Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines

Abstract

Hybrid cells created by fusion of antigen presenting and tumour cells have been shown to induce potent protective and curative anti-tumour immunity in rodent cancer models. The application of hybrid cell vaccines for human tumour therapy and the timely intervention in disease control are limited by the requirement to derive sufficient autologous cells to preserve homologous tumour antigen presentation. In this study, the efficiency of various methods of electrofusion in generating hybrid human cells have been investigated with a variety of human haemopoietic, breast and prostate cell lines. Cell fusion using an electrical pulse is enhanced by a variety of stimuli to align cells electrically or bring cells into contact. Centrifugation of cells after an exponential pulse from a Gene Pulser electroporation apparatus provided the highest yield of mixed cell hybrids by FACS analysis. An extensive fusogenic condition generated in human cells after an electrical pulse contradicts the presumption that prior cell contact is necessary for cell fusion. Alignment of cells in a concurrent direct current charge and osmotic expansion of cells in polyethylene glycol also generated high levels of cell fusion. Waxing of one electrode of the electroporation cuvette served to polarize the fusion chamber and increase cell fusion 5-fold. Optimisation of a direct current charge in combination with a fusogenic pulse in which fusion of a range of human cells approached or exceeded 30% of the total pulsed cells. The yield of hybrid prostate and breast cancer cells with dendritic cells was similar to the homologous cell fusion efficiencies indicating that dendritic cells were highly amenable to fusion with human tumour cells under similar electrical parameters. Elimination of unfused cells by density gradient and culture is possible to further increase the quantity of hybrid cells. The generation and purification of quantities of hybrid cells sufficient for human vaccination raises the possibility of rapid, autologous tumour antigen presenting vaccines for trial with common human tumours.

In vitrodendritic cell-induced T cell responses to B cell chronic lymphocytic leukaemia enhanced by IL-15 and dendritic cell–B-CLL electrofusion hybrids

“…+ cells have resulted in hybrid cells capable of efficient growth [22] and whole cell vaccines have been produced in this manner for several different tumours [19][20][21]. Use of human allogeneic DCs fused with tumour cells has shown more promising results [32,33].…”

“…The electrofusion method was based upon findings of Scott-Taylor et al . [22]. B-CLL B cells and dendritic cells were resuspended in 0·3 M sodium sucrose solution at a cell density of 5 ¥ 10 5 /ml; 0·4 ml of B-CLL B cells and dendritic cells were added to a 0·8-ml electro-plated cuvette (Bio-Rad).…”

Dendritic Cell-Ewing’s Sarcoma Cell Hybrids Enhance Antitumor Immunity

“…The fusion of DCs and A673s has the advantage that the antigen does not need to be identified [28,30,34,35]. Hybrid DCs can present all the antigens of the entire tumor cell to effector cells, especially the tumor antigens that have not been identified but might be crucial to tumor immunity.…”

“…Electrofusion was carried out by an ECM 830 (BTX Instruments, Human Disease Genomics Center, Peking University). Fused cells were allowed to rest for 5 minutes [30,36]. Then, they would either immediately undergo fluorescence activated cell sorting (FACS) analysis for assessing percentage of hybrids or be cultured overnight.…”

Therapeutic Applications of Electroporation

“…The hybridoma is then able to continue to grow in culture, and a large amount of specific desired antibodies can be recovered after processing. Electrofusion has proved to be a successful approach in the production of vaccines Orentas et al (2001);Scott-Taylor et al (2000), antibodies Schmidt et al (2001), and reconstructed embryos in mammalian cloning Gaynor et al (2005).…”