Methods for generating modified human primary blood dendritic cell lines

Abstract

Modified primary blood and monocyte-derived dendritic cells that express functional Tax protein from T-cell leukemia virus, as well as methods for making and using the same, are provided. The present invention provides a method for generating modified primary blood dendritic cells, comprising: The method includes: i) providing a sample of cells comprising immature dendritic cells, e.g., PBMCs; ii) treating the cells in culture with an effective amount of phytohemagglutinin (PHA); iii) treating the cells in culture with an effective amount of IL-2; iv) expressing a functional Tax protein from a T-cell leukemia virus in the cells and culturing the cells; and v) depleting T cells from the cultured cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 243,177, filed October 19, 2015, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials The concurrently submitted computer readable sequence listing is incorporated by reference in its entirety herein and is identified as follows: one 60,990 Byte ASCII (text) file entitled "sequence_listing_ST25.txt", created on October 19, 2016.

[0003] The field of the invention relates generally to the fields of biology and medicine, and also to methods and related compositions for the production of dendritic cells, which are useful in the treatment of diseases such as cancer. [Background technology]

[0004] Among professional antigen-presenting cells (APCs), dendritic cells (DCs) retain the unique ability to prime naive T lymphocytes in mediating antigen-specific adaptive immune responses. Palucka, K. & Banchereau, J. Cancer immunotherapy via dendritic cells. Nature Reviews Cancer 12, 265-277 (2012). DCs also possess the ability to induce activation and proliferation of γσ T cells and natural killer (NK) cells, bridging innate immunity to adaptive immune responses. Tyler, CJ, Doherty, DG, Moser, B. & Eberl, M. human Vγ9 / Vδ2 T cells: Innate adaptors of the immune system. Cellular immunology 296, 10-21 (2015); van Beek, JJ, Wimmers, F., Hato, SV, de Vries, IJM & Skold, AEDendritic cell cross talk with innate and innate-like effector cells in antitumor immunity: implications for DC vaccination. Critical Reviews™ in Immunology 34: 517-536 (2014). DCs evolve from the bone marrow and migrate via the blood circulation to lymphoid and non-lymphoid tissues. In the blood, DCs are largely immature and may take up foreign antigens from invading pathogens. Upon uptake of foreign antigens, immature DCs undergo a complex maturation and activation process through the activation of many pathogen recognition receptors (PRRs), such as Toll-like receptors (TLRs), and cytokine receptors.Pradere. 52, 20-33 (2012); Hammer, GE & Ma, A. Molecular control of steady-state dendritic cell maturation and immune homeostasis. Annual Review of Immunology 31, 743-791 (2013). Mature and activated DCs characteristically express cell surface costimulatory molecules such as CD80, CD83 and CD86, assemble antigen peptide-MHC complexes and migrate to secondary lymphoid tissues to promote antigen-specific T cell expansion.

[0005] DCs constitute 1% of immune cells in blood. Two main types of blood DCs have been characterized, including CD11c+ myeloid DCs (mDCs) and CD303+ plasmacytoid DCs (pDCs). Mildner, A. & Jung, S. Development and function of dendritic cell subsets. Immunity 40, 642-656 (2014). pDCs are important in mediating anti-inflammatory responses by secreting large amounts of interferon alpha (IFNα) upon pathogen encounter, whereas mDCs play a key role in antigen presentation to prime naive T cells. Osugi,Y.,Vuckovic,S.& Hart,DNMyeloid blood CD11c+ dendritic cells and monocyte-derived dendritic cells differ in their ability to stimulate T lymphocytes.Blood 100,2858-2866(2002);Kassianos,AJ,Jongbloed,SL,Hart,DN& Radford,KJIsolation of human blood DC subtypes.Dendritic Cell Protocols,45-54(2010).The CD11c+ subset of mDCs is relatively abundant, and the minor subset or CD141+mDCs show the potential to cross-present extracellular antigens on MHC-I to CD8 T cells and divide into cytotoxic T lymphocytes (CTLs).Jongbloed,SLet al.Human CD141+ (BDCA-3)+ dendritic cells(DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens.The Journal of Experimental Medicine 207,1247-1260(2010);Bachem,A.et al.Superior antigen cross-presentation and XCR1 expression define human CD11c+ CD141+ cells as homologues of mouse CD8+ dendritic cells.The Journal of Experimental Medicine 207,1273-1281(2010).By analyzing transcriptional profiling by comparative evaluation of human and mouse DC subsets, it is proposed that human CD141+DCs are closely related to their mouse CD8+DC counterparts as the major subset of DCs in mediating antigen cross-presentation. Because DCs are rarely infected by invading pathogens, DC-mediated cross-presentation and induction of terminally differentiated cytotoxic effector T cells is therefore an essential step in developing adaptive immunity against pathogenic viruses and cancer cells.

[0006] Due to the small number of blood DCs, current DC methods frequently utilize antigen-loaded monocyte-derived DCs (MoDCs) through a complex maturation and activation process with a mixture of cytokines and PRR stimulants. Kvistborg, P., Bogh, M., Pedersen, A., Claesson, M. & Zocca, M. Fast generation of dendritic cells. Cellular immunology 260, 56-62 (2009); Jolanda, I., de Vries, M., Adema, GJ, Punt, CJ & Figdor, CGPhenotypical and functional characterization of clinical-grade dendritic cells. Adoptive Immunotherapy: Methods and Protocols, 113-125 (2005). This method has been widely used in clinical cancer vaccine trials, but the clinical benefit of such an approach is rather limited. This approach apparently has several drawbacks. Compared with primary blood DCs, MoDCs are more closely related to in vitro-derived macrophages. Robbins, SH et al. Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling. Genome Biology 9, R17 (2008). In addition, in vitro developed MoDCs have limited growth potential and can hardly be maintained in culture for long periods of time, so repeated MoDC preparations are necessary for several series of vaccine deliveries. To address these limitations, several human DC models have been developed to understand DC biology and evaluate their potential use as cancer vaccines.van Helden, SF, van Leeuwen, FN & Figdor, CG Human and murine model cell lines for dendritic cell biology evaluated. Immunology Letters 117, 191-197 (2008). For example, the MUTZ-3 myeloid leukemia cell line can be matured and differentiated in vitro into DC-like cells. Masterson, AJ et al. MUTZ-3, a human cell line model for the cytokine-induced differentiation of dendritic cells from CD34+ precursors. Blood 100, 701-703 (2002). MUTZ-3 cells can be stably maintained at the pre-DC stage in culture to provide adequate amounts of inducible DC-like cells for the development of cancer vaccines. Santegoets,SJ,van den Eertwegh,AJ,van de Loosdrecht,AA,Scheper,RJ& de Gruijl,TDHuman dendritic cell line models for DC differentiation and clinical DC vaccination studies.Journal of Leukocyte Biology 84,1364-1373(2008);Santegoets,SJet al.In vitro priming of tumor-specific cytotoxic T lymphocytes using allogeneic Dendritic cells derived from the human MUTZ-3 cell line.Cancer Immunology, Immunotherapy 55, 1480-1490(2006).

[0007] Human T-cell leukaemia virus type 1 (HTLV-1) primarily infects CD4+ T cells and causes malignant transformation of T cells and the development of adult T-cell leukaemia-lymphoma (ATL) in 3-5% of infected individuals. Ishitsuka,K.& Tamura,K.Human T-cell leukaemia virus type I and adult T-cell leukaemia-lymphoma.The Lancet Oncology 15,e517-e526(2014). Other HTLV viruses are known. For example, HTLV-2 is related to its pathogenic counterpart, HTLV-1, but HTLV-2 has not been associated with the development of leukemia in humans. Ciminale,V.,Rende,F.,Bertazzoni,U.& Romanelli,MGHTLV-1 and HTLV-2:highly similar viruses with distinct oncogenic properties.Front.Microbiol.5(398):1-9(2014). The viral Tax protein of HTLV-1 is thought to play a central role in the process leading to adult T-cell leukemia. Azran et al., Role of Tax protein in human T-cell leukemia virus type-I leukemogenicity, Retrovirology 20041:20(2004). HTLV-1 also infects dendritic cells and makes the cell-free virus infectious to CD4+ T cells.Jones, KS, Petrow-Sadowski, C., Huang, YK, Bertolette, DC & Ruscetti, FW Cell-free HTLV-1 infects dendritic cells leading to transmission and transformation of CD4+ T cells. Nature medicine 14, 429-436 (2008); Jain, P. et al. DC-SIGN mediates cell-free infection and transmission of human T-cell lymphotropic virus type 1 by dendritic cells. Journal of Virology 83, 10908-10921 (2009). A very small percentage of HTLV-1-infected patients develop a neuroinflammatory disorder called HTLV-1-associated myelopathy and tropical spastic paraplegia (HAM / TSP). Yamano, Y. & Sato, T. Clinical pathophysiology of human T-lymphotropic virus-type 1-associated myelopathy / tropical spastic paraparesis. Frontiers in Microbiology 3, 389 (2012). The development of Tax-reactive CD8+ CTLs plays a role in the pathogenesis of HAM / TSP, whereas Tax-specific CTLs are recognized to exert protective immunity against ATL. Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need to develop novel methods for generating dendritic cells, particularly from blood, for use as dendritic cell vaccines, and for activating cytotoxic T lymphocytes that can be used therapeutically in subjects. The present invention satisfies this need and also provides additional advantages.

[0009] This background information is provided for informational purposes only. No admission is necessarily intended, nor should any of the preceding information be construed as construing, that it constitutes prior art against the present invention. [Means for solving the problem]

[0010] It is to be understood that both the foregoing general description of the embodiments and the following detailed description are exemplary and explanatory and are not intended to limit the scope of the embodiments.

[0011] Provided herein are methods for culturing primary dendritic cells with the viral protein Tax from a non-leukemia-causing retrovirus, human T-cell leukemia virus type II (HTLV-2), a virus related to its pathogenic counterpart HTLV-1.

[0012] It is shown herein that Tax can harness dendritic cells, resulting in their maturation / activation and subsequent induction of divided cytotoxic T lymphocyte responses. In this disclosure, HTLV-2 Tax is utilized as a molecular tool to establish primary blood dendritic cell lines and monocyte-derived dendritic cells. Provided are human primary blood dendritic cells and monocyte-derived dendritic cells that allow for the generation of highly potent anti-cancer cytotoxic lymphocytes, along with methods for their production and use.

[0013] In one aspect, the invention provides modified primary blood dendritic cells that express a functional Tax protein from a human T-cell leukemia virus. In some embodiments, the Tax protein is from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, HTLV-3, and HTLV-4.

[0014] In some embodiments, the modified primary blood dendritic cells have a constitutive maturation and activation phenotype. The modified primary blood dendritic cells are CD205+ and CD11c+. In some embodiments, the modified primary blood dendritic cells express one or more dendritic cell maturation and activation markers selected from the group consisting of CD83, CD80, CD86, CD70, CCR7, and HLA-DR. In some embodiments, the modified primary blood dendritic cells are TLR3+ and TLR4+. In some embodiments, the modified primary blood dendritic cells express a truncated form of TLR7. In some embodiments, the dendritic cells express TLR9. In some embodiments, the modified primary blood dendritic cells produce IL-1A and TNFα. In some embodiments, the modified primary blood dendritic cells produce IL-15. In some embodiments, the modified primary blood dendritic cells produce negligible levels of IL-10 and TGFβ.

[0015] In some embodiments, the modified primary blood dendritic cells express one or more of c-Myc, Mcl-1, Bcl-xL, Bcl-2, phosphorylated pRb, phosphorylated cdc, phosphorylated Stat1, phosphorylated Stat3, and phosphorylated Stat5. In some embodiments, the transcription factors NF-κB, Stat3, and AP-1 are active in the modified primary blood dendritic cells. In some embodiments, the modified primary blood dendritic cells are capable of inducing proliferation of naive lymphocytes in the absence of exogenously added IL-2.

[0016] In some embodiments, the modified primary blood dendritic cells are genetically engineered to express one or more HLA proteins. In some embodiments, the HLA protein is HLA-A2.1.

[0017] In some embodiments, the modified primary blood dendritic cells harbor one or more exogenously added antigens or antigen fragments or variants thereof. In some embodiments, the antigen is a cancer antigen. In some embodiments, the cells express human telomerase reverse transcriptase or an antigen fragment or variant thereof. In some embodiments, the antigen comprises a fusion protein comprising a proteasome targeting sequence of IκBα and a fragment of human telomerase reverse transcriptase. In some embodiments, the fusion protein comprises SEQ ID NO: 13. In some embodiments, the modified primary blood dendritic cells present a cancer antigen in an HLA-restricted manner. In some embodiments, the cancer antigen is introduced by viral transformation. In some embodiments, the virus is a lentivirus. In some embodiments, the modified primary blood dendritic cells are capable of priming naive PBMCs to generate cytotoxic lymphocytes that recognize the antigen. In some embodiments, the modified primary blood dendritic cells are capable of priming naive PBMCs to generate cytotoxic lymphocytes that are capable of killing cells in a non-HLA restricted manner.

[0018] In another aspect, the present invention provides a method of generating modified primary blood dendritic cells, comprising: i) providing a sample of cells comprising immature dendritic cells; ii) expressing a functional Tax protein from a T-cell leukemia virus in said cells; The method includes the steps of:

[0019] In some embodiments, the method further comprises iii) culturing the cells to induce their maturation and activation. The culturing step for inducing maturation and activation of the modified primary blood dendritic cells is not limiting. In some embodiments, the cells are cultured in the presence of one or more cytokines. In some embodiments, the cells are cultured in a medium comprising an effective amount of IL-2. In some embodiments, the method further comprises depleting T cells from the cultured cells.

[0020] In another aspect, the present invention provides a method of generating modified primary blood dendritic cells, comprising: i) providing a sample of cells comprising immature dendritic cells; ii) expressing a functional Tax protein from a T-cell leukemia virus in said cell and culturing said cell; iii) culturing the cells to induce differentiation into mature dendritic cells; iv) depleting T cells from cultured cells; The method includes the steps of:

[0021] In another aspect, the present invention provides a method of generating modified primary blood dendritic cells, comprising: i) providing a sample of cells comprising immature dendritic cells, e.g. a sample of PBMCs; ii) treating the cells in culture with an effective amount of phytohemagglutinin (PHA); iii) treating the cells in culture with an effective amount of IL-2; iv) expressing a functional Tax protein from a T-cell leukemia virus in said cell and culturing said cell; v) depleting T cells from cultured cells; The method includes the steps of:

[0022] In some embodiments, the PBMCs are isolated from leukopacks. In some embodiments, the concentration of PHA ranges from about 1-5 μg / ml. In some embodiments, the cells are treated with PHA for about 12 hours to about 36 hours. In some embodiments, the cells are treated with about 100-200 units / ml of IL-2 and cultured in medium containing IL-2 for a period of time. In some embodiments, the cells are cultured in medium containing IL-2 for a period of about 4-5 days. In some embodiments, the cells are manipulated to express functional Tax protein after step iii).

[0023] In some embodiments, the cells are transduced with a virus encoding a functional Tax protein. In some embodiments, the virus is a lentivirus. In some embodiments, the cells are transduced in the presence of about 10 μg / ml of polybrene.

[0024] In some embodiments, the transduced cells are further cultured in a medium containing about 100-200 units / ml of IL-2. In some embodiments, the transduced cells are further cultured in a medium containing IL-2 for about 2-3 months. In some embodiments, the transduced cells are cultured in a medium containing human serum and about 100-200 units / ml of IL-2. In some embodiments, the transduced cells are cultured in a medium containing human serum and about 100-200 units / ml of IL-2 for about 2-3 months. In some embodiments, the cells are cultured in complete RPMI 1640 medium containing 5% heat-inactivated human AB serum and about 100-200 units / ml of IL-2. In some embodiments, the cells are cultured in a medium containing complete RPMI 1640 medium containing 10% fetal bovine serum and about 100-200 units / ml of IL-2.

[0025] In some embodiments, T cells are depleted by a composition comprising a molecule that binds to T cells. In some embodiments, T cells are depleted by an antibody. In some embodiments, the antibody is an anti-CD3 antibody. In some embodiments, the antibody is conjugated to a magnetic bead.

[0026] In another aspect, the invention provides modified monocyte-derived dendritic cells, the dendritic cells expressing a functional Tax protein from a T-cell leukemia virus. In some embodiments, the Tax protein is from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, HTLV-3, and HTLV-4. The modified monocyte-derived dendritic cells are CD205+.

[0027] In some embodiments, the modified monocyte-derived dendritic cells have a constitutive maturation and activation phenotype. In some embodiments, the modified monocyte-derived dendritic cells express one or more dendritic cell maturation and activation markers selected from the group consisting of CD83, CD80, CD86, and CD70. In some embodiments, the modified monocyte-derived dendritic cells further express 4-1BBL and / or CD4. In some embodiments, the modified monocyte-derived dendritic cells are allowed to expand in culture for up to 3 months. In some embodiments, the cells are TLR3+ and TLR4-. In some embodiments, the modified monocyte-derived dendritic cells express a truncated form of TLR7. In some embodiments, the cells produce IL-1A and TNFα. In some embodiments, the modified monocyte-derived dendritic cells produce TGFβ. In some embodiments, the modified monocyte-derived dendritic cells produce IL-15. In some embodiments, the modified monocyte-derived dendritic cells express one or more of the following: c-Myc, Mcl-1, Bcl-xL, Bcl-2, phosphorylated pRb, phosphorylated cdc2, phosphorylated Stat1, phosphorylated Stat3, and phosphorylated Stat5.

[0028] In some embodiments, the modified monocyte-derived dendritic cells are capable of inducing proliferation of naive lymphocytes in the absence of exogenously added IL-2. In some embodiments, the modified monocyte-derived dendritic cells are genetically engineered to express one or more HLA proteins. In some embodiments, the modified monocyte-derived dendritic cells are genetically engineered to express HLA-A2.1.

[0029] In some embodiments, the modified monocyte-derived dendritic cells express one or more antigens or antigen fragments or variants thereof. In some embodiments, the antigen is a cancer antigen. In some embodiments, the modified monocyte-derived dendritic cells express human telomerase reverse transcriptase or an antigen fragment or variant thereof. In some embodiments, the antigen comprises a fusion protein comprising a proteasome targeting sequence of IκBα and a fragment of human telomerase reverse transcriptase. In some embodiments, the fusion protein comprises SEQ ID NO: 13. In some embodiments, the cells present the cancer antigen in an HLA-restricted manner.

[0030] In some embodiments, the modified monocyte-derived dendritic cells are capable of priming a population of naive lymphocytes, e.g., PBMCs, to generate cytotoxic lymphocytes that recognize antigens, hi some embodiments, the modified monocyte-derived dendritic cells are capable of priming a population of naive lymphocytes, e.g., PBMCs, to generate cytotoxic lymphocytes that are capable of killing cells in a non-HLA restricted manner.

[0031] In another aspect, the present invention provides a method for generating monocyte-derived dendritic cells, comprising: i) providing a sample of cells comprising immature monocyte-derived dendritic cells; ii) expressing a functional Tax protein from a T-cell leukemia virus in said cells; The method includes the steps of:

[0032] In some embodiments, the method further comprises culturing the monocyte cells to induce their differentiation into immature dendritic cells prior to part i). In some embodiments, differentiation is induced by contacting the monocytes with a culture medium comprising an effective amount of a composition to induce differentiation of the monocytes into immature dendritic cells, including, but not limited to, GM-CSF; GM-CSF and IL-4; GM-CSF and IL-13; GM-CSF and IL-15; and IFNα. In some embodiments, the monocytes are cultured in a medium comprising an effective amount of GM-CSF and IL-4 to induce differentiation into immature dendritic cells.

[0033] In some embodiments, the method further comprises a step after part ii) comprising culturing the cells to induce their maturation and activation. The culturing step for inducing maturation and activation of monocyte-derived dendritic cells is not limiting. In some embodiments, the cells are cultured in the presence of one or more cytokines. In some embodiments, the cells are further cultured in a medium comprising effective amounts of GM-CSF and IL-4.

[0034] In another aspect, the present invention provides a method for generating monocyte-derived dendritic cells, comprising: i) providing adherent monocytic cells; ii) culturing the cells in a medium containing effective amounts of GM-CSF and IL-4; iii) expressing a functional Tax protein from a T-cell leukemia virus in said cell; iv) culturing the functional Tax protein expressing cells in a medium containing effective amounts of GM-CSF and IL-4; The method includes the steps of:

[0035] In some embodiments, after step iv), the method further comprises culturing the cells in a medium comprising an effective amount of IL-2. In some embodiments, IL-4 and / or GM-CSF are fused to Fc4. In some embodiments, the adherent monocytes are derived from PBMCs, e.g., about 4 million PBMCs. In some embodiments, the culturing of part ii) comprises culturing the cells in a medium comprising conditioned medium from GM-CSF-Fc4 / IL-4-Fc4 producing 293 cells. In some embodiments, the cells are cultured in a medium comprising conditioned medium at about a 1:10 dilution for about 7 days. In some embodiments, the cells are transduced with a virus encoding a functional Tax protein from a T-cell leukemia virus. In some embodiments, the cells are transduced in the presence of about 10 μg / ml polybrene.

[0036] In some embodiments, the cells are cultured in a medium comprising RPMI 1640 with about 10% fetal bovine serum. In some embodiments, the culturing step iv) comprises culturing the Tax protein expressing cells in RPMI 1640 medium with 10% FBS in the presence of GM-CSF-Fc4 and IL-4-Fc4 for about 5-7 days, and maintaining the cells in culture in the presence of about 100-200 u / ml of IL-2.

[0037] In some embodiments, the method further comprises activating the monocyte-derived dendritic cells. In some embodiments, the activating comprises stimulating the cells with an effective amount of TNFα and LPS. In some embodiments, the cells are stimulated for about 2 days.

[0038] In another aspect, the invention provides a method for producing cytotoxic T lymphocytes comprising culturing primary blood or monocyte derived dendritic cells with cells comprising naive lymphocytes for a period of time, thereby producing cytotoxic T lymphocytes.

[0039] In another aspect, the present invention provides a method for producing cytotoxic T lymphocytes, comprising: i) culturing dendritic cells of the invention with cells comprising naive lymphocytes for a first period of time to produce a mixed culture of cells; ii) treating the mixed culture of cells with IL-2 and continuing to culture the cells for a second period of time; Including, This results in the production of cytotoxic T lymphocytes.

[0040] In some embodiments, the cells comprising naive lymphocytes comprise naive PBMCs. In some embodiments, the naive lymphocytes are isolated from leukopacks. In some embodiments, the naive lymphocytes and dendritic cells are allogeneic. In some embodiments, the ratio of dendritic cells to naive PBMCs is about 1:100. In some embodiments, the culturing of step i) is performed without the addition of exogenous cytokines. In some embodiments, the first period of time is about 2-3 days. In some embodiments, the concentration of IL-2 is about 100-200 u / ml. In some embodiments, the second period of time is 2-6 weeks. In some embodiments, the cytotoxic T lymphocytes are antigen-specific and induce cytolysis of target cells in an HLA-restricted manner.

[0041] In some embodiments, the cytotoxic T lymphocytes comprise CD3+ / CD56+ T cells that may kill target cells in a non-HLA-restricted manner. In some embodiments, the dendritic cells express 4-1BBL. In some embodiments, the dendritic cells have been modified to express 4-1BBL. In some embodiments, the non-HLA-restricted killing of the cells is mediated by ligand binding of NKG2D.

[0042] In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of cytotoxic T lymphocytes produced by the method of the present invention.

[0043] In another aspect, the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of cytotoxic T lymphocytes, wherein the cytotoxic T lymphocytes are produced using primary blood dendritic cells and / or monocyte-derived dendritic cells of the invention.

[0044] In another aspect, the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of cytotoxic T lymphocytes, wherein the cytotoxic T lymphocytes are produced by a method of the invention.

[0045] In another aspect, the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of primary blood dendritic cells and / or monocyte-derived dendritic cells of the invention.

[0046] In another aspect, the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of primary blood dendritic cells and / or monocyte-derived dendritic cells produced by the methods of the invention.

[0047] In some embodiments, the disease being treated is cancer. In some embodiments, the cancer cells express MICA / B. In some embodiments, the cancer is breast cancer. In some embodiments, the subject being treated is a human.

[0048] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0049] Those skilled in the art will appreciate that the figures described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way. [Brief description of the drawings]

[0050] [Figure 1] Immunophenotypic analysis of established ihv-DC cell lines. (a) ihv-DC1. (b) ihv-DC2. (c) Images of GM-CSF / IL-4 stimulated / differentiated DCs from adherent monocytes of healthy blood donors. (d) Immunophenotype of MoDC-Tax cells generated from Tax-GFP lentivirus-transduced MoDCs. [Diagram 2] Expression profiles of TLRs, cytokines and intracellular signaling molecules in ihv-DCs. (a) Expression of TLR3 and TLR4 in ihv-DCs and MoDC-Tax cells examined by FACS. (b) Immunoblot analysis of TLR1, TLR2, TLR7 and TLR9 in established DCs using relevant antibodies. Cytokine expression profiles were determined by qRT-PCR in ihv-DC1 cells (c), ihv-DC2 cells (d), MoDC-Tax cells (e) and TNFα / LPS-activated MoDCs (f). (g) Expression levels of c-Myc in different DCs were determined by qRT-PCR. (h) Immunoblot analysis of the expression of Tax-GFP, Bcl-2, Bcl-xL and Mcl-1 in established DCs. PBLs (peripheral blood lymphocytes from normal donors) and MT4s were used as controls. (i) The phosphorylation status of Stat1, Stat3, Stat5, pRb or cdc2 was examined using specific anti-phospho-specific antibodies. (j) EMSA assays for detection of transcriptional activity of NF-κB, Stat3 and AP-1 using nuclear extracts prepared from ihv-DCs. [Diagram 3]Induction of antigen-specific CTLs by ihv-DCs. (a) Immunophenotype of ihv-DC2-activated CTLs by two-color FACS analysis. (b) Perforin and granzyme B levels in ihv-DC-activated CTLs generated from six blood donors were assessed by immunoblotting using relevant antibodies. (c) Cytokine expression profile of ihv-DC2-activated CTLs by qRT-PCR. (d) MT4L cells (MT4 cells expressing luciferase) were transduced with HLA-A2.1 lentivirus to generate MT4L-A2.1 cells. HLA-A2 expression in MT4L and MT4L-A2.1 cells was verified by FACS using anti-HLA-A2-APC. (e) Cell viability assay to determine cell lysis of modified MT4 cells by ihv-DC2-activated CTLs. (f) NIH3T3 cells were transduced with luciferase and HLA-A2 lentiviruses with or without Tax-FLAG lentivirus to generate 3T3L-A2.1 and 3T3L-A2.1 / Tax cells. HLA-A2 staining in 3T3 cells by FACS is shown in the left panel, while Tax expression was determined by immunofluorescence imaging using anti-FLAG antibody (right panel). Cell viability assays were performed to determine cell lysis of 3T3L-A2.1 and 3T3L-A2.1 / Tax cells by ihv-DC2-activated CTLs using luciferase assay (g) and cell image analysis (h). **: p<0.01 as determined by two-tailed Student's t-test. [Figure 4]HLA-restricted cancer killing by ihv-DC2-activated CTLs. Cell viability assays to determine cell lysis of A375 cells (a), PANC-1 cells (b) and H1299 cells (c) by ihv-DC2-activated CTLs using luciferase assay and cell image analysis (d). (e) FACS analysis of HLA-A2 expression in A375, PANC-1, H1299 and H1299-HLA-A2.1 cells. H1299-HLA-A2.1 cells were generated by lentiviral transduction of HLA-A2.1 into H1299 cells. Cell viability assays were performed to determine cell lysis of H1299 / HLA-A2.1 cells by ihv-DC1-activated CTLs using luciferase assay (f) and cell image analysis (g). **: p<0.01 determined by 2-tailed Student's t-test. [Diagram 5] Expression of hTERT fragments in ihv-DC1 cells to generate hTERT-specific CTLs. (a) HLA-A2 expression in ihv-DC1 cells transduced with HLA-A2.1 lentivirus. (b) Six HLA-A2-restricted CTL epitopes in hTERT (aa301-700) were displayed. (c) pTERT was generated by fusion of IκBα with TERT (aa301-700) and delivered into ihv-DC1 cells via lentiviral transduction. Expression of pTERT was verified by anti-IκBα immunoblot using whole cell extracts from ihv-DCs pretreated with DMSO or MG132 (5 μM) for 1 h. (d) Images of FLAG-TERT (aa301-700) expression in HLA-A2+ NIH3T3 cells using anti-FLAG antibody staining. Cell viability assays were performed to determine cell lysis of 3T3L-A2.1 / FLAG-TERT(aa301-700) cells by ihv-DC1- or ihv-DC1 / pTERT-activated CTLs using luciferase assay (e) and cell image analysis (f). **: p<0.01 determined by two-tailed Student's t-test. [Figure 6]Non-HLA-restricted cancer cell killing by ihv-DC1-activated CD3+ / CD56+ CTLs. (a) Immunophenotype of ihv-DC1-activated CTLs. (b) MICA / B expression in various cancer cell lines examined by FACS. Cell viability assays were performed to determine cell lysis of H1299 cells (c), HeLa cells (d) and A375 cells (e) by ihv-DC1-activated CTLs using luciferase assay. Cell image analysis of various cancer cell lines (f) and normal fibroblasts (g) incubated with ihv-DC1-activated CTLs at the indicated E:T ratios. **: p<0.01 determined by 2-tailed Student's t-test. [Figure 7] ihv-DC1-activated CTLs kill cancer cells through the interaction of NKG2D and its ligands. (a) K562 cells were mixed with ihv-DC1-activated CTLs at the indicated E:T ratios in the presence of isotype IgG or anti-NKG2D antibodies (50 μg / ml) for 4 h. Cytolysis was determined by luciferase assay. (b) K562 cells were mixed with ihv-DC1-activated CTLs at a fixed E:T ratio (2.5:1) in the presence of isotype IgG and anti-NKG2D antibodies at the indicated doses for 4 h. (c) A375 cells were mixed with ihv-DC1-activated CTLs at the indicated E:T ratios in the presence of isotype IgG and anti-NKG2D antibodies (50 μg / ml). Cytolysis was determined by luciferase assay. (d) 3T3L cells were transduced with hMICA lentivirus and the expression of hMICA was examined by FACS. Cell viability assays were performed to determine cytolysis of 3T3L / vector and 3T3L / hMICA cells by ihv-DC1-activated CTLs using luciferase assay (e) and cell image analysis (f). **: p<0.01 as determined by two-tailed Student's t-test. [Figure 8]ihv-DC1-activated CTLs kill breast cancer cells in an NKG2D-dependent manner. Cell viability assays were performed to determine cytolysis of MCF-7, MCF-7 / ADR, and HCC1954 cells by ihv-DC1-activated CTLs by luciferase assay (a) and cell image analysis (b). (c) MICA / B expression in MCF-7, MCF-7 / ADR, and HCC1954 cells examined by FACS. (d) MCF-7, MCF-7 / ADR, and HCC1954 cells were incubated with ihv-DC1-activated CTLs at the indicated E:T ratios in the presence of isotype IgG antibodies (100 μg / ml) or NKG2D blocking antibodies (100 μg / ml). Cytolysis of various cancer cell lines by ihv-DC1-activated CTLs was determined by luciferase assay. [Figure 9] ihv-DC1-activated CTLs inhibit the growth of HCC1954 tumors in NSG mice. Breast cancer xenografts and CTL treatment were described in Methods. (a) Tumor volumes in control and CTL-treated mice. (b) Images of tumors harvested at the end point. (c) Tumor weights at the end point. (d) HE staining of lung tissues in control and CTL-treated groups. (e) Anti-human CD3 staining of tumors and various tissues using IHC method. [Figure 10] ihv-DC1-activated CTLs inhibit the growth of MCF-7 / ADR tumors in NSG mice. (a) Tumor volume. (b) Tumor weight at endpoint. (c) Mouse body weight. (d) HE staining of lung tissues of mice in the control and CTL-treated groups. (e) Anti-human CD3 staining of tumors and various tissues using IHC method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The inventors have surprisingly discovered a method for preparing modified dendritic cells. The modified dendritic cells can be either primary blood dendritic cells or monocyte-derived dendritic cells. The modified dendritic cells express a functional Tax protein from a T-cell leukemia virus. The present invention provides modified dendritic cells, methods of making and using the cells, and pharmaceutical and vaccine compositions comprising the cells.

[0052] In some embodiments, the antigen-loaded dendritic cells of the present invention are useful in the treatment or prevention of disease, or as a vaccine for the activation of T cells, and can be used in therapy. For example, antigen-loaded dendritic cells can be used to generate an immune response against an antigen. These cells can be used as a vaccine to prevent future infections or diseases, or to activate the immune system to treat ongoing diseases, including but not limited to pathogen infections or cancer. Antigen-loaded dendritic cells can be formulated for use as a vaccine or pharmaceutical composition with a suitable carrier, such as a physiological buffer or other injectable liquid. The vaccine or pharmaceutical composition can be administered in a therapeutically effective amount sufficient to generate an immune response.

[0053] Reference will now be made in detail to the presently preferred embodiments of the invention, together with the figures and the following examples, which serve to explain the principles of the invention. These embodiments are described in sufficient detail to enable one skilled in the art to practice the invention, and it is understood that other embodiments may be utilized, and that structural, biological, and chemical changes may be made without departing from the spirit and scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0054] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology that are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2001. ndedition (1989); Current Protocols in Molecular Biology (FMAusubel et al. eds. (1987)); the series Methods in Enzymology (Academic Press, Inc.); PCR: A Practical Approach (M. MacPherson et al. IRL Press at Oxford University Press (1991)); (1995)); Antibodies, A Laboratory Manual (Harlow and Lane eds. (1988)); Using Antibodies, A Laboratory Manual (Harlow and Lane eds. (1999)); and Animal Cell Culture (RIFreshney ed. (1987)).

[0055] Definitions of common terms in molecular biology can be found, for example, in Benjamin Lewin, Genes VII, 2000 (ISBN019879276X), published by Oxford University Press; Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, 1994 (ISBN0632021829), published by Blackwell Publishers; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, 1995 (ISBN0471186341), published by Wiley, John & Sons, Inc.

[0056] For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of a word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its accompanying claims, unless a contrary meaning is clearly intended (e.g., in the document in which the term is originally used). The use of "or" means "and / or" unless otherwise stated. As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. Furthermore, when the term "comprising" is used in the detailed description of one or more embodiments, those skilled in the art will understand that in some specific instances, the embodiment or embodiments may alternatively be described using the words "consisting essentially of" and / or "consisting of."

[0057] As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used.

[0058] An "effective amount" is an amount effective to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications or dosages. For example, the term "effective amount" in relation to activated lymphocytes refers to an amount administered to produce a desired effect, such as inhibiting the growth of or killing cancer cells. In relation to dendritic cells, an "effective amount" refers to an amount capable of inducing an immune response, such as inducing the activation of T lymphocytes. Activated T lymphocytes can function in an HLA or non-HLA restricted manner. The exact amount depends on the specific agent, the subject being treated, and can be ascertained by one of skill in the art using known methods and techniques for determining effective doses.

[0059] As used herein, "treat" and all of its forms and tenses (including, for example, treating, treated, and treatment) can refer to therapeutic or prophylactic treatment. In certain embodiments of the invention, those in need of treatment include those already having a pathological condition of the invention (including, for example, cancer), where treating refers to administering a therapeutically effective amount of a composition to a subject (including, for example, a human or other mammal in need of treatment) such that a sign or symptom of the pathological condition of the invention is ameliorated in the subject. Such improvement may be any observable or measurable improvement. Thus, one of skill in the art will understand that treatment may improve the patient's condition but may not be a complete cure of the pathological condition. In other certain embodiments of the invention, those in need of treatment include those already having cancer as well as those prone to having cancer or those in whom cancer metastasis is to be prevented.

[0060] "Cancer" refers to the abnormal presence of cells that exhibit relatively autonomous proliferation, such that cancer cells exhibit an abnormal growth phenotype characterized by a significant loss of cell proliferation control. Cancerous cells can be benign or malignant. Various types of cancer are known, and the cancer to be treated is not limited. Cancer cells, as used herein, include not only primary cancer cells, but any cells derived from cancer cells. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. Cancers include, but are not limited to, solid tumors and hematological malignancies.

[0061] The term "antigen" is well understood in the art and includes a substance that is immunogenic, i.e., an immunogen, as well as an antigenic epitope. In some embodiments, the modified primary blood dendritic cells and monocyte-derived dendritic cells of the invention can be loaded with one or more antigens for presentation to other cells, e.g., T lymphocytes. Antigen-loaded dendritic cells are useful as vaccines and for in vitro stimulation of T cells.

[0062] It is recognized that the use of any antigen is contemplated for use in the present invention, including, but not limited to, self-antigens (normal or disease-related), infectious antigens (e.g., microbial antigens, viral antigens, etc.), or some other foreign antigens (e.g., food components, pollen, etc.). The term "antigen" or alternatively "immunogen" also applies to a collection of more than one immunogen, such that an immune response to multiple immunogens can be modulated simultaneously. The term also includes any of a variety of different formulations of immunogens or antigens.

[0063] In some embodiments, the antigen is from a cancer cell or a pathogen, hi some embodiments, the cancer cell is a breast cancer cell.

[0064] Antigens can be loaded into immature or mature dendritic cells. When antigens are loaded into immature dendritic cells, the immature dendritic cells can then be matured by the process of self-loading or by other maturation methods described herein or alternative maturation methods known to those skilled in the art. The loading of antigens in dendritic cells is not limiting. In some embodiments, antigens can be loaded using methods described herein, including transfection, transformation, electroporation, and peptide pulsing. In some embodiments, cancer antigens are introduced by viral transformation. In some embodiments, the virus is a lentivirus. In some embodiments, antigens are delivered to dendritic cells described herein in the form of nucleic acids that are isolated or derived from cancer cells or pathogens. In some embodiments, antigens are encoded by a virus that is capable of infecting dendritic cells. "Derived from" includes, but is not limited to, recombinant variants of naturally occurring sequences, including fusion to unrelated or related sequences.

[0065] In some embodiments, the antigen(s) may be loaded as the antigen itself (e.g., proteins, peptides, epitopes, cell lysates, viral particles, etc.) or as nucleic acid(s) encoding the antigen(s). Methods for loading dendritic cells with peptide and protein antigens, cells, cell or tissue lysates, viruses or viral particles, nucleic acids, etc. are known to those of skill in the art.

[0066] The antigen may be from any source. However, in some embodiments, the antigen or antigen(s) is autologous to the subject. Autologous to the subject means that the antigen is obtained or derived from the subject. As a non-limiting example, the antigen may be from a cancer cell or tumor tissue obtained from the subject. The cancer antigen may be loaded into dendritic cells as cancer cells, cancer cell or tissue lysates, extracts from cancer cells or tissues, purified or clonal components of cancer cells or tissues, total RNA or total mRNA, or selected RNA or mRNA from such cells or tissues, whether present in an extract, purified, amplified, or translated in vitro. In one embodiment, the cancer antigen is modified into a viral vector, such as a lentiviral vector, for administration to immature or mature dendritic cells. In some embodiments, the antigen may be obtained or derived from a pathogen or pathogen-infected cells present in the subject.

[0067] In some embodiments, the antigen is a cancer antigen and comprises human telomerase reverse transcriptase or an antigenic fragment or variant thereof. In some embodiments, the cancer antigen comprises a fusion protein comprising a proteasome targeting sequence of IκBα and a sequence from a cancer cell. In some embodiments, the IκBα has the nucleotide and amino acid sequences set forth in SEQ ID NO:11 and the nucleotide sequence set forth in SEQ ID NO:12, respectively. In some embodiments, the sequence from a cancer cell comprises a fragment of human telomerase reverse transcriptase (hTERT). In some embodiments, the hTERT has the amino acid sequence set forth in SEQ ID NO:9 and the nucleotide sequence set forth in SEQ ID NO:10. In some embodiments, the cancer antigen comprises a full-length IκBα and a hTERT fragment (aa301-700) fusion protein. In some embodiments, the fusion protein comprises SEQ ID NO:13.

[0068] As used herein, the term "cytokine" refers to any one of numerous factors that exert a variety of effects on cells, eg, inducing growth or proliferation. Non-limiting examples of cytokines that may be used alone or in combination in the practice of the present invention include interleukin-2 (IL-2), stem cell factor (SCF), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin-1 beta (IL-1β), interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), prostaglandin E2 (PGE2), MIP-11, leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO), and flt3 ligand. Cytokines are commercially available from several vendors, e.g., Genzyme (Framingham, Mass.), Genentech (South San Francisco, Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems (Minneapolis, Minn.), and Immunex (Seattle, Wash.). Although not always explicitly stated, it is contemplated that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the present invention.

[0069] The term "dendritic cells (DC)" refers to a population of morphologically similar cell types found in various lymphoid and non-lymphoid tissues. Steinman, Ann. Rev. Immunol. 9:271-296 (1991). Dendritic cells constitute the most potent and preferred APC in the organism. Dendritic cells can be distinguished from monocytes and possess a distinct phenotype. For example, a specific differentiation marker, the CD14 antigen, is not found in dendritic cells but is possessed by monocytes. It has been shown that mature DCs can provide all of the signals necessary for T cell activation and proliferation. Also, mature dendritic cells are not phagocytes, whereas monocytes and immature dendritic cells are strongly phagocytic cells. Immature DCs are capable of capturing antigens by endocytosis, phagocytosis, macropinocytosis or adsorptive pinocytosis and receptor-mediated antigen uptake, and are phenotypically CD80-positive. - or CD80 低 , CD83 - or CD83 低 , CD86 低 and have a high intracellular concentration of MHC class II molecules. Mature DCs have an unclear morphology, a lower endocytic capacity, and are phenotypically CD80-positive compared to immature DCs. 高 , CD83 高 , CD86 高 It is.

[0070] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or mRNA is translated into a peptide, polypeptide, or protein.

[0071] The term "genetically engineered" means containing and / or expressing foreign genes or nucleic acid sequences that in turn modify the genotype or phenotype of the cell or its progeny.

[0072] The term "isolated" means separated from cellular components, or in other cases polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof, or components with which the cell is essentially normally associated. For example, with respect to mammalian cells, in some embodiments, the isolated mammalian cells are normally found in the body and have been separated or removed from the body. For example, white blood cells collected by leukapheresis are "isolated" and dendritic cells differentiated from monocytes or other cells in vitro are "isolated". Dendritic cells can also be further isolated and purified from a heterogeneous population of cells, e.g., lymphocytes, with which they are associated.

[0073] The term "major histocompatibility complex" or "MHC" refers to a complex of genes that encode cell surface molecules required for antigen presentation to T cells and rapid graft rejection. In humans, the MHC is also known as the "human leukocyte antigen" or "HLA" complex. The proteins encoded by the MHC are known as "MHC molecules" and are classified into class I and class II MHC molecules. Class I MHC molecules contain membrane heterodimeric proteins composed of an α chain encoded in the MHC non-covalently associated with β2-microglobulin. Class I MHC molecules are expressed by nearly all nucleated cells and have been shown to function in antigen presentation to CD8 T cells. Class I molecules include HLA-A, B, and C in humans. Class II MHC molecules also contain membrane heterodimeric proteins composed of non-covalently associated α and β chains. Class II MHC molecules are expressed by CD4 T cells and CD8 T cells. + They have been shown to function in T cells and in humans include HLA-DP, DQ, and DR.

[0074] "Pathogen" as used herein refers to any disease-causing organism, such as a bacterium, fungus, parasite or virus, and also to attenuated derivatives thereof.

[0075] A "pharmaceutical composition" is intended to include the combination of an active agent (e.g., antigen-loaded dendritic cells and / or activated lymphocytes) with a carrier, inert or active, to produce a composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0076] As used herein, the term "pharmaceutical acceptable carrier" includes standard pharmaceutical carriers, such as heat-inactivated serum + 10% DMSO + 5% dextrose, phosphate buffered saline solution, water, and emulsions, such as oil / water or water / oil emulsions, as well as any of the various types of wetting agents. The composition may also include adjuvants, stabilizers, and preservatives. For examples of carriers, stabilizers, and adjuvants, see Remington's Pharm.Sci.18.sup.th Ed. (Mack Publ.Co., Easton (1990)).

[0077] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to a polymeric form of nucleotides of any length. A polynucleotide may contain deoxyribonucleotides, ribonucleotides, and / or their analogs. Nucleotides may have any three-dimensional structure and may perform any function, known or unknown. The term "polynucleotide" includes, for example, single-stranded, double-stranded, and triple-helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. In addition to native nucleic acid molecules, the nucleic acid molecules of the present invention may also include modified nucleic acid molecules.

[0078] The term "peptide" is used in the broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, such as esters, ethers, etc. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics. Peptides of three or more amino acids are commonly referred to as oligopeptides when the peptide chain is short. When the peptide chain is long, the peptide is commonly referred to as a polypeptide or protein.

[0079] As used herein, "subject" refers to any bird, fish, reptile, amphibian, or mammal. In some embodiments, the subject is a primate, rodent, dog, or cat. In some embodiments, the subject is a human.

[0080] As used herein, "monocytes" refers to CD14+ white blood cells that have the ability to differentiate into dendritic cells. Monocytes may differentiate into immature dendritic cells in response to GM-CSF and IL-4. Monocytes may be from any animal, and in some embodiments are human monocytes. Monocytes may be provided and incubated in compositions such as, but not limited to, blood, blood fractions (e.g., white blood cells (WBCs), buffy coat, peripheral blood mononuclear cells (PBMCs), and in compositions further enriched for monocytes. In one embodiment, monocytes are provided together with other peripheral blood mononuclear cells (PBMCs), e.g., as a leukopheresis product. In another embodiment, monocytes are enriched from PBMCs or isolated directly from peripheral blood. Methods for isolating monocytes or PBMCs containing monocytes are known to those of skill in the art. In some embodiments, monocytes are collected together with other PBMCs by leukopheresis. Methods for leukopheresis are known in the art. In some embodiments of the invention, PBMCs, including monocytes, are collected from a subject by leukapheresis at a hospital, clinic, doctor's office, etc. Leukapheresis is a procedure in which leukapheresis is performed to remove leukapheresis cells from a subject's blood, the remainder of which is then transfused back to the subject. Leukapheresis products are typically blood fractions enriched in PBMCs and have low concentrations of contaminating red blood cells, granulocytes, and platelets. Methods and devices for performing leukapheresis are well known in the art. For more information on leukapheresis, see, for example, gambrobct.com / Products_&_Services / . Examples of leukapheresis devices include COBESpectra™ manufactured by GAMBRO BCT and CS3000 plus Blood Cell Separator manufactured by Baxter Fenwal.

[0081] A "functional Tax protein" is a protein that is capable of performing one or more functions of a wild-type Tax protein from a T-cell leukemia virus (HTLV). A functional Tax protein is defined by its activity in inducing NF-κB activation in a cell. The modified primary blood dendritic cells and monocyte-derived dendritic cells of the invention comprise a functional Tax protein. In some embodiments, the Tax protein is from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, HTLV-3, and HTLV-4. In some embodiments, the functional Tax protein is a full-length protein from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, HTLV-3, and HTLV-4. In some embodiments, the Tax protein is from HTLV-1. In some embodiments, the Tax protein is from HTLV-1 and comprises the amino acid sequence of SEQ ID NO:1 and the nucleotide sequence of SEQ ID NO:2. In some embodiments, the Tax protein is from HTLV-2. In some embodiments, the Tax protein is from HTLV-2 and comprises the amino acid sequence of SEQ ID NO:3 and the nucleotide sequence of SEQ ID NO:4. In some embodiments, the Tax protein is from HTLV-3. In some embodiments, the Tax protein is from HTLV-3 and comprises the amino acid sequence of SEQ ID NO:5 and the nucleotide sequence of SEQ ID NO:6. In some embodiments, the Tax protein is from HTLV-4. In some embodiments, the Tax protein is from HTLV-4 and comprises the amino acid sequence of SEQ ID NO:7 and the nucleotide sequence of SEQ ID NO:8. In some embodiments, the Tax protein is from HTLV-2, a virus that does not cause leukemia in humans.

[0082] In some embodiments, the nucleic acid sequence of the functional Tax protein contains a nucleotide sequence that is highly identical, at least 90% identical, to a nucleotide sequence encoding a Tax polypeptide. In some embodiments, the nucleic acid sequence of the functional Tax protein comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in any of SEQ ID NOs: 2, 4, 6 or 8.

[0083] In some embodiments, a polynucleotide encoding a functional Tax protein may comprise the coding sequence for the full-length polypeptide or a fragment thereof by itself; or the coding sequence for the full-length polypeptide or a fragment thereof in reading frame with other coding sequences. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites, and sequences that stabilize mRNA.

[0084] In some embodiments, a nucleotide sequence encoding a functional Tax protein comprises a nucleic acid molecule comprising a polynucleotide having: (a) a nucleotide sequence encoding a Tax protein having the amino acid sequence in any of SEQ ID NOs: 1, 3, 5 or 7; or (b) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence complementary to the nucleotide sequence in (a).

[0085] In some embodiments, a functional Tax protein for use in the present invention comprises SEQ ID NO:3. In some embodiments, the Tax protein comprises a biologically active fragment or variant of SEQ ID NO:3. In some embodiments, the Tax protein comprising a biologically active fragment or variant has at least 80% identity to SEQ ID NO:3. In some embodiments, the biologically active fragment or variant has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the polypeptide of SEQ ID NO:3.

[0086] In some embodiments, the functional Tax protein comprises a fusion protein comprising a full-length Tax protein or a biologically active fragment thereof. A fragment is a polypeptide having an amino acid sequence that is the same in whole as part, but not all, of the amino acid sequence of the Tax polypeptides described above. In some embodiments, the fragment may comprise at least about 150 contiguous amino acids identified in any of SEQ ID NOs: 1, 3, 5, or 7. In some embodiments, the fragment is at least about 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, or 330 contiguous amino acids identified in any of SEQ ID NOs: 1, 3, 5, or 7.

[0087] In some embodiments, fragments include truncated polypeptides having the amino acid sequence of, e.g., Tax, except for a deletion of a contiguous series of residues including the amino terminus, or a deletion of a contiguous series of residues including the carboxy terminus, or a deletion of two contiguous series of residues, one including the amino terminus and one including the carboxy terminus.

[0088] In one embodiment, the fragment comprises about the last 290, 300, 310, 320, or 330 amino acids of any of SEQ ID NOs: 1, 3, 5 or 7.

[0089] In one embodiment, the fragment comprises the first 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320 or 330 amino acids of any of SEQ ID NOs: 1, 3, 5 or 7.

[0090] Biologically active or functional fragments or variants of Tax protein include polypeptides or fragments thereof having an amino acid sequence that is at least 80% identical to any of SEQ ID NOs: 1, 3, 5 or 7, which have at least 80% identity to the corresponding fragments of any of SEQ ID NOs: 1, 3, 5 or 7. Included in this group are variants of the given sequences and fragments. In some embodiments, the variants vary from the reference by conservative amino acid substitutions, i.e., substituting a residue with another of similar characteristics. Exemplary substitutions are between Ala, Val, Leu and Ile; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; and between basic residues Lys and Arg, or aromatic residues Phe and Tyr. In some embodiments, the polypeptide is a variant in which several, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids have been substituted, deleted or added, in any combination.

[0091] The percent identity of particular sequences may be determined using conventional means using known computer programs, such as the BestFit program (Wisconsin Sequence Analysis Package, Version 10 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711).

[0092] As described in the methods herein, immature dendritic cells are manipulated to express functional Tax protein from a T-cell leukemia virus. The functional Tax protein present in dendritic cells as described herein according to the present invention is not expressed as a result of infection of immature dendritic cells with a T-cell leukemia virus (i.e., unmodified or wild-type virus). Instead, nucleic acid encoding a functional Tax protein is delivered to the cells by other mechanisms, such as modifying a nucleic acid construct containing a functional Tax protein and delivering the construct to the immature dendritic cells. In some embodiments, nucleic acid encoding a functional Tax protein can be modified in a viral vector and used to transduce cells. In some embodiments, cells are transduced in the presence of about 10 μg / ml polybrene.

[0093] Viral vectors that can be used to transduce cells are not limited. In some embodiments, viral vectors typically include highly attenuated, non-replicating viruses. Viral vectors include DNA viral vectors, such as those based on adenoviruses, including but not limited to adeno-associated viruses, herpes simplex viruses, avian viruses, such as Newcastle disease viruses, poxviruses, such as vaccinia viruses, and parvoviruses; and RNA viral vectors, including but not limited to retroviral vectors. Vaccinia vectors and methods useful in immunization protocols are described in U.S. Pat. No. 4,722,848. Retroviral vectors include lentiviruses, such as human immunodeficiency viruses. Naldini et al. (1996) Science 272:263-267. Replication-defective retroviral vectors that carry the nucleotide sequence of interest as part of the retroviral genome can be used. Such vectors have been described in detail. (Miller, et al. (1990) Mol. Cell. Biol. 10:4239; Kolberg, R. (1992) J. NIH Res. 4:43; Cornetta, et al. (1991) Hum. Gene Therapy 2:215).

[0094] Adenovirus and adeno-associated virus vectors useful in the present invention may be produced according to methods already taught in the art. (See, e.g., Karlsson, et al. (1986) EMBO 5:2377; Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzcyzka (1992) Current Top. Microbiol. Immunol. 158:97-129; Gene Targeting: A Practical Approach (1992) ed. A.L. Joyner, Oxford University Press, NY). Several different approaches are feasible.

[0095] Alphavirus vectors, such as Venezuelan Equine Encephalitis (VEE) virus, Semliki Forest virus (SFV) and Sindbis virus vectors, can be used for efficient gene transfer. Replication-deficient vectors are available.

[0096] Further references describing viral vectors that may be used in the methods of the invention include: Horwitz, MS, Adenoviridae and Their Replication, in Fields, B., et al. (eds.) Virology, Vol. 2, Raven Press New York, pp. 1679-1721, 1990; Graham, F. et al., pp. 109-128 in Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols, Murray, E. (ed.), Humana Press, Clifton, NJ (1991); Miller, et al. (1995) FASEB Journal 9: 190-199, Schreier (1994) Pharmaceutica Acta Helvetiae 68: 145-159; Schneider and French (1993) Circulation 88: 1937-1942; Curiel, et al. (1992) Human Gene Therapy 3:147-154; WO95 / 00655; WO95 / 16772; WO95 / 23867; WO94 / 26914; WO95 / 02697 (January 26, 1995); and WO95 / 25071.

[0097] In some embodiments, the viral vector is a retrovirus / lentivirus, adenovirus, adeno-associated virus, alphavirus, vaccinia virus, or herpes simplex virus, hi some embodiments, the viral vector is a lentiviral vector.

[0098] I. Modified Primary Blood Dendritic Cells and Methods of Production A. Modified Primary Blood Dendritic Cells In one embodiment, the invention provides modified primary blood dendritic cells, the dendritic cells expressing a functional Tax protein from a T-cell leukemia virus. According to the invention, the primary blood dendritic cells are CD205+ and CD11c+. In some embodiments, the modified primary blood dendritic cells are produced by the methods as described herein.

[0099] In some embodiments, the modified primary blood dendritic cells have a constitutive maturation and activation phenotype. In some embodiments, the dendritic cells express one or more dendritic cell maturation and activation markers. In some embodiments, the dendritic cells comprise one or more maturation and activation markers selected from the group consisting of CD83, CD80, CD86, CD70, CCR7, and HLA-DR.

[0100] In some embodiments, the modified primary blood dendritic cells may be further characterized by the presence or absence of various markers, by their ability to produce various cytokines and grow in suspension culture. In some embodiments, the modified primary blood dendritic cells are TLR3+ and TLR4-. In some embodiments, the modified primary blood dendritic cells contain a truncated form of TLR7. In some embodiments, the modified primary blood dendritic cells express TLR9. In some embodiments, the modified primary blood dendritic cells produce IL-1A and TNFα. In some embodiments, the modified primary blood dendritic cells produce IL-15.

[0101] The presence or absence of certain markers can be assayed using known techniques, such as fluorescence activated cell sorting (FACS).

[0102] In some embodiments, the modified primary blood dendritic cells produce negligible levels of IL-10 and TGF.beta.. In some embodiments, the dendritic cells are capable of growth in suspension culture.

[0103] The source of the cells is not particularly limiting. In some embodiments, the modified primary blood dendritic cells may be derived from blood cells from birds, fish, reptiles, amphibians, or mammals. In some embodiments, the cells are derived from blood cells from primates, rodents, such as rats, mice, or guinea pigs, dogs, or cats. In some embodiments, the cells are derived from blood cells from humans.

[0104] In some embodiments, the modified primary blood dendritic cells further express c-Myc. In some embodiments, the modified primary blood dendritic cells further express one or more of Mcl-1, Bcl-xL, Bcl-2, phosphorylated pRb, phosphorylated cdc2, phosphorylated Stat1, phosphorylated Stat3, and phosphorylated Stat5. In some embodiments, the transcription factors NF-κB, Stat3, and AP-1 are active.

[0105] The modified primary blood dendritic cells may be engineered to express one or more HLA proteins. HLA is a major histocompatibility complex (MHC) antigen specific to humans. HLA-A, HLA-B, and HLA-C are types of human MHC class I cell surface receptors. The receptors are heterodimers and are composed of a heavy α chain and a smaller β chain. The α chain is encoded by a variant HLA-A, HLA-B, or HLA-C gene, and the β chain is in a variant β2 microglobulin molecule. The β2 microglobulin protein is encoded by a separate region of the human genome. In some embodiments, the modified primary blood dendritic cells may be engineered to express one or more of HLA-A, HLA-B, or HLA-C. In one embodiment, the modified primary blood dendritic cells are engineered to express HLA-A. In some embodiments, the modified primary blood dendritic cells may be engineered to express HLA-A2.1. In some embodiments, the amino acid sequence of HLA-A2.1 is SEQ ID NO:15.

[0106] The modified primary blood dendritic cells may be loaded with one or more antigens. In some embodiments, the modified primary blood dendritic cells harbor one or more antigens or antigen fragments or variants thereof. In some embodiments, the antigen is a cancer antigen. In some embodiments, the antigen is an antigen from a pathogenic organism.

[0107] In some embodiments, the modified primary blood dendritic cells induce proliferation of naive lymphocytes. In some embodiments, the modified primary blood dendritic cells are capable of inducing proliferation of naive lymphocytes in the absence of exogenously added IL-2.

[0108] In some embodiments, the modified primary blood dendritic cells include the cell lines ihv-DC1 and ihv-DC2 described in Example 1.

[0109] In some embodiments, the modified primary blood dendritic cells are capable of priming naive lymphocytes (e.g., a population of PBMCs) to generate cytotoxic lymphocytes that recognize antigens. In some embodiments, the modified primary blood dendritic cells present antigens in an HLA-restricted manner. In some embodiments, the modified primary blood dendritic cells are capable of priming naive lymphocytes (e.g., a population of PBMCs) to generate cytotoxic lymphocytes that are capable of killing cells in a non-HLA-restricted manner.

[0110] B. Methods for Producing Modified Primary Blood Dendritic Cells The modified primary blood dendritic cells of the present invention can be prepared by a variety of methods, including but not limited to, hi some embodiments, the modified primary blood dendritic cells are prepared by the methods described herein.

[0111] In one embodiment, the present invention provides a method of generating modified primary blood dendritic cells, comprising: i) providing a sample of cells comprising immature dendritic cells; ii) expressing a functional Tax protein from a T-cell leukemia virus in said cells; The method includes the steps of:

[0112] In some embodiments, the method further comprises iii) culturing the cells to induce maturation and activation thereof. The culturing step for inducing maturation and activation of the modified primary blood dendritic cells is not limiting. In some embodiments, the cells are cultured in the presence of one or more cytokines. In some embodiments, the cells are cultured in a medium comprising an effective amount of IL-2. In some embodiments, the concentration of IL-2 comprises at least about 10 units / ml. In some embodiments, the concentration of IL-2 is at least about 50 units / ml. In some embodiments, the concentration of IL-2 is at least about 100 units / ml. In some embodiments, the concentration of IL-2 ranges from about 100 to 200 units / ml. IL-2 may be provided in recombinant form. Recombinant IL-2 may be obtained from the NIH AIDS Reagent Program (Germantown, Maryland).

[0113] In some embodiments, the method further comprises depleting T cells from the cultured cells.

[0114] In one embodiment, the present invention provides a method of generating modified primary blood dendritic cells, comprising: i) providing a sample of cells comprising immature dendritic cells; ii) treating the cells in culture with an effective amount of phytohemagglutinin (PHA); iii) treating the cells in culture with an effective amount of IL-2; iv) expressing a functional Tax protein from a T-cell leukemia virus in said cell; v) depleting T cells from cultured cells; The method includes the steps of:

[0115] In some embodiments, the sample of cells containing immature dendritic cells comprises a sample of PBMCs. In some embodiments, the PBMCs are from a subject who will subsequently be treated with dendritic cells or T lymphocytes activated by dendritic cells. In some embodiments, the PBMCs are isolated from a leukopack. In some embodiments, the PBMCs are prepared from a healthy blood donor. For example, a leukopack from a healthy blood donor may be obtained from a blood bank. The PBMCs may be isolated using the Ficoll-Paque method. For example, 10 ml of blood from a leukopack may be added to 10 ml of CaCl2 in a 50 ml conical tube. 2+ / Mg 2+ Free PBS buffer may be mixed. 15 ml of Ficoll-Paque PLUS (GE Healthcare) may be pipetted into a separate 50 ml tube. 20 ml of diluted blood may be layered on top of the Ficoll. Samples may be spun at 400×g for 30 minutes at room temperature (RT). The PBMC layer may be collected at the diluted plasma / Ficoll interface and PBS buffer may be added for a total volume of 40 ml, then spun at 200×g for 10 minutes at RT. The pellet may be washed with PBS and spun again at 200×g for 10 minutes at RT. Cell numbers may be counted and a new 2 million PBMCs may be stimulated with PHA (5 μg / ml) and IL-2 (100 u / ml).

[0116] In some embodiments, the concentration of PHA in part ii) is at least about 1 μg / ml. In some embodiments, the concentration of PHA is in the range of about 1-30 μg / ml. In some embodiments, the concentration of PHA in part ii) is in the range of about 1-5 μg / ml. In some embodiments, the concentration of PHA in part ii) is at least about 5 μg / ml. In some embodiments, the cells are treated with PHA for about 12 hours to about 36 hours. In some embodiments, the cells are treated with PHA for about 24 hours.

[0117] In some embodiments, the cells are treated with IL-2 after treatment with PHA. In some embodiments, the cells are treated with at least about 10 units / ml of IL-2. In some embodiments, the cells are treated with at least about 50 units / ml of IL-2. In some embodiments, the cells are treated with about 10-500 units / ml of IL-2 and cultured in a medium containing IL-2 for a period of time prior to expression of functional Tax protein. In some embodiments, the cells are treated with about 100-200 units / ml of IL-2 and cultured in a medium containing IL-2 for a period of time. In some embodiments, the cells are cultured in a medium containing IL-2 for a period of time ranging from 2 to 10 days. In some embodiments, the cells are cultured in a medium containing IL-2 for a period of time ranging from about 100 to 200 units / ml of IL-2 for a period of time ranging from about 4 to 5 days.

[0118] The cells are engineered to express a functional Tax protein from a T-cell leukemia virus. The functional Tax protein present in the dendritic cells described herein according to the invention is not expressed as a result of infection of immature dendritic cells with a T-cell leukemia virus (i.e., unmodified or wild-type virus). Instead, a nucleic acid encoding a functional Tax protein is delivered to the cells by other mechanisms, such as modifying a nucleic acid construct containing a functional Tax protein and delivering the construct to immature dendritic cells. In some embodiments, the Tax protein is from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, HTLV-3 and HTLV-4. In some embodiments, the Tax protein is from HTLV-2, a virus that does not cause leukemia in humans.

[0119] In some embodiments, the nucleic acid encoding a functional Tax protein can be engineered into a viral vector and used to transduce cells. In some embodiments, the cells are transduced in the presence of about 10 μg / ml of polybrene. In some embodiments, the viral vector is a retrovirus / lentivirus, adenovirus, adeno-associated virus, alphavirus, vaccinia virus, or herpes simplex virus. In some embodiments, the viral vector is a lentivirus vector.

[0120] In some embodiments, functional Tax protein is prepared in a lentiviral vector and cells are cultured and transduced as follows: The tax gene from HTLV-2 is fused with a fragment encoding enhanced green fluorescent protein (EGFP) and the Tax2-GFP fusion fragment can be cloned into the lentiviral vector. In some embodiments, the Tax2-GFP fusion has the amino acid sequence of SEQ ID NO: 16 and the nucleotide sequence of SEQ ID NO: 17. To generate Tax2-GFP lentivirus, the lentiviral Tax2-GFP vector can be co-transfected with a packaging plasmid mix containing expression plasmids for VSV-G, Gag-Pol and Rev (Invitrogen) into 293 cells using SuperFect transfection reagent (Qiagen). The viral supernatant can be collected and subjected to ultracentrifugation at 25,000 rpm / 4°C for 2 hours. The viral pellet can be resuspended in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma) and stored at -80°C. 2 million freshly isolated PBMCs can be stimulated with PHA (5 μg / ml) for 24 hours. The following day, the cells can be washed once with complete RPM11640 medium, followed by the addition of recombinant IL-2 (100 units / ml). The activated PBMCs can be cultured for 4-5 days. 2 million activated PBMCs can be mixed with Tax2-GFP lentivirus at an MOI (very large number of infection) of about 20 and the addition of polybrene (6 μg / ml). The cell-lentivirus mix is ​​incubated for 16 hours at 32°C / 5% CO2 in a cell culture incubator. The following day, the transduced cells can be washed once with complete RPM11640 medium, then replaced in a culture flask with complete RPM11640 medium supplemented with recombinant IL-2 (100 units / ml). In some embodiments, the 10% FBS in the complete medium may be replaced by 5% heat-inactivated human AB serum from Sigma.

[0121] In some embodiments, the transduced cells are further cultured in a medium comprising an effective amount of IL-2 to facilitate their maturation and activation. In some embodiments, the concentration of IL-2 comprises at least about 10 units / ml. In some embodiments, the concentration of IL-2 is at least about 50 units / ml. In some embodiments, the concentration of IL-2 is at least about 100 units / ml. In some embodiments, the transduced cells are cultured in a medium comprising about 100-200 units / ml of IL-2. In some embodiments, the transduced cells are further cultured for about 2-3 months in a medium comprising an effective amount of IL-2. In some embodiments, the medium comprises 100-200 units / ml of IL-2 and serum. In some embodiments, the medium comprises about 10% fetal bovine serum and 100-200 units / ml of IL-2. In some embodiments, the medium comprises about 5% human serum and 100-200 units / ml of IL-2. In some embodiments, the transduced cells are cultured for about 2-3 months in a medium comprising IL-2 and human serum. In some embodiments, the medium comprises 5% heat-inactivated human AB serum and 100-200 units / ml of IL-2. The medium generally comprises a basal medium. The basal medium is not limiting. In some embodiments, the basal medium is complete RPMI 1640 medium.

[0122] T cells can be depleted from cultured cells using known methods. In some embodiments, T cells are depleted by a composition comprising a molecule that binds to T cells. In some embodiments, T cells are depleted by an antibody. In some embodiments, the antibody is an anti-CD3 antibody. In some embodiments, the antibody is conjugated to magnetic beads. In some embodiments, T cells are depleted by cell sorting or flow cytometry.

[0123] In some embodiments, T cells are depleted as follows: 10 million transduced cells can be incubated with 100 μl of anti-CD3 magnetic beads (Invitrogen) for 30 minutes at 4° C., 2-3 weeks after lentiviral transduction. Cells that do not bind to the beads under the magnetic bar can be aspirated and cultured in complete RPM11640 medium supplemented with IL-2 in a 6-well culture plate. In some embodiments, the CD3-negative selected cells can be tested by FACS to determine cell purity. In some embodiments, a further round of anti-CD3 negative selection can be performed as desired when the removed cell population is not pure.

[0124] In some embodiments, the depleted population of T cells may be further cultured in a medium containing an effective amount of IL-2. In some embodiments, the concentration of IL-2 is about 100-200 units / ml. In some embodiments, the CD3-negative cell population is cultured in complete RPM11640 medium supplemented with 100-200 units / ml of IL-2. In some embodiments, the CD3-negative cell population may be analyzed by a panel of antibodies using FACS to determine the immunophenotype, about 2 months after CD3 negative selection.

[0125] In some embodiments, the modified primary blood dendritic cells produced by the above methods have a constitutive maturation and activation phenotype, hi some embodiments, the modified primary blood dendritic cells comprise maturation and activation markers selected from the group consisting of CD83, CD80, CD86, CD70, CCR7, 4-1BBL, and HLA-DR, and combinations thereof.

[0126] In some embodiments, the method further comprises loading primary blood dendritic cells with one or more antigens or antigen fragments or variants thereof. In some embodiments, the method comprises transducing cells with a viral vector encoding one or more antigens or antigen fragments or variants thereof. In some embodiments, the antigen is a cancer antigen.

[0127] In some embodiments, the primary blood dendritic cells express human telomerase reverse transcriptase or an antigenic fragment or derivative thereof. In some embodiments, the human telomerase reverse transcriptase amino acid sequence comprises SEQ ID NO:9 and the nucleotide sequence comprises SEQ ID NO:10.

[0128] In some embodiments, the nucleic acid sequence of human telomerase reverse transcriptase contains a nucleotide sequence that is highly identical, at least 90% identical, to a nucleotide sequence encoding a human telomerase reverse transcriptase polypeptide. In some embodiments, the nucleic acid sequence of human telomerase reverse transcriptase comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO:10.

[0129] In some embodiments, a polynucleotide encoding human telomerase reverse transcriptase may comprise the coding sequence for the full-length polypeptide or a fragment thereof by itself; or the coding sequence for the full-length polypeptide or a fragment thereof in reading frame with other coding sequences. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites, and sequences that stabilize mRNA.

[0130] In some embodiments, the nucleotide sequence encoding human telomerase reverse transcriptase comprises a nucleic acid molecule comprising a polynucleotide having: (a) a nucleotide sequence encoding human telomerase reverse transcriptase having the amino acid sequence in SEQ ID NO:9; or (b) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence complementary to the nucleotide sequence in (a).

[0131] In some embodiments, the human telomerase reverse transcriptase for use in the present invention comprises SEQ ID NO: 9. In some embodiments, the human telomerase reverse transcriptase comprises an antigenic fragment or variant of SEQ ID NO: 9. In some embodiments, the human telomerase reverse transcriptase comprising an antigenic fragment or variant has at least 80% identity to SEQ ID NO: 9. In some embodiments, the antigenic fragment or variant has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the polypeptide of SEQ ID NO: 9.

[0132] In some embodiments, the human telomerase reverse transcriptase comprises a fusion protein comprising full-length human telomerase reverse transcriptase or an antigenic fragment or derivative thereof. A fragment is a polypeptide having an amino acid sequence that is entirely the same as part, but not all, of the human telomerase reverse transcriptase described above. In some embodiments, a fragment may comprise at least about 250 contiguous amino acids as identified in SEQ ID NO:9. In some embodiments, a fragment is at least about 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or 1050 contiguous amino acids as identified in SEQ ID NO:9.

[0133] In some embodiments, a fragment includes a truncated polypeptide having the amino acid sequence of, e.g., human telomerase reverse transcriptase, except for a deletion of a contiguous series of residues including the amino terminus, or a deletion of a contiguous series of residues including the carboxy terminus, or a deletion of two contiguous series of residues, one including the amino terminus and one including the carboxy terminus.

[0134] Antigenic fragments or variants of human telomerase reverse transcriptase include polypeptides or fragments thereof having an amino acid sequence at least 90% identical to SEQ ID NO:9, which has at least 90% identity with the corresponding fragment of SEQ ID NO:9. This group includes variants of the given sequences and fragments. In some embodiments, the variants vary from the reference by conservative amino acid substitutions, i.e., replacing a residue with another one of similar characteristics. Exemplary substitutions are between Ala, Val, Leu and Ile; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; and between basic residues Lys and Arg, or aromatic residues Phe and Tyr. In some embodiments, the polypeptide is a variant in which several, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are substituted, deleted or added in any combination.

[0135] In some embodiments, the antigen comprises a fusion protein. In some embodiments, the fusion protein comprises IκBα fused to an antigen. In some embodiments, the amino acid sequence of IκBα comprises SEQ ID NO:11 and the nucleotide sequence comprises SEQ ID NO:12. In some embodiments, the fusion protein comprises a proteasome targeting sequence of IκBα and comprises an antigen, such as a cancer antigen. In some embodiments, the cancer antigen is a fragment of human telomerase reverse transcriptase. In some embodiments, the fusion protein comprises IκBα fused to a fragment of human telomerase reverse transcriptase, comprising the amino acid sequence of SEQ ID NO:13 and comprising the nucleotide sequence of SEQ ID NO:14.

[0136] In some embodiments, the method further comprises genetically engineering the primary blood dendritic cells to express one or more HLA proteins. In some embodiments, the method comprises genetically engineering the primary blood dendritic cells to express one or more of HLA-A, HLA-B, or HLA-C. In some embodiments, the primary blood dendritic cells are genetically engineered to express HLA-A2.1. In some embodiments, the amino acid sequence of HLA-A2.1 is SEQ ID NO: 15.

[0137] In some embodiments, the functionality of the modified primary blood dendritic cells can be determined by their ability to prime naive lymphocytes. In some embodiments, this is performed by an assay called the mixed leukocyte reaction (MLR). In some embodiments, about 2 million unstimulated (naive) allogeneic PBMCs are mixed with the modified primary blood dendritic cells (e.g., 2×10 6 cells) at a ratio of about 100:1 without the addition of any cytokines or growth factors. 4 The cells are mixed with the proliferating cells. After 10-14 days, IL-2 can be added into the cell culture. The immunophenotype and tumor killing activity of the proliferating cells can be determined by FACS and cytotoxicity assays, respectively.

[0138] In some embodiments, the invention provides primary blood dendritic cells prepared by any of the above methods.

[0139] II. Modified Monocyte-Derived Dendritic Cells and Methods of Production A. Modified Monocyte-Derived Dendritic Cells In some embodiments, the present invention provides modified monocyte-derived dendritic cells that express a functional Tax protein from a T-cell leukemia virus. The monocyte-derived dendritic cells are CD205+.

[0140] The source of the cells is not particularly limiting. In some embodiments, the modified monocyte-derived dendritic cells may be derived from monocyte cells from birds, fish, reptiles, amphibians, or mammals. In some embodiments, the cells are derived from monocyte cells from primates, rodents, such as rats, mice, or guinea pigs, dogs, or cats. In some embodiments, the cells are derived from monocyte cells from humans.

[0141] In some embodiments, the modified monocyte-derived dendritic cells have a constitutive maturation and activation phenotype. In some embodiments, the modified monocyte-derived dendritic cells express one or more dendritic cell maturation and activation markers. In some embodiments, the dendritic cell maturation and activation markers are selected from the group consisting of CD83, CD80, CD86, and CD70.

[0142] In some embodiments, the modified monocyte-derived dendritic cells may be further characterized by their ability to produce various cytokines, and the presence or absence of various markers, their growth characteristics. In some embodiments, the modified monocyte-derived dendritic cells express a truncated form of TLR7. In some embodiments, the modified monocyte-derived dendritic cells produce IL-1A and TNFα. In some embodiments, the modified monocyte-derived dendritic cells produce TGFβ. In some embodiments, the modified monocyte-derived dendritic cells produce IL-15. In some embodiments, the modified monocyte-derived dendritic cells express one or more of the following: c-Myc, Mcl-1, Bcl-xL, Bcl-2, phosphorylated pRb, phosphorylated cdc2, phosphorylated Stat1, phosphorylated Stat3, and phosphorylated Stat5. In some embodiments, the modified monocyte-derived dendritic cells further express 4-1BBL and / or CD4. In some embodiments, the modified monocyte-derived dendritic cells are TLR3+ and TLR4-. In some embodiments, the modified monocyte-derived dendritic cells are capable of growth in suspension culture, hi some embodiments, the monocyte-derived dendritic cells are capable of expansion in culture for up to three months.

[0143] In some embodiments, the modified monocyte-derived dendritic cells are capable of inducing proliferation of naive lymphocytes in the absence of exogenously added IL-2.

[0144] The modified monocyte-derived dendritic cells may be engineered to express one or more HLA proteins. In some embodiments, the modified monocyte-derived dendritic cells may be engineered to express one or more of HLA-A, HLA-B, or HLA-C. In some embodiments, the modified monocyte-derived dendritic cells are engineered to express HLA-A. In some embodiments, the modified monocyte-derived dendritic cells may be engineered to express HLA-A2.1. In some embodiments, the amino acid sequence of HLA-A2.1 is SEQ ID NO: 15.

[0145] The modified monocyte-derived dendritic cells can be loaded with one or more antigens. In some embodiments, the modified monocyte-derived dendritic cells present one or more antigens or antigen fragments or variants thereof. In some embodiments, the antigen is a cancer antigen. In some embodiments, the antigen is an antigen from a pathogenic organism.

[0146] The loading of the modified monocyte-derived dendritic cells with antigen is not limiting. In some embodiments, the antigen can be loaded using methods described herein, including transfection, transformation, electroporation, and peptide pulsing. In some embodiments, the antigen is introduced by viral transformation. In some embodiments, the virus is a lentivirus.

[0147] In some embodiments, the modified monocyte-derived dendritic cells are capable of inducing proliferation of naive lymphocytes.

[0148] In some embodiments, the modified monocyte-derived dendritic cells are capable of priming naive lymphocytes (e.g., a population of PBMCs) to generate cytotoxic lymphocytes that recognize antigens. In some embodiments, the modified monocyte-derived dendritic cells present antigens in an HLA-restricted manner. In some embodiments, the modified monocyte-derived dendritic cells are capable of priming naive lymphocytes (e.g., a population of PBMCs) to generate cytotoxic lymphocytes that are capable of killing cells in a non-HLA-restricted manner.

[0149] B. Methods for Producing Modified Monocyte-Derived Dendritic Cells The modified monocyte-derived dendritic cells of the present invention can be prepared by a variety of methods, including but not limited to, in some embodiments, the modified monocyte-derived dendritic cells are prepared by the methods described herein.

[0150] In one embodiment, the present invention provides a method for generating monocyte-derived dendritic cells, comprising: i) providing a sample of cells comprising immature monocyte-derived dendritic cells; ii) expressing a functional Tax protein from a T-cell leukemia virus in said cells; The method includes the steps of:

[0151] In some embodiments, the method further comprises culturing monocyte cells to induce their differentiation into immature dendritic cells prior to part i). In some embodiments, differentiation is induced by contacting with culture medium comprising an effective amount of a composition that induces differentiation of monocytes into immature dendritic cells, such as, but not limited to, GM-CSF; GM-CSF and IL-4; GM-CSF and IL-13; GM-CSF and IL-15; and IFNα. In some embodiments, the monocytes are cultured in medium comprising an effective amount of GM-CSF and IL-4 that induces differentiation into immature dendritic cells.

[0152] In some embodiments, the method further comprises a step after part ii) comprising culturing the cells to induce their maturation and activation. The culturing step for inducing maturation and activation of monocyte-derived dendritic cells is not limiting. In some embodiments, the cells are cultured in the presence of one or more cytokines. In some embodiments, the cells are further cultured in a medium comprising effective amounts of GM-CSF and IL-4.

[0153] Various methods for collecting monocytes and PBMCs, which contain monocytes, from a subject are known to those of skill in the art. For example, for detailed information on leukapheresis for collecting PBMCs and elutriation for purification of monocytes, see gambrobct.com / Products_&_Services / . In one embodiment, PBMCs are obtained by collecting blood in a heparinized syringe, diluting with PBS, layering on Histopaque 1077 (Sigma), centrifuging, and collecting PBMCs at the interface. See Woodhead et al. (2000) International Immunol 12:1051-1061. Further methods for collecting, purifying, or fractionating PBMCs are known to those of skill in the art.

[0154] In some embodiments, the method uses enriched monocyte medium. Methods of enriching monocytes are known to those skilled in the art and include, but are not limited to, density gradient centrifugation (e.g., dilute Ficoll density gradient centrifugation, dilute Percoll density gradient centrifugation, etc.), elutriation, adherence to plastic, tangential flow filtration, fluorescence activated cell sorting (FACS), immune cell separation techniques (antibody panning to select monocytes or remove non-monocytes (e.g., leukocytes, macrophages, granulocytes, etc.), differential lysis, magnetic cell sorting, etc.), culture in plastic culture bags coated with plastic microcarrier beads, etc. For example, O'oherty et al. (1993) J Exp Med 178:1067-1076; Young et al. (1990) J Exp Med 171:1315-1332; Freudenthal et al. (1990) PNAS 87:7698-7702; Bernhard et al. (1995) Cancer Res 55:1099-1104;Caux et al.(1992)Nature 360:258-261;Read et al.(2003) “Evaluation of a Closed Automated System to Isolate Peripheral Blood Monocytes for Dendritic Dell(DC)Immunotherapy”Ninth annual meeting of the ISCT;Mu et al.(2003)Scand J Immunol 58:578-586;Maffei et See, e.g., J. Hematother. Stem Cell Res. 12:289-299; and WO2004 / 000444, the contents of which are incorporated by reference. For example, magnetic cell sorting can be used to enrich for morphological monocytes by positive selection (CD14+ cells) or by negative selection (i.e., removal of non-monocytic cells; e.g., CD3+, CD19+, and CD2+ cells).

[0155] In some embodiments, monocytes are enriched from the leukapheresis product by elutriation, an automated method for isolating monocytes from a subject's leukapheresis. Methods of leukapheresis are known in the art. For example, elutriation can be performed in a Gambro BCT Elutra™ Cell Separation System (Gambro BCT, Lakewood, Colo.). Elutriation buffer can be prepared by adding 1000 mL of 5% human albumin serum (HSA) to a 4 L bag of Hank's Balanced Salt Solution (HBSS). Cells can be fractionated by elutriation according to the manufacturer's protocol. In one embodiment, a modified version of the manufacturer's (Gambro) protocol is used for elutriation, where the final rotor off fraction is the 4th fraction instead of the 5th fraction. CBC with differential analysis can be performed on the monocyte fraction to verify purity and recovery. In some embodiments, monocyte purity can be assessed by immunophenotyping with CD14.

[0156] In some embodiments, monocytes are enriched prior to differentiation of the cells into dendritic cells. In particular, the PBMCs may be further purified or monocytes may be enriched from the PBMCs during this incubation. In one embodiment, monocytes may be enriched from the PBMCs after an incubation period and selection of adherent monocytes by culture in a container, preferably a plastic container.

[0157] In one embodiment, monocytes are cultured in a medium comprising a composition that induces differentiation of monocytes into immature or mature dendritic cells. Compositions that induce differentiation of monocytes into immature dendritic cells are known to those skilled in the art. Such compositions include, but are not limited to, GM-CSF+IL-4; GM-CSF+IL-13; GM-CSF+IL-15; IFNα; and GM-CSF+TNFα. In some embodiments, the composition that induces differentiation is GM-CSF+IL-4. In some embodiments, the concentrations of GM-CSF and IL-4 may range from about 400 to 2000 units / ml of each cytokine. In some embodiments, the concentrations of GM-CSF and IL-4 range from 500 to 1000 units / ml of each cytokine. In one embodiment, monocytes are contacted with GM-CSF and IL-4 for about 4 to 7 days, and in some embodiments, for about 5 to 6 days, during which time the monocytes differentiate into immature dendritic cells. Conditioned medium containing effective amounts of GM-CSF and IL-4 may also be utilized. In some embodiments, culturing comprises culturing the cells in medium containing conditioned medium from GM-CSF-Fc4 / IL-4-Fc4 producing 293 cells. In some embodiments, the cells are cultured in medium containing conditioned medium at about a 1:10 dilution.

[0158] Following differentiation of monocytes into immature dendritic cells and expression of functional Tax protein, in some embodiments, the immature dendritic cells may be matured into mature dendritic cells. In one embodiment, the immature dendritic cells are matured by contact with a medium comprising an effective amount of IL-2. In some embodiments, the concentration of IL-2 comprises at least about 10 units / ml. In some embodiments, the concentration of IL-2 is at least about 50 units / ml. In some embodiments, the concentration of IL-2 is at least about 100 units / ml. In some embodiments, the concentration of IL-2 is in the range of about 100-200 units / ml.

[0159] In another embodiment, the present invention provides a method of generating monocyte-derived dendritic cells, comprising: i) providing adherent monocytic cells; ii) culturing the cells in a medium containing effective amounts of GM-CSF and IL-4; iii) expressing a functional Tax protein from a T-cell leukemia virus in said cell; iv) culturing the functional Tax protein expressing cells in a medium containing effective amounts of GM-CSF and IL-4; The method includes the steps of:

[0160] In some embodiments, the adherent monocyte cells are isolated from PBMCs. In some embodiments, the adherent monocytes are isolated from about 4 million PBMCs. In some embodiments, after expressing a functional Tax protein and culturing in a medium containing an effective amount of GM-CSF and IL-4, the method further comprises culturing the cells in a medium containing IL-2 (e.g., containing about 100-200 units / ml).

[0161] In some embodiments, IL-4 and / or GM-CSF are fused to Fc4. In some embodiments, IL-4 and / or GM-CSF may be provided by conditioned medium. In some embodiments, the culturing of part ii) comprises culturing the cells in medium comprising conditioned medium from GM-CSF-Fc4 / IL-4-Fc4 producing 293 cells. In some embodiments, the cells are cultured in medium comprising conditioned medium at about a 1:10 dilution for about 7 days.

[0162] The cells are engineered to express a functional Tax protein from a T-cell leukemia virus. The functional Tax protein present in dendritic cells as described herein according to the present invention is not expressed as a result of infection of immature dendritic cells with a T-cell leukemia virus (i.e., unmodified or wild-type virus). Instead, a nucleic acid encoding a functional Tax protein is delivered to the cell by other mechanisms, such as modifying a nucleic acid construct containing a functional Tax protein and delivering the construct to immature dendritic cells. In some embodiments, the Tax protein is from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, HTLV-3 and HTLV-4. In some embodiments, the Tax protein is from HTLV-2, a virus that does not cause leukemia in humans.

[0163] In some embodiments, the nucleic acid encoding a functional Tax protein can be modified in a viral vector and used to transduce the cells. In some embodiments, the cells are transduced in the presence of an effective amount of polybrene, for example about 10 μg / ml of polybrene. In some embodiments, the viral vector is a retrovirus / lentivirus, adenovirus, adeno-associated virus, alphavirus, vaccinia virus, or herpes simplex virus. In some embodiments, the viral vector is a lentivirus vector.

[0164] The base medium for culturing the cells is not limiting. In some embodiments, the medium comprises RPMI1640 medium with about 10% fetal bovine serum. In some embodiments, the culturing of part iv) comprises culturing the Tax protein expressing cells in RPMI1640 medium with 10% FBS in the presence of an effective amount of GM-CSF-Fc4 and IL-4-Fc4 for about 5-7 days, followed by maintaining the cells in culture in the presence of about 100-200 units / ml of IL-2.

[0165] In some embodiments, the method further comprises activating the monocyte-derived dendritic cells. In some embodiments, the monocyte-derived dendritic cells are stimulated with effective amounts of TNFα and LPS. In some embodiments, the TNFα is administered in an amount of about 10-250 ng / ml and the LPS is administered in an amount of about 1-5 μg / ml. In some embodiments, the cells are stimulated for 1-5 days. In some embodiments, the cells are stimulated for about 2 days.

[0166] In some embodiments, the monocyte-derived dendritic cells produced by the above methods have a constitutively mature and activated phenotype. In some embodiments, the monocyte-derived dendritic cells express one or more dendritic cell maturation and activation markers. In some embodiments, the monocyte-derived dendritic cell maturation and activation markers are selected from the group consisting of CD83, CD80, CD86, and CD70. In some embodiments, the monocyte-derived dendritic cells further express 4-1BBL and / or CD4.

[0167] In some embodiments, monocyte-derived dendritic cells are allowed to expand in culture for up to three months.

[0168] In some embodiments, the method further comprises loading the monocyte-derived dendritic cells with one or more antigens or antigen fragments or variants thereof. In some embodiments, the method comprises transducing the cells with a viral vector encoding one or more antigens or antigen fragments or variants thereof. In some embodiments, the antigen is a cancer antigen. In some embodiments, the monocyte-derived dendritic cells express human telomerase reverse transcriptase or an antigenic fragment or variant thereof. In some embodiments, the cancer antigen comprises a fusion protein comprising a proteasome targeting sequence of IκBα and a fragment of human telomerase reverse transcriptase. In some embodiments, the fusion protein comprises SEQ ID NO: 13.

[0169] In some embodiments, the method further comprises genetically engineering the monocyte-derived dendritic cells to express one or more HLA proteins. In some embodiments, the method comprises transducing the cells with a viral vector encoding one or more HLA proteins. In some embodiments, the method comprises genetically engineering the monocyte-derived dendritic cells to express one or more of HLA-A, HLA-B, or HLA-C. In some embodiments, the dendritic cells are genetically engineered to express HLA-A2.1. In some embodiments, the amino acid sequence of HLA-A2.1 is SEQ ID NO: 15.

[0170] In some embodiments, the functionality of modified monocyte-derived dendritic cells can be determined by their ability to prime naive lymphocytes. In some embodiments, this is performed by an assay called the mixed leukocyte reaction (MLR). In some embodiments, about 2 million unstimulated (naive) allogeneic PBMCs are mixed with modified monocyte-derived dendritic cells (e.g., 2×10 6 cells) at a ratio of about 100:1 without the addition of any cytokines or growth factors. 4 The cells are mixed with IL-2 (cells). After 10-14 days, IL-2 can be added to the cell culture. The immunophenotype and tumor killing activity of the proliferating cells can be determined by FACS and cytotoxicity assays, respectively.

[0171] In some embodiments, the invention provides monocyte-derived dendritic cells prepared by any of the above methods.

[0172] III. Methods for generating cytotoxic T lymphocytes In another embodiment, the present invention provides a method for producing cytotoxic T lymphocytes comprising culturing primary blood dendritic cells of the present invention with cells comprising naive lymphocytes for a period of time, thereby producing cytotoxic T lymphocytes.

[0173] In another embodiment, the present invention provides a method for producing cytotoxic T lymphocytes comprising culturing monocyte-derived dendritic cells of the present invention with cells comprising naive lymphocytes for a period of time, thereby producing cytotoxic T lymphocytes.

[0174] In another embodiment, the present invention provides a method for producing cytotoxic T lymphocytes, comprising: i) culturing the primary blood dendritic cells of the present invention together with cells comprising naive lymphocytes for a first period of time to produce a mixed culture of cells; ii) treating the mixed culture of cells with an effective amount of IL-2 and continuing to culture the cells for a second period of time; thereby producing cytotoxic T lymphocytes.

[0175] In another embodiment, the present invention provides a method for producing cytotoxic T lymphocytes, comprising: i) culturing the monocyte-derived dendritic cells of the invention with cells, including naive lymphocytes, for a first period of time to produce a mixed culture of cells; ii) treating the mixed culture of cells with an effective amount of IL-2 and continuing to culture the cells for a second period of time; thereby producing cytotoxic T lymphocytes.

[0176] In some embodiments, the cells comprising naive lymphocytes comprise naive PBMCs. In some embodiments, the naive lymphocytes are isolated from leukopacks. In some embodiments, the naive lymphocytes and dendritic cells are allogeneic.

[0177] The ratio of dendritic cells to naive PBMCs is not particularly limited. In some embodiments, the ratio of dendritic cells to naive PBMCs is about 1:10, about 1:25, about 1:50, about 1:100, about 1:250 or about 1:500. In some embodiments, the ratio of dendritic cells to naive PBMCs is about 1:100.

[0178] In some embodiments, the culturing in step i) is performed without the addition of exogenous cytokines. In some embodiments, the first period of time is about 2 to 3 days.

[0179] In some embodiments, the concentration of IL-2 comprises at least about 10 units / ml. In some embodiments, the concentration of IL-2 is at least about 50 units / ml. In some embodiments, the concentration of IL-2 is at least about 100 units / ml. In some embodiments, the concentration of IL-2 is about 100-200 units / ml. In some embodiments, the second period of time is about 2-6 weeks.

[0180] In some embodiments, the cytotoxic T lymphocytes are antigen-specific and induce cytolysis of target cells in an HLA-restricted manner.

[0181] In some embodiments, the cytotoxic T lymphocytes comprise CD3+ / CD56+ T cells that may kill target cells in a non-HLA-restricted manner. In some embodiments, the dendritic cells express 4-1BBL. In some embodiments, the dendritic cells have been modified to express 4-1BBL. In some embodiments, the non-HLA-restricted killing of the cells is mediated by ligand binding of NKG2D.

[0182] IV. Methods of Treatment with Cytotoxic T Lymphocytes In another embodiment, the present invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of cytotoxic T lymphocytes, wherein the cytotoxic T lymphocytes are produced using modified primary blood dendritic cells of the present invention.

[0183] In another embodiment, the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of cytotoxic T lymphocytes, wherein the cytotoxic T lymphocytes are produced using modified primary blood dendritic cells produced by the method of the invention.

[0184] In another embodiment, the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of cytotoxic T lymphocytes, wherein the cytotoxic T lymphocytes are produced using modified monocyte-derived dendritic cells of the invention.

[0185] In another embodiment, the invention provides a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of cytotoxic T lymphocytes, wherein the cytotoxic T lymphocytes are produced using modified monocyte-derived dendritic cells produced by the method of the invention.

[0186] In some embodiments, about 1×10 8 ~1×10 10 of cytotoxic T lymphocytes are administered prior to infusion. In some embodiments, about 5×10 9 of cells are administered prior to infusion.

[0187] In some embodiments, the disease to be treated is cancer. In some embodiments, the cancer cells express MICA / B. In some embodiments, the cancer is breast cancer.

[0188] In another embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of cytotoxic T lymphocytes produced by the methods of the present invention.

[0189] The cytotoxic T lymphocytes are administered in a pharma- ceutically acceptable carrier. In some embodiments, the cytotoxic T lymphocytes are frozen in the presence of a cryoprotectant, e.g., for storage and / or transportation, and then thawed prior to administration. See Part V below for exemplary cryoprotectant compositions.

[0190] The cells may be administered to a subject or patient using methods known in the medical arts. In some embodiments, the cells are injected into the subject. In some embodiments, the cells are transplanted into the subject. The cells may be administered as a single dose or multiple doses over a period of time. In some embodiments, the cells are administered about every 1-2 weeks, about every 3 weeks, about every 4 weeks, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, about every 3 months, about every 4 months, about every 5 months, about every 6 months, or about every year.

[0191] In some embodiments, the method of the present invention is combined with one or more other known treatments. In the treatment of cancer, one or more other known treatments can include radiation, surgery, chemotherapy or administration of other anti-cancer drugs, and combinations thereof. In some embodiments, the treatment of the present invention is combined with chemotherapy drugs.

[0192] V. DENDRITIC CELL VACCINES AND METHODS OF USE The dendritic cells and methods for their preparation described herein are particularly useful for preparing vaccines. As such, related dendritic cell compositions and vaccines are provided herein. In one embodiment, the vaccine is autologous to the subject. In some embodiments, the vaccine is allogeneic to the subject. In some embodiments, the dendritic cell vaccine is loaded with antibodies from cancer cells or pathogens present in the subject.

[0193] Dendritic cells, whether loaded with mature or immature antigens, can be frozen in a composition comprising a cryoprotectant. Numerous cryoprotectants are known to those skilled in the art. Examples of cryoprotectants include, but are not limited to, dimethylsulfoxide (DMSO), glycerol, ethanol, methanol, acetamide, glycerol monoacetate, propanediol, polyethylene glycol, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, D-lactose, i-inositol, choline chloride, amino acids, albumin (preferably human serum albumin), polyvinylpyrrolidone, dextran, sucrose, ficoll, inorganic salts, and hydroxyethyl starch. In a preferred embodiment, the cryoprotectant is DMSO. In some embodiments, the concentration of DMSO is 2-20%, more preferably 5-15%, and most preferably approximately 10%. The freezing medium may also contain one or more polyol compounds derived from carbohydrates, such as glucose, dextrose, sucrose, etc., preferably at a dextrose concentration of 2-30%, more preferably 5-10%, and most preferably 5%. Methods for freezing dendritic cells are known to those of skill in the art. See, for example, U.S. Patent Application Publication No. 2004 / 0253574, the contents of which are incorporated by reference. In some embodiments, the cryoprotectant is dimethyl sulfoxide (DMSO). In some embodiments, the concentration of DMSO is 5%-20%. Most preferably, the concentration of DMSO in the composition is approximately 10%.

[0194] Other suitable formulations for administration include isotonic sterile aqueous injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizers, preservatives, immunostimulants, cytokines, and adjuvants.

[0195] In one embodiment, the therapeutic cells are administered at approximately 1×10 in heat-inactivated autologous plasma and 10% dextrose. 8The cells are suspended at a final concentration of 1000 cells / ml. The cells are then diluted 1:1 with a mixture of heat-inactivated autologous plasma and 20% DMSO in heat-inactivated autologous plasma containing 5% dextrose and 10% DMSO. The final filled formulation is stored in a container suitable for cryopreservation. The vaccine is then frozen and stored at ≦150° C. in dry liquid nitrogen cryogen. The vaccine can be ready for administration after thawing without the need for washing and resuspension.

[0196] In some embodiments, the present invention provides the use of antigen-loaded dendritic cells according to the present invention for the preparation of a frozen medicament for the treatment or prevention of a disease, such as cancer or a pathogen infection, wherein the medicament is in a pharma- ceutically acceptable vehicle and is ready for administration upon thawing.

[0197] In another embodiment, the present invention provides a method of vaccinating a subject, comprising: i) thawing the frozen dendritic cell vaccine of the present invention; ii) administering the thawed vaccine to a subject; The method includes the steps of:

[0198] In another embodiment, the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of modified primary blood dendritic cells of the invention.

[0199] In another embodiment, the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of modified monocyte-derived dendritic cells of the invention.

[0200] Dendritic cell vaccines may be administered in a variety of ways, including, but not limited to, by injection (e.g., subcutaneous, intradermal, intravenous, intralymphatic, intraarticular, intramuscular, intraperitoneal), by continuous injection, sustained release from a graft, etc. In some embodiments, the dendritic cell vaccine is administered at 2-4 week intervals. The dendritic cell vaccine may be administered with a physiologically acceptable carrier, buffer, diluent, adjuvant, immunomodulator, etc. In some embodiments, the dendritic cell vaccine is autologous to the recipient or HLA-matched patient.

[0201] The dose of cells (e.g., activated T cells, or dendritic cells) administered to a subject is an effective amount effective to achieve a desired beneficial therapeutic response in the subject over time, e.g., inhibiting the growth of cancer cells or inhibiting an infection. In one embodiment, the dose is approximately 10 7 ~10 10 The dendritic cells are cells. A biological response modifier is optionally added for treatment with the dendritic cells or activated T cells of the present invention. For example, in some embodiments, the cells are optionally administered with an adjuvant, or a cytokine, such as GM-CSF, IL-12, or IL-2. In some embodiments, the dendritic cells are irradiated prior to administration to the subject. In some embodiments, about 1×10 per injection is administered. 8 ~Approx. 1×10 10 of cells are administered.

[0202] In some embodiments, the disease to be treated is cancer. In some embodiments, the cancer cells express MICA / B. In some embodiments, the cancer is breast cancer.

[0203] The cells may be administered to a subject or patient using methods known in the medical arts. In some embodiments, the cells are injected into the subject. In some embodiments, the cells are transplanted into the subject. The cells may be administered as a single dose or multiple doses over a period of time. In some embodiments, the cells are administered about every 1-2 weeks, about every 3 weeks, about every 4 weeks, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, about every 3 months, about every 4 months, about every 5 months, about every 6 months, or about every year.

[0204] In some embodiments, the method of the present invention is combined with one or more other known treatments. In the treatment of cancer, one or more other known treatments can include radiation, surgery, chemotherapy or administration of other anti-cancer drugs, and combinations thereof. In some embodiments, the treatment of the present invention is combined with chemotherapy drugs.

[0205] VI. Sample Embodiments This section describes exemplary compositions and methods of the invention, presented without limitation, as a series of paragraphs, some or all of which may be listed in alphanumeric order for clarity and efficiency. Each of these paragraphs may be suitably combined with one or more of the other paragraphs and / or disclosure content from anywhere in this application, including materials incorporated by reference. Some of the following paragraphs explicitly refer to and further qualify other paragraphs, providing some examples of suitable combinations without limitation.

[0206] 1. Modified dendritic cells expressing a functional Tax protein from the T-cell leukemia virus.

[0207] 2. The dendritic cell according to item 1, wherein the Tax protein is from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, HTLV-3 and HTLV-4.

[0208] 3. The dendritic cell according to any one of items 1 to 2, wherein the Tax protein is derived from HTLV-2.

[0209] 4. The dendritic cell according to any one of claims 1 to 3, which has a constitutively mature and activated phenotype.

[0210] 5. The dendritic cell according to any one of items 1 to 4, which is a human cell.

[0211] 6. The dendritic cell according to any one of items 1 to 5, which is CD205+.

[0212] 7. The dendritic cell according to any one of items 1 to 6, which is CD11c+.

[0213] 8. The dendritic cell according to any one of items 1 to 7, which expresses one or more dendritic cell maturation and activation markers.

[0214] 9. The dendritic cell according to paragraph 8, wherein the dendritic cell maturation and activation markers are selected from the group consisting of CD83, CD80, CD86, CD70, CCR7 and HLA-DR.

[0215] 10. The dendritic cell according to any one of items 1 to 10, which is capable of growing in suspension culture.

[0216] 11. The dendritic cell according to any one of items 1 to 10, which is TLR3+ and TLR4-.

[0217] 12. The dendritic cell according to any one of items 1 to 11, which expresses a truncated form of TLR7.

[0218] 13. The dendritic cell according to any one of items 1 to 12, which expresses TLR9.

[0219] 14. The dendritic cell according to any one of items 1 to 13, which produces IL-1A and TNFα.

[0220] 15. The dendritic cell according to any one of items 1 to 14, which produces IL-15.

[0221] 16. The dendritic cell according to any one of items 1 to 15, which produces IL-10 and TGFβ at negligible levels.

[0222] 17. A dendritic cell according to any one of items 1 to 16, which expresses c-Myc.

[0223] 18. The dendritic cell according to any one of items 1 to 17, in which transcription factors NF-κB, Stat3 and AP-1 are active.

[0224] 19. The dendritic cell according to any one of items 1 to 18, which is capable of inducing proliferation of naive lymphocytes in the absence of exogenously added IL-2.

[0225] 20. The dendritic cell according to any one of items 1 to 19, which has been genetically engineered to express HLA protein of 20.1 or higher.

[0226] 21. The dendritic cell according to any one of items 1 to 20, which has been genetically engineered to express HLA-A2.1.

[0227] 22. A dendritic cell according to any one of items 1 to 21, which presents one or more antigens or antigen fragments or mutants thereof.

[0228] 23. The dendritic cell according to any one of items 1 to 22, wherein the antigen is a cancer antigen.

[0229] 24. A dendritic cell according to any one of items 1 to 23, which expresses human telomerase reverse transcriptase or an antigen fragment or mutant thereof.

[0230] 25. The dendritic cell according to item 24, wherein the antigen comprises a fusion protein comprising full-length IκBα and a fragment comprising amino acids 301 to 700 of human telomerase reverse transcriptase.

[0231] 26. The dendritic cell of clause 25, wherein the fusion protein has SEQ ID NO:13.

[0232] 27. The dendritic cell according to any one of items 1 to 26, which presents the cancer antigen in an HLA-restricted manner.

[0233] 28. The dendritic cell according to any one of items 1 to 27, wherein the cancer antigen is introduced by viral transformation.

[0234] 29. The dendritic cell according to any one of items 1 to 28, wherein the virus is a lentivirus.

[0235] 30. A dendritic cell according to any one of items 1 to 29, which is capable of priming naive PBMCs to produce cytotoxic lymphocytes that recognize an antigen.

[0236] 31. A dendritic cell according to any of items 1 to 30, capable of priming naive PBMCs to generate cytotoxic lymphocytes capable of killing cells in a non-HLA restricted manner.

[0237] 32. A modified monocyte-derived dendritic cell, said dendritic cell expressing a functional Tax protein from T-cell leukemia virus.

[0238] 33. The dendritic cell of paragraph 32, wherein the Tax protein is from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, HTLV-3 and HTLV-4.

[0239] 34. The dendritic cell according to any one of items 32 to 33, wherein the Tax protein is from HTLV-2.

[0240] 35. The dendritic cell according to any one of items 32 to 34, which has a constitutive mature and activated phenotype.

[0241] 36. The dendritic cell according to any one of items 32 to 35, which is a human cell.

[0242] 37. The dendritic cell according to any one of items 32 to 36, which is CD205+.

[0243] 38. The dendritic cell according to any one of items 32 to 37, which expresses one or more dendritic cell maturation and activation markers.

[0244] 39. The dendritic cell of paragraph 38, wherein the dendritic cell maturation and activation markers are selected from the group consisting of CD83, CD80, CD86, and CD70.

[0245] The dendritic cell according to clause 38, further expressing 40.4-1BBL and / or CD4.

[0246] 41. The dendritic cell according to any one of items 32 to 40, which is capable of growth in suspension culture.

[0247] 42. The dendritic cell according to any one of items 32 to 41, which is capable of growing in culture for up to 3 months.

[0248] 43. The dendritic cell according to any one of items 32 to 42, which is TLR3+ and TLR4-.

[0249] 44. The dendritic cell according to any one of items 32 to 43, which expresses a truncated form of TLR7.

[0250] 45. The dendritic cell according to any one of items 32 to 44, which produces IL-1A and TNFα.

[0251] 46. ​​The dendritic cell according to any one of items 32 to 45, which produces TGFβ.

[0252] 47. The dendritic cell according to any one of items 32 to 46, which produces IL-15.

[0253] 48. The dendritic cell according to any one of items 32 to 47, which expresses c-Myc.

[0254] 49. The dendritic cell according to any one of items 32 to 48, which is capable of inducing proliferation of naive lymphocytes in the absence of exogenously added IL-2.

[0255] Item 50. The dendritic cell according to any one of Items 32 to 49, which is genetically engineered to express an HLA protein of 50.1 or more.

[0256] 51. The dendritic cell according to any one of items 32 to 50, which has been genetically engineered to express HLA-A2.1.

[0257] 52. The dendritic cell according to any one of items 32 to 51, which expresses one or more antigens or antigen fragments or mutants thereof.

[0258] 53. The dendritic cell according to any one of items 32 to 52, wherein the antigen is a cancer antigen.

[0259] 54. The dendritic cell according to any one of items 32 to 53, which expresses human telomerase reverse transcriptase or an antigen fragment or mutant thereof.

[0260] 55. The dendritic cell of paragraph 54, wherein the antigen comprises a fusion protein comprising a proteasome targeting sequence of IκBα and a fragment of human telomerase reverse transcriptase.

[0261] 56. The dendritic cell of clause 55, wherein the fusion protein has SEQ ID NO:13.

[0262] 57. The dendritic cell according to any one of items 32 to 56, which presents the cancer antigen in an HLA-restricted manner.

[0263] 58. The dendritic cell according to any one of items 32 to 57, wherein the antigen is introduced by viral transformation.

[0264] 59. The dendritic cell according to any one of items 32 to 58, wherein the virus is a lentivirus.

[0265] 60. The dendritic cell according to any of clauses 32 to 59, which is capable of priming naive PBMCs to generate cytotoxic lymphocytes that recognize an antigen.

[0266] 61. A dendritic cell according to any of clauses 32 to 60, capable of priming naive PBMCs to generate cytotoxic lymphocytes capable of killing cells in a non-HLA restricted manner.

[0267] 62. A method for generating modified primary blood dendritic cells, comprising: i) providing a sample of cells comprising immature dendritic cells, e.g., PBMCs; ii) treating the cells in culture with an effective amount of phytohemagglutinin (PHA); iii) treating the cells in culture with an effective amount of IL-2; iv) expressing a functional Tax protein from a T-cell leukemia virus in said cells and culturing said cells; v) depleting T cells from the cultured cells; The method comprising:

[0268] 63. The method of claim 62, wherein the PBMCs are isolated from leukopacks.

[0269] 64. The method according to any one of items 62 to 63, wherein the concentration of the PHA is in the range of about 1 to 5 μg / ml.

[0270] 65. The method according to any one of items 62 to 64, wherein the concentration of the PHA is about 5 μg / ml.

[0271] 66. The method according to any one of items 62 to 65, wherein the cells are treated with PHA for about 12 hours to about 36 hours.

[0272] 67. The method of any of paragraphs 62 to 66, wherein the cells are treated with PHA for about 24 hours.

[0273] 68. The method of any of paragraphs 62 to 67, wherein the cells are treated with IL-2 after treatment with PHA.

[0274] 69. The method of any of paragraphs 62 to 68, wherein the cells are treated with IL-2 after treatment with PHA.

[0275] 70. The method according to any of items 62 to 69, wherein the cells are treated with about 100 to 200 units / ml of IL-2 and cultured in a medium containing IL-2 for a period of time.

[0276] 71. The method of any of paragraphs 62 to 70, wherein the cells are cultured in a medium containing IL-2 for a period ranging from 2 to 10 days.

[0277] 72. The method according to any of paragraphs 62 to 71, wherein the cells are cultured in a medium containing about 100 to 200 units / ml of IL-2 for a period of about 4 to 5 days.

[0278] 73. The method of any of paragraphs 71 to 72, wherein the cells are manipulated to express a functional Tax protein after step iii).

[0279] 74. The method of clause 73, wherein the cells are transduced with a virus encoding a functional Tax protein from a T-cell leukemia virus.

[0280] 75. The method of clause 74, wherein the Tax protein is from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, and HTLV-3.

[0281] 76. The method of clause 75, wherein the Tax protein is from HTLV-2.

[0282] 77. The method of any one of clauses 74 to 76, wherein the virus is a lentivirus.

[0283] 78. The method of any of paragraphs 74 to 77, wherein the cells are transduced in the presence of about 10 μg / ml of polybrene.

[0284] 79. The method of any of paragraphs 73 to 78, wherein the transduced cells are further cultured in a medium containing about 100 units / ml of IL-2.

[0285] 80. The method according to any one of paragraphs 73 to 79, wherein the transduced cells are further cultured in a medium containing IL-2 for about 2 to 3 months.

[0286] 81. The method of any of paragraphs 73 to 78, wherein the transduced cells are cultured in a medium containing human serum and IL-2.

[0287] 82. The method of claim 81, wherein the transduced cells are cultured in a medium containing human serum for about 2 to 3 months.

[0288] 83. The method of clause 82, wherein the cells are cultured in a medium containing 5% heat-inactivated human AB serum.

[0289] 84. The method according to any one of items 70 to 80, wherein the medium comprises complete RPMI 1640 medium containing 10% fetal bovine serum.

[0290] 85. The method of any of paragraphs 81 to 83, wherein the medium comprises complete RPMI 1640 medium.

[0291] 86. The method of any of paragraphs 62 to 85, wherein the T cells are depleted by a composition comprising a molecule that binds to T cells.

[0292] 87. The method of clause 86, wherein the T cells are depleted by antibody.

[0293] 88. The method of claim 87, wherein the antibody is an anti-CD3 antibody.

[0294] 89. The method according to any one of items 87 to 88, wherein the antibody is conjugated to a magnetic bead.

[0295] 90. The method of any of paragraphs 62 to 89, wherein the dendritic cells have a constitutive mature and activated phenotype.

[0296] 91. The method according to any one of items 62 to 90, wherein the dendritic cells are human cells.

[0297] 92. The method according to any one of items 62 to 91, wherein the dendritic cells are CD205+.

[0298] 93. The method according to any one of items 62 to 92, wherein the dendritic cells are CD11c+.

[0299] 94. The method of any of paragraphs 62 to 93, wherein the dendritic cells express one or more dendritic cell maturation and activation markers.

[0300] 95. The method of paragraph 94, wherein the dendritic cell maturation and activation markers are selected from the group consisting of CD83, CD80, CD86, CD70, CCR7 and HLA-DR.

[0301] 96. The method of any of paragraphs 62 to 95, wherein the dendritic cells are capable of growth in suspension culture.

[0302] 97. The method according to any one of items 62 to 96, wherein the dendritic cells are TLR3+ and TLR4-.

[0303] 98. The method of any of items 62 to 97, wherein the dendritic cells express a truncated form of TLR7.

[0304] 99. The method according to any one of items 62 to 98, wherein the dendritic cells express TLR9.

[0305] 100. The method according to any one of items 62 to 99, wherein the dendritic cells produce IL-1A and TNFα.

[0306] 101. The method according to any one of items 62 to 100, wherein the dendritic cells produce IL-15.

[0307] 102. The method according to any one of items 62 to 101, wherein the dendritic cells produce negligible levels of IL-10 and TGFβ.

[0308] 103. The method according to any one of items 62 to 102, wherein the dendritic cells express c-Myc.

[0309] 104. The method according to any one of items 62 to 103, wherein the transcription factors NF-κB, Stat3 and AP-1 are active.

[0310] 105. The method according to any of items 62 to 104, wherein the dendritic cells are capable of inducing proliferation of naive lymphocytes in the absence of exogenously added IL-2.

[0311] 106. The method of any of paragraphs 62 to 105, wherein the dendritic cells are genetically engineered to express one or more HLA proteins.

[0312] 107. The method of any of paragraphs 62 to 106, wherein the dendritic cells are genetically engineered to express HLA-A2.1.

[0313] 108. The method of any of paragraphs 62 to 107, wherein the dendritic cells present one or more antigens or antigen fragments or variants thereof.

[0314] 109. The method according to any one of items 62 to 108, wherein the antigen is a cancer antigen.

[0315] 110. The method of any of paragraphs 62 to 109, wherein the cell expresses human telomerase reverse transcriptase or an antigenic fragment or variant thereof.

[0316] 111. The method of claim 110, wherein the cancer antigen comprises a fusion protein comprising a proteasome targeting sequence of IκBα and a fragment of human telomerase reverse transcriptase.

[0317] 112. The method of claim 111, wherein the fusion protein has SEQ ID NO:13.

[0318] 113. The method of any of paragraphs 62 to 112, wherein the cells present the cancer antigen in an HLA-restricted manner.

[0319] 114. The method according to any one of items 62 to 113, wherein the cancer antigen is introduced by viral transformation.

[0320] 115. The method of clause 114, wherein the virus is a lentivirus.

[0321] 116. The method according to any of paragraphs 62 to 115, wherein the dendritic cells are capable of priming naive PBMCs to generate cytotoxic lymphocytes that recognize an antigen.

[0322] 117. A method for generating monocyte-derived dendritic cells, comprising: providing a sample of cells comprising immature dendritic cells derived from monocytes; and expressing a functional Tax protein from a T-cell leukemia virus in said cells. The method comprising:

[0323] 118. A method for generating monocyte-derived dendritic cells, comprising: i) providing adherent monocytic cells; ii) culturing the cells in a medium containing effective amounts of GM-CSF and IL-4; iii) expressing a functional Tax protein from a T-cell leukemia virus in said cell; iv) culturing said functional Tax protein expressing cells in a medium containing effective amounts of GM-CSF and IL-4; The method comprising:

[0324] 119. The method of claim 118, further comprising culturing the cells in a medium containing IL-2 after step iv).

[0325] 120. The method according to any one of items 117 to 119, wherein IL-4 and / or GM-CSF are fused to Fc4.

[0326] 121. The method of any of paragraphs 117 to 120, wherein the adherent monocytes are derived from approximately 4 million PBMCs.

[0327] 122. The method of any of paragraphs 117 to 121, wherein the culturing of part ii) comprises culturing the cells in a medium comprising conditioned medium from GM-CSF-Fc4 / IL-4-Fc4 producing 293 cells.

[0328] 123. The method of paragraph 122, wherein the cells are cultured in medium containing the conditioned medium at about a 1:10 dilution for about 7 days.

[0329] 124. The method of any of paragraphs 117 to 123, wherein the cells are transduced with a virus encoding a functional Tax protein from a T-cell leukemia virus.

[0330] 125. The method of paragraph 124, wherein the Tax protein is from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, and HTLV-3.

[0331] 126. The method of clause 124, wherein the Tax protein is from HTLV-2.

[0332] 127. The method of any of clauses 123 to 126, wherein the virus is a lentivirus.

[0333] 128. The method of any of paragraphs 123 to 127, wherein the cells are transduced in the presence of about 10 μg / ml of polybrene.

[0334] 129. The method according to any of paragraphs 117 to 128, wherein the medium comprises RPMI 1640 medium having about 10% fetal bovine serum.

[0335] 130. The method according to any of items 117 to 129, wherein the culturing step iv) comprises culturing the Tax protein-expressing cells in RPMI1640 medium containing 10% FBS for about 5 to 7 days in the presence of GM-CSF-Fc4 and IL-4-Fc4, and subsequently maintaining the cells in culture in the presence of about 100 units / ml of IL-2.

[0336] 131. The method according to any one of items 117 to 130, further comprising activating the monocyte-derived dendritic cells.

[0337] 132. The method of paragraph 131, wherein said activating comprises stimulating said cells with TNFα and LPS.

[0338] 133. The method of claim 132, wherein the TNFα is administered in an amount of about 10 to 250 ng / ml, and the LPS is administered in an amount of about 1 to 5 μg / ml.

[0339] 134. The method of any of items 131 to 133, wherein the cells are stimulated for 1 to 5 days.

[0340] 135. The method of paragraph 134, wherein the cells are stimulated for about 2 days.

[0341] 136. The method of any of paragraphs 117 to 135, wherein the Tax protein is from a human T-cell leukemia virus selected from the group consisting of HTLV-1, HTLV-2, HTLV-3 and HTLV-4.

[0342] 137. The method of any of paragraphs 117 to 136, wherein the Tax protein is from HTLV-2.

[0343] 138. The method of any of paragraphs 117 to 137, wherein the dendritic cells have a constitutive mature and activated phenotype.

[0344] 139. The method according to any one of items 117 to 138, wherein the dendritic cells are human cells.

[0345] 140. The method of any one of items 117 to 139, wherein the dendritic cells are CD205+.

[0346] 141. The method of any of paragraphs 117 to 140, wherein the dendritic cells express one or more dendritic cell maturation and activation markers.

[0347] 142. The method of any one of paragraphs 117 to 141, wherein the dendritic cell maturation and activation marker is selected from the group consisting of CD83, CD80, CD86, and CD70.

[0348] 143. The method of clause 142, wherein the dendritic cells further express 4-1BBL and / or CD4.

[0349] 144. The method of any of paragraphs 117 to 143, wherein the dendritic cells are capable of growth in suspension culture.

[0350] 145. The method of any of paragraphs 117 to 144, wherein the dendritic cells are allowed to grow in culture for up to 3 months.

[0351] 146. The method according to any one of items 117 to 145, wherein the dendritic cells are TLR3+ and TLR4-.

[0352] 147. The method of any of paragraphs 117 to 146, wherein the dendritic cells express a truncated form of TLR7.

[0353] 148. The method according to any one of items 117 to 147, wherein the dendritic cells produce IL-1A and TNFα.

[0354] 149. The method according to any one of items 117 to 148, wherein the dendritic cells produce TGFβ.

[0355] 150. The method according to any one of items 117 to 149, wherein the dendritic cells produce IL-15.

[0356] 151. The method according to any one of items 117 to 150, wherein the dendritic cells express c-Myc.

[0357] 152. The method of any of paragraphs 117 to 151, wherein the dendritic cells are capable of inducing proliferation of naive lymphocytes in the absence of exogenously added IL-2.

[0358] 153. The method of any of paragraphs 117 to 152, wherein the dendritic cells are genetically engineered to express one or more HLA proteins.

[0359] 154. The method of any of paragraphs 117 to 153, wherein the dendritic cells are genetically engineered to express HLA-A2.1.

[0360] 155. The method of any of paragraphs 117 to 154, wherein the dendritic cells express one or more antigens or antigen fragments or variants thereof.

[0361] 156. The method according to any one of items 117 to 155, wherein the antigen is a cancer antigen.

[0362] 157. The method of any of paragraphs 117 to 156, wherein the dendritic cells express human telomerase reverse transcriptase or an antigenic fragment or variant thereof.

[0363] 158. The method of claim 157, wherein the cancer antigen comprises a fusion protein comprising a proteasome targeting sequence of IκBα and a fragment of human telomerase reverse transcriptase.

[0364] 159. The method of claim 158, wherein the fusion protein has sequence number 13.

[0365] 160. The method of any of paragraphs 154 to 159, wherein the cells present the antigen in an HLA-restricted manner.

[0366] 161. The method of any of paragraphs 154 to 160, wherein the antigen is introduced by viral transformation.

[0367] 162. The method of clause 161, wherein the virus is a lentivirus.

[0368] 163. The method according to any of paragraphs 117 to 162, wherein the dendritic cells are capable of priming naive PBMCs to generate cytotoxic lymphocytes that recognize an antigen.

[0369] 164. A dendritic cell prepared by the method according to any one of items 62 to 163.

[0370] 165. A method for producing cytotoxic T lymphocytes, comprising: Culturing the dendritic cells according to any one of items 1 to 61 together with cells containing naive lymphocytes for a certain period of time. thereby producing cytotoxic T lymphocytes.

[0371] 166. A method for producing cytotoxic T lymphocytes, comprising: i) culturing the dendritic cells according to any one of paragraphs 1 to 61 together with cells comprising naive lymphocytes for a first period of time to produce a mixed culture of cells; ii) treating the mixed culture of cells with an effective amount of IL-2 and continuing to culture the cells for a second period of time; thereby producing cytotoxic T lymphocytes.

[0372] 167. The method of any of items 165 to 166, wherein the cells containing naive lymphocytes include naive PBMCs.

[0373] 168. The method of any of paragraphs 165 to 167, wherein the naive lymphocytes are isolated from leukocytic cells.

[0374] 169. The method of any of paragraphs 165 to 168, wherein the naive lymphocytes and the dendritic cells are allogeneic.

[0375] 170. The method of any of paragraphs 165 to 169, wherein the ratio of dendritic cells to naive PBMCs is about 1:100.

[0376] 171. The method according to any one of items 166 to 170, wherein the culturing in step i) is carried out without adding exogenous cytokines.

[0377] 172. The method of any of paragraphs 166 to 172, wherein the first period is about 2 to 3 days.

[0378] 173. The method according to any one of items 166 to 172, wherein the concentration of IL-2 is about 100 to 200 units / ml.

[0379] 174. The method of any one of paragraphs 166 to 173, wherein the second period is 2 to 6 weeks.

[0380] 175. The method of any of paragraphs 166 to 174, wherein the cytotoxic T lymphocytes are antigen-specific and induce cytolysis of target cells in an HLA-restricted manner.

[0381] 176. The method of any of paragraphs 166 to 175, wherein the cytotoxic T lymphocytes comprise CD3+ / CD56+ T cells that may kill target cells in a non-HLA-restricted manner.

[0382] 177. The method of any of paragraphs 166 to 176, wherein the dendritic cells express 4-1BBL.

[0383] 178. The method of any of paragraphs 166 to 177, wherein the dendritic cells have been modified to express 4-1BBL.

[0384] 179. The method of any of paragraphs 176 to 178, wherein the non-HLA-restricted killing of the cells is mediated by ligand binding of NKG2D.

[0385] 180. A pharmaceutical composition comprising an effective amount of cytotoxic T lymphocytes produced by the method according to any one of items 165 to 179.

[0386] 181. A method for treating a disease or condition in a subject, comprising administering to the subject an effective amount of cytotoxic T lymphocytes, the cytotoxic T lymphocytes being produced using dendritic cells according to any one of paragraphs 1 to 61.

[0387] 182. A method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of cytotoxic T lymphocytes, wherein the cytotoxic T lymphocytes are produced by a method according to any one of paragraphs 165 to 179.

[0388] 183. A method for treating a disease or condition in a subject, comprising administering to the subject an effective amount of a dendritic cell according to any one of paragraphs 1 to 61.

[0389] 184. The method according to any one of items 181 to 183, wherein the disease is cancer.

[0390] 185. The method of claim 184, wherein the cancer cells express MICA / B.

[0391] 186. The method according to any one of items 181 to 185, wherein the cancer is breast cancer.

[0392] 187. The method of any one of paragraphs 181 to 186, wherein the subject is a mammal.

[0393] 188. The method of any one of paragraphs 181 to 187, wherein the subject is a human.

[0394] 189. A method for generating modified primary blood dendritic cells, comprising: i) providing a sample of cells comprising immature dendritic cells; ii) expressing a functional Tax protein from a T-cell leukemia virus in said cell; and The method comprising:

[0395] 190. A method for generating modified primary blood dendritic cells, comprising: i) providing a sample of cells comprising dendritic precursor cells; ii) expressing a functional Tax protein from a T-cell leukemia virus in said cells and culturing said cells; iii) culturing the cells to induce differentiation into dendritic cells; iv) depleting T cells from the cultured cells; The method comprising:

[0396] 191. A method for generating monocyte-derived dendritic cells, comprising: i) providing adherent monocytic cells; ii) expressing in said cell a functional Tax protein from a T-cell leukemia virus; iii) culturing the cells to induce differentiation into dendritic cells; The method comprising:

[0397] Application of the teachings of the present invention to a particular problem is within the capabilities of one of ordinary skill in the art in light of the teachings contained herein. Examples of compositions and methods of the present invention are evident in the non-limiting examples that follow. EXAMPLES

[0398] Example 1. Development of human primary blood and monocyte derived dendritic cell lines HTLV-2 Tax was utilized as a molecular tool for its role in regulating DC function. To prevent potential loss of DCs during the culture of Tax-transduced PBMCs, transduced cells were negatively selected using anti-CD3 magnetic beads to deplete T cells. Two CD3-negative Tax-GFP+ cell lines from transduced PBMCs of 10 blood donors were continuously evolved and grown for 6 months without loss of growth potential. These two DC cell lines established from two different blood donors expressed dendritic cell-associated markers. These cells were lineage-negative and expressed CD11c and CD205, as well as DC maturation and activation molecules including CD83, CD80, CD86, CD70, CCR7, and HLA-DR (Figures 1a and 1b). These two cell lines were designated ihv-DC1 and ihv-DC2 and represented a subset of human CD11c+ / CD205+ blood dendritic cells that maintained a constitutive mature and activated phenotype.

[0399] ihv-DCs grew in suspension in normal culture conditions, with some cells adhering and displaying typical dendrites. To demonstrate the ability of Tax in promoting DC growth, adherent monocytes from PBMCs were first stimulated with GM-CSF / IL-4 to generate monocyte-derived dendritic cells (MoDCs) (Fig. 1c). Some of these MoDCs displayed dendrites, and MoDCs were subsequently transduced with Tax lentivirus. Transduced MoDC-Tax cells were detached during culture, and these cells were allowed to proliferate for up to 3 months. MoDC-Tax cells displayed a mature and activated phenotype (Fig. 1d). These results suggested that Tax was able to promote the growth of both blood- and monocyte-derived DCs that had lost cell adhesion in normal culture conditions.

[0400] result Expression of TLRs, cytokines and intracellular signaling molecules in ihv-DCs The intracellular dsRNA-sensing TLR3 receptor was expressed, whereas TLR4 was negative in both ihv-DC and MoDC-Tax cells (Fig. 2a). A truncated form of TLR7 was detected in both ihv-DC and MoDC-Tax, but not in MoDC (Fig. 2b), suggesting that Tax upregulated TLR7 in DCs. TLR9 was highly expressed in ihv-DC2 and weakly in MoDC (Fig. 2b). Regardless of TLR expression status, these receptors were no longer required to induce DC maturation and activation, as these established DC cell lines persistently expressed high levels of costimulatory molecules, such as CD80, CD86, CD70, and CD83.

[0401] Proinflammatory cytokines, such as IL-1A and TNFα, were produced in all ihv-DCs and MoDCs, whereas immunoregulatory factors, including IL-10 and TGFβ, were expressed at negligible levels in ihv-DCs and MoDC-Tax cells produced TGFβ (Fig. 2c-2f). IL-15, which favors the development of CD8+ cytotoxic lymphocytes, was produced at high levels in all established DC cell lines and activated MoDCs (Fig. 2c-2f). Taken together, the TLR and cytokine expression profiles in ihv-DCs correlated well with the phenotype of mature and activated blood DCs generated by conventional methods.

[0402] Tax had the ability to drive cell cycle progression as evidenced by overexpression of c-Myc in both ihv-DC and MoDC-Tax cells (Fig. 2g). The pro-survival Bcl-2 protein, Bcl-xL, was specifically upregulated only in DCs that expressed Tax (Fig. 2h). The cell cycle regulators, pRb and cdc2, were phosphorylated in ihv-DC (Fig. 2i). Phosphorylated forms of Stat1, Stat3, and Stat5 were detected in all DCs (Fig. 2l), and activation of transcription factors including NF-κB, Stat3, and AP-1 was also observed in ihv-DC (Fig. 2j). This suggested that Tax immortalized blood DCs by a mechanism similar to Tax-mediated immortalization of CD4+ T cells through induction of tumorigenic activation and cell cycle progression.

[0403] Induction of antigen-specific CTLs by ihv-DCs HLA-A and -B alleles from ihv-DCs were cloned and sequenced. Sequence analysis showed that ihv-DC1 cells were HLA-A30 / B71, whereas ihv-DC2 cells were HLA-A2.1 / B40. As HLA-A2 is one of the most frequent HLA alleles, it was determined first to test the functionality of ihv-DC2 in inducing cytotoxic lymphocytes. A mixed leukocyte reaction (MLR) assay was set up by mixing ihv-DC2 cells with naive PBMCs isolated directly from HLA-A2+ healthy blood donors at a ratio of 1:100. It was observed that ihv-DC2 cells induced a dramatic increase in donor lymphocytes in the absence of recombinant IL-2 in the first 2-3 days. The reactive lymphocytes continued to proliferate in the presence of recombinant IL-2 for 4-6 weeks. Two weeks after MLR, ihv-DC-activated, proliferating lymphocytes generated from PBMCs of different donors displayed the predominant immunophenotype of effector CD8+ T cells, expressed abundant amounts of the cytotoxic components, perforin and granzyme B, and produced large amounts of IFNγ and TNFα (Fig. 3a, 3b, and 3c). ihv-DC cells were completely eliminated as evidenced by the absence of Tax-GFP green fluorescent signal in MLR cultures. ihv-DC2-activated CTLs were evaluated for recognition of the Tax antigen. HLA-A2.1 was first expressed in HLA-A2-negative MT4 cells (Tax+HTLV-1-transformed T cells) (Fig. 3d). It was found that ihv-DC2-activated CTLs induced cytolysis of MT4-A2.1 cells more potently than that of MT4 cells (Fig. 3e). A target cell model was developed using NIH3T3 cells modified by sequential lentiviral transduction of luciferase, beta-2-microglobulin (B2M) and HLA-A2.1 with or without Tax-FLAG (Fig. 3f). Tax was localized mainly in cytoplasmic puncta (Fig. 3f). It was shown that ihv-DC-activated CTL induced cytolysis of approximately 90% of Tax-expressing HLA-A2+3T3 cells at an E:T ratio of 10:1 (Fig. 3g and 3h).

[0404] Because Tax-immortalized DCs overexpressed and activated proto-oncogene proteins, such as c-Myc, to promote cell cycle progression and proliferation, it is likely that CTLs activated by ihv-DCs could recognize cancer cells in which these oncoproteins are often deregulated. To test this hypothesis, we selected two HLA-A2+ cancer cell lines, A375 (metastatic melanoma) and PANC-1 (pancreatic cancer), and one HLA-A2-negative cell line, H1299 (lung cancer). It was shown that ihv-DC2-activated CTLs effectively induced cytolysis of A375 and PANC-1 cells but had no apparent killing activity in H1299 cells (Figures 4a-4d). HLA-A2.1 cDNA was then introduced into H1299 cells (Fig. 4e), and it was found that HLA-A2.1+H1299 cells became susceptible to killing mediated by ihv-DC2-activated CTLs (Fig. 4f, 4g). Collectively, these results supported the notion that ihv-DC2-activated CTLs had the capacity to mediate HLA-restricted target cell killing in Tax-expressing and cancer cells.

[0405] Expression of hTERT fragments in ihv-DCs generating hTERT-specific CTLs One significant advantage of established dendritic cell lines is that, unlike primary DCs, DC cell lines can be genetically engineered to improve their function in antigen presentation. To test this idea, HLA-A2.1 was introduced into HLA-A2-negative ihv-DC1 cells to generate ihv-DC1-A2.1 cells. Nearly 100% of ihv-DC1-A2.1 cells were HLA-A2+ (Fig. 5a). Known universal tumor antigens were selected and introduced into ihv-DC1-A2.1 cells. hTERT is overexpressed in more than 90% of human cancers and constitutes a myriad of recognized CTL epitopes24. To facilitate antigen processing, a fusion construct, pTERT, was designed, which consisted of the proteasome targeting sequence of IκBα and a fragment of hTERT (aa301-700) covering six HLA-A2-restricted CTL epitopes (Fig. 5b, 5c). As ihv-DCs showed constitutive activity of NF-κB, pTERT degraded at a high rate as expected, potentially producing abundant amounts of TERT peptide in the formation of complexes with HLA molecules. pTERT was introduced into ihv-DC1-A2.1 cells via lentiviral transduction. It was found that pTERT was barely detectable in DMSO-treated ihv-DC1 / pTERT cells, and pretreatment of these DCs with MG132, a chemical inhibitor of the proteasome, induced the accumulation of pTERT (Fig. 5c). Next, a target cell model was designed using modified 3T3L-A2.1 cells by lentiviral transduction of FLAG-TERT (aa301-700) (Fig. 5d). ihv-DC1-A2.1 and ihv-DC1-A2.1 / pTERT cells were used to prime naive PBMCs to generate cytotoxic lymphocytes. It was found that ihv-DC1-A2.1 / pTERT-activated CTLs induced cytolysis of modified NIH3T3 cells much more potently than ihv-DC1-A2.1-activated CTLs (Figures 5e, 5f). These findings supported the idea that established ihv-DCs could be further modified to deliver a given tumor antigen into cells.

[0406] Non-HLA-restricted cancer cell killing by ihv-DC1-activated CTLs Unlike ihv-DC2 cells, ihv-DC1-activated CTLs constituted a major population of CD3+ / CD56+ T cells (Fig. 6a), which resembled the composition of cytokine-induced killer cells (CIKs) that kill cancer cells through the interaction of NKG2D and its ligands in a non-HLA-restricted manner. NKG2D ligands, including MICA / MICB, were selectively expressed on the surface of cancer cells and virus-infected cells, but were not detected or expressed even at very low levels in normal cells25,26. This unique feature serves as a selective target for CTL-mediated killing in cancer cells. As shown in Fig. 6b, MICA / B was expressed in H1299, HeLa, and A375 cells, but was not detected in normal human fibroblasts (NHFs). It was found that ihv-DC1-activated CTLs potently induced cytolysis of all these cancer cells, regardless of HLA type (Fig. 6c-6f). In contrast, these CTLs had no killing activity in NHF cells (FIG. 6g).

[0407] To further explore this cytotoxic mechanism, blocking antibodies were utilized against NKG2D. K562 cells were known to be resistant to CTL-mediated killing because they lack surface expression of HLA molecules but were susceptible to NK cells killing target cells through the interaction of NKG2D and its ligands. A fixed amount of anti-NKG2D antibody (50 μg / ml) effectively protected K562 cells from killing by ihv-DC1-activated CTLs at lower E:T ratios (1.25:1 and 2.5:1) (Figure 7a). At higher E:T ratios (5:1 and 10:1), anti-NKG2D antibody appeared to be less effective in protecting K562 cells from killing (Figure 7a). Next, a fixed E:T ratio (2.5:1) and increasing amounts of anti-NKG2D antibody were used. This blocking antibody was shown to prevent ihv-DC1-CTL-mediated killing of K562 cells in a dose-dependent manner (Figure 7b). Similarly, anti-NKG2D antibody (50 μg / ml) was found to rescue melanoma cells from cytolysis by ihv-DC1-activated CTLs at lower E:T ratios (Figure 7c). NIH3T3 cells were further modified by expressing luciferase with or without human MICA (Figure 7d). Because human HLA alleles were absent from NIH3T3 cells, it was unlikely that killing of these target cells by human CTLs was mediated through TCR recognition. It was indeed found that ihv-DC1-activated CTLs induced cytolysis of 3T3L / hMICA cells more potently than killing of 3T3L / vector cells (Figures 7e, 7f). Collectively, these findings indicated that ihv-DC1-activated CTLs, with a predominant composition of CD3+ / CD56+ T cells, had the ability to induce cytolysis of cancer cells through the interaction of NKG2D and its ligands expressed in cancer cells and also kill target cells in an HLA-dependent manner, as shown in Figure 5. Furthermore, the high density of NKG2D expressed in ihv-DC1-activated CTLs could potentially overcome the neutralizing effect from soluble NKG2D ligands.

[0408] IHV-DC1-Based Immunotherapy of Human Breast Cancer in the NSG Mouse Model To explore the application of ihv-DC-based immunotherapy, ihv-DC1 cells were selected for their unique ability to generate abundant amounts of CD3+ / CD56+ T cells that kill cancer cells in a non-HLA-restricted manner. Three breast cancer cell lines were chosen, including MCF-7, MCF-7 / ADR (a multiple chemotherapeutic drug-resistant derivative of MCF-7), and HER2 / Neu+HCC1954 cells. It was observed that ihv-DC1-activated CTLs very potently induced cytolysis of these breast cancer cells (Fig. 8a, 8b). MICA / B was highly expressed in all tested breast cancer cell lines (Fig. 8c). NKG2D-blocking antibodies (100 μg / ml) efficiently prevented cancer cell killing from CTLs (Fig. 8d).

[0409] These promising in vitro results prompted us to investigate T cell therapy in an orthotopic breast cancer NSG mouse model. In the first animal experiment, ihv-DC1-activated CTLs dramatically suppressed the growth of established HCC1954 tumors in mammary fat pads after T cell therapy described in the above method (Fig. 9a-9c). More notably, ihv-DC1-activated CTLs efficiently inhibited lung metastasis of HCC1954 tumors. No metastatic sites were found in all mice in the CTL-treated group, while micrometastases were detected in the lungs from all mice in the non-treated group (Fig. 9d). Anti-human CD3 staining was then applied to evaluate the in vivo trafficking pattern of the injected CTLs. It was shown that human CD3+ T cells were detected in the tumor mass, spleen and lung tissues, and some CD3+ cells were found in the liver in CTL group mice (Fig. 9e). No infiltrating T cells were found in the heart and kidney in CTL group mice (Fig. 9e). The second animal experiment utilized the MCF-7 / ADR breast cancer NSG mouse model. Similar to the HCC1954 NSG model, it was observed that ihv-DC1-activated CTLs inhibited the growth of MCF-7 / ADR tumors in mammary fat pads (Fig. 10a-10b). No weight loss or other side effects were observed in CTL group mice over the 60-day experimental period (Fig. 10c). More importantly, ihv-DC1-activated CTLs effectively inhibited the lung metastasis of MCF-7 / ADR tumors in CTL group mice (Fig. 10d). The injected CTLs infiltrated the tumor mass, liver, spleen, and lung tissues, but not the heart and kidneys (Fig. 10e). These data therefore demonstrated the therapeutic efficacy of ihv-DC1-activated CTLs in the mouse model and also showed the desired in vivo trafficking pattern.

[0410] Herein we describe the generation of an immortalized, constitutively activated human primary blood dendritic cell (DC) line, ihv-DC, by expressing the viral Tax protein that induces cell cycle progression through tumorigenic activation. ihv-DC are a subset of CD11c+ / CD205+DC that persistently express costimulatory molecules and can prime donor-derived lymphocytes to generate CD8+ effector cytotoxic T lymphocytes (CTLs) that can recognize and kill Tax-expressing and cancer cells in an HLA-restricted manner. ihv-DC can be engineered to express human telomerase reverse transcriptase (hTERT) fragments in priming T cells to generate HLA-A2-restricted hTERT-specific CTLs. In addition to priming T cells to mediate HLA-restricted target cell killing, one of the ihv-DC lines, ihv-DC1, promotes robust expansion of CD3+ / CD56+ CTLs with broad anticancer activity through the interaction of NKG2D with its ligand expressed on cancer cells. ihv-DC1-activated CTLs potently inhibit the growth of established tumors and suppress lung metastasis of breast cancer cells in an orthotopic NSG mouse model. More importantly, ihv-DC1-activated CTLs can infiltrate primary tumor loci and metastatic sites, mimicking natural in vivo CTL trafficking characteristics. These established human blood DCs facilitate the development of cell-based immunotherapies.

[0411] A novel method is described herein to generate human blood dendritic cell lines by selecting PHA / IL-2 stimulated Tax-transduced PBMCs from blood donors. The established ihv-DCs can be reliably grown and maintained in long-term culture without losing their maturation and activation phenotype. A method is also described herein to expand monocyte-derived dendritic cells (MoDCs) by expressing Tax in GM-CSF / IL-4-induced DCs differentiated from adherent monocytes. In some embodiments, the second method allows for the growth of mature and activated MoDCs for up to three months. This method can generate a reasonable number of activated MoDCs from millions of PBMCs, which is a significant advantage over conventional MoDC methods by eliminating the undesirable apheresis procedure. Moreover, unlike primary DCs, established DCs can be easily modified by expressing a given tumor antigen intracellularly to present multiple antigenic epitopes in priming CD8 T cells and enhance the generation of anti-cancer immunity.

[0412] The ability of Tax in immortalizing and activating blood DCs has not been reported before. Without being bound by theory, the achievement of the effort is attributed to cell selection and culture techniques at several levels. First, PHA / IL-2 stimulates T cell proliferation, and activated T cells may secrete soluble factors that potentially allow blood immature DCs to be transduced by Tax lentivirus, since primary immature DCs are highly resistant to lentiviral transduction. Second, the depletion of T cells at the early selection stage is a necessary step to allow a small number of DCs to grow healthy, since the majority of Tax-transduced lymphocytes proliferate much faster than Tax-transduced DCs. Third, most Tax-transduced DCs may experience a "growth turning point" at 3 months after transformation, and some of them regain their growth strength after passing this stage. Therefore, a continuous culture effort is required to help Tax-transduced DCs become immortal.

[0413] One important phenotype is that ihv-DCs are constitutively activated without the need for further stimulation. Tax seemingly has the ability to promote DC maturation and activation. It is speculated that Tax-induced activation of NF-κB signaling plays an important role in the process of DC maturation and activation. Upon encountering an invading pathogen, the pathogen components stimulate TLRs, such as TLR3 and TLR4, in immature DCs. TLR3 or TLR4 binding activates the receptor-associated TRAF6, which then triggers the activation of downstream signaling molecules, including the IκB kinase complex, a central regulator of NF-κB signaling. Kobayashi, T. et al. TRAF6 is a critical factor for dendritic cell maturation and development. Immunity 19, 353-363 (2003); Hull, C., McLean, G., Wong, F., Duriez, PJ & Karsan, A. Lipopolysaccharide signals an endothelial apoptosis pathway through TNF receptor-associated factor 6-mediated activation of c-Jun NH2-terminal kinase. The Journal of Immunology 169, 2611-2618 (2002). It is well recognized that NF-κB is a key mediator in inducing DC maturation and activation.Rescigno, M., Martino, M., Sutherland, CL, Gold, MR & Ricciardi-Castagnoli, P. Dendritic cell survival and maturation are regulated by different signaling pathways, The Journal of Experimental Medicine 188, 2175-2180 (1998); Ardeshna, KM, Pizzey, AR, Devereux, S. & Khwaja, A. The PI3 kinase, p38 SAP kinase, and NF-κB signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells, Blood 96, 1039-1046 (2000). TRAF6 is also required to activate MAP kinases and result in AP-1 activation. Bradley, JR & Pober, JS Tumor necrosis factor receptor-associated factors (TRAFs). Oncogene 20, 6482-6491 (2001). Furthermore, Stat3 activity is important for IL-15-derived DCs to acquire antigen-presenting capacity and mediate the responses of divided CD8+ T cells. Okada, S., Han, S., Patel, ES, Yang, L.-J. & Chang, L.-J. STAT3 signaling contributes to the high effector activities of interleukin-15-derived dendritic cells. Immunology and Cell Biology 93, 461-471 (2015).Consistent with these reports, it has been demonstrated that ihv-DCs exhibit constitutive activity of NF-κB, AP-1 and Stat3, which is induced by Tax and, optionally, by inflammatory cytokines such as IL-1 and TNFα, contributing to their maturation and activation.

[0414] Both ihv-DC1 and ihv-DC2 cells have the ability to prime naive PBMCs to generate antigen-specific CTLs that induce cytolysis of target cells in an HLA-restricted manner. However, ihv-DC1 cells are seemingly different from ihv-DC2 cells because they can also induce and increase CD3+ / CD56+ T cells that can kill target cells in a non-HLA-restricted manner. By analyzing the expression profiles of DC markers, costimulatory molecules and cytokines, it has been shown that the level of 4-1BBL and the CD11c / 4-1BBL ratio tend to play an important role in determining the fate of the composition of cytotoxic lymphocytes. Compared to ihv-DC1 cells, ihv-DC2 cells express lower levels of 4-1BBL and higher levels of CD11c, promoting the generation of CD3+ / CD56- T cells. Conversely, ihv-DC1 cells induce the generation and expansion of CD3+CD56+T cells by expressing higher levels of 4-1BBL and lower levels of CD11c. To test this possibility, the expression of 4-1BBL was enhanced in DCs expressing low levels of 4-1BBL. We indeed found that DCs with much higher levels of 4-1BBL primed naive PBMCs to generate CD3+ / CD56+T cells as well as NK cells (data not shown). This phenomenon will be investigated in future studies. Therefore, ihv-DC1 cells are unique because they are able to prime naive PBMCs to generate abundant amounts of CD3+ / CD56+ effector T cells that can kill various cancer cells regardless of HLA type.

[0415] In theory, DC-primed T cells are antigen-specific and kill target cells in an HLA-restricted manner in an autologous transplantation setting. However, the number of tumor antigen-specific lymphocytes in healthy donors is extremely low, so it is a difficult task to generate an adequate number of tumor antigen-specific CTLs for cancer treatment using an autologous transplantation setting. Notably, up to 10% of naive lymphocytes exhibit alloreactivity and recognize tumor antigen / HLA complexes in a non-syngeneic setting. Rossjohn, J. & McCluskey, J. How a home-grown T cell receptor interacts with a foreign landscape, Cell 129, 19-20 (2007); Gras, S., Kjer-Nielsen, L., Chen, Z., Rossjohn, J. & McCluskey, J. The structural bases of direct T-cell allorecognition: implications for T-cell-mediated transplant rejection. Immunology and Cell Biology 89, 388-395 (2011); Morelli, A. E. & Thomson, A. W. Dondritic cells: regulators of alloimmunity and opportunities for tolerance induction. Immunological Reviews 196, 125-146 (2003). IHV-DC1-activated allogeneic CTLs were found to be antigen-specific and to recognize target cells presenting Tax antigen / HLA-A2 complexes and tumor antigen / HLA-A2 complexes. In addition, similar to NK cells, ihv-DC1-activated CTLs kill a broad range of human cancers through their ligand, NKG2D, which targets a group of stress-induced surface molecules typically expressed on cancer cells and virus-infected cells. Nausch, N. & Cerwenka, A. NKG2D ligands in tumor immunity. Oncogene 27, 5944-5958 (2008). Raulet, DH, Gasser, S., Gowen, BG, Deng, W. & Jung, H.Regulation of ligand for the NKG2D activating receptor.Annual Review of Immunology 31,413-441(2013);Verneris,MR,Karami,M.,Baker,J.,Jayaswal,A.& Negrin,RSRole of NKG2D signaling in the cytotoxicity of activated and expanded CD8+ T cells.Blood 103,3065-3072(2004);Karimi,M.et al.Silencing human NKG2D,DAP10,and DAP12 reduces cytotoxicity of activated CD8+ T cells and NK cells.The Journal of Immunology 175,7819-7828(2005). Through the interaction of NKG2D in CTLs with its ligands in cancer cells, ihv-DC1-induced CTLs release cytotoxic components including perforin and granzymes to cause cancer cell death. In this regard, ihv-DC1-activated CTLs function similarly to NK cells or cytokine-induced killer cells (CIKs) that kill cancer cells in a non-HLA-restricted manner. Marten, A. et al. Enhanced lytic activity of cytokine-induced killer cells against multiple myeloma cells after co-culture with idiotype-pulsed dendritic cells. Haematologica 86, 1029-1037 (2001); Wang, Y.-F. et al. Cytokine-induced killer cells co-cultured with complete tumor antigen-loaded dendritic cells, have enhanced selective cytotoxicity on carboplatin-resistant retinoblastoma cells.Oncology Reports 29,1841-1850(2013);Wongkajornsilp,A.et al.Sunitinib indirectly enhanced anti-tumor cytotoxicity of cytokine-induced killer cells and CD3+ CD56+ subset through the co-culturing dendritic cells.PloS One 8,e78980(2013);Castillo,E.F.,Stonier,S.W.,Frasca,L.& Schluns,K.S.Dendritic cells support the in vivo development and maintenance of NK cells via IL-15 trans-presentation.The Journal of Immunology 183,4948-4956(2009);Denman,C.J.et al.Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells.PloS One 7,e30264(2012);Domogala,A.,Madrigal,J.A.& Saudemont,A.Natural killer cell immunotherapy:from bench to bedside.Frontiers in Immunology 6,264(2015);Childs,R.W.& Carlsten,M.Therapeutic approaches to enhance natural killer cell cytotoxicity against cancer:the force awakens.Nature Reviews Drug Discovery 14,487-498(2015). Moreover, these CTLs still retain the ability to recognize target cells via tumor antigen / HLA complexes. Moreover, under HLA-matched conditions, these CTLs attack cancer cells via two mechanisms: TCR recognition in the TAA / HLA complex and interaction with NKG2D and its ligands; potentially conferring optimal therapeutic efficiency.

[0416] In summary, the ihv-DC model confers several salient features: (a) ihv-DCs are activated and mature DCs, thus eliminating the need for complex maturation and activation processes to obtain functional DCs; (b) ihv-DC cells constitutively express abundant amounts of costimulatory receptors, including CD80, CD86, and CD70, which stimulate antigen-specific CD8+ cytotoxic T cells to proliferate; (c) ihv-DCs also express the chemokine receptor CCR7, a lymphoid tissue homing receptor; (d) the ihv-DC model can provide a particularly useful tool for studying dendritic cell biology. These DCs can be genetically engineered to investigate the role of existing or putative costimulatory receptors in mediating or enhancing protective anti-cancer or anti-viral immunity.

[0417] Materials and Methods Cell lines, antibodies and cytokines. H1299, PANC-1, HCC1954, MCF-7, HeLa and NIH3T3 cell lines were obtained from ATCC. MCF-7 / ADR cell line was previously described. Wu, L. et al. The reversal effects of 3-bromopyruvate on multidrug resistance in vitro and in vivo derived from human breast MCF-7 / ADR cells. PloS One 9, e112132 (2014). A375 and NHF cell lines were kindly provided by Drs. Isaiah J. Fidler and Jieyu Zhu, respectively. MT4 cells were obtained from the NIH AIDS Reagent Program.

[0418] Recombinant IL-2 was obtained from the AIDS Research and Reference Reagent Program. TNFα was purchased from R&D, and LPS was from Sigma. Perforin (PRF1) and granzyme B (GZMB) antibodies were from Santa Cruz Biotechnology, and anti-beta-actin was from Sigma (St. Louis, MO). Anti-phospho-Stat proteins and anti-Bcl-2 family proteins were obtained from Cell Signaling. NKG2D blocking and isotype IgG control antibodies as well as various APC- or FITC-labeled antibodies were purchased from Biolegend. GM-CSF-Fc4 and IL-4-Fc4 proteins were generated from 293 cells transduced with lentiviral fusion gene constructs using a PCR-based cloning method with GM-CSF, IL-4, and IgG4 cDNAs obtained from Open Biosystems.

[0419] Lentiviral vectors, virus production and transformation. The Tax gene from HTLV-2 was fused to a fragment encoding enhanced green fluorescent protein (EGFP) and the tax2-gfp fusion fragment was cloned into a lentiviral vector in which the human elongation factor promoter drives transgene expression. Lentiviral vectors expressing HLA-A2 (cDNA from A375 cells), B2M (cDNA from normal lymphocytes) and MICA (cDNA from A375 cells) were constructed using a PCR-based cloning method. To generate the fusion gene constructs, pTERT (aa301-700), IκBα (cDNA from normal lymphocytes) and hTERT (aa301-700) (cDNA from U2OS cells) were amplified by high-fidelity PCR, enzymatically digested and inserted into the lentiviral vector.

[0420] To generate recombinant lentivirus, lentiviral constructs were transfected into 293 cells using SuperFect transfection reagent (Qiagen) along with a packaging plasmid mix containing expression plasmids for VSV-G, Gag-Pol and Rev (Invitrogen). Viral supernatants were collected and subjected to ultracentrifugation at 25,000 rpm / 4°C for 2 h. Viral pellets were resuspended and stored at -80°C.

[0421] To generate luciferase-expressing target cell lines, PANC-1, A375, HeLa, H1299, MCF-7, MCF-7 / ADR, HCC1954, MT4 and NIH3T3 cells were transduced with luciferase lentivirus at a multiplicity of infection (MOI) of 10. Expression of luciferase in these cell lines was verified by luciferase activity assay using a kit from Promega according to the manufacturer's recommended protocol. pTERT-expressing DCs were generated by pTERT lentiviral transduction for ihv-DCs at MOI20.

[0422] Generation of ihv-DC cell lines. Leukopacks were obtained from the New York Blood Bank. Human peripheral blood mononuclear cells (PBMCs) were isolated from the leukopacks and stimulated with PHA (5 μg / ml) for 24 h, followed by the addition of recombinant IL-2 (100 units / ml). Activated PBMCs were cultured for 4-5 days and then transduced with Tax2-GFP lentivirus in the presence of polybrene (10 μg / ml). Transduced cells were continuously cultured in complete RPMI1640 medium containing 10% fetal bovine serum (Sigma) and 100 units / ml of recombinant IL-2. Alternatively, transduced cells were cultured in RPMI1640 medium supplemented with 5% heat-inactivated human AB serum (Sigma). Two to three weeks after transduction, transduced cells were negatively selected by anti-CD3 magnetic beads (Life technologies) and depleted of T cells. CD3-negative cells were continuously maintained in culture and analyzed for immunophenotype approximately 3 months after transduction. Two DC cell lines were established from two blood donors and designated ihv-DC1 and ihv-DC2. ihv-DC1 cells lost CD40 during the course of culture, and CD40 was restored by CD40 lentiviral transduction. ihv-DCs were grown in RPMI1640 medium supplemented with 10% FBS or 5% heat-inactivated human AB serum in the presence of IL-2 (50-100 units / ml).

[0423] To generate monocyte-derived DCs, adherent monocytes from 4 million PBMCs were stimulated with GM-CSF-Fc4 / IL-4-Fc4 using conditioned medium of GM-CSF-Fc4 / IL-4-Fc4-producing 293 cells at a 1:10 dilution for 7 days. Differentiated DCs were subsequently transduced with Tax2-GFP lentivirus. Tax-expressing MoDCs (MoDC-Tax) were cultured in RPMI1640 medium with 10% FBS in the presence of GM-CSF-Fc4 / IL-4-Fc4 for 5-7 days and then continued to be maintained in culture in the presence of recombinant IL-2 at 100 units / ml. Activated MoDCs were generated by stimulating GM-CSF-Fc4 / IL-4-Fc4-differentiated MoDCs with TNFα (50 ng / ml) and LPS (1 μg / ml) for 2 days. Mature and activated MoDCs displayed typical dendrites and were harvested for RNA and protein extraction.

[0424] Immunophenotypic analysis by flow cytometry. The immunophenotype of ihv-DC cells, MoDC-Tax and DC-activated lymphocytes was analyzed by FACS. Cells were stained with allophycocyanin (APC)-conjugated antibodies (Biolegend) as indicated in the figures, according to the manufacturer's instructions. Stained cells were subjected to FACS analysis. For intracellular staining of TLR3, ihv-DC cells were stained with APC-conjugated anti-TLR3 antibody or IgG isotype control antibody after permeabilization using an intracellular staining kit from eBioscience, according to the manufacturer's instructions.

[0425] Allogeneic mixed leukocyte reaction (MLR) assay. ihv-DCs were mixed with naive PBMCs isolated from leukopacks at a ratio of 1:100. The mixed cells were kept in culture for 2-3 days without the addition of exogenous cytokines, followed by the addition of recombinant IL-2 (100u-200 units / ml). The proliferation of ihv-DC-reactive lymphocytes was monitored by FACS, and the presence of ihv-DCs in the mixed cultures was monitored using fluorescence microscopy. After 2-3 weeks of MLR, ihv-DC-activated lymphocytes were analyzed by FACS and tested for cytotoxic activity in target cells.

[0426] Cytotoxicity assay. ihv-DC-activated lymphocytes were generated 18-21 days after MLRs served as effectors, while various cell lines modified to express luciferase were used as targets. Cancer cells were first placed in 24-well plates for 2 h to allow complete attachment. ihv-DC-activated lymphocytes were then placed on cancer cells at the indicated E:T ratios. At 4 and 16 h after effector and target cell co-culture, viable cells were washed with PBS buffer to remove cell debris and subjected to luciferase activity assay using a kit from Promega. Cytotoxic activity of ihv-DC-activated lymphocytes was therefore determined by comparing luciferase activity in target cells treated without or with cytotoxic lymphocytes at the indicated E:T ratios.

[0427] Electrophoretic mobility gel shift assay (EMSA). Nuclear extracts were prepared from various cell lines using NE-PER nuclear and cytoplasmic extraction reagent (Pierce). Oligonucleotides were 5'-end labeled with biotin (Integrated DNA Technologies) and annealed to their complementary strands. Binding activity was tested by EMSA using a light shift chemiluminescence EMSA kit (Pierce) according to a previously reported protocol (Jain, P. et al. DC-SIGN mediates cell-free infection and transmission of human T-cell lymphotropic virus type 1 by dendritic cells. Journal of Virology 83, 10908-10921 (2009).). The oligonucleotide probes are for STAT3 (5'-GATCCTTCTGGGAATTCCTAGATC-3') (SEQ ID NO: 18), NF-κB (5'-GAT-CCGGCAGGGGAATCTCCCTCTC-3') (SEQ ID NO: 19) and AP-1 (5'-CGCTTGATGACTCAG-CCGGAA-3') (SEQ ID NO: 20).

[0428] Immunofluorescence imaging. Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min and 0.1% Triton® X-100 for 10 min. After washing, cells were incubated with 5% goat serum albumin as blocking solution. Primary antibody, anti-Flag (1:100, sigma, USA), was applied at a dilution of 1:200, followed by secondary antibody, anti-rabbit Alexa Fluor488 (Cell Signaling, USA). After washing three times with cold TBST buffer, cell nuclei were stained with DAPI (1:2000, Cell Signaling, USA). Stained cells were examined using fluorescence microscopy (Nikon E800, Japan).

[0429] Quantitative real-time PCR (qRT-PCR). Total RNA was isolated using the RNeasy kit (Qiagen) and its concentration was determined using a NanoDrop1000 spectrophotometer (Thermo Scientific). The amount and integrity of total RNA was assessed in 1% formaldehyde-agarose gels. cDNA was synthesized using the Omniscript reverse transcriptase kit (Qiagen) according to the manufacturer's recommended protocol. Triplicate template samples were subjected to real-time quantitative PCR (Stratagene Mx3005P system) using Power SYBR Green (Applied Biosystems). Primer sequences are listed in Table 1. [Table 1] TIFF0007604000000002.tif172164

[0430] Animals and xenograft models. Animal experiments were performed using approved IACUC protocols. Female NSG mice (4-6 weeks, weighing 18-22 g) were obtained from the Animal Center of the University of Maryland School of Medicine and housed in a specific pathogen-free room. MCF-7 / ADR cells (4 million), HCC1554 cells (1 million) were collected and resuspended in an equal volume of Matrigel (BD, USA) in Hanks buffer, and then injected into the mammary fat pads of female NSG mice. After cancer cell implantation, mice were randomized into two groups (CTL and control). Treatment began when tumors were palpable. Treatment group mice were injected with ihv-DC1 activated CTLs (q5d x 8, 20 million in 100 μL + 1,000u recombinant IL-2) via the tail vein of the mice, and control group mice were injected with Hanks buffer (q5d x 8, iv, 100 μL + 1,000u recombinant IL-2) via the tail vein of the mice. The body weight of the animals was measured every 2 days. Tumor growth was recorded starting from the first day of treatment and the volume of the xenograft was measured every 2 days. Tumor volume (V) was calculated according to the formula V = πa *b2 / 6: where a and b are the longest and shortest diameters, respectively.

[0431] Immunohistochemistry and HE staining. Tissue sections were incubated at 60°C for 10 min and then deparaffinized in xylene, followed by ETOH and DI water. After deparaffinization, tissue sections were immersed in preheated antigen unmasking solution (Vector, CA, USA), placed in a pressure cooker for 5-10 min, and then cooled to room temperature under cold water. Anti-human CD3 antibody (1:100) was used to stain the preheated tissue sections. Endogenous peroxide activity was blocked by incubation of the tissue sections with 3% hydrogen peroxide in PBS for 10 min. Biotinylated goat anti-mouse IgG antibody (H+L) (1:200) (Vector, CA, USA) was applied as the secondary antibody. Hematoxylin counterstaining was also performed using VECTASTAIN Elite ABC HRP Kit (Vector, CA, USA) as the chromogen. The protocol is detailed in Table 2. HE staining (paraffin embedding) was performed by the Pathology Electron Microscopy and Histology Laboratory, University of Maryland School of Medicine. [Table 2]

[0432] While the present teachings have been described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be recognized by those skilled in the art.

[0433] Throughout this disclosure, various publications, patents and published patent applications are referenced by an identifying citation. The disclosures of these publications, patents and published patent applications are hereby incorporated by reference into this disclosure in order to more fully describe the state of the art to which this invention pertains.

Claims

1. 1. Use of modified human blood dendritic cells in the manufacture of a medicament for treating cancer, wherein the dendritic cells have been genetically engineered to contain a nucleic acid sequence for a wild-type full-length Tax protein from human T-cell leukemia virus, thereby expressing the Tax protein, the dendritic cells are CD205+ and CD11c+, the dendritic cells are not infected with a T-cell leukemia virus, the dendritic cells express one or more dendritic cell maturation and activation markers, and protein expression of CD83, CD80, and CD86 is increased compared to immature dendritic cells due to expression of the functional Tax protein, the Tax protein is derived from HTLV-2, and the nucleic acid sequence for the Tax protein is provided to primary human blood dendritic cells to generate modified human blood dendritic cells, wherein the cells present one or more antigens or antigen fragments or variants thereof.

2. The use according to claim 1 , wherein the dendritic cells have a constitutive mature and activated phenotype.

3. The use according to claim 1, wherein the dendritic cell maturation and activation markers are further selected from the group consisting of CD70, CCR7 and HLA-DR.

4. The use according to claim 1, wherein the cells are capable of inducing proliferation of naive lymphocytes in the absence of exogenously added IL-2.

5. The use according to claim 1, wherein the cells are genetically engineered to contain a nucleic acid sequence of one or more HLA proteins, thereby expressing the one or more HLA proteins.

6. The use according to claim 5, wherein the cells are genetically engineered to contain a nucleic acid sequence of HLA-A2.1, thereby expressing one or more HLA-A2.

1.

7. The use according to claim 1 , wherein the antigen is a cancer antigen.

8. The use according to claim 7, wherein the antigen is human telomerase reverse transcriptase or an antigenic fragment thereof or a variant thereof.

9. The use of claim 8, wherein the antigen comprises a fusion protein comprising full-length human IκBα and a fragment comprising amino acids 301-700 of SEQ ID NO:9 of human telomerase reverse transcriptase.

10. The use according to claim 9, wherein the fusion protein comprises SEQ ID NO:

13.

11. The use according to claim 1, wherein the cells present the antigen in an HLA-restricted manner.

12. The use according to claim 1 , wherein the antigen is introduced by viral transformation.

13. The use according to claim 12, wherein the virus is a lentivirus.

14. The use according to claim 1, wherein the dendritic cells are capable of priming naive lymphocytes to generate cytotoxic lymphocytes that recognize an antigen.

15. The use according to claim 1, wherein said dendritic cells are capable of priming naive lymphocytes to generate cytotoxic lymphocytes capable of killing cells in a non-HLA restricted manner.

16. 1. Use of modified monocyte-derived human dendritic cells in the manufacture of a medicament for treating cancer, wherein said dendritic cells comprise a nucleic acid sequence of a wild-type full-length Tax protein from human T-cell leukemia virus, thereby expressing said Tax protein, said dendritic cells are CD205+, said Tax protein is derived from HTLV-2, and said cells present one or more antigens or antigen fragments or variants thereof.

17. The use according to claim 16, wherein the dendritic cells express one or more dendritic cell maturation and activation markers selected from the group consisting of CD83, CD80, CD86, and CD70.

18. 13. Use of cytotoxic T lymphocytes in the manufacture of a medicament for treating cancer, comprising culturing the dendritic cells of claim 1 together with cells including naive lymphocytes for a period of time, thereby producing cytotoxic T lymphocytes.

19. The use according to claim 18, wherein the cells comprising naive lymphocytes comprise naive PBMCs.

20. 20. The use according to claim 19, wherein said naive lymphocytes and said dendritic cells are allogeneic.

21. The use according to claim 20, wherein said cytotoxic T lymphocytes are antigen-specific and induce cytolysis of target cells in an HLA-restricted manner.

22. The use according to claim 20, wherein said cytotoxic T lymphocytes comprise CD3+ / CD56+ T cells capable of killing target cells in a non-HLA-restricted manner.

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