Promising Immunotherapeutic Strategies for Cancer and Infectious Disease and Evaluation of Cell-Loaded Hydrogels for Type 1 Diabetes

Abstract

Advances in the area of vaccine immune responses has paved the way for new alternative delivery systems and novel painless routes of administration. Particulate delivery systems encapsulating protein antigens have been proved to improve the delivery and efficacy of vaccine responses. For the purpose of this dissertation, three separate projects with unique aims and hypotheses have been described. The first two projects focus on preparation and evaluation of a microparticulate vaccine incorporating antigens for an infectious disease [respiratory syncytial virus (RSV)] and cancer (melanoma), which have no licensed vaccines till date. In project 1, we have developed and tested a vaccine against RSV we have used a unique strategy of encapsulating virus-like particles prepared with the F protein, present on the surface of the respiratory syncytial virus. We used a biodegradable matrix composed of cellulose polymers for biocompatibility and better uptake by immune cells. Adjuvants have long been administered with vaccines to improve the longevity and magnitude of an antigen-specific immune response. Hence, we carried out an in vitro screening assay using dendritic cells stimulated with RSV F-VLP vaccine with/without adjuvants such as alum, MF59, MPL, R848, poly (I:C) and flagellin. We observed significant enhancement of immune markers such as nitric oxide, CD80, CD86 and MHC II with several adjuvants. Another important aspect of vaccine delivery is the route of vaccination. The transdermal route of vaccination has gained popularity in research, owing to the rich source of immune cells in the dermis and epidermis. In our study, we vaccinated mice using hollow microneedles and the microparticulate formulation with a TLR-4 agonist, MPL A. We observed high CD4+ and CD8+ T cell populations and lower lung viral titers in the groups vaccinated with MPL and RSV F-VLP in microparticulate form, post-challenge with live RSV A2 virus. Immunotherapeutic approaches to treat cancer have been successful owing to the selectivity and potency to target and eliminate tumors. Isolation of cancer antigens that generate specific immune responses and eliminate tumors has been challenging. Project 2 focuses on a mechanistic screening approach that utilizes tumor associated antigens from B16F10 metastatic melanoma cells. In our study, we have investigated the immunogenicity of the B16F10 antigens by performing an in vitro mechanistic study to evaluate the response of particulate antigens and adjuvants as a critical step in the development of an effective therapeutic vaccine. The development of a vaccine against cancer is a big challenge since tumor cells express self-proteins and hence are poorly immunogenic. Our strategy utilizes the spray drying technique to prepare vaccine microparticles using whole cell lysate. We systematically studied activation of the immune system in vitro with the formulated microparticles. With an aim to increase the potency of our vaccine candidate, several toll-like receptor (TLR) and non-TLR adjuvants were also studied. MHC I/II and co-stimulatory expression measured by flow cytometry was found to upregulated in dendritic cells stimulated with vaccine and adjuvant microparticles. In a real-life scenario, the patient’s own tumor cells will be used as the vaccine which will be administered to them in via the oral or transdermal routes, and an in vitro screening assay could help determine whether the appropriate antigen and adjuvant combination would be suitable for future use. In future, we hope that specific antigens will be identified so that a strong specific immune response can be generated. Cell encapsulation technology raises great hopes in medicine and biotechnology. Transplantation of encapsulated pancreatic islets represents a promising approach to the final cure of type 1 diabetes mellitus. We have also discussed a proof-of-concept study to treat type-1 diabetes mellitus wherein live beta cells embedded in a thermosensitive hydrogel matrix were injected in diabetic mice to observe reduction in glucose over a prolonged period. We aimed to evaluate a novel live cell therapy for type 1 diabetes using poloxamer 407 solution which at optimum concentration is liquid at room temperature, and gels at physiological conditions. We tested a thermosensitive hydrogel such as poloxamer 407 containing beta TC-6 cells to be delivered in a mouse model which would ensure continuous release of insulin for a prolonged period. The cells were found to be viable in the hydrogel matrix (25% w/w) as demonstrated by the Live/Dead and MTS assay. A preliminary in vitro immunogenicity study of the gel formulation containing beta TC-6 cells was evaluated using dendritic cells, which proved the gel protects cells from immune attack. The cells in aqueous formulation was injected intraperitoneally, into diabetic mice following which the formulation formed a depot. Blood glucose levels were found to decrease over 2 days, after which another dose of cells was given to match baseline glucose levels. After 35 days, the mice were sacrificed and immune effector and memory cells were quantified to observe the effect of formulation in vivo. No significant changes in cell counts were seen among groups.