Cancer is a group of diseases caused by aberrant continuously proliferating cells capable of metastasis. Despite significant advances in preventive, diagnostic and treatment measures, cancer is one of the major causes of death in the United States, second only to heart diseases. Main treatment approaches are surgery, radiotherapy, chemotherapy, and the recently expanding immunotherapeutic approaches. The main challenge in treating cancer is the ability of cancer cells to mutate and develop resistance to drug treatments therefore lowering the efficacy of chemotherapy in preventing metastatic tumors. Cancer vaccines are a treatment modality that employs the potential of the immune system to recognize and eliminate tumor cells by unmasking tumor cell antigens and generating an effective anti-tumor immune response with an immune memory capable of preventing metastases formation. This dissertation describes and evaluates an innovative cell-particle hybrid cancer vaccine construct involving irradiated tumor cell surface-engineered with polymeric particles using streptavidin-biotin cross-linking. The tumor cells were biotinylated indirectly using biotin-linked antibodies targeting a surface integrin and the particles were loaded with an immune adjuvant and coated with streptavidin. The tumor cells served as the source of tumor antigens and the anchored particles served to confine loaded immune adjuvant to the tumor cells. The vaccine construct was designed to co-deliver tumor antigens and the immune adjuvant to the same antigen presenting cell, a criteria that has been suggested recently to be important for optimal cancer vaccine potency. The first report on this cell-particle construct was published in my master’s thesis defended in May 2013. In that report, the feasibility of assembling the cell-particle hybrid was demonstrated. However, loading of the immune adjuvant, CpG ODN (cytosine phosphate guanine oligonucleotide), into streptavidin-coated particles was not optimal. In the current studies, this problem was addressed and the cancer vaccine potential of the cell-particle construct was assessed. We first evaluated a new TLR4 (toll like receptor 4) agonist, PET lipid A (pentaeryhtritol lipid A), for its potential use in cancer vaccines with the intention to incorporate it in the cell-particle hybrid. PET lipid A is a fully synthetic lipid A analog that has been demonstrated to have immunostimulatory properties. We evaluated the potential use of PET lipid A in cancer vaccine applications and the effect of particulate formulations on its adjuvant properties. Results showed improved in vitro immunostimulatory properties for particle based formulations. Upon testing the immunostimulatory properties of PET lipid A in vivo, moderate enhancement in antigen specific cytotoxic T cells stimulation was observed when PET lipid A was delivered in particles, which then translated into a corresponding trend toward increased survival in a prophylactic tumor study. PET lipid A was concluded to be a weak potential cancer vaccine adjuvant and was not chosen as the immune adjuvant to use in the cell-particle hybrid assembly. Instead, CpG ODN (TLR9 agonist) was chosen due to its strong record of efficacy as a cancer vaccine adjuvant. The second part of this research project aimed at addressing the challenges we encountered previously in achieving acceptable CpG ODN loading of the final streptavidin-coated PLGA (Polylactic-co-glycolic acid) particles. The approach taken was to modify the method used earlier to make the particles in order to circumvent CpG ODN loss. In the modified method the number of steps required to make streptavidin-coated CpG ODN-loaded PLGA particles was reduced and the fabrication media was altered to allow simultaneous particle fabrication and activation of surface carboxyl groups. The modified method resulted in 5-fold higher loading in the final streptavidin-coated particles compared to the original method. Subsequent to establishing the feasibility of constructing the cell-particle hybrid and characterizing the assembled hybrid in vitro, the in vivo cancer vaccine potential of the designed construct was examined. Two independent murine tumor models were chosen for this purpose, namely prostate cancer and melanoma. The proposed cell-particle hybrid vaccine construct had significant therapeutic outcomes in the prostate cancer tumor model where mice vaccinated with cell-particle hybrids were the only group to show significant improvement in survival compared to untreated controls whereas no other vaccine formulation had such an effect. Unfortunately, no prophylactic benefit was observed from any of the vaccine formulations used in the melanoma tumor model involving irradiated GM-CSF (granulocyte macrophage colony stimulating factor)-secreting B16.F10 cells. In vitro examination of the immunostimulatory properties of all cell lines used in these studies revealed that transfected and parent B16.F10 cells (representing murine melanoma) were possibly immunoinhibitory whereas RM11 (representing murine prostate cancer) cells lacked such immunosuppressive effect in vitro. Our objective was to design and evaluate a new cancer vaccine construct that improved the immunostimulatory properties of irradiated tumor cell based vaccines. The approach taken was to surface engineer tumor cells with immune adjuvant loaded polymeric particles. We reported a simple method for fabricating streptavidin-coated PLGA particles and a versatile method of tumor cell surface engineering. We found that the efficacy of tumor cell-based vaccines can be inconsistent across tumor models and the in vitro immunosuppressive effect of tumor cells might be a contributing factor.

Cancer vaccines represent a promising treatment modality for a world-wide health problem. Whether as an adjuvant or as a stand-alone therapy, cancer vaccines represent a tumor-specific and systemic treatment potentially capable of eliminating metastatic lesions without the severe side-effects often associated with chemotherapy. Specifically, whole cell tumor vaccines have shown promise in preclinical and clinical settings and the studies presented here represent the beginnings of an approach to improve the antitumor potency of these vaccines. This project demonstrates as "proof of concept" the feasibility of manufacturing tumor cell-particle hybrids. The coupled use of these two components, whole tumor cells and cargo-carrying biodegradable particles, as one entity in a cancer vaccine system is a new line of research. Stable cell-particle hybrids were assembled using avidin-biotin chemistry where cargo-carrying PLGA particles (500 nm diameter) were coated with streptavidin and allowed to bind to tumor cells that had been indirectly labeled with biotin (using an integrin-specific biotinylated antibody). That successful cell-particle hybrids were assembled was determined by multiple means, including flow cytometry, laser scanning confocal microscopy and scanning electron microscopy. Two murine tumor cell lines (representing melanoma and prostate cancer) were investigated in this study and successfully demonstrated the general applicability of the assembly method. Particles appeared to be localized on the cell surface (rather than endocytosed) as determined by microscopic imaging. The cell-particle hybrid was shown to be stable to irradiation, an important consideration since whole tumor cells need to be treated with ionizing radiation prior to being used as vaccines in order to render them nonproliferative and immunogenic. We also characterized loading and release profiles of CpG, a prospective vaccine adjuvant, into PLGA particles. We conclude that we have developed a method for manufacturing cell-particle hybrids comprising PLGA nanoparticles and irradiated tumor cells. The next step would be to use CpG-loaded particles in the assembled hybrid and test the anti-tumor immune efficiency of this cancer vaccine formulation in either a melanoma or prostate cancer model.