBMC with unloaded DC (group 2) or MUCl loaded-DC(-)IL-4 (group 3) enhanced CTL protection against tumor growth. Group 4 received only CTL on day 0, as a control for the remaining groups with in vivo stimulation. Groups 5 - 8 received CTL on day 0 plus APC on two different schedules. MUCl loaded-PBMC were injected IP on days 0 and 14 (group 5) or days 0, 2, 4, 9, 14, and 19 (group 6) as a control for DC. MUCl loaded-DC (-) IL-4 were injected IP on days 0 and 14 (group 7) or days 0, 2, 4, 9, 14, and 19 (group 8) to determine the optimum schedule of DC administration.

Tumors were allowed to develop for thirty-one days after the MCF-7 injection on day 0 (Figure 4). Group 1, which received MCF-7 alone, exhibited 9% protection (2 mice without tumors out of 23). A statistically significant difference was shown in the number of tumors produced in the control group, which received MCF-7 alone vs. all other treatment groups (p < 0.05). An exception was group 2, which received PBMC on day 1 and unloaded DC on days 0, 2, 4, 9, 14 and 19, exhibiting 33% protection (2 mice without tumors out of 6) (Figure 4). This implies that all treatments except group 2 were effective. Group 3, which received PBMC on day 0 and MUCl loaded-DC (-) IL-4 on days 0, 2, 4, 9, 14 and 19, exhibited 53% protection (8 mice without tumors out of 15). This shows that PBMC may be stimulated in vivo by MUCl loaded- DC (-) IL-4 to kill tumor cells. Group 4, which received CTL only on day 0, exhibited 50% protection (5 mice without tumors out of 10). This shows that CTL generated in vitro exhibit similar tumor cell killing as the in vivo stimulated PBMC, group 4. Group 5, which received CTL on day 0 and MUCl loaded-PBMC on days 0 and 14, exhibited 50% protection (5 mice without tumors out of 10). This shows that MUCl loaded-PBMC on days 0 and 14 did not enhance protection CTL alone. Group 6, which received CTL on day 0 and MUCl loaded- PBMC on days 0, 2, 4, 9, 14 and 19, exhibited 56% protection (5 mice without tumors out of 9). This shows that MUCl loaded-PBMC added on additional days did not enhance protection CTL alone. Group 7, which received CTL on day 0 and MUCl loaded-DC (-) IL-4 on days 0 and 14, exhibited 73% protection development (8 mice without tumors out of 11). This shows that MUCl loaded-DC (-) IL-4 on days 0 and 14 further enhanced tumor cell killing. Group 8, which received CTL on day 0 and MUCl loaded-DC (-) IL-4 on days 0, 2, 4, 9, 14 and 19, exhibited 100% protection (1 1 mice without tumors out of 1 1). This shows that additional days of MUCl loaded-DC (-) IL-4 enhanced tumor cells killing. A statistically significant difference was shown in the number of tumors produced in group 8 vs. all other treatment groups (p < 0.05). The exception was that group 8 was not significantly different than group 7, which received the same cells but for fewer days (Figure 4). The results are representative of two studies with cells from two subjects.

These results demonstrate that DC generated without IL-4 ³⁰' ³¹ were superior in activating CTL to kill tumor cells. Development of a type 2 response by the DC generated with IL-4 may explain their reduced ability to stimulate T lymphocytes.^23"28 These results also demonstrate that non-stimulated PBMC and unloaded DC are ineffective in protecting against tumor growth without T lymphocytes. In addition, these results show that loaded DC can stimulate PBMC in vivo to generate CTL that can kill tumor cells. In fact, in vivo activated T lymphocytes were as effective as those generated in vitro. MUC1 loaded-PBMC were ineffective in enhancing CTL to prevent tumor formation. On the other hand, MUC1 loaded-DC (-) IL-4 on days 0, 14 (group 7) or days 0, 2, 4, 9, 14 and 19 (group 8) enhanced protection compared to the other groups. There was no significant difference between groups receiving CTL plus MUC1 loaded-DC(-)IL-4 on days 0, 14 (group 7) or days 0, 2, 4, 9, 14 and 19 (group 8) statistically, but the group that received six days of stimulation showed 100% tumor protection. This shows that multiple and/or extended stimulation of CTL with MUC1 loaded-DC (-) IL-4 is optimum.

The fact that MCF-7 cell-injected mice were protected from tumor growth when MUC1 loaded- DC(-)IL-4 were injected along with CTL, implies that MUC1 loaded-DC(-)IL-4 enhanced the in vivo killing of tumor cells and are required, in addition to CTL, to obtain 100% protection from tumor growth. A similar in vivo study with mouse cells showed DC enhanced CTL killing of tumor.³² The present study is novel in that human cells were used.

These results show that DC loaded with a TAA, MUC1, in vitro (MUC1 loaded-DC(-)IL-4), and administered to animals along with CTL initiated a protective anti-tumor immune response that was superior to the individual cell components in providing protection against tumor development. This implies that MUC1 loaded-DC (-) IL-4 enhanced adoptive immunotherapy with CTL. The mechanism of enhancement of killing by CTL stimulated with DC may be to increase survival of T-cells. Survival of T-cells are important for eliminating cancer cells ³³ and it has been shown that DC enhance survival of T-cells.¹⁹ Alternately, cytokines, such as IL-21,³⁴ IL-7 ³⁵ or IL-15,³⁶ also enhance survival of T-cells and thus might replace DC. Clinical relevance of this modality was shown by a complete remission in a subject with pancreatic cancer induced by DC and CTL.³⁷ This work needs to be confirmed in other clinical trials. Tumor antigen-loaded DC, IL-21, IL-7 or IL-15 or other modalities to enhance survival of CTL, such as adding costimulatory molecules to T-cell receptors or chimeric T-cell receptors,³⁸ should be considered for adoptive immunotherapy with CTL.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The term "or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. In certain embodiments, the present invention may also include methods and compositions in which the transition phrase "consisting essentially of or "consisting of may also be used.

As used herein, words of approximation such as, without limitation, "about", "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

CLAIMS:

1. A method of improving a cytotoxic T cell immune response to a cancer antigen comprising:

obtaining peripheral blood mononuclear cells from a subject;

incubating adherent peripheral blood mononuclear cells with the cancer antigen and GM-

CSF in the absence of IL-4 to mature the adherent peripheral blood mononuclear cells into matured dendritic cells; and

isolating the matured dendritic cells, wherein the matured dendritic cells can stimulate cytotoxic T cells specific for the cancer antigen in vivo.

2. The method of claim 1, wherein the GM-CSF is added to the dendritic cells on at least one of day 0, 3, and 7.

3. The method of claim 1, wherein the dendritic cells are further incubated with at least one of tumor necrosis factor alpha or poly I:C prior to cytotoxic T cell stimulation.

4. The method of claim 1, wherein the cancer antigen is a MUC-1 peptide.

5. The method of claim 1, wherein the cancer antigen has SEQ ID No.: 1.

6. The method of claim 1, wherein the cancer antigen is selected from at least one of MelanA (MART -I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-family members, BAGE-family members, GAGE-family members, RAGE-family members, pl5(58), CEA, NY- ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl 85erbB2, pl 80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, pl6, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1,

SDCCAG16, TA-90, TAAL6, TAG72, TLP, and TPS.

7. The method of claim 1, wherein a media for incubating the dendritic cells is serum free.

8. The method of claim 1, wherein the dendritic cells matured in the absence of IL-4 stimulate a stronger cytotoxic T cell immune response than dendritic cells stimulated in the presence of IL-4.

9. The method of claim 1, further comprising the step of contacting the matured dendritic cells with peripheral blood mononuclear cells that comprise immature T cells in the presence of the cancer antigen under conditions in which the immature T cells are converted into cancer antigen-specific active cytotoxic T cells.

10. A method of improving a cytotoxic T cell immune response to a cancer antigen comprising:

obtaining dendritic cells from a subject;

incubating the dendritic cells with the cancer antigen and GM-CSF in the absence of IL-4 to mature the dendritic cells;

isolating the matured dendritic cells, wherein the matured dendritic cells stimulate cytotoxic T cells specific for the cancer antigen in vivo; and

contacting the matured dendritic cells with peripheral blood mononuclear cells that comprise immature T cells in the presence of the cancer antigen under conditions in which the immature T cells are converted into cancer antigen-specific active cytotoxic T cells.

1 1. The method of claim 10, wherein the GM-CSF is added to the dendritic cells on at least one of day 0, 3, and 7.

12. The method of claim 10, wherein the dendritic cells are further incubated with at least one of tumor necrosis factor alpha or poly I:C prior to cytotoxic T cell stimulation.

13. The method of claim 10, wherein the cancer antigen is a MUC-1 peptide.

14. The method of claim 10, wherein the cancer antigen has SEQ IS NO.: l .

15. The method of claim 10, wherein the cancer antigen is selected from at least one of MelanA (MART -I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-family members, BAGE-family members, GAGE-family members, RAGE-family members, pl5(58), CEA, NY- ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl 85erbB2, pl 80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, pl6, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1,

SDCCAG16, TA-90, TAAL6, TAG72, TLP, and TPS.

16. The method of claim 10, wherein a media for incubating the dendritic cells is serum free.

17. The method of claim 10, wherein the dendritic cells matured in the absence of IL-4 stimulate a stronger cytotoxic T cell immune response than dendritic cells stimulated in the presence of IL-4.

18. A composition for activating a cytotoxic T cell response comprising:

isolated adherent peripheral blood mononuclear cells from a subject incubated with a cancer antigen and GM-CSF matured in the absence of IL-4, wherein the matured adherent peripheral blood mononuclear cells stimulate cytotoxic T cells specific for the cancer antigen in vivo.

19. The composition of claim 18, wherein the GM-CSF is added to the dendritic cells on at least one of day 0, 3, and 7.

20. The composition of claim 18, wherein the dendritic cells are further incubated with at least one of tumor necrosis factor alpha or poly I:C prior to cytotoxic T cell stimulation.

21. The composition of claim 18, wherein the cancer antigen is a MUC-1 peptide.

22. The composition of claim 18, wherein the cancer antigen HAS SEQ ID NO. : 1.

23. The composition of claim 18, wherein the cancer antigen is selected from at least one of MelanA (MART -I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-family members, BAGE-family members, GAGE-family members, RAGE-family members, pl5(58), CEA, NY- ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, pl 85erbB2, pl 80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, pl6, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1,

SDCCAG16, TA-90, TAAL6, TAG72, TLP, and TPS.

24. The composition of claim 18, wherein a media for incubating the dendritic cells is serum free.

25. The composition of claiml 8, wherein the dendritic cells matured in the absence of IL-4 stimulate a stronger cytotoxic T cell immune response than dendritic cells stimulated in the presence of IL-4.

26. An isolated dendritic cell made by a method comprising: obtaining dendritic cells from a subject; incubating the dendritic cells with the cancer antigen and GM-CSF in the absence of IL-4 to mature the dendritic cells; and

isolating the matured dendritic cells, wherein the matured dendritic cells stimulate cytotoxic T cells specific for the cancer antigen in vivo.