Sean G. Smith

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.

Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag

Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous. Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ∼98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichroism, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31±0.05 mg/mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app)=69±1 μM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics.

Future directions in bladder cancer immunotherapy: towards adaptive immunity

The clinical management of bladder cancer has not changed significantly in several decades. In particular, intravesical bacillus Calmette-Guérin (BCG) immunotherapy has been a mainstay for high-risk nonmuscle invasive bladder cancer since the late 1970s/early 1980s. This is despite the fact that bladder cancer has the highest recurrence rates of any cancer and BCG immunotherapy has not been shown to induce a tumor-specific immune response. We and others have hypothesized that immunotherapies capable of inducing tumor-specific adaptive immunity are needed to impact bladder cancer morbidity and mortality. This article summarizes the preclinical and clinical development of bladder cancer immunotherapies with an emphasis on the last 5 years. Expected progress in the near future is also discussed.

Colony stimulating factors secreted by irradiated autologous tumor cell vaccines inhibit immunity

Autologous tumor cell-based vaccines (ATCVs) have a number of potential advantages including multivalency and patient specificity. ATCVs contain many potential antigens, both known and unknown which potentiate polyclonal responses capable of responding to a more diverse population of tumor cells. In addition, because ATCVs are created from a patients own tumor, all potential immunogenic epitopes are patient specific and each patient is immunized against her complete and individualized antigen repertoire. This is particularly important for breast cancer as each tumor can contain up to 100 different mutant genes so no two tumors are identical. The major disadvantage of ATCVs, is poor immunogenicity. In order to develop an effective, immunogenic ATCV against breast cancer, we wanted to understand the features of immunogenicity. In this study, BALB/c female mice were given priming and booster vaccinations, ten days apart, with 1 million irradiated (100Gy) EMT6 and/or 4T1cells. Ten days after the booster vaccination, mice were challenged with live tumor cells. 80% of mice vaccinated with EMT6 cells were completely protected against a live EMT6 challenge. However, mice vaccinated with irradiated 4T1 cells failed to provide any protection against a live 4T1 challenge. Most interestingly, when mice were vaccinated with a mixture of irradiated EMT6 and 4T1 cells at the same site, the protective response against EMT6 challenge was significantly diminished as 60% of mice developed tumors. Furthermore, when irradiated EMT6 and 4T1 cells were administered on opposite sides, protection from an EMT6 challenge was also significantly diminished with 88% of mice developing tumors. This finding implied that non-immunogenic irradiated 4T1 cells released one or more immunosuppressive factors that inhibited anti-EMT6 immunity. Thus, we investigated the levels of different immunosuppressive cytokines, G-CSF, M-CSF, GM-CSF, IL-6, MCP-1, TGF-β and VEGF released by both 4T1 and EMT6 cells before and after irradiation. Irradiated 4T1 cells secreted high levels of colony stimulating factors. Specifically, at 24 and 48 hours after irradiation, 4T1 cells secreted 60 and 705 pg/ml of M-CSF; 912 and 5190 pg/ml of G-CSF; 29 and 180 pg/ml of GM-CSF. We believe that high levels of colony stimulating factors induce the accumulation of large amounts of myeloid derived suppressor cells (MDSCs) in the tumor site and lymphoid organs which, in turn, suppress anti-EMT-6 immunity. Future studies will determine if blocking colony stimulating factors will decrease the accumulation of MDSCs and subsequently increase anti-tumor immunity.

Abstract B020: Effector cells in chitosan/interleukin-12 immunotherapy of bladder tumors in mice

Introduction: Intravesical immunotherapy with mycobacterium bovis baccillus Calmette-Guerin (BCG) has been the standard of care for high grade non-muscle invasive bladder tumors for nearly 40 years. While BCG has been successful at lowering recurrence rates and preventing progression, it does not engage adaptive memory and is associated with recurrence rates as high as 50%. Thus the search for treatment strategies that promote a durable anti-tumor memory response is warranted. One such strategy developed by our group is the intravesical administration of a simple coformulation of the TH1 polarizing cytokine interleukin-12 (IL-12) and the biopolymer chitosan (chitosan/IL-12). Chitosan is not bioactive but is cationic and viscous in solution and acts as a delivery vehicle for IL-12. It is presumed to enhance three aspects of delivery. 1) Chitosan9s positive charge interacts with the urothelium9s tight junctions to transiently increase its permeability. 2) Mucoadhesive forces between the urothelium9s mucosa and chitosan prolong the contact of IL-12 with the urothelium. 3) The viscous nature of chitosan limits expulsion of IL-12 during voiding of the bladder. Our group has recently demonstrated that four weekly intravesical administrations of chitosan/IL-12 can 1) eliminate established orthotopic in >90% of mice, 2) provide protective local immunity, and 3) promote durable systemic immunity. The purpose of this study is to investigate the mechanisms of intravesical chitosan/IL-12 immunotherapy. Specifically, we look at the role of natural killer cells (NK), CD8+ cytotoxic T-cells, and CD4+ T-helper cells in chitosan/IL-12 therapy of orthotopic tumors as well as their importance for maintaining systemic immunity to those bladder tumors. Methods: Orthotopic bladder tumors were generated via intravesical implantation of 75,000 MB49 cells into female C57BL/6J mice. Briefly, anesthetized mice were catheterized with a 24G x 3/4” Teflon catheter and the cells injected after a 10 minute wash with Poly-L-Lysine. Upon onset of hematuria, usually one week post implantation, mice received four twice-weekly intravesical administrations of either saline or chitosan/IL-12 immunotherapy (1 µg IL-12 in 100µl of 10 mg/ml chitosan). For immune subset depletion studies, four daily administrations of 100 µg anti-CD4 (GK1.5), anti-CD8 (2.43), or anti-NK1.1 (PK136) antibodies were given prior to implantation. Depletion was maintained for the duration of the study with 100 µg antibody given twice per week. For systemic rechallenge studies, mice were injected with 300,000 MB49 in the right flank. Results: Mice were depleted of NK, CD8+, or CD4+ lymphocytes prior to tumor inoculation to investigate the importance of immune cell types on the efficacy of intravesical chitosan/IL-12. Mice depleted of CD8+ T-cells (n=9) failed to eliminate orthotopic tumors, but did extend survival over the saline treated control. Mice depleted of CD4+ or NK+ immune cells elicited a more mixed result with 4/8 and 3/8 mice respectively surviving long term, free of tumors. Mice that were not depleted, but were given chitosan/IL-12 therapy eliminated their tumors in 7/9 instances. In a separate study to investigate the importance of T-cell subtypes in maintaining systemic immunity, mice previously cured of orthotopic disease were depleted of CD8+ or CD4+ T-cells and then rechallenged subcutaneously. Eight out of 10 CD8-depleted mice rejected the tumors, while only 1/9 CD4-depleted mice rejected the rechallenge. All naive mice grew subcutaneous tumors (n=9). All mice previously cured of orthotopic disease and not depleted rejected the subcutaneous rechallenge (n=4). Conclusions: The elimination of established orthotopic tumors in response to chitosan/IL-12 therapy involves multiple immune cells types. As expected CD8+ T-cells appear to be the primary effectors with CD4+ and NK+ cells playing lesser, but still important roles. Interestingly, the relative importance of CD8+ and CD4+ T-cells seems to reverse for the systemic memory response to tumor rechallenge; almost all subcutaneously rechallenged mice depleted of CD4 grew tumors, but nearly all CD8 depleted mice rejected tumors. Further mechanistic studies are warranted to explain and build upon these results. Citation Format: Sean Smith, Jack Baltz, Bhanu Koppolu, Sruthi Ravindranathan, Khue Nguyen, David Zaharoff. Effector cells in chitosan/interleukin-12 immunotherapy of bladder tumors in mice. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B020.

Abstract P4-04-16: Characterization of an autologous tumor cell vaccine against breast cancer in mice

The vast majority of breast cancer-related deaths are due to progressive recurrences of non-metastatic disease. Current adjuvant therapies aimed at preventing breast cancer recurrence are ineffective for tens of thousands of women each year. In addition, current adjuvant therapies carry significant co-morbidities. Active, specific immune-based strategies have the potential to train a patient9s immune system to recognize and eliminate occult metastases. Here, we explore autologous tumor cell vaccines (ATCVs) as a strategy to prevent tumor recurrence and improve survival. Because >90% of breast cancer patients undergo resection, autologous tumor cells are readily available for the production of personalized vaccine. Patient-specific vaccination is particularly attractive for breast cancer which is highly heterogeneous. The major disadvantage of ATCVs, is poor immunogenicity. Thus, in order to develop an effective, immunogenic ATCV against breast cancer, we wanted to understand the features of immunogenicity. In this study, we evaluated the immunogenicities of two murine breast cancer cell lines, EMT6 and 4T1. For tumor protection studies, BALB/c female mice were given priming and booster vaccinations, ten days apart, with 1,000,000 irradiated (100Gy) cells. Ten days after the booster vaccination, mice were challenged with live tumor cells. Mice vaccinated with EMT6 cells were completely protected against a live EMT6 challenge in 80% of mice. However, mice vaccinated with irradiated 4T1 cells failed to provide any protection against a live 4T1 challenge. Most interestingly, when mice were vaccinated with a mixture of irradiated EMT6 and 4T1 cells, the protective response against EMT6 challenge was significantly diminished as 60% of mice developed tumors. This finding implied that non-immunogenic 4T1 cells released one or more immunosuppressive factors that inhibited anti-EMT6 immunity. Thus, we investigated the levels of different immunosuppressive cytokines, GM-CSF, IL-6, MCP-1, TGF-β and VEGF released by both 4T1 and EMT6 cells before and after irradiation. Of the different cytokines measured, we found that only GM-CSF is produced at significantly higher levels by 4T1 cells than EMT6 cells. Since at higher levels (>200pg/ml) GM-CSF can induce the accumulation of large amounts of myeloid derived suppressor cells (MDSCs) in the tumor site and lymphoid organs, we believe it to be the reason for the failure of the hybrid vaccine. Future studies will determine if blocking GM-CSF production by 4T1 cells will decrease the accumulation of MDSCs and subsequently increase anti-tumor immunity. Citation Format: Ravindranathan S, Kurtz SL, Smith SG, Koppolu B, Nguyen KG, Zaharoff DA. Characterization of an autologous tumor cell vaccine against breast cancer in mice. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-16.

Abstract 4404: A novel chitosan-based hydrogel for intratumoral release of immunotherapeutic cytokines

Controlled delivery of IL-12 has been shown to stimulate the immune system to eliminate primary tumors and elicit tumor-specific, protective immunity. Recently, we have developed a novel in situ gelling chitosan-based hydrogel that appears capable of further improving cytokine delivery within the tumor microenvironment while preventing systemic dissemination of potentially toxic, pro-inflammatory cytokines. Change in viscosity during gelation was measured using a Brookfield DV-III Ultra Rheometer, revealing a 300-fold increase in viscosity. Manipulation of chitosan9s chemical properties such as molecular weight, concentration, and degree of acetylation was found to control the time of gelation, which ranged from ten seconds to one minute. A protein release study was conducted using fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) as a surrogate for IL-12. Dialysis tubes (with a molecular weight cutoff of 100kDa) were loaded with FITC-BSA co-formulated with hydrogel and FITC-BSA in PBS alone. The tubes were placed in a PBS bath and samples were taken at 48, 96, and 168 hours. The degree of release from the tubes was determined by measuring the fluorescence of protein in samples using a spectrofluorimeter. In vitro drug release studies demonstrated zero-order release of FITC-BSA from the hydrogel over a one-week period. Comparison to FITC-BSA in PBS indicates that the hydrogel exhibits a controlled kinetics profile. Bioactivity of IL-12 when co-formulated with the hydrogel was determined by quantifying the production of interferon gamma (IFNγ) by NK-92MI cells, a line of human Natural Killer immune cells. NK-92MI cells were grown in the presence of human IL-12 with and without the hydrogel, and IFNγ production was measured via ELISA. The bioactivity assay indicated that IL-12 bioactivity was not compromised when mixed with the hydrogel. Specifically, after a 24 hour incubation period, the amount of IFNγ produced by the NK-92MI cells was found to be similar between groups treated with IL-12 alone and IL-12/hydrogel co-formulation. After bioactivity was confirmed, mice (n = 3 per group) were subcutaneously injected with the mouse urothelial carcinoma cell line, MB49 (∼300,000 cells per mouse). After sufficient (approximately 50mm3) tumor growth, the mice were injected with either a hydrogel/IL-12 formulation or saline, which served as a control. Tumor volume was measured on days 7, 9, 14 and 19 after tumor implantation. In the anti-tumor study, mice treated with the IL-12/hydrogel co-formulation experience reduction in tumor size, while those treated with saline experience expected tumor growth. These studies demonstrate that a novel chitosan-based hydrogel allows for sustained delivery of antitumor cytokines without altering bioactivity. Citation Format: Ethan D. Lowry, Christopher Wallace, Bhanu prasanth Koppolu, Sean Smith, David Zaharoff. A novel chitosan-based hydrogel for intratumoral release of immunotherapeutic cytokines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2015-4404