CpG therapy is superior to BCG in an orthotopic bladder cancer model and generates CD4+ T-cell immunity.

Bacillus Calmette Guérin (BCG) immunotherapy has been successful in extending tumor remission in bladder cancer, the fifth most common cancer in men. However, relapses are frequent and some patients develop resistance to BCG. CpGs were previously demonstrated to be effective in the murine MB49 model. In this paper, we modeled a more aggressive orthotopic bladder cancer than previously studied. Moreover, we compared standard BCG immunotherapy side-by-side with the Toll-like receptor-9 agonist CpG. MB49 tumor-bearing mice were treated with BCG or CpG and survival as well as tumor progression were observed over time. Urine, blood, and tumors were collected and analyzed. Mice were rechallenged and evaluated for tumor-specific immunity. In this study, CpGs induced a complete response of large aggressive orthotopic MB49 bladder tumors, resulting in tumor-specific systemic immunity. Further, data indicated that this potent antitumor effect required T cells. A comparison of CpGs and BCG in both a highly and less aggressive orthotopic tumor model, and in a subcutaneous tumor model, demonstrated that CpGs were superior to BCG. In the orthotopic model, BCG induced a local cytokine storm during treatment initiation whereas CpG affected a more refined cytokine pattern over time. Increased levels of cytokines in serum correlated with enhanced survival in the subcutaneous model. Further, immune cell depletion studies demonstrated that CpG-induced protective immunity was CD4+ T-cell dependent. Taken together, our data suggest that CpGs are superior to BCG for bladder cancer immunotherapy. Thus, this potent new drug may be an attractive therapeutic alternative and should be evaluated in bladder cancer patients.

Human bladder carcinoma is dominated by T-regulatory cells and Th1 inhibitory cytokines.

PURPOSE: Immunotherapy has faced limited success, although many solutions have been proposed. Recently regulatory T cells have made a comeback in the immunological arena and the role of these cells in patients with cancer is in focus. It is under evaluation whether the immunological status of patients with cancer may affect their sensitivity to immunotherapy. We are developing immunostimulating gene therapy for treating bladder cancer. In this study we constructed an immunological profile of patients with bladder carcinoma to understand which obstacles must be circumvented. MATERIALS AND METHODS: Biopsies and blood were used to identify immune cell populations by FACS(R), histochemistry and proliferation assays, and cytokine production by polymerase chain reaction. RESULTS: Results indicate that bladder carcinoma is a Tr1 dominated tumor, as shown by the infiltration of T-regulatory cells expressing FOXP3, and the presence of tumor necrosis factor-beta and interleukin-10 mRNA copies. We further noted that circulating patient T cells were unresponsive to polyclonal T-cell activation compared to healthy donor cells. Moreover, CD4+CD25+ T cells were increased in patient blood and could suppress the expansion of allogeneic T cells from healthy donors. CONCLUSIONS: Patients with bladder carcinoma show an immunosuppressive regulatory profile, including nonresponsive T cells. Clinical protocols able to effectively counteract these mechanisms are warranted.

Dendritic cells engineered to express CD40L continuously produce IL12 and resist negative signals from Tr1/Th3 dominated tumors.

TNFalpha-matured dendritic cells (DCs) pulsed with tumor antigens are being evaluated as cancer vaccines. It has been shown that DCs produce IL12 during a limited time span and subsequently enter a stage of IL12 exhaustion. If DCs are generated ex vivo, the patient could receive IL12-exhausted DCs which may be detrimental for stimulating anti-tumor Th1 responses. Furthermore, many cancer patients exhibit a cytokine profile skewed toward IL10 and TGFbeta. This immunological profile, called the Tr1/Th3 response, is associated with the presence of regulatory T-cells. Tr1/Th3 responses potently inhibit DC maturation, thereby regulating Th1 responses. In the present study, we produced genetically engineered DCs that continuously express Th1-related cytokines such as IL12, and resist negative signals from Tr1/Th3-dominated bladder carcinoma cells. Human immature DCs were genetically engineered by adenoviral vectors to express CD40L, or were treated with TNFalpha as a positive control for maturation. The expression of different Th1/Th3 and inflammatory cytokines was monitored. IL12 and IFNgamma were expressed by CD40L-engineered DCs, while TNFalpha-matured DCs lacked IFNgamma and exhibited low IL12 expression. The addition of recombinant IL10 to genetically engineered DCs did not abolish their Th1 profile. Likewise, coculture with tumor cell lines expressing TGFbeta with or without recombinant IL10 did not revert to the engineered DCs. We further demonstrate that the resistance of CD40L-expressing DCs to TGFbeta and IL10 may be due to decreased levels of TGFbeta and IL10 receptors. Thus, CD40L-engineered DCs are robust Th1-promoting ones that are resistant to Tr1/Th3-signaling via IL10 and TGFbeta.

CpG oligonucleotide therapy cures subcutaneous and orthotopic tumors and evokes protective immunity in murine bladder cancer.

Bacillus Calmette-Guerin (BCG) instillation is standard immunotherapy for superficial bladder carcinoma. However, many patients become refractory to BCG, giving impetus to the development of alternative therapies. CpG oligodeoxynucleotide (ODN) therapy has been shown to promote T(H)1-oriented antitumor responses in various tumor models. To investigate its therapeutic effect in bladder cancer, we used different CpG ODNs to treat C57BL/6 mice bearing the subcutaneous murine bladder tumor MB49. CpG type B ODN 1668 was superior at inhibiting tumor growth, leading to complete regression of large tumors. More importantly, CpG ODN 1668 also regressed orthotopically growing MB49 tumors for the first time. Rechallenge of CpG ODN-cured mice with MB49 showed that a majority of the mice were protected long term, demonstrating that CpG ODN therapy evokes a memory response. Adenoviral vectors (Ad) encoding CD40L, tumor necrosis factor-related activation-induced cytokine, lymphotactin, interleukin (IL) 2, and IL-15 were also investigated. AdCD40L and AdIL-15 transduction could abolish MB49 tumorigenicity, and these vectors were combined with CpG ODN 1668 to investigate any enhanced effects. No such effects were seen. All groups of mice treated with CpG ODNs, alone or in combination with adenoviral vector, exhibited increased serum concentrations of IL-12, indicative of a T(H)1 response. Our results show that CpG ODN therapy cures established subcutaneous and orthotopic bladder cancer via a T(H)1-mediated response and provides long-lasting protective immunity.

Adenovirus CD40 ligand gene therapy counteracts immune escape mechanisms in the tumor Microenvironment.

Tumors exhibit immune escape properties that promote their survival. These properties include modulation of Ag presentation, secretion of immunosuppressive factors, resistance to apoptosis, and induction of immune deviation, e.g., shifting from Th1- to Th2-type responses. These escape mechanisms have proven to hamper several immunotherapeutic strategies, and efforts need to be taken to revert this situation. We have studied the immunological effects of introducing CD40 ligand (CD40L), a potent dendritic cell activation molecule, into the tumor micromilieu by adenoviral gene transfer. For this purpose, a murine bladder cancer model (MB49) was used in C57BL/6 mice. The MB49 cells are known to induce IL-10 in the tumor environment. IL-10 potently inhibits the maturation of dendritic cells and thereby also the activation of CTLs. In this paper we show that CD40L immunogene therapy suppresses IL-10 and TGF-beta production (2-fold decrease) and induces a typical Th1-type response in the tumor area (200-fold increase in IL-12 production). The antitumor responses obtained were MB49 cell specific, and the cytotoxicity of the stimulated CD8(+) cells could be blocked by IL-10. Adenovirus CD40L therapy was capable of regressing small tumors (five of six animals were tumor free) and inhibiting the progression of larger tumors even in the presence of other escape mechanisms, such as apoptosis resistance. Furthermore, CD40L-transduced MB49 cells promoted the maturation of dendritic cells (2-fold increase in IL-12) independently of IL-10. Our results argue for using adenovirus CD40L gene transfer, alone or in combination with other modalities, for the treatment of Th2-dominated tumors.