Sruthi Ravindranathan

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.

Neoadjuvant immunotherapy with chitosan and interleukin-12 to control breast cancer metastasis

Metastasis accounts for approximately 90% of breast cancer-related deaths. Therefore, novel approaches which prevent or control breast cancer metastases are of significant clinical interest. Interleukin-12 (IL-12)-based immunotherapies have shown promise in controlling metastatic disease, yet modest responses and severe toxicities due to systemic administration of IL-12 in early trials have hindered clinical application. We hypothesized that localized delivery of IL-12 co-formulated with chitosan (chitosan/IL-12) could elicit tumor-specific immunity and provide systemic protection against metastatic breast cancer while minimizing systemic toxicity. Chitosan is a biocompatible polysaccharide derived primarily from the exoskeletons of crustaceans. In a clinically relevant resection model, mice bearing spontaneously metastatic 4T1 mammary adenocarcinomas received intratumoral injections of chitosan/IL-12, or appropriate controls, prior to tumor resection. Neoadjuvant chitosan/IL-12 immunotherapy resulted in long-term tumor-free survival in 67% of mice compared to only 24% or 0% of mice treated with IL-12 alone or chitosan alone, respectively. Antitumor responses following chitosan/IL-12 treatment were durable and provided complete protection against rechallenge with 4T1, but not RENCA renal adenocarcinoma, cells. Lymphocytes from chitosan/IL-12-treated mice demonstrated robust tumor-specific lytic activity and interferon-γ production. Cell-mediated immune memory was confirmed in vivo via clinically relevant delayed-type hypersensitivity (DTH) assays. Comprehensive hematology and toxicology analyses revealed that chitosan/IL-12 induced transient, reversible leukopenia with no changes in critical organ function. Results of this study suggest that neoadjuvant chitosan/IL-12 immunotherapy prior to breast tumor resection is a promising translatable strategy capable of safely inducing to tumor-specific immunity and, in the long term, reducing breast cancer mortality due to progressive recurrences.

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

ABSTRACT 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.

Current status of autologous breast tumor cell-based vaccines

Approximately nine out of ten breast cancer-related deaths are attributable to metastasis. Yet, less than 4% of breast cancer patients are initially diagnosed with metastatic cancer. Therefore, the majority of breast cancer-related deaths are due to recurrence and progression of non-metastatic disease. There is tremendous clinical opportunity for novel adjuvant strategies, such as immunotherapies, that have the potential to prevent progressive recurrences. In particular, autologous tumor cell-based vaccines (ATCVs) can train a patient’s immune system to recognize and eliminate occult disease. ATCVs have several advantages including safety, multivalency and patient specificity. Furthermore, because lumpectomy or mastectomy is indicated for the vast majority of breast cancer patients, resected tumors offer a readily available, patient-specific source of tumor antigen. Disadvantages of ATCVs include poor immunogenicity and production inconsistencies. This review summarizes recent progress in the development of autologous breast tumor vaccines and offers insight for overcoming existing limitations.

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.

Tumor-derived granulocyte colony-stimulating factor diminishes efficacy of breast tumor cell vaccines

BackgroundAlthough metastasis is ultimately responsible for about 90% of breast cancer mortality, the vast majority of breast-cancer-related deaths are due to progressive recurrences from non-metastatic disease. Current adjuvant therapies are unable to prevent progressive recurrences for a significant fraction of patients with breast cancer. Autologous tumor cell vaccines (ATCVs) are a safe and potentially useful strategy to prevent breast cancer recurrence, in a personalized and patient-specific manner, following standard-of-care tumor resection. Given the high intra-patient and inter-patient heterogeneity in breast cancer, it is important to understand which factors influence the immunogenicity of breast tumor cells in order to maximize ATCV effectiveness.MethodsThe relative immunogenicity of two murine breast carcinomas, 4T1 and EMT6, were compared in a prophylactic vaccination-tumor challenge model. Differences in cell surface expression of antigen-presentation-related and costimulatory molecules were compared along with immunosuppressive cytokine production. CRISPR/Cas9 technology was used to modulate tumor-derived cytokine secretion. The impacts of cytokine deletion on splenomegaly, myeloid-derived suppressor cell (MDSC) accumulation and ATCV immunogenicity were assessed.ResultsMice vaccinated with an EMT6 vaccine exhibited significantly greater protective immunity than mice vaccinated with a 4T1 vaccine. Hybrid vaccination studies revealed that the 4T1 vaccination induced both local and systemic immune impairments. Although there were significant differences between EMT6 and 4T1 in the expression of costimulatory molecules, major disparities in the secretion of immunosuppressive cytokines likely accounts for differences in immunogenicity between the cell lines. Ablation of one cytokine in particular, granulocyte-colony stimulating factor (G-CSF), reversed MDSC accumulation and splenomegaly in the 4T1 model. Furthermore, G-CSF inhibition enhanced the immunogenicity of a 4T1-based vaccine to the extent that all vaccinated mice developed complete protective immunity.ConclusionsBreast cancer cells that express high levels of G-CSF have the potential to diminish or abrogate the efficacy of breast cancer ATCVs. Fortunately, this study demonstrates that genetic ablation of immunosuppressive cytokines, such as G-CSF, can enhance the immunogenicity of breast cancer cell-based vaccines. Strategies that combine inhibition of immunosuppressive factors with immune stimulatory co-formulations already under development may help ATCVs reach their full potential.

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.