title: Generation of dendritic cell-tumor cell hybrids by electrofusion for clinical vaccine application.
abstract: Vaccination with hybrids comprising fused dendritic cells (DCs) and tumor cells is a novel cancer immunotherapy approach designed to combine tumor antigenicity with the antigen-presenting and immune-stimulatory capacities of DCs. For clinical purposes, we have incorporated a large-scale process for the generation of clinical-grade DCs together with novel electrofusion technology. The electrofusion system provides for ease and standardization of method, efficient DC-tumor cell hybrid formation, and large-quantity production of hybrids in a high-volume (6-ml) electrofusion chamber. In addition, we have evaluated DC electrofusion with a variety of allogeneic human tumor cell lines with the rationale that these tumor cell partners would prove a ready, suitable source for the generation of DC-tumor cell hybrid vaccines. The DC production process can generate 6x10(8) to 2x10(9) DCs from a single leukapheresis product (approximately 180 ml). As determined by FACS analysis, electrofusion of 6x10(7) total cells (1:1 ratio of DC and tumor cells) resulted in a consistent average of 8-10% DC-tumor cell hybrids, irrespective of the tumor type used. Hybrids were retained in the population for 48 h postfusion and following freezing and thawing. Upon pre-irradiation of the tumor cell partner for vaccine purposes, the overall fusion efficiency was not altered at doses up to 200 Gy. Evaluation of DC-tumor cell hybrid populations for their ability to stimulate T-cell responses demonstrated that electrofused populations are superior to mixed populations of DCs and tumor cells in generating a primary T-cell response, as indicated by IFN-gamma release. Moreover, hybrids comprising HLA-A*0201 DCs and allogeneic melanoma tumor cells (Colo 829 cell line) stimulated IFN-gamma secretion by antigen-specific CD8+ T cells, which are restricted for recognition of a melanoma gp100 peptide antigen (gp100(209-217)) within the context of the DC HLA haplotype. Maturation of the DC-Colo 829 cell hybrid population served to further improve this T-cell gp100-specific response. Overall, our results are promising for the large-scale generation of electrofused hybrids comprising DCs and allogeneic tumor cells, that may prove useful in human vaccine trials.
authors.affiliation: Arizona Cancer Center, 1515 N. Campbell Avenue, PO Box 245024, Tucson 85748, USA. ktrevor@azcc.arizona.edu
authors.forename: Katrina T
authors.initials: KT
authors.lastname: Trevor
authors.forename: Cathleen
authors.initials: C
authors.lastname: Cover
authors.forename: Yvette W
authors.initials: YW
authors.lastname: Ruiz
authors.forename: Emmanuel T
authors.initials: ET
authors.lastname: Akporiaye
authors.forename: Evan M
authors.initials: EM
authors.lastname: Hersh
authors.forename: Didier
authors.initials: D
authors.lastname: Landais
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Taylor
authors.forename: Alan D
authors.initials: AD
authors.lastname: King
authors.forename: Richard E
authors.initials: RE
authors.lastname: Walters
mesh_terms: D017951:Antigen Presentation; D000938:Antigen-Presenting Cells / Q000276:immunology* / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000276:immunology / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D019496:Cancer Vaccines / Q000627:therapeutic use*; D002459:Cell Fusion; D002470:Cell Survival / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D004558:Electric Stimulation; D015234:HLA-A Antigens / Q000276:immunology / Q000378:metabolism; D015789:HLA-A2 Antigen; D006801:Humans; D006822:Hybrid Cells; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D009363:Neoplasm Proteins / Q000276:immunology / Q000378:metabolism; D009369:Neoplasms / Q000276:immunology / Q000378:metabolism / Q000628:therapy*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014407:Tumor Cells, Cultured / Q000276:immunology / Q000378:metabolism; D058951:gp100 Melanoma Antigen
chemical_list: D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D058951:gp100 Melanoma Antigen