**3. Preclinical experience of glioma-radio-immune-modulatory therapy**

In the Lund clinical study, named "Brain-Immuno-Gene-Tumour-Therapy" (BRIGTT), patients were immunized with their own tumour cells, cultivated from their surgical specimens and transfected with human IFN gene (Salford, et al. 2002). The cells taken from the surgically removed tumour were grown in culture. The day before immunization the karyotyped tumour cells were infected with an Adenovirus expressing human IFN. At the day after transfection, the immunization of the patient takes place soon after the cells have been irradiated with Cs-137 gamma radiations to an absorbed dose of 100 Gy (Baureus-Koch, et al. 2004). By subcutaneous (s.c.) implantation of these cells in the arm of the patient it is expected that the host immune system is activated against the tumour. The activated CD8(+) T-cells will pass the BBB and attack the cancer cells present at the primary tumour site as well as the distant metastases "*guerrilla cells*" (Salford, et al. 2006; Salford, et al. 2001; Salford, et al. 2002; Salford, et al. 2004; Siesjö, et al. 1993; Visse, et al. 1999). Results from the first eight human treatments in the phase 1—2 BRIGTT study show that immunization with transfected tumour cells is safe for the patients and improves survival (A. Persson, et al. 2005; Salford, et al. 2005; Salford, et al. 2011; Salford, et al. 2004).

In order to further enhance the effect of this immunotherapy we investigated the effect of combining it with a single fraction radiation therapy in an animal model. The results of

loading onto MHC class I molecules. The proteasome in tumour cells is a sensitive target for radiation, resulting in decreased processing of endogenous self antigens. The processing of tumour antigens is, however, increased by radiation, which enhance the accumulation of

Radiation therapy also causes an increase in production of the cytokine IFN in the target region which up-regulates low levels of MHC class I, creating a tumour microenvironment conducive for CD8(+) T cell infiltration and their recognition of tumour cells (Lugade, et al.

It has been demonstrated that antigen presentation by MHC class I is increased for many days by single fraction radiation therapy. The most pronounced effect was recorded at 7 days after irradiation with an absorbed dose of 8 Gy. This might be one of the reasons why the efficacy of tumour immunotherapy is most effective in combination with single fraction radiation therapy (Reits, et al. 2006). Maximum loading of the tumour micro-environment with cancer antigen occurred 2 days after radiation therapy and coincided with the optimal

It has been demonstrated that the radiation modulation of MHC-I mediated antitumor immunity also depends on the antigen presenting pathways of the dendritic cells (Liao, et al. 2004). The dendritic cells either initiate an effective cytotoxic response against antigenbearing cells, or produce tolerance, depending on the context in which those antigens are presented (Zou 2005). It has been shown that cell death caused by radiation therapy release tumour antigen, which facilitates an effective cytotoxic response of the dendritic cells (Hatfield, et al. 2005). Radiation therapy activation of dendritic cells (DC), induce secretion of interleukin-1 beta (IL-1), which is required for the adequate polarization of IFN

**3. Preclinical experience of glioma-radio-immune-modulatory therapy** 

In the Lund clinical study, named "Brain-Immuno-Gene-Tumour-Therapy" (BRIGTT), patients were immunized with their own tumour cells, cultivated from their surgical specimens and transfected with human IFN gene (Salford, et al. 2002). The cells taken from the surgically removed tumour were grown in culture. The day before immunization the karyotyped tumour cells were infected with an Adenovirus expressing human IFN. At the day after transfection, the immunization of the patient takes place soon after the cells have been irradiated with Cs-137 gamma radiations to an absorbed dose of 100 Gy (Baureus-Koch, et al. 2004). By subcutaneous (s.c.) implantation of these cells in the arm of the patient it is expected that the host immune system is activated against the tumour. The activated CD8(+) T-cells will pass the BBB and attack the cancer cells present at the primary tumour site as well as the distant metastases "*guerrilla cells*" (Salford, et al. 2006; Salford, et al. 2001; Salford, et al. 2002; Salford, et al. 2004; Siesjö, et al. 1993; Visse, et al. 1999). Results from the first eight human treatments in the phase 1—2 BRIGTT study show that immunization with transfected tumour cells is safe for the patients and improves survival (A. Persson, et al.

In order to further enhance the effect of this immunotherapy we investigated the effect of combining it with a single fraction radiation therapy in an animal model. The results of

antigen/MHC class I complexes on the cell surface (Pajonk &Mcbride 2001 ).

time for CD8(+) T cell transfer (Bin Zhang, et al. 2007).

**2.3 Radiation effecting dendritic cells DC function** 

producing CD8(+) T-cells (Aymeric, et al. 2010).

2005; Salford, et al. 2005; Salford, et al. 2011; Salford, et al. 2004).

2008).

these preclinical experiments, which were performed already 2001, showed that a single fraction of RT combined with immunotherapy resulted in a significantly increased survival time of rats with intra-cranially implanted N29 or N32 glioblastoma. Further there were significant numbers of complete remissions of the most infiltrative N29 tumour implanted in Fischer-344 rats (B.R.R. Persson, et al. 2010). Other researchers have also reported substantial tumour regression by single fraction radiation therapy combined with various regimes of immune therapy (Bradley 1999; Chakraborty, et al. 2003; Demaria, et al. 2005a; Friedman 2002; Garnett, et al. 2004; Graf, et al. 2002; Lumniczky, et al. 2002).

#### **3.1 The Lund experience of combined single fraction RT and Immunization with IFN secreting tumour cells**
