A Family of Bicistronic Vectors to Enhance Both Local and Systemic Antitumor Effects of HSVtk or Cytokine Expression in a Murine Melanoma Model

Abstract

The herpes simplex virus-thymidine kinase/ganciclovir (HSVtk/GCV) system produces both direct and immune-mediated tumor cell killing. Here, we compare the efficacy of HSVtk/GCV with cytokines, alone and in combination, on the tumorigenicity and immunogenicity of B16 cells. With respect to single gene modifications, only HSVtk/GCV, or high-level interleukin-2 (IL-2) secretion, completely prevented tumor growth, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) generated the best levels of long-term systemic protection. To augment both local killing and immune activation, we constructed bicistronic constructs that express HSVtk and a cytokine within the same cell. Co-expression of HSVtk with IL-2 or GM-CSF enhanced the local antitumor activity of any gene alone. In a tumor-prevention model, HSVtk killing, in an environment preprimed with GM-CSF, generated the best long-term immune protection. However, in a short-term therapy model, continued IL-2 expression was most effective against 3-day established tumors. This probably reflects differences in the activities of IL-2 and GM-CSF in generating short-term, nonspecific immune stimulation compared to long-term immunological memory, respectively. As a prelude to in vivo delivery experiments, we also demonstrated that these bicistronic cassettes can be packaged normally into retroviral (5 x 10(5) virus/ml from pooled populations) and adenoviral vectors (5 x 10(9) virus/ml) and function as predicted within virally infected cells. This family of bicistronic vectors can be used to stimulate synergy between suicide and cytokine genes, overcomes the problems of delivering two genes on separate vectors, and should allow easier preparation of vectors for the delivery of multiple genes to patients' tumor cells.

Anti-tumour activity against B16-F10 melanoma with a GM-CSF secreting allogeneic tumour cell vaccine

“…We have experienced difficulties in achieving levels of GM-CSF secretion as reported by Dranoff and colleagues 1 but our levels are similar to those attained by several other groups. [30][31][32] A possible explanation for the discrepancy in GM-CSF levels is that Dranoff and colleagues 1 used the MFG vector which lacks an internal promoter for the selectable marker gene, presence of this promoter could lead to interference with the promoter for the GM-CSF gene thus leading to lower levels of secretion.…”

“…Moreover, another group, working with autologous vaccines secreting similar levels of GM-CSF to our vaccine, have also found their vaccine ineffective in the therapeutic setting. 30 It is therefore possible that the modest level of anti-tumour activity generated by the GM-CSF secreting allogeneic vaccine is supported by the alloresponse that the allogeneic vaccine would induce.…”

Tumour cell expression of B7 costimulatory molecules and interleukin-12 or granulocyte–macrophage colony-stimulating factor induces a local antitumour response and may generate systemic protective immunity

“…40 Indeed, GM-CSF is particularly effective in generating systemic immunity against a number of poorly immunogenic tumours. [41][42][43] It is expected that the combination of B7 costimulation and GM-CSF would be especially beneficial, since each promotes one of the two main routes by which activation of cytotoxic T cells occurs -direct antigen presentation and crosspriming by professional APCs.…”

Cancer gene therapy: hard lessons and new courses

“…21 In addition, immune stimulation through cell killing can also enhance local tumour killing 22 and can help to generate systemic immunity to other tumour deposits. 23 These effects can be active either over a short period of time through activation of non-specific immune effector mechanisms 22,24 or can set the foundations of long-lived, T cell-mediated immunity to tumour cells. 17,25 The most effective way to stimulate T cell-mediated immunity to tumours is to kill cells in such a way as to convince the immune system that they are the source of an aggressive, infection-like situation.…”