<p>Bacillus Calmette Guérin (BCG), or attenuated Mycobacterium bovis, is the gold standard of immunotherapy in the clinic to treat superficial bladder cancer. However, setbacks remain due to a high recurrence rate, side effects, and BCG-refractory disease. In this thesis, we explored the use of novel immunotherapeutic agents such as CpG oligodeoxynucleotides (CpG ODNs) or synthetic ODNs containing unmethylated CpG dinucleotides. Since unmethylated CpG motifs are predominant in bacterial but not vertebrate DNA, they function as a “danger signal” leading to a potent immune response.</p><p>To be able to test various immunotherapeutic agents, we optimized subcutaneous (s.c.), metastatic, and orthotopic models using the murine bladder-49 (MB49) cancer cell line. In the orthotopic model, we show that poly-L-lysine promotes MB49 attachment to the bladder leading to 100% tumor take. In addition, Clorpactin (sodium oxychlorosene) potently enhances adenoviral transduction in the bladder.</p><p>Utilizing the MB49 model, we compare CpG ODNs with BCG and demonstrate the increased efficacy of CpG ODNs which could cure both s.c. and aggressive orthotopic bladder cancer. In our model, type B ODNs were most optimal and the antitumor response required T cells in order to induce regression and tumor-specific immunity. We also combined CpG ODNs with adenoviral vectors (Ad) expressing the immunostimulatory molecules CD40L, TRANCE, lymphotactin, IL2 or IL15. However, we show that CpG ODNs are effective as a monotherapy and adenoviral vectors did not enhance the effect.</p><p>AdCD40L was also used to genetically modify human dendritic cells (DCs). AdCD40L-transduced DCs not only had a higher and prolonged expression of the Th1 cytokine IL12 compared to TNFα-matured DCs, but CD40L-activated DCs could also resist the suppressive effects of IL10 and TGFβ. Since TNFα is commonly used in clinical DC vaccination protocols and because tumors often secrete immunosuppressive cytokines, these data have important implications for optimizing cancer immunotherapy.</p>

Bacillus Calmette Guérin (BCG), or attenuated Mycobacterium bovis, is the gold standard of immunotherapy in the clinic to treat superficial bladder cancer. However, setbacks remain due to a high recurrence rate, side effects, and BCG-refractory disease. In this thesis, we explored the use of novel immunotherapeutic agents such as CpG oligodeoxynucleotides (CpG ODNs) or synthetic ODNs containing unmethylated CpG dinucleotides. Since unmethylated CpG motifs are predominant in bacterial but not vertebrate DNA, they function as a “danger signal” leading to a potent immune response. To be able to test various immunotherapeutic agents, we optimized subcutaneous (s.c.), metastatic, and orthotopic models using the murine bladder-49 (MB49) cancer cell line. In the orthotopic model, we show that poly-L-lysine promotes MB49 attachment to the bladder leading to 100% tumor take. In addition, Clorpactin (sodium oxychlorosene) potently enhances adenoviral transduction in the bladder. Utilizing the MB49 model, we compare CpG ODNs with BCG and demonstrate the increased efficacy of CpG ODNs which could cure both s.c. and aggressive orthotopic bladder cancer. In our model, type B ODNs were most optimal and the antitumor response required T cells in order to induce regression and tumor-specific immunity. We also combined CpG ODNs with adenoviral vectors (Ad) expressing the immunostimulatory molecules CD40L, TRANCE, lymphotactin, IL2 or IL15. However, we show that CpG ODNs are effective as a monotherapy and adenoviral vectors did not enhance the effect. AdCD40L was also used to genetically modify human dendritic cells (DCs). AdCD40L-transduced DCs not only had a higher and prolonged expression of the Th1 cytokine IL12 compared to TNFα-matured DCs, but CD40L-activated DCs could also resist the suppressive effects of IL10 and TGFβ. Since TNFα is commonly used in clinical DC vaccination protocols and because tumors often secrete immunosuppressive cytokines, these data have important implications for optimizing cancer immunotherapy.

This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy. Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement. CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success. When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction. The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.