DC vaccination approaches for brain tumors have been implemented in human clinical trials (5
). Several clinical trials for GMB using DC vaccines have demonstrated cellular and humoral anti-tumor immune responses; and albeit safe, the clinical efficacy of DC vaccination for GBM remains limited (3
). Our group has shown that in situ
Ad-Flt3L/TK-mediated immunogene therapy elicits an influx of DCs, macrophages, CD4+ T cells, and CD8+ T cells into the brain tumor microenvironment (17
) and stimulates effective anti-GBM immune response resulting in tumor regression and long-lasting CD8+ T cell mediated anti-tumor immunological memory in several mouse and rat orthotopic brain tumor models (16
). Based on these data, a Phase I clinical trial for GBM was recently cleared by the FDA and is slated to commence in 2011.
The combined conditional cytotoxic/immuno-stimulatory gene therapy approach, utilizes HSV1-TK to kill actively dividing brain tumor cells in the presence of GCV, thereby releasing endogenous brain tumor antigens and also innate immune adjuvants, such as high-mobility group protein B1 (HMGB1) (18
). HMGB1 belongs to a class of innate immune adjuvants called damage associated molecular pattern molecules (DAMPs) which mediate signaling by binding to a family of receptors called pattern recognition receptors (PPRs), thereby promoting innate and adaptive immune responses (41
). DCs express a large repertoire of PRRs and several studies have shown that signaling through of PPRs leads to DC activation, which is characterized by high levels of MHC-antigen complexes on the DC cell surface, upregulation of co-stimulatory molecules such as CD80 and CD86, and the production of cytokines such as IL-12 and IFNα. The production of these cytokines is directly involved in priming Th1 based immune responses (40
). We have previously shown that HMGB1 is released from dying brain tumor cells in response to treatment with Ad-TK/GCV or TMZ, and acts as an endogenous TLR2 agonist to activate bone-marrow derived, brain tumor-infiltrating DCs (18
). The other arm of our therapeutic strategy involves expression of Flt3L within the tumor microenvironment. Flt3L recreates a missing immune circuit from the brain, by inducing the expansion and migration of DCs into the brain tumor milieu where they encounter and phagocytose newly released endogenous brain tumor antigens (16
Herein we tested the hypothesis that combining Ad-Flt3L/TK in situ immunogene therapy with peripheral DC vaccination, would lead to enhanced therapeutic efficacy in a syngeneic brain tumor model. Our data demonstrate that the therapeutic combination, i.e., in situ immunogene therapy with DC vaccination led to long term survival in 90% of rats bearing large, syngeneic brain tumors; showing a ~40% increase in survival compared to Ad-Flt3L/TK immunogene therapy alone and ~80% increase in survival compared to DC vaccination alone. Our results also showed enhanced anti-tumor humoral immune response, i.e., ~2.5 fold increase in the levels of circulating anti-tumor antibodies compared to either treatment alone.
The enhanced therapeutic efficacy observed could result from expression of Flt3L within the brain tumor microenvironment which could enhance the trafficking of systemically delivered DC vaccines to the draining lymph nodes, where they present tumor antigens to naïve T cells and induce the clonal expansion of tumor-specific CTLs. To this effect, it has been previously shown that Flt3L elicits recruitment of DC populations, including fully differentiated DCs in lymphoid organs (40
) and traffic of subcutaneously delivered DC vaccines to the inguinal draining lymph nodes in mice (42
). We demonstrated that intracranial delivery of Ad-Flt3L leads to circulating Flt3L in rat serum (19
), thus supporting hypothesis that Flt3L could also act systemically and enhance the trafficking of DCs to draining lymph nodes. Increased trafficking of systemically delivered DCs to draining lymph nodes could lead to enhanced priming of anti-tumor immune responses, inducing increased levels of tumor regression and long-term survival as observed in this study.
An additional explanation for the increased therapeutic efficacy of in situ
immunogene therapy in combination with DC vaccination is that DAMPs, i.e. HMGB1, are known to enhance the activation status of DCs and facilitate Th1 immune responses (40
). We have previously shown that treatment of intracranial brain tumors with TK/GCV results in increased levels of circulating HMGB1 in the sera of mice (18
) and rats (21
). Therefore HMGB1 released into the systemic circulation could further activate systemically delivered DCs, thus enhancing their ability to prime an adaptive, anti-tumor immune response.
When used to treat small, established intracranial tumors, DC vaccination exhibited higher levels of long-term survival (~30%) when compared to treatment of large intracranial tumors (~10% long-term survival). These data suggest that the efficiency of a DC vaccination approach is associated with the degree of tumor burden in the host. Furthermore, our data demonstrate that DC vaccination is highly efficacious (100% long-term survival) when administered before tumor implantation, suggesting that DC vaccines would be more effective at preventing tumor recurrences after initial surgical debulking, chemotherapy, and radiotherapy.
Effective uptake and loading of tumor-associated antigens onto DCs’ MHC complexes and expansion of DC subgroups that can efficiently prime naïve T cells play a critical role in the effectiveness of DC vaccination. Therefore, it is critically important to optimize the preparation of tumor cell antigens and dendritic cells. In this study, we compared the immunogenicity and levels of phagocytosis of apoptotic, autophagic, and necrotic tumor cells lysates. In line with previous evidence (26
), our data indicate that triggering autophagy and/or apoptosis to generate tumor cell lysates increases the immunogenicity of tumor cells and enhances the delivery of tumor-associated antigens to DCs. Furthermore, we have previously reported that treatment of GBM cells with Ad-TK/GCV or TMZ results in the release of the endogenous TLR2 ligand HMBG1 from GBM cells (18
). Autophagic GBM cells have also been shown to release HMGB1, without causing lysis of the cell membrane and classical necrosis (45
). Previous reports demonstrated that DCs loaded with purified autophagosomes from autophagic tumor cells induced tumor-specific immune responses (26
), suggesting that autophagosomes contain a wide range of tumor-associated antigens and immune adjuvants, i.e., HMGB1 (46
). There is experimental evidence that suggests that intratumoral delivery of a recombinant cytotoxin composed of Pseudomonas exotoxin fused to IL-13 into mice bearing human xenografts causes cell death not only by apoptosis, but also by necrosis. Furthermore, in this paper, Kawakami et al. demonstrate that delivery of the IL13-PE cytotoxin induced phagocytes which may play a role in cytotoxin mediated tumor regression (47
). Additionally, it has been previously demonstrated that the fusion of the recombinant Pseudomonas exotoxin to a model tumor antigen may enhance vaccine potency (48
GM-CSF combined with IL-4 has been previously used to generate DCs from both murine and human bone marrow progenitor cells (30
). Flt3L has been introduced as an alternative means to generate DCs (35
) and recent reports demonstrated that Flt3L combined with IL-6 enhances the expansion of Th1-polarizing DCs, a requirement for the efficient induction of anti-tumor immune responses (50
). Thus, we compared the characteristics of GM-CSF+IL4- and Flt3L+IL6-generated DCs to establish the optimal parameters for ex vivo
expansion of DCs to be used in the vaccination paradigms described. Our results showed that ex vivo
conditioning with Flt3L resulted in higher levels of DCs when compared to GM-CSF+IL4; these results are in line with results previously obtained with canine DC cultures (49
). Levels of Th1 polarizing cytokines such as IL-12 and IFN-γ were higher when DCs were cultured with Flt3L+IL6. These data are in line with evidence that Flt3L induces DCs that preferentially secrete Th1-polarizing cytokines, when compared with GM-CSF cultured DCs (35
). Consistent with these results, inhibition of tumor progression and therapeutic efficacy of DC vaccination was higher when we used Flt3L+IL6-generated DCs. Addition of IL-6 in combination with Flt3L further enhanced the proliferation of BMDCs. Flt3L+IL6 induced DCs displayed decreased levels of cell surface markers CD80, CD86, MHCII and exhibited high phagocytic capacity consistent with an immature phenotype.
The advantage of combining immunogene therapy (Ad-Flt3L/Ad-TK) with DC vaccination is that gene therapy can be administered into the tumor bed/cavity at the time of surgical resection to immediately initiate anti-brain tumor immune responses whilst the autologous DC vaccine with autologous tumor lysate is being prepared ex vivo. After the DC vaccine preparation is completed, it can be administered using the appropriate vaccination regime to enhance the anti-tumor immunity and therapeutic efficacy. In summary, the data presented demonstrate that immunogene therapy not only elicits tumor regression and anti-tumor immunity, it also potentiates the therapeutic efficacy elicited by DC vaccination and support the implementation of novel Phase I clinical trials to assess the safety and efficacy of this combined therapeutic strategy.