Source: http://www.google.com/patents/US20050003484?dq=645576
Timestamp: 2015-11-29 18:39:34
Document Index: 149430132

Matched Legal Cases: ['art127', 'ART1', 'art1', 'art1', 'art127', 'art127', 'art127', 'art127', 'art127', 'art127', 'ART-1']

Patent US20050003484 - Modified antigen-presenting cells - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe invention relates to antigen-presenting cells having specificity against a selected antigen and methods for making the cells. The invention also relates to a method of selecting efficient antigen-presenting cells using reporter fusion constructs. The highly efficient antigen-presenting cells of the...http://www.google.com/patents/US20050003484?utm_source=gb-gplus-sharePatent US20050003484 - Modified antigen-presenting cellsAdvanced Patent SearchPublication numberUS20050003484 A1Publication typeApplicationApplication numberUS 10/850,294Publication dateJan 6, 2005Filing dateMay 20, 2004Priority dateNov 20, 2001Also published asCA2566903A1, EP1769066A2, EP1769066A4, US7955845, WO2005113589A2, WO2005113589A3Publication number10850294, 850294, US 2005/0003484 A1, US 2005/003484 A1, US 20050003484 A1, US 20050003484A1, US 2005003484 A1, US 2005003484A1, US-A1-20050003484, US-A1-2005003484, US2005/0003484A1, US2005/003484A1, US20050003484 A1, US20050003484A1, US2005003484 A1, US2005003484A1InventorsNaoto Hirano, Marcus Butler, Lee NadlerOriginal AssigneeDana Farber Cancer InstituteExport CitationBiBTeX, EndNote, RefManPatent Citations (9), Referenced by (28), Classifications (33), Legal Events (2) External Links: USPTO, USPTO Assignment, EspacenetModified antigen-presenting cells
US 20050003484 A1Abstract
The invention relates to antigen-presenting cells having specificity against a selected antigen and methods for making the cells. The invention also relates to a method of selecting efficient antigen-presenting cells using reporter fusion constructs. The highly efficient antigen-presenting cells of the invention will provide a therapeutic strategy of modulating immune responses for a variety of diseases. Images(20) Claims(70)
1. A vertebrate cell comprising a nucleic acid encoding a first exogenous immunoregulatory molecule and an exogenous nucleic acid encoding an antigen, wherein said antigen is presented on the surface of said cell, and wherein said cell functions as a professional antigen presenting cell. 2. The vertebrate cell of claim 1, wherein said cell further comprises a nucleic acid encoding a second exogenous immunoregulatory molecule. 3. The vertebrate cell of claim 1 wherein said antigen is expressed as a fusion polypeptide with a heterologous polypeptide. 4. The vertebrate cell of claim 3 wherein said antigen is fused in frame at its N-terminus to said heterologous reporter polypeptide. 5. The vertebrate cell of claim 3 wherein said antigen is located at the C terminus of said fusion polypeptide. 6. The vertebrate cell of claim 3 wherein said heterologous polypeptide is a reporter polypeptide. 7. The vertebrate cell of claim 6 wherein said heterologous reporter polypeptide comprises a selectable marker that permits the selection and purification of cells comprising said nucleic acid encoding an antigen. 8. The vertebrate cell of claim 6 wherein said heterologous reporter polypeptide comprises a Green Fluorescent Protein. 9. The vertebrate cell of claim 6 wherein said heterologous reporter polypeptide comprises a portion of a cell surface protein that is expressed on the surface of a cell. 10. The vertebrate cell of claim 9 wherein said cell surface protein that is expressed on the surface of a cell permits the selection of cells expressing said reporter polypeptide by binding to an antibody specific for said cell surface protein. 11. The vertebrate cell of claim 9 wherein said heterologous reporter polypeptide comprises a polypeptide which permits said cell to survive in selective medium. 12. The vertebrate cell of claim 3 wherein said antigen is fused to said heterologous reporter polypeptide through a linker polypeptide. 13. The vertebrate cell of claim 12 wherein said linker is cleavable by a cell-associated protease. 14. The vertebrate cell of claim 13 wherein said cell-associated protease is an endogenous protease. 15. The vertebrate cell of claim 13 wherein said cell-associated protease is an exogenous protease expressed by an exogenous nucleic acid encoding said protease. 16. The vertebrate cell of claim 12 wherein said linker encodes said protease. 17. The vertebrate cell of claim 3 wherein said antigen expressed as a fusion polypeptide with a heterologous polypeptide is 8 to 10 amino acids in length. 18. The vertebrate cell of claim 1, further comprising a nucleic acid encoding an exogenous class I molecule. 19. The vertebrate cell of claim 18 wherein said class I molecule is an HLA molecule. 20. The vertebrate cell of claim 18 wherein said class I molecule is an H-2 molecule. 21. The vertebrate cell of claim 1 wherein said antigen is a tumor-specific antigen. 22. The vertebrate cell of claim 1 wherein said cell is a human cell. 23. The vertebrate cell of claim 1 wherein said cell is selected from the group consisting of a dendritic cell, a macrophage, a B cell, a mast cell, a parenchymal cell, a kupffer cell, or a fibroblast cell. 24. The vertebrate cell of claim 1 wherein said cell is an immortalized cell. 25. A vertebrate cell comprising a nucleic acid encoding a first exogenous immunoregulatory molecule, a nucleic acid encoding a second exogenous immunoregulatory molecule, and an exogenous nucleic acid encoding an antigen, wherein said antigen is presented on the surface of said cell, and wherein said cell functions as a professional antigen presenting cell. 26. A vertebrate cell comprising an exogenous antigen, a nucleic acid encoding an exogenous antigen-presenting molecule, and a nucleic acid encoding a first exogenous immunoregulatory molecule, wherein said antigen is presented on the surface of said cell, and wherein said cell functions as a professional antigen presenting cell. 27. The vertebrate cell of claim 26, wherein said cell further comprises a second exogenous immunoregulatory molecule. 28. The vertebrate cell of claim 26, wherein said nucleic acid encoding said first and second exogenous immunoregulatory molecules encodes CD80 and CD83, respectively. 29. The vertebrate cell of claim 26, wherein said nucleic acid encoding an exogenous antigen presenting molecule encodes an exogenous class I molecule. 30. The vertebrate cell of claim 26, wherein said exogenous class I molecule is an HLA molecule. 31. The vertebrate cell of claim 26, wherein said exogenous class I molecule is an H-2 molecule. 32. The vertebrate cell of claim 26, wherein said antigen is a tumor-specific antigen. 33. The vertebrate cell of claim 26, wherein said cell is a human cell. 34. The vertebrate cell of claim 26, wherein said cell is selected from the group consisting of a dendritic cell, a macrophage, a B cell, a mast cell, a parenchymal cell, a kupffer cell, or a fibroblast cell. 35. The vertebrate cell of claim 26, wherein said cell is an immortalized cell. 36. A vertebrate cell comprising an exogenous antigen, a nucleic acid encoding an exogenous antigen-presenting molecule, a nucleic acid encoding a first exogenous immunoregulatory molecule, and a nucleic acid encoding a second exogenous immunoregulatory molecule, wherein said antigen is presented on the surface of said cell, and wherein said cell functions as a professional antigen presenting cell. 37. A method of making an artificial antigen presenting cell, said method comprising: a) contacting a population of vertebrate cells with a nucleic acid encoding an exogenous antigen-presenting molecule, and a nucleic acid encoding a first exogenous immunoregulatory molecule; b) contacting said population of vertebrate cells with an exogenous antigen; and c) selecting a cell that comprises said nucleic acid encoding an exogenous antigen-presenting molecule, and said first exogenous immunoregulatory molecule, and which presents said antigen at the cell surface bound to said exogenous antigen-presenting molecule, wherein said vertebrate cell functions as a professional antigen presenting cell. 38. The method of claim 37, wherein step (a) further comprises contacting said population of vertebrate cells with a nucleic acid encoding a second exogenous immunoregulatory molecule. 39. The method of claim 37 wherein said population of vertebrate cells does not express an endogenous class I molecule that binds antigen. 40. The method of claim 39 wherein said exogenous antigen-presenting molecule is a class I molecule. 41. The method of claim 40 wherein said class I molecule is an HLA molecule. 42. The method of claim 40 wherein said class I molecule is an H-2 molecule. 43. The method of claim 37, further comprising the steps of establishing a clonal population of the cell selected in step (b), exposing said population to cytotoxic T cells which specifically recognize said antigen, and monitoring cell death in said population. 44. The method of claim 37 wherein said immunoregulatory molecule is selected from the group consisting of a costimulatory molecule, an accessory molecule, a cytokine, a chemokine, an adhesion molecule, and combinations thereof. 45. The method of claim 44 wherein said costimulatory molecule is CD80 or CD83. 46. A method of activating an immune effector cell against a given polypeptide, said method comprising: a) contacting a professional antigen presenting cell of claim 1 with an immune effector cell, thereby activating said immune effector cell. 47. The method of claim 46 wherein said immune effector cell is selected from the group consisting of lymphocytes, macrophages and neutrophils. 48. A method of activating an immune effector cell against a given polypeptide, said method comprising: a) contacting a cell of claim 26 with an immune effector cell, thereby activating said immune effector cell. 49. The method of claim 48 wherein said immune effector cell is selected from the group consisting of lymphocytes, macrophages and neutrophils. 50. A method of modulating an immune response in a subject, said method comprising the step of administering a vertebrate cell of claim 1 to said subject. 51. The method of claim 50 wherein said immune effector cell is obtained from said subject. 52. The method of claim 50 wherein said immune effector cell is obtained from an individual other than said subject. 53. A method of modulating an immune response in a subject, said method comprising the step of administering a cell of claim 26 to said subject. 54. The method of claim 53 wherein said immune effector cell is obtained from said subject. 55. The method of claim 53 wherein said immune effector cell is obtained from an individual other than said subject. 56. A method of modulating an immune response in a subject, said method comprising the step of contacting an immune effector cell with a cell of claim 1, thereby activating said immune effector cell, and transplanting said effector cell into said subject. 57. The method of claim 56 wherein said immune effector cell is obtained from said subject. 58. The method of claim 56 wherein said immune effector cell is obtained from an individual other than said subject. 59. A method of modulating an immune response in a subject, said method comprising the step of contacting an immune effector cell with a cell of claim 26, thereby activating said immune effector cell, and transplanting said effector cell into said subject. 60. The method of claim 59 wherein said immune effector cell is obtained from said subject. 61. The method of claim 59 wherein said immune effector cell is obtained from an individual other than said subject. 62. A kit comprising a plurality of populations of antigen presenting cells, wherein each member of said plurality expresses the same antigen, and wherein each said population expresses a different antigen-presenting molecule. 63. The kit of claim 62, further comprising a nucleic acid encoding a first immunoregulatory molecule. 64. The kit of claim 63, further comprising a nucleic acid encoding a second immunoregulatory molecule. 65. A kit comprising a nucleic acid encoding an antigen, and a plurality of populations of antigen presenting cells, wherein each said population expresses a different antigen presenting molecule. 66. The kit of claim 65, further comprising a nucleic acid encoding a first immunoregulatory molecule or an antigen-presenting molecule. 67. The kit of claim 66, further comprising a nucleic acid encoding a second immunoregulatory molecule. 68. A kit comprising a plurality of populations of antigen presenting cells, wherein each member of said plurality comprises the same antigen, and wherein each said population expresses a different antigen-presenting molecule. 69. The kit of claim 68, further comprising a nucleic acid encoding a first immunoregulatory molecule. 70. The kit of claim 69, further comprising a nucleic acid encoding a second immunoregulatory molecule.
RELATED APPLICATION [0001] This application is a continuation of International Application PCT/US02/37123, with an international filing date of Nov. 20, 2002, which in turn claims priority from provisional application 60/331,928 filed Nov. 20, 2001. The entire teaching of the above application is incorporated herein by reference.
FIELD OF THE INVENTION [0002] The invention relates to compositions and methods comprising modified antigen-presenting cells for modulating an antigen-specific immune response. BACKGROUND [0003] Antigen molecules are recognized by the immune system after internal processing by antigen-presenting cells (APCs) (Lanzavecchia, 1996, Curr. Opin. Immunol., 8:348-54). In order to present an antigen, the antigen is broken down into small peptidic fragments by enzymes contained in vesicles in the cytoplasm of the antigen-presenting cells (for reviews, see: Wick, et al., 1999, Immunol. Rev., 172:153-62; Lehner, et al., 1998, Curr. Biol., 8: R605-8; Braciale, 1992, Curr. Opin. Immunol., 4:59-62). The enzymes are part of a complex of proteolytic enzymes called a proteosome. Most cells have several different types of proteosomes with differing combinations of specificities, which they use to recycle their intracellular proteins. The peptides produced by the proteosomes are generated in the cytosol and must be transported into the Golgi compartment in order to associate with newly synthesized class I molecules. This is accomplished by a heterodimeric protein called TAP (for transporter associated with antigen processing) (Townsend, et al., 1993, Eur. J. Immunogenetics, 19:45-55), which is associated with the ER and actively transports peptides into the Golgi, where they are linked to cellular major histocompatibility complex (MHC) molecules (known as HLA in human). [0004] There are two types of MHC molecules used for antigen presentation, class I and class II molecules. MHC class I molecules are expressed on the surface of all cells and MHC class II are expressed on the surface of a specialized class of cells called professional antigen-presenting cells. MHC class II molecules bind primarily to peptides derived from proteins made outside of an antigen-presenting cell, but can present self (endogenous) antigens. In contrast, MHC class I molecules bind to peptides derived from proteins made inside a cell, including proteins expressed by an infectious agent (e.g., such as a virus) in the cell and by a tumor cell. When the MHC class I proteins reach the surface of the cell these molecules will thus display any one of many peptides derived from the cytosolic proteins of that cell, along with normal “self” peptides being synthesized by the cell. Peptides presented in this way are recognized by T-cell receptors which engage T-lymphocytes in an immune response against the antigens (cellular immunity). [0005] Antigen binding also requires the interaction of a number of co-receptor/ligand molecules that interact with ligand/receptors on the T cell. CD4 and CD8 act as co-receptors (one type only present per T cell) that interact with the TCR on the appropriate T cell to form a receptor/co-receptor complex. The receptor/co-receptor complex binds to the relevant MHC molecules on the APC. CD4 binds to class II molecules and CD8 binds to class I molecules. Various adhesion molecules (e.g., LFA-1, LFA2 (CD2), LFA3 (CD58), ICAM1, ICAM2, ICAM3), costimulatory molecules (e.g., CD80: B7-1 and B7-2) and accessory molecules (e.g., CD83) are also involved in facilitating T cell binding to APCs. [0006] Conventional immunization techniques, such as those using killed or attenuated viruses, often fail to elicit an appropriate CTL response which is effective against an intracellular infection. Thus, there remains a need for the development of vaccines that stimulate appropriate responses (i.e., cell-mediated as well as antibody-mediated immune responses), in order to prevent disease. [0007] Induction of primary MHC class I restricted CTL by pure soluble antigenic proteins in vitro has not been reported. The most common tool for ex vivo induction of primary CTL are small (8-11-mer) synthetic peptides (Stauss, et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89:7871-5); Carbone, et al., 1988, J. Exp. Med., 167:1767-79). These synthetic peptides associate with class I molecules on the cell surface without the requirement for endogenous processing. When presented on the surface of an appropriate APC (such as a dendritic cell) they can then induce a primary CTL response. However, frequently these CTL do not protect against challenge with pathogens that endogenously synthesize the protein from which the peptide was derived because of their low T-cell receptor avidity (Speiser, et al., 1992, J. Immunol., 149:972-80) and because they induce reactivity with a single epitope of the target antigen. [0008] Another way of activating an efficient immune response against a specific antigen is to stimulate T cells with APCs engineered to express a specific antigen. U.S. Pat. Nos. 5,962,320, 6,187,307, 6,194,205 and patent publication WO 97/29183 disclose a method of making engineered APCs by transfecting professional or non-professional APCs with selected antigens to regulate the immune response of a subject. [0009] WO 96/27392, U.S. Pat. Nos. 5,225,042, 6,251,627 and 5,962,320 disclose engineered APCs transfected with MHC molecules. SUMMARY OF THE INVENTION [0010] The present invention provides modified antigen-presenting cells (APCs) expressing one or more selected antigens for generating or enhancing an antigen-specific immune response, and methods for making such APCs. The selected antigens are highly expressed on the surface of the cells. The APCs can also be modified to express one or more other immunomodulatory molecules. The APCs of the invention can be used in the treatment of a variety of diseases including microbe infections, cancers and pathologies associated with transplantation. [0011] In one embodiment, the invention provides an animal cell comprising a nucleic acid encoding an exogenous antigen-presenting molecule (e.g., a class I or a class II molecule) and a nucleic acid encoding an antigen fused in frame at its N-terminus to a heterologous reporter polypeptide, wherein the animal cell functions as a professional APC. The heterologous polypeptide aids in the efficient presentation of the antigen on the surface of the cell. In a preferred embodiment, the antigen is fused to the heterologous polypeptide through a linker polypeptide which is cleavable by a cell-associated protease, separating the antigen from the heterologous polypeptide. The cell-associated protease can be an endogenous protease (e.g., such as trypsin) or an exogenous protease (not naturally expressed by the cell) which is expressed by a nucleic acid encoding the exogenous protease which is introduced into the cell. The linker itself can encode a protease (i.e., the linker can be a self-cleaving linker). Most preferably, the C-terminus of the antigen-heterologous polypeptide fusion, or antigen-linker-heterologous polypeptide fusion, is the C-terminus of a minimal antigen sequence, i.e., the C-terminus of the smallest peptide which binds to an antigen-presenting molecule and which upon binding elicits an immune response (e.g., such as an antigen-specific cytotoxic T cell response). [0012] The heterologous polypeptide can be used to provide a selectable marker enabling selection and purification of cells comprising the antigen-encoding nucleic acid. In one aspect, the heterologous polypeptide is a reporter polypeptide such as Green Fluorescent Protein (GFP) or Enhanced Green Fluorescent Protein (EGFP). In another aspect, the heterologous polypeptide comprises a portion of a cell surface protein which is expressed on the surface of a cell, enabling cells which comprise the nucleic acid to be selected for by screening for cells which bind to an antibody specific for the portion of the cell surface protein. The heterologous polypeptide also can provide a function (e.g., such as G418 resistance) which enables cells to survive in a particular type of selection medium (e.g., G418). While “function” in the sense of a reporter or selectable molecule is desirable, the primary function of the heterologous fusion polypeptide that is fused N-terminal to the antigen sequence is to aid in the efficient presentation of the antigen at the cell surface in association with a class I molecule. Thus, cells comprising the nucleic acid can be identified and selected based on their ability to function as APCs (e.g., generating an antigen-specific immune response). [0013] The APC in the above embodiment further can comprise a nucleic acid encoding an exogenous immunoregulatory molecule. [0014] In another embodiment, the invention provides an animal cell comprising a nucleic acid encoding an exogenous immunoregulatory molecule and a nucleic acid encoding an antigen which is expressed on the surface of the cell. The animal cell functions as a professional APC. Preferably, as above, sequence of the antigen is fused in frame at its N-terminus with a heterologous polypeptide and aids in the efficient presentation of the antigen at the cell surface in association with a class I molecule. In one aspect, the heterologous polypeptide is a reporter polypeptide. Preferably, the C-terminus of the antigen is the C-terminus of the antigen-heterologous polypeptide fusion. [0015] Preferably, the immunoregulatory molecule is selected from the group consisting of a costimulatory molecule, an accessory molecule, a cytokine, a chemokine, an adhesion molecule, and combinations thereof. More preferably, the costimulatory molecule is CD80. Still more preferably, the accessory molecule is CD83. [0016] The APC in the above embodiment further may comprise a nucleic acid encoding an antigen-presenting molecule such as a class I or class II molecule. In one aspect, the nucleic acid encodes an exogenous antigen-presenting molecule (e.g., an antigen-presenting molecule not naturally found in the cell). [0017] In one embodiment, antigen-presenting molecule is a class I molecule which is an HLA molecule. In another embodiment, the class I molecule is an H-2 molecule. [0018] Preferably, the antigen presented by the APC is a tumor-specific antigen. [0019] The APC can be a dendritic cell, a macrophage, a B cell, a mast cell, a parenchymal cell, a kupffer cell, or a fibroblast cell. Preferably, the APC is an immortalized cell. Most preferably, the APC is a human cell. [0020] The invention also provides a method for producing a modified APC comprising contacting a population of animal cells with a nucleic acid encoding an antigen which is efficiently presented on the surface of a cell, and selecting a cell which comprises the nucleic acid, presents the antigen on its surface; and functions as a professional APC. [0021] In one aspect, the antigen is fused in frame to a heterologous polypeptide (such as a reporter polypeptide), preferably via a linking polypeptide which is cleavable by a cell-associated protease, as described above. In a preferred aspect, the population of cells also is contacted with a nucleic acid encoding an exogenous antigen-presenting molecule (e.g., a class I or class II molecule not naturally expressed by the cell). [0022] In another embodiment, the invention provides a method for producing a modified APC comprising contacting a population of animal cells with a nucleic acid encoding an exogenous immunoregulatory molecule and a nucleic acid encoding an antigen which is presented on the surface of a cell, and selecting a cell which comprises the nucleic acid encoding the antigen and the nucleic acid encoding the immunoregulatory molecule, presents the antigen on its surface and which functions as a professional APC. [0023] As above, the antigen can be fused in frame to a heterologous polypeptide such as a reporter polypeptide and is preferably linked to the heterologous polypeptide by a linker polypeptide cleavable by a cell-associated protease, such as trypsin. The method further may comprise contacting the population of cells with a nucleic acid encoding an exogenous antigen-presenting molecule such as a class I or class II molecule and selecting one or more cells which express the exogenous immunoregulatory molecule, the antigen, and the antigen-presenting molecule. [0024] Preferably, the immunoregulatory molecule is selected from the group consisting of a costimulatory molecule, an accessory molecule, a cytokine, a chemokine, an adhesion molecule, and combinations thereof. [0025] The contacting in step may be performed by providing the nucleic acids in any of: a viral particle (e.g., an adenovirus or retrovirus), a liposome, and a particle comprising a ligand specific for a receptor expressed by the cells. The nucleic acids also can be provided as naked nucleic acids. Cells can be contacted with the nucleic acid encoding the immunoregulatory molecule, the nucleic acid encoding the exogenous antigen-presenting molecule, and the nucleic acid encoding the antigen simultaneously or sequentially in any order. [0026] In a preferred embodiment, the method for producing a modified APC further comprises establishing clonal populations of the one or more selected cells, exposing the populations to cytotoxic T cells which specifically recognize the antigen and monitoring cell death in the populations. [0027] One embodiment of the invention provides a method for activating an immune effector cell against a selected peptide comprising providing any of the modified APCs described above, and contacting the APCs with an immune effector cell, thereby activating the immune effector cell. Preferably, the immune effector cell is selected from the group consisting of lymphocytes, macrophages, and neutrophils. [0028] The invention also provides a method for modulating an immune response in a subject comprising administering a therapeutically effective amount of any of the modified APCs described above to the subject. In one aspect, the method comprises contacting an immune effector cell with any of the modified APCs described above, thereby activating the immune effector cell, and transplanting the immune effector cell to the subject. The immune effector cell can be obtained from the same subject who is to receive the modified APC or from a different subject. In one aspect, when immune effector cells are obtained from a different subject, the different subject has an antigen-presenting molecule which matches that of the first subject (e.g., the subject has a matching MHC class I determinant). Preferably, the APCs comprise human cells. [0029] The invention further provides kits comprising a plurality of different APCs expressing the same antigen-heterologous polypeptide fusion or antigen-linker-heterologous polypeptide fusion, but each cell expressing a different antigen-presenting molecule. Alternatively, the kit can comprise a plurality of different APCs, each cell expressing a different antigen-presenting molecule and at least one nucleic acid encoding an antigen-heterologous polypeptide fusion or antigen-linker-polypeptide fusion for introducing into the cell. The kit also can comprise one or more nucleic acid molecules encoding one or more immunoregulatory molecules, or antigen-presenting molecules.
BRIEF DESCRIPTION OF DRAWINGS [0030] The objects and features of the invention can be better understood with reference to the following detailed description and accompanying drawings. [0031] FIG. 1 shows a schematic diagram of a cloning vector for generating GFP-antigen fusions according to one aspect of the invention. [0032] FIG. 2 shows schematic diagrams of two exemplary embodiments of antigen-heterologous fusion polypeptides for generating antigen presenting cells as described herein, relative to a control construct (bottom). “EGFP” refers to the enhanced GFP polypeptide. “Flu58-66” and “Mart127-35” refer to nonapeptide antigen sequences corresponding to amino acids 58-66 of Influenza virus MP1 antigen and amino acids 27-35 of the MART1 melanoma-associated tumor antigen, respectively. [0033] FIG. 3 shows the expression of the polypeptides shown schematically in FIG. 2, as measured by fluorescence of the EGFP fusion partner. [0034] FIG. 4 shows that EGFP-flu is expressed, processed and presented in the leukemia cell line K562/A2 that expresses CD80 and CD83, as measured by T-cell activation assay (induction of IFN-γ secretion). [0035] FIG. 5 shows HPLC analyses of eluted antigenic peptides expressed on the surface of K562/A2/CD80/CD83 cells expressing the EGFP-Flu (MP1), EGFP and EGFP-Mart1 constructs shown in FIG. 2, and mass spectroscopy comparison of the eluted influenza virus MP58-66 antigen versus synthetic MP58-66. The transduced and processed peptide expressed on the transduced cells has a similar mass spectroscopy spectrum to the synthetic antigenic peptide. [0036] FIG. 6 shows the results of a comparison of memory CD8 T cell activation by two populations of K562/A2/CD80/CD83 antigen presenting cells that were each either pulse-loaded with influenza MP1 peptide (58-66) (at doses of 0.1 μg/ml, 1.0 μg/ml and 10 μg/ml) or transduced with an EGFP-flu58-66 construct. The cells transduced with the EGFP-flu construct were stimulated more potently by the cells expressing the EGFP-flu construct than by any of the peptide-pulsed cells. [0037] FIG. 7 shows the results of flow sorting of memory cytotoxic T lymphocytes stimulated by K562/A2/CD80/CD83 cells transduced with EGFP-flu. The population of Flu-specific CTLs is dramatically induced by the transduced EGFP-flu, relative to the induction of CTLs specific for a control antigen. [0038] FIG. 8 shows the results of a comparison of naive CD8 T cell activation by two populations of K562/A2/CD80/CD83 antigen presenting cells that were each either pulse-loaded with Mart1 peptide (27-35) (at doses of 0.1 μg/ml, 1.0 μg/ml and 10 μg/ml) or transduced with an EGFP-Mart127-35 construct. In each case, the cells transduced with the EGFP-Mart127-35 construct were stimulated more potently by the cells expressing the EGFP-Mart127-35 construct than by any of the peptide-pulsed cells. [0039] FIG. 9 shows the results of flow sorting of naive cytotoxic T lymphocytes stimulated by K562/A2/CD80/CD83 cells transduced with EGFP-Mart127-35. The population of Mart127-35-specific CTLs is dramatically induced by the transduced EGFP-Mart127-35, relative to the induction of CTLs specific for a control antigen. [0040] FIG. 10 shows the results of experiments examining the proteasome-dependency of the processing and presentation of Flu peptide by K562/A2/CD80/CD83/EGFP and/EGFP-flu cells. K562/A2/CD80/CD83 cells transduced with either EGFP or EGFP-Flu construct were treated with proteasome inhibitor, with or without pulsed control peptide (“pol peptide”) or flu peptide (“flu peptide”). Treated cells were monitored for their ability to activate IFN-γ expression by T cells (ELISPOT assay). Cells expressing EGFP alone failed to activate T cells, with or without proteasome inhibitor, while EGFP-transduced cells pulse loaded with flu peptide activated T cells efficiently. In contrast, cells expressing EGFP-flu activated T cells; this activation was inhibited by proteasome inhibitor, while flu peptide pulse loaded cells activated T cells even when treated with proteasome inhibitor. Thus, processing of EGFP-flu to present the flu antigen appears to be proteasome dependent. [0041] FIG. 11 shows schematic diagrams of additional antigen-heterologous fusion polypeptide constructs for antigen presentation. Antigens include Her2/neu369-377 (for breast and ovarian cancer therapies), TERT1540-548 (for multiple disease therapies), PR1169-177 (for chronic myelogenous leukemia therapies), HIV polymerase476-484 (for AIDS therapies), and CYPIB 1190-198 (for multiple disease therapies). [0042] FIG. 12 shows the expression of transduced HLA-A2, CD80 and CD83 genes in MEA1 cells. [0043] FIG. 13 shows a schema for CTL generation according to the invention. [0044] FIG. 14 shows the expansion of antigen specific T cells; specific for the Flu, MP58 antigen. [0045] FIG. 15 shows the expansion of antigen specific T cells, specific for the MART-1, M27 antigen. [0046] FIG. 16 shows the cytotoxicity of peptide pusled T2 targets. [0047] FIG. 17 shows the results of ELISPOT measurements of y-interferon secretion. [0048] FIG. 18 shows the phenotype of multimer stained “young” CTL cells. [0049] FIG. 19 shows the re