Trevor Katrina T, Cover Cathleen, Ruiz Yvette W, Akporiaye Emmanuel T, Hersh Evan M, Landais Didier, Taylor Rachel R, King Alan D, Walters Richard E
Arizona Cancer Center, 1515 N. Campbell Avenue, PO Box 245024, Tucson 85748, USA.
Cancer Immunol Immunother. 2004 Aug;53(8):705-14. doi: 10.1007/s00262-004-0512-1. Epub 2004 Mar 26.
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.
用包含融合树突状细胞（DC）和肿瘤细胞的杂种进行疫苗接种是一种新型癌症免疫疗法，旨在将肿瘤抗原性与DC的抗原呈递和免疫刺激能力相结合。出于临床目的，我们采用了大规模生产临床级DC的工艺以及新型电融合技术。该电融合系统使方法简便且标准化，能高效形成DC-肿瘤细胞杂种，并可在大容量（6毫升）电融合室中大量生产杂种。此外，我们评估了DC与多种异基因人类肿瘤细胞系的电融合，理由是这些肿瘤细胞伙伴将被证明是生成DC-肿瘤细胞杂种疫苗的现成且合适的来源。DC生产工艺可从单个白细胞分离产品（约180毫升）中产生6×10⁸至2×10⁹个DC。通过流式细胞术分析确定，6×10⁷个总细胞（DC与肿瘤细胞1:1比例）的电融合无论使用何种肿瘤类型，均能产生平均8 - 10%的DC-肿瘤细胞杂种。杂种在融合后48小时以及冻融后仍保留在群体中。为制备疫苗对肿瘤细胞伙伴进行预照射时，剂量高达200 Gy时总体融合效率未改变。对DC-肿瘤细胞杂种群体刺激T细胞反应能力的评估表明，如通过γ-干扰素释放所示，电融合群体在产生初始T细胞反应方面优于DC和肿瘤细胞的混合群体。此外，包含HLA-A*0201 DC和异基因黑色素瘤肿瘤细胞（Colo 829细胞系）的杂种刺激了抗原特异性CD8⁺T细胞分泌γ-干扰素，这些T细胞在DC HLA单倍型背景下受限识别黑色素瘤gp100肽抗原（gp100(209 - 217)）。DC-Colo 829细胞杂种群体的成熟进一步改善了这种T细胞对gp100的特异性反应。总体而言，我们的结果对于大规模生成包含DC和异基因肿瘤细胞的电融合杂种很有前景，这可能在人类疫苗试验中有用。
