Our antitumor studies demonstrate that intravesical chitosan/IL-12 is superior to both intravesical IL-12 alone and BCG, the standard-of-care immunotherapy for the treatment of superficial bladder cancer in a murine model. Intravesical treatments with chitosan/IL-12 consistently resulted in 88 to 100% of mice being cured of established, aggressive orthotopic bladder tumors. IL-12 alone was less effective (38 to 60% survival), a finding similar to those in published studies using either recombinant IL-12 protein (16
) or IL-12 gene therapy (17
) in orthotopic bladder cancer models. Transfection of MB49 cells with the luciferase construct allowed for visualization of tumor growth as early as 4 days post-tumor implantation. Tumors did not become palpable or show any evidence of hematuria for at least one week, and often up to 10 days post-tumor implantation. The enhanced sensitivity of bioluminescence imaging was useful for excluding mice in which tumors do not “take.”
In our study, BCG had minimal antitumor activity and no long-term survival advantage against orthotopic MB49 tumors. In previous studies, BCG treatment initiated the day after tumor implantation conferred a nominal 10 to 15% survival advantage (30
). Therefore, the survival advantage mediated by intravesical BCG in this model is abrogated when treatment is withheld until tumors are better established (day 5 post-tumor implantation).
Immunological memory is particularly important for bladder cancer, which has the highest recurrence rate of any cancer. Thus, perhaps more important than the high rate of tumor regression mediated by intravesical chitosan/IL-12 is the complete protection from tumor rechallenge it confers, suggesting that chitosan/IL-12 immunotherapy can generate durable immunological memory. In contrast, others have demonstrated that intravesical BCG does not mediate protective immunity, and no CTL activity could be measured from spleens of BCG-cured mice (32
). In yet another study, intravesical IL-12 gene therapy, but not BCG, conferred protection from tumor rechallenge (17
Our finding that intravesical chitosan/IL-12 produced greater serum levels of IL-12 and IFN-γ than intravesical IL-12 alone supports the hypothesis that chitosan’s unique properties can enhance local IL-12 delivery. It is likely that chitosan enhances both retention through mucoadhesive interactions and penetration through increased epithelial permeability. In previous reports, chitosan has been shown to enhance local delivery of drugs to and across multiple mucosal surfaces, including nasal (34
), buccal (35
), intestinal (36
), and vaginal (37
). Chitosan has also been shown to enhance the permeability of the bladder to small hydrophilic drugs (38
). In the present study, this enhanced permeability may have allowed IL-12 better access to resident immune cells in the submucosa. The relative importance of increasing epithelial permeability vs. IL-12 retention is currently under investigation.
The difference in IFN-γ levels between intravesical and s.c. administrations is not as great as the difference in IL-12 levels. In our experience, serum IFN-γ reaches a plateau at moderate IL-12 doses. For instance, at 24 h, serum IFN-γ levels with 1 μg of IL-12 s.c. are similar to levels with 5 μg of IL-12 s.c. (10,897 ± 4,004 pg/mL vs. 13,431 ± 4,894 pg/mL). It should be noted that although IL-12 was detected in serum following intravesical chitosan/IL-12 administration, the concentrations were 516 times lower than levels measured following s.c. injections.
Urinary cytokine analysis revealed that chitosan/IL-12 induced significant production of important TH
1 cytokines (IL-12p70, IFN-γ, and TNFα) (). The induction of multiple urinary cytokines has long been thought to play a role in intravesical BCG immunotherapy (25
). A previous study looked extensively into a range of urinary cytokines produced by tumor-free mice in response to intravesical BCG (29
). Our measurements of urinary TNFα, IFN-γ, and IL-10 in response to BCG were similar. However, we measured significantly lower values for IL-12p70 and IL-6. Two procedural differences between the studies may account for this discrepancy. First, our mice had MB49 tumors, which Lattime et al. showed to be highly immunosuppressive (41
). Second, our instillation volume was 100 μL rather than the 200 μL used by Saban et al. (29
). The larger volume, which is close to the maximum capacity of the murine bladder, would have likely disrupted the integrity of the bladder wall and possibly resulted in a more effective BCG infection. In yet another study, no increases of urinary IFN-γ or IL-12 were detected between 4 h and 24 h after repeated BCG instillation (43
). In the present study, chitosan/IL-12 induced much higher levels of TH
1 cytokines than BCG. The ability of chitosan/IL-12 to induce far greater levels of cytokines than BCG correlates with its superior antitumor activity. Based on these results, urinary cytokines may be an important prognostic indicator of clinical efficacy in potential trials involving intravesical chitosan/IL-12.
Immunohistochemical staining of treated bladder tumors provided further insight into the mechanism of action of chitosan/IL-12. In our experience, 2 intravesical treatments with chitosan/IL-12 were necessary to observe initial signs of tumor regression; i.e., stable or decreasing bioluminescence and reversal of hematuria. Thus, the optimal time to look for differences in tumor infiltrates was after the second treatment. Indeed, tumors treated twice with chitosan/IL-12 showed moderate to intense staining for CD3 and F4/80 infiltrates (). Within the CD3 compartment, CD8 staining was greater than CD4 staining (). This is in contrast to preclinical and clinical studies with BCG in which CD4+
cells outnumber CD8+
). Our results point to CD8+
T cells and macrophages as potential effector cells mediating the regression of MB49 tumor. Subset depletion studies are planned to confirm this hypothesis.
Combining immunohistochemistry results with urinary cytokine measurements, it appears likely that IL-12 induces a TH
1 cytokine cascade that is produced initially by resident immune cells (most likely T cells and macrophages). These cytokines can (a
) have direct cytotoxic and antitumor effects; (b
) recruit additional immune cells to the tumor; (c
) enhance the proliferation of pre-activated T cells; and (d
) reverse immune suppression. In particular, IFN-γ has anti-angiogenic properties (44
), is directly cytotoxic when combined with other cytokines such as TNFα (10
), can upregulate MHC I and II molecules on tumor cells, including the MB49 cell line (45
), and can activate macrophage effectors (46
) and rescue immunosuppressive tumor-associated macrophages (47
). Chitosan enhances all of these effects through better local delivery of IL-12, which leads to higher cytokine production.
In additional immunohistochemistry studies, we found that a residual population of T cells and macrophages remains in the bladder submucosa after a tumor is completely eradicated (). These populations begin to approach normal (naïve) levels 6 months after tumor cure. These residual effectors may be responsible for the complete protection from tumor rechallenge among cured mice. The relative contributions of innate (macrophages) and adaptive (antigen-specific CD8+ cells) immune systems during this rejection are currently being studied. Nevertheless, because of the high rate of recurrence of bladder cancer, the ability of an intravesical immunotherapy to maintain a resident effector population in the bladder is a distinct benefit.
Additional sections of naïve and cured bladder tissues were stained with hematoxylin and eosin to document any pathological changes associated with chitosan/IL-12 treatment. There were no pathological differences between the cured and naïve bladders as determined by a board certified pathologist. This suggests that chitosan/IL-12 is likely a well tolerated immunotherapy with no long-term pathology. Confirmatory toxicology and pathology studies are planned.
To date, the only clinical trial to date using intravesical IL-12 Failed to show clinically relevant antitumor or immunological effects in patients with recurrent superficial bladder cancer (18
) which contrasts the preclinical data reported here (, ) and elsewhere (16
). There are at least two possible explanations for this discordance. First, the dose of IL-12 delivered intravesically in humans may have been insufficient to induce an effective immune/antitumor response. The findings of this (supplemental Fig. 2
) and other studies (16
) have reported dose-dependent effects of intravesical IL-12-based therapies in murine models. In the clinical study, the highest dose used was 200μg which is equivalent, based on bladder volume, to approximately 0.07μg in mice. At this dose, even when formulated with chitosan, IL-12 would not be expected to improve survival in the MB49 model. Although this is not a perfect comparison because of the aggressiveness of the murine tumor, it does indicate that there may be a threshold dose for IL-12 to induce a clinically relevant immune response. Based on our study, it is reasonable to predict that chitosan would substantially reduce this threshold dose in humans.
A second possible explanation is that IL-12 may be less potent in humans than in mice. Systemic (i.v. or s.c.) IL-12 therapy has shown remarkable antitumor effects in numerous murine models that have not been duplicated in clinical trials although those trials have reported sporadic complete responses, significant percentages of stable disease (10
) as well as enhanced NK cytolytic activity and T cell proliferation (50
). However, two on-study deaths in a Phase II study have overshadowed the modest antitumor and immunological effects generated by systemically administered IL-12. Taken together, the modest antitumor efficacy and well documented toxicity in humans indicate that the therapeutic window for systemically administered IL-12 is more narrow for humans than mice.
The disappointment of early trials brings into question the translatability of any new IL-12-based monotherapy, such as the one introduced here. However, it is important to note that local administrations of IL-12 can limit systemic toxicity and offer an opportunity to expand the therapeutic window of IL-12. Although clinical success of local IL-12 therapies is far from guaranteed, promising approaches deserve consideration for human testing without prejudices based on earlier disappointments from systemic IL-12 trials.
In sum, intravesical chitosan/IL-12 is a promising immunotherapy for the treatment of superficial bladder cancer. We found chitosan/IL-12 to be superior to intravesical IL-12 alone and BCG in terms of survival and induction of TH1 urinary cytokines. Furthermore, chitosan/IL-12 immunotherapy, unlike BCG, mediates complete protection from tumor rechallenge. Taken together, we believe these studies form the rationale for clinical studies employing intravesical chitosan/IL-12 for the treatment of superficial bladder cancer.