Both soluble and membrane-bound forms of Flt3 ligand enhance tumor immunity following "suicide" gene therapy in a murine colon carcinoma model

Abstract

In prodrug-activated ("suicide") gene therapy, tumor cells are transfected with the gene for an enzyme that converts an inactive prodrug, such as ganciclovir (GCV), to a toxic compound. Transfected cells are killed on administration of GCV, as also are untransfected "bystander" cells. The ability of the dendritic cell stimulatory cytokine Flt3 ligand (Flt3-L) to modulate prodrug-activated gene therapy has been investigated. Transfectants of the murine colon carcinoma MC26 were generated expressing soluble (FLS) and membrane-bound forms of Flt3-L. They were inoculated together with wild-type MC26 cells and cells expressing herpes simplex virus-1 (HSV1) thymidine kinase into BALB/c mice, which were then administered GCV. Expression of Flt3-L or FLS prevented regrowth of tumor in most mice, which was comparable to the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), while tumors recurred in all mice receiving "suicide" gene therapy alone. Recurring tumor cells were resistant to direct killing by GCV but sensitive to "bystander" killing in vitro. Mice without tumor recurrence were rechallenged with unmodified MC26 cells. Of those mice given transfectants expressing GM-CSF, Flt3-L, or FLS, approximately 50% were immune to rechallenge. These mice also showed cytotoxic and proliferative responses to MC26 cells. These experiments show that both soluble and membrane-bound forms of Flt3-L were able to induce a protective immune response to colon carcinoma cells in a fashion similar to GM-CSF.

Fig. 1

Tumor growth in mice inoculated with MC26 cells + 10% MC26-TK alone (solid diamond), or in combination with 10% GM-CSF (solid square), 10% MC26–Flt3-L (solid triangle), or 10% MC26-FLS (solid circle) and administered GCV

Fig. 2

Proliferation of MC26 cells isolated from recurring tumors, cultured in the presence of increasing proportions of MC26-TK cells. Cells were cultured in the absence (solid diamond) or the presence of GCV at concentrations of 1 μg/ml (solid square), 2 μg/ml (solid triangle), or 4 μg/ml (open circle)

Fig. 3

Tumor growth in previously tumor-free mice rechallenged with unmodified MC26 cells following initial tumor induction with MC26 cells + 10% MC26-TK + 10% MC26–GM-CSF (solid square), + 10% MC26–Flt3-L (solid triangle), + 10% MC26-FLS (solid circle). Tumor growth is compared with that in naïve mice receiving MC26 cells + 10% MC26-TK alone (solid diamond)

Fig. 4

Cytotoxic activity against unmodified MC26 cells of spleen cells from control mice and mice resistant to tumor rechallenge. Effector spleen cells were taken from control untreated mice and mice initially inoculated with 10⁶ tumor cells comprising 80% MC26 + 10% MC26-TK + either 10% MC26–GM-CSF, 10% MC26–Flt3-L, or 10% MC26-FLS, and subsequently rechallenged with 10⁶ unmodified MC26 tumor cells . Assays were performed 200 days after tumor rechallenge. Results are expressed as % specific ⁵¹Cr release ± SD. Effector to target ratio 20:1. Student’s t-test: control vs GM-CSF (p<0.02); control vs FLS (p<0.05)

Fig. 5

Cytotoxic activity against unmodified MC26 cells and KBALB cells of spleen cells (n=3) and TILs (n=1) from mice inoculated with 10⁶ tumor cells comprising MC26 + MC26-TK + MC26-FLS, and receiving GCV treatment, but in which the tumor subsequently recurred. Results are expressed as percentage specific ⁵¹Cr release ± SD. Effector to target ratios are as indicated; for TILs these were 100:1. Student’s t-test: MC26 vs KBALB, p<0.05

Fig. 6

Proliferation of spleen cells in response to irradiated MC26 cells. Spleen cells were from mice resistant to rechallenge with unmodified MC26 cells having received MC26–Flt3-L, MC26-FLS, or MC26–GM-CSF cells. Mice receiving MC26-TK with recurrent tumor were also tested (TK). Proliferation assays were conducted in the presence of 10-nM rofecoxib. Results are expressed as mean ± SD relative to control naïve mice. Flt3-L, FLS, and GM-CSF groups all showed significantly greater proliferation than in control mice (p<0.05)