**3.1.5 Summary of the LUND experiment**

The results of the Lund experiments reveal that a single fraction radiotherapy session of 5 or 15 Gy combined with immunization by i.p. injection of irradiated syngeneic tumour cells induces a significant anti-tumour response to intra cranial implanted glioblastoma tumours

**RT 5 Gy + Imu**

**RT 15 Gy + Imu**

**0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32**

**Time after inoculation / days**

**Mann-Whitney 2-tailed test versus Control**

**Tumour weight g** 

**Controls No Treatment**

and a combination of radiation therapy and immunization (upper panel).

**Num. Rats** 

Fig. 4. Survival plot of intra-cerebral implanted N32 tumours: Controls (Lower panel); Immunization with syngeneic N32 tumours cells (2nd panel); radiation therapy (3rd panel),

> **Median Survival time days**

**Control** 12 19 3 0.19 0.16 **IMU IFN** 13 19 6 NS 0.25 0.23 **RT 5 Gy** 6 19.5 2 NS 0.18 0.10 **RT 15 Gy** 13 23 2 P<0.001\*\*\* 0.16 0.13 **RT 5 Gy +IMU IFN** 7 19 2 NS 0.14 0.09 **RT 15 Gy + IMU IFN 13 27 3 P<0.0001\*\*\* 0.30 0.28**  Table 4. Number of rats, mean survival time, and the significance of Mann-Whitney 2-tailed test versus control is shown in columns 2-4. In the last column is given the tumour weight at time of death of intra cerebral N32 tumours treated with syngeneic IFN transfected tumour

cells (IMU IFN), radiation therapy (RT) and their combination (RT + IMU IFN).

The results of the Lund experiments reveal that a single fraction radiotherapy session of 5 or 15 Gy combined with immunization by i.p. injection of irradiated syngeneic tumour cells induces a significant anti-tumour response to intra cranial implanted glioblastoma tumours

**Immunized IFN**

**RT 15 Gy**

**RT 5 Gy**

**3.1.5 Summary of the LUND experiment** 

*Number of living rats with N32 tumors*

**Type of treatment** 

in Fischer-344 rats. In the rats, which were inoculated with N32 tumour cells, the combination of 15 Gy single fraction radiation therapy with immunization of IFN- secreting syngeneic cells resulted in increased survival time by about 40% (p<0.001). But none of these rats survived longer than 30 days. In the group inoculated with N29 tumour cells and treated with 5 Gy RT combined with immunization the survival time was significantly increased by 87% (p=0.003), and 75% of the animals survived for more than 170 days. The difference in response of N29 and N32 cell lines indicate that there is difference in immune response in different clones of glioma.

#### **3.2 The Hungarian experience of single fraction RT and Immunization with (GM-CSF, IL-4, IL-12) in a mouse glioma (Gl261) brain tumour model**

In Hungary a study was performed in a mouse glioma (Gl261) brain tumour model with single fraction radiotherapy combined with administration of cytokine-producing cancer cell vaccines (Lumniczky, et al. 2002). Their brain tumour bearing mice were treated with various cytokine producing vaccines made by in vitro transduction of Gl261 tumour cells with different genes such as: IL-4, IL-6, IL-7, GM-CSF, TNF. Immunotherapy alone with vaccines producing either IL-4 or GM-CSF resulted in complete remission in 20–40% of the mice. By combining immunotherapy using (GM-CSF, IL-4, IL-12) producing vaccines with local tumour radiotherapy (single fraction 6 Gy X-ray radiations) about 80–100% of the glioma-bearing mice were cured. The high efficiency of the combined treatment was maintained even under suboptimal conditions when neither of the individual modalities alone cured any of the mice (Lumniczky, et al. 2002). Their results are in good agreement the survival rate of 75% (p<0.05) achieved in the Lund study of N29 tumours in rats treated with IFN- secreting vaccine combined with 5 Gy single fraction RT (B. R. R. Persson, et al. 2010).

#### **3.3 The U.S. experience of radiation therapy combined with vaccination of mice with Glioma or mammary carcinoma**

#### **3.3.1 Combining radiation therapy with blockade of the CTLA-4 pathway**

The cytotoxic T lymphocyte-associated protein CTLA-4 is involved in the immune regulatory mechanisms that control the early stage of the T cell response. It has previously been demonstrated that blockade of the CTLA-4 protein enhance anti-tumour responses both in experimental systems and in clinical trials (Chambers, et al. 2001; Egen, et al. 2002).

In a mouse model of the poorly immunogenic metastatic mouse mammary carcinoma 4T1, however, neither anti-tumour response nor survival-time was affected by using an anti-CTLA-4 monoclonal antibody for blocking the CTLA-4 protein. But anti-CTLA-4 monoclonal antibody administration combined with one 12 Gy fraction of radiation therapy, inhibited the growth of the primary irradiated tumour. Also the survival-time of the mice was significantly increased by this combined treatment (Demaria, et al. 2004; Demaria, et al. 2003; Demaria, et al. 2005b).

Another investigation of the effects of systemic CTLA-4 blockade with monoclonal antibody (9H10) to CTLA-4 employed in a mice model with well-established glioma, showed that CTLA-4 blockade confers long-term survival in 80% of treated mice (Fecci, et al. 2007). Thus the combination of local RT with CTLA-4 blockade might be applied as radio-immunemodulating therapeutic strategy also against glioma.

Radiation Immune Modulation Therapy of Glioma 375

rejecting challenge tumours. Antitumor immunity was associated with an increased number of tumour-infiltrating lymphocytes (TILs) in brain tumours and increased tumour-specific production of IFN. Since anti-CD137 therapy is already used in clinical trials it was suggested to be studied further in combination with local hypo-fractionated (2x4 Gy)

The expression profiles of CD4(+) and CD8(+) T cells and Treg from patients with newly diagnosed *glioblastoma multiforme* are quite different when compared with normal healthy volunteers (Learn, et al. 2006). But how various absorbed dose or various fractionation pattern or methods of radiation delivery can affect T-cell populations and alternative regulatory molecules in glioma patients is still under debate (Chiba, et al. 2010; Teitz-

**4.1 Effects of concomitant temozolomide and radiation therapies on WT1-specific** 

Like many other solid tumours, glioma have been found to express a protein characteristic for Wilms' tumour 1 (WT1) (Hashiba, et al. 2007). A peptide based immunotherapy targeting the WT1 gene has successfully been used in patients with recurrent glioma. The clinical response indicates that CD8(+) cytotoxic T lymphocytes (CTLs) are the main effectors of this WT1 vaccination (Oka, et al. 2004). A phase II clinical trial of the WT1 vaccination for patients with recurrent malignant glioma resulted in a partial response rate of 9.5% but none complete response. The median length of period with progression-free

In planning for a clinical trial of WT1 vaccination involving patients with newly diagnosed malignant glioma, it is also aimed to combine concurrent radiation /TMZ therapy with WT1 immunotherapy. The critical question is, however, if the depletion of lymphocytes caused by the current standard radiation/TMZ treatment is a drawback for a combination with WT1 immunotherapy. Therefore a clinical study was performed in order to determine how the concomitant radiation/TMZ therapy affects the WT1-specific T-cells and other T-cells in terms of their frequencies and total numbers. This study concluded that, even after the decrease of the absolute numbers of lymphocytes, the fraction of WT1 specific T-cells was stable. They concluded that it may the possible to apply WT1 immunotherapy after the end

In another clinical study of 8 patients with primary glioma it was found that concomitant radiation/TMZ therapy integrated with autologous dendritic cell-based immunotherapy was feasible and well tolerated. The median progression-free survival (PFS) was 75% and at 6 months and 50% at 18 months. The median time of survival for all patients is 24 months. One patient was still free from progression or recurrence at 34 months (Ardon, et al. 2010).

A single fraction of high dose radiation therapy has been demonstrated to dramatically increase the priming of T-cell in draining lymphoid tissues, which increased the action of the CD8(+) T cells and lead to reduction and eradication of the primary tumour or distant

**4.2 Treatment recurrent malignant glioma with hypo-fractionated radiotherapy** 

radiation therapy for clinical translation (Newcomb, et al. 2010).

Tennenbaum, et al. 2008; Verastegui, et al. 2003).

survival was 20 weeks (Izumoto, et al. 2008).

**combined with immune therapy** 

of 6 weeks of radiation/TMZ therapy (Chiba, et al. 2010).

**T-cells in malignant glioma** 

**4. Clinical studies of combining radiation and immune therapy** 
