Intravesical bacillus Calmette-Guerin (BCG) has been used successfully to treat superficial bladder cancer for 3 decades. However, 20-30% of patients will fail initial BCG therapy and 30-50% of patients will develop recurrent tumors within 5 years. Alternative or complementary strategies for the management of superficial bladder cancer are needed. Interleukin-12 (IL-12) is a potent TH1 cytokine with robust antitumor activity and the ability to potentiate immunological memory. Unfortunately, intravesical IL-12 did not demonstrate anti-tumor efficacy in a recent clinical study of patients with recurrent superficial bladder cancer. We hypothesized that co-formulation of IL-12 with chitosan a biocompatible, mucoadhesive polysaccharide could improve intravesical IL-12 delivery and provide an effective and durable alternative for the treatment of superficial bladder cancer. In antitumor studies, 88 to 100% of mice bearing orthotopic bladder tumors were cured after 4 intravesical treatments with chitosan/IL-12. In contrast, only 38 to 60% of mice treated with IL-12 alone, and 0% treated with BCG, were cured. Antitumor responses following chitosan/IL-12 treatments were durable and provided complete protection from intravesical tumor rechallenge. Urinary cytokine analysis showed that chitosan/IL-12 induced multiple TH1 cytokines at levels significantly higher than either IL-12 alone or BCG. Immunohistochemistry revealed moderate to intense tumor infiltration by T cells and macrophages following chitosan/IL-12 treatments. Bladder submucosa from cured mice contained residual populations of immune cells that returned to baseline levels after several months. Intravesical chitosan/IL-12 is a well-tolerated, effective immunotherapy that deserves further consideration for testing in humans for the management of superficial bladder cancer. Sustained, local delivery of immunomodulatory cytokines is under investigation for its ability to enhance vaccine and anti-tumor responses clinically. This project evaluated the ability of chitosan, a biocompatible polysaccharide, to (1) control the dissemination of a cytokine, GM-CSF, and (2) enhance the immunoadjuvant properties of GM-CSF. While cytokines have previously been delivered in lipid-based adjuvants and other vehicles, these do not have the clinical safety profile or unique properties of chitosan. We found that chitosan solution maintained a measurable depot of recombinant GM-CSF (rGM-CSF) at a subcutaneous injection site for up to 9 days. In contrast, when delivered in a saline vehicle, rGM-CSF was undetectable in 1224 h. Furthermore, a single s.c. injection of 20 μg rGM-CSF in chitosan solution (chitosan/rGM-CSF(20 μg)) transiently expanded lymph nodes up to 4.6-fold and increased the number of MHC class II expressing cells and dendritic cells by 7.4-fold and 6.8-fold, respectively. These increases were significantly greater than those measured when rGM-CSF was administered in saline at the standard preclinical dose and schedule, i.e. 4 daily s.c. injections of 20 μg. Furthermore, lymph node cells from mice injected with chitosan/rGM-CSF(20 μg) induced greater allogeneic T cell proliferation, indicating enhanced antigen presenting capability, than lymph node cells from mice injected with rGM-CSF alone. Finally, in vaccination experiments, chitosan/rGM-CSF was superior to either chitosan or rGM-CSF alone in enhancing the induction of antigen-specific CD4+ proliferation, peptide-specific CD8+ pentamer staining and cytotoxic T cell lysis. Altogether, chitosan/rGM-CSF outperformed standard rGM-CSF administrations in dendritic cell recruitment, antigen presentation and vaccine enhancement. We conclude that chitosan solution is a promising delivery platform for the sustained, local delivery of rGM-CSF. In a related study, chitosan solution enhances both humoral and cell-mediated immune responses to subcutaneous vaccination of an antigen. The development of safe, novel adjuvants is necessary to maximize the efficacy of new and/or available vaccines. Chitosan is a non-toxic, biocompatible, biodegradable, natural polysaccharide derived from the exoskeletons of crustaceans and insects. Chitosans biodegradability, immunological activity and high viscosity make it an excellent candidate as a depot/adjuvant for parenteral vaccination. To this end, we explored chitosan solution as an adjuvant for subcutaneous vaccination of mice with a model protein antigen. We found that chitosan enhanced antigen-specific antibody titers over five-fold and antigen-specific splenic CD4+ proliferation over six-fold. Strong increases in antibody titers together with robust delayed-type hypersensitivity (DTH) responses revealed that chitosan induced both humoral and cell-mediated immune responses. When compared with traditional vaccine adjuvants, chitosan was equipotent to incomplete Freunds adjuvant (IFA) and superior to aluminum hydroxide. Mechanistic studies revealed that chitosan exhibited at least two characteristics that may allow it to function as an immune adjuvant. First, the viscous chitosan solution created an antigen depot. More specifically, less than 9% of a protein antigen, when delivered in saline, remained at the injection site after 8 h. However, more than 60% of a protein antigen delivered in chitosan remained at the injection site for 7 days. Second, chitosan induced a transient 67% cellular expansion in draining lymph nodes. The expansion peaked between 14 and 21 days after chitosan injection and diminished as the polysaccharide was degraded. These mechanistic studies, taken together with the enhancement of a vaccine response, demonstrate that chitosan is a promising and safe platform for parenteral vaccine delivery.
| Heffernan, Michael J; Zaharoff, David A; Fallon, Jonathan K et al. (2011) In vivo efficacy of a chitosan/IL-12 adjuvant system for protein-based vaccines. Biomaterials 32:926-32 |
| Zaharoff, David A; Hance, Kenneth W; Rogers, Connie J et al. (2010) Intratumoral immunotherapy of established solid tumors with chitosan/IL-12. J Immunother 33:697-705 |
| Zaharoff, David A; Hoffman, Benjamin S; Hooper, H Brooks et al. (2009) Intravesical immunotherapy of superficial bladder cancer with chitosan/interleukin-12. Cancer Res 69:6192-9 |
