Immunological mechanisms of intravesical chitosan/interleukin-12 immunotherapy against murine bladder cancer

There is a critical unmet clinical need for bladder cancer immunotherapies capable of inducing durable antitumor immunity. We have shown that four intravesical treatments with a simple co-formulation of interleukin-12 and the biopolymer chitosan not only destroy orthotopic bladder tumors, but also promote a potent long-lasting systemic immune response as evidenced through tumor-specific in vitro killing assays, complete protection from rechallenge, and abscopal antitumor responses at distant non-treated tumors. This study investigates the immunological kinetics underlying these results. We show through depletion studies that CD8+ T cells are required for initial tumor rejection, but CD4+ T cells protect against rechallenge. We also show that even a single intravesical treatment can eliminate tumors in 50% of mice with 6/9 and 7/8 mice eliminating tumors after three or four treatments respectively. We then performed immunophenotyping studies to analyze shifts in immune cell populations after each treatment within the tumor itself as well as in secondary lymphoid organs. These studies demonstrated an initial infiltration of macrophages and granulocytes followed by increased CD4+ and CD8+ effector-memory cells. This was coupled with a decreased level of regulatory T cells in peripheral lymph nodes as well as decreased myeloid-derived suppressor cell infiltration in the bladder. Taken together, these data demonstrate the ability of properly delivered interleukin-12-based therapies to engage adaptive immunity within the tumor itself as well as throughout the body and strengthen the case for clinical translation of chitosan/interleukin-12 as an intravesical treatment for bladder cancer

Modulation of Interleukin-12 activity in the presence of heparin

Glycosaminoglycans (GAGs), especially heparin and heparan sulfate (HS), modulate the functions of numerous cytokines. The aims of this multidisciplinary research were to characterize heparin binding to interleukin-12 (IL-12) and determine the mechanism(s) by which heparin influences IL-12 bioactivity. Heparin and HS were found to bind human IL-12 (hIL-12) with low micromolar affinity and increase hIL-12 bioactivity by more than 6-fold. Conversely, other GAGs did not demonstrate significant binding, nor did their addition affect hIL-12 bioactivity. Biophysical studies demonstrated that heparin induced only minor conformational changes while size-exclusion chromatography and small angle X-ray scattering studies indicated that heparin induced dimerization of hIL-12. Heparin modestly protected hIL-12 from proteolytic degradation, however, this was not a likely mechanism for increased cytokine activity in vitro. Flow cytometry studies revealed that heparin increased the amount of hIL-12 bound to cell surfaces. Heparin also facilitated hIL-12 binding and signaling in cells in which both hIL-12 receptor subunits were functionally deleted. Results of this study demonstrate a new role for heparin in modulating the biological activity of IL-12

Effect of Chitosan Properties on Immunoreactivity

Chitosan is a widely investigated biopolymer in drug and gene delivery, tissue engineering and vaccine development. However, the immune response to chitosan is not clearly understood due to contradicting results in literature regarding its immunoreactivity. Thus, in this study, we analyzed effects of various biochemical properties, namely degree of deacetylation (DDA), viscosity/polymer length and endotoxin levels, on immune responses by antigen presenting cells (APCs). Chitosan solutions from various sources were treated with mouse and human APCs (macrophages and/or dendritic cells) and the amount of tumor necrosis factor-α (TNF-α) released by the cells was used as an indicator of immunoreactivity. Our results indicate that only endotoxin content and not DDA or viscosity influenced chitosan-induced immune responses. Our data also indicate that low endotoxin chitosan (<0.01 EU/mg) ranging from 20 to 600 cP and 80% to 97% DDA is essentially inert. This study emphasizes the need for more complete characterization and purification of chitosan in preclinical studies in order for this valuable biomaterial to achieve widespread clinical application

Immunological mechanisms of intravesical chitosan/interleukin-12 immunotherapy against murine bladder cancer

There is a critical unmet clinical need for bladder cancer immunotherapies capable of inducing durable antitumor immunity. We have shown that four intravesical treatments with a simple co-formulation of interleukin-12 and the biopolymer chitosan not only destroy orthotopic bladder tumors, but also promote a potent long-lasting systemic immune response as evidenced through tumor-specific in vitro killing assays, complete protection from rechallenge, and abscopal antitumor responses at distant non-treated tumors. This study investigates the immunological kinetics underlying these results. We show through depletion studies that CD8(+) T cells are required for initial tumor rejection, but CD4(+) T cells protect against rechallenge. We also show that even a single intravesical treatment can eliminate tumors in 50% of mice with 6/9 and 7/8 mice eliminating tumors after three or four treatments respectively. We then performed immunophenotyping studies to analyze shifts in immune cell populations after each treatment within the tumor itself as well as in secondary lymphoid organs. These studies demonstrated an initial infiltration of macrophages and granulocytes followed by increased CD4(+) and CD8(+) effector-memory cells. This was coupled with a decreased level of regulatory T cells in peripheral lymph nodes as well as decreased myeloid-derived suppressor cell infiltration in the bladder. Taken together, these data demonstrate the ability of properly delivered interleukin-12-based therapies to engage adaptive immunity within the tumor itself as well as throughout the body and strengthen the case for clinical translation of chitosan/interleukin-12 as an intravesical treatment for bladder cancer

Effect of extension of the heparin binding pocket on the structure, stability, and cell proliferation activity of the human acidic fibroblast growth factor

Acidic human fibroblast growth factor (hFGF1) plays a key role in cell growth and proliferation. Activation of the cell surface FGF receptor is believed to involve the glycosaminoglycan, heparin. However, the exact role of heparin is a subject of considerable debate. In this context, in this study, the correlation between heparin binding affinity and cell proliferation activity of hFGF1 is examined by extending the heparin binding pocket through selective engineering via charge reversal mutations (D82R, D84R and D82R/D84R). Results of biophysical experiments such as intrinsic tryptophan fluorescence and far UV circular dichroism spectroscopy suggest that the gross native structure of hFGF1 is not significantly perturbed by the engineered mutations. However, results of limited trypsin digestion and ANS binding experiments show that the backbone structure of the D82R variant is more flexible than that of the wild type hFGF1. Results of the temperature and urea-induced equilibrium unfolding experiments suggest that the stability of the charge-reversal mutations increases in the presence of heparin. Isothermal titration calorimetry (ITC) data reveal that the heparin binding affinity is significantly increased when the charge on D82 is reversed but not when the negative charge is reversed at both positions D82 and D84 (D82R/D84R). However, despite the increased affinity of D82R for heparin, the cell proliferation activity of the D82R variant is observed to be reduced compared to the wild type hFGF1. The results of this study clearly demonstrate that heparin binding affinity of hFGF1 is not strongly correlated to its cell proliferation activity. Keywords: Fibroblast growth factor, Heparin binding, Stability, Cell proliferatio