The mTOR protein has a critical role in integrating diverse environmental signals that ultimately affect cell growth and proliferation. Inhibition of mTOR function has a large number of cellular effects that include inhibition of proliferation. Temsirolimus is a rapamycin analogue and a potent mTOR inhibitor that is FDA approved for the treatment of advanced RCC. We characterised effects of temsirolimus when used in combination with a novel cancer vaccine. It is surprising that temsirolimus can enhance anti-tumour immunity, as rapamycin is widely used in the clinic to suppress the immune system following solid-organ transplantation. In an experimental murine model of RCC (RENCA), the combination of an HSP-based cancer vaccine and temsirolimus was more effective against established tumours than either agent alone. This observation was confirmed in an experimental murine model of melanoma (B16), which is another classic immunoresponsive malignancy. In both models, untreated animals were needed to be killed ~20 days following tumour implantation. To make the study rigorous, tumours were allowed to grow for 10 days until they were palpable and had an established vascular supply before starting treatment.
Temsirolimus had direct anti-proliferative effects on RENCA cells in vitro. However, there was no direct effect on the growth of B16 cells, suggesting that immune modulation may have produced the anti-tumour effects seen in the animal studies. To provide definitive evidence that mTOR inhibition can positively modulate an immune response, a tumour prevention model was used to isolate the immune effects of combination therapy. Mice were challenged with tumour cells for more than 100 days following treatment with cancer vaccine and temsirolimus. As the tumour cells were never directly exposed to temsirolimus, all anti-tumour effects could be attributed to immunostimulation. The combination therapy completely inhibited tumour growth, whereas treatment with cancer vaccine alone did not. Therefore, temsirolimus enhanced anti-tumour immunity.
The anti-tumour studies demonstrated that the net effect of immune modulation with mTOR inhibition was to enhance the cancer vaccine. However, mTOR inhibitors are known to have both immune-stimulating and immune-suppressing effects. To better understand these opposing functions, the immune effects of mTOR inhibition were characterised in our model. A detailed understanding of immune modulation may shed light on the mechanism of action of pharmacological mTOR inhibition, and identify strategies to enhance a particularly mTOR function and achieve varied clinical objectives.
Temsirolimus and rapamycin inhibited the proliferation of both activated CD8 and CD4 T cells in vitro
. This observation is consistent with previous reports that rapamycin directly inhibits T-cell activation and proliferation (Mondino and Mueller, 2007
; Song et al, 2007
). In our study, the in vivo
effects of mTOR inhibition differed for CD8 and CD4 T cells. When temsirolimus was administered to mice, the percent of CD8 lymphocytes in lymph nodes did not change, whereas the percent of CD4 lymphocytes decreased. CD4 lymphocytes include both effector and regulatory lymphocytes. Despite an overall decrease in CD4 lymphocytes, the percent of CD4 lymphocytes expressing FoxP3 increased in groups that received temsirolimus. This observation is consistent with reports that regulatory T cells are less sensitive to the anti-proliferative effects of mTOR inhibition (Battaglia et al, 2006
; Segundo et al, 2006
). In transplant patients treated with mTOR inhibition, an increase in regulatory T cells is believed to be an important mechanism for immune suppression. Therefore, the effects of mTOR inhibition on lymphocyte proliferation and regulatory T-cells are expected to favour immune suppression.
However, effector T cells generated in vivo during treatment with mTOR inhibitor had greater activity as measured using an ELISPOT assay. In a confirmatory study, the increased IFN-γ response was localised to CD8 T-cells. Furthermore, temsirolimus enhanced in vivo killing by cytotoxic T lymphocytes. These results suggest that the primary immune response generated by the cancer vaccine was enhanced by temsirolimus. It is possible that the regulatory T cells that increase during temsirolimus treatment ultimately suppress the CD8 T cell response; however, another possibility is that the increase in regulatory T cells is a negative feedback response to the enhanced cytotoxic T-cell function.
Previous reports do not produce a clear picture of the effects of mTOR inhibition on DC function. For example, rapamycin has been shown to suppress DC maturation by downregulating the IL-4 receptor complex and decreasing the production of IL-2 and tumour necrosis factor (Hackstein et al, 2003
). Also, human DCs matured from monocytes in the presence of rapamycin had decreased ability to take up antigen, including decreased endocytosis and phagocytosis (Monti et al, 2003
). However, other recent reports suggest that mTOR inhibition may enhance the innate immune response in DCs (Weichhart et al, 2008
) and stimulate autophagy, which can enhance antigen presentation (Jagannath et al, 2009
). We found no evidence that temsirolimus promotes anti-tumour immunity through its effects on DCs. Temsirolimus did not change the expression of baseline DC markers. In fact, BM-derived DCs stimulated with cancer vaccine and temsirolimus had decreased ability to stimulate CD8 T cell when compared with DCs stimulated with vaccine alone. The mTOR inhibition had a suppressive effect on overall DC function. Therefore, the effects of mTOR inhibition on DCs do not explain the enhanced anti-tumour immunity seen in our models.
We also characterised the effects of temsirolimus on CD8 memory cell formation. To minimise the effects of temsirolimus during the T-cell expansion phase, temsirolimus was started during T-cell contraction. This treatment schedule focuses the effects of temsirolimus on the transition from effector CD8 T cells to memory cells. Mice were re-stimulated with tumour antigen 33 days following primary vaccination when all transferred T cells should be long-lived memory cells. Samples collected 7 days after the re-stimulation show that CD8 T cells from animals treated with both vaccine and temsirolimus have an enhanced IFN-γ
response when compared with controls; however, CD8 T-cell proliferation was not significantly enhanced by temsirolimus. This is consistent with a recent report that rapamycin treatment during T-cell contraction does not alter the number of CD8 T cells, but rather accelerates memory differentiation and produces T cells with phenotypic characteristics of highly functioning memory cells (Araki et al, 2009
). We found that combined treatment with cancer vaccine and temsirolimus produced memory cells with greater CD62L expression than control treatments, indicating that temsirolimus promotes formation of central memory, which is associated with enhanced anti-tumour activity (Klebanoff et al, 2005
The RCC and melanoma are classic immunoresponsive tumours. Novel cancer vaccines are being actively developed for these and other malignancies. Our results provide a rationale for combining mTOR inhibitors with novel and established immune therapies. Temsirolimus is currently approved for the treatment of RCC, and is therefore particularly attractive for use with cancer vaccines that are being investigated for RCC. Future studies will need to develop optimal dosing strategies and identify cancer vaccines that are best suited for use with mTOR inhibitors.
Temsirolimus is an anti-neoplastic agent that is currently approved for patient use. It has immune-modulating activity. In animal models for studying tumour vaccines, temsirolimus enhanced vaccine activity by enhancing effector T-cell function and enhancing the production of CD8 memory T cells.