Cancer is an age-old disease and a leading cause of mortality worldwide. It starts with the development of abnormal cells that multiply to form tumours and may lead to metastasis causing damage to healthy tissues and organs of the body. Lung cancer is the most common type of cancer with significant morbidity and mortality globally and is of major concern for the society. Decades of research and innovation in the treatment of cancer led to the development of certain conventional therapies such as surgery, radiotherapy, and chemotherapy that are being effectively used. However, these traditional therapies have a disadvantage of destroying healthy cells and tissues in its close proximity, leading to other severe diseases and side effects. Therefore, a much-advanced approach to treat cancer without causing severe side effects became an important area of research. Improvements in the field of anti-cancer therapies led to a strategy to combine traditional treatment approaches to destroy cancer cells. Additionally, treatments such as targeted therapies, hormone therapy, and precision medicine came into existence. Alternatively, the immune system was analysed to determine an effective approach that could control proliferation of cancer cells and cause limited or no damage to other cells. This area of cancer therapeutics, known as immunotherapy, targets the immune system to enhance its ability of recognizing invasive neoplasms. Our immune system in response to an infection immediately activates immune cells, creating a protective immunity for decades. Unfortunately, this immune response often fails when fighting against cancer and is unable to entirely eliminate tumour cells or develop effective immunity. Cancer vaccines, a branch of immunotherapy that has potential therapeutic benefits can build immunity and ultimately lead to prevention from future illness. These have been employed to treat the disease using tumour associated antigens and various adjuvants or immune stimulators and only a few have been in clinical trials. The potential of such cancer vaccines can be exploited to further develop treatment strategies to prevent tumour occurrence and provide long-lasting immunity. This study was designed to primarily explore the potential of a novel immune stimulator, RNA:DNA hybrid and an established TLR9 agonist, CpG 7909. Furthermore, these agonists were used to develop a preventative cancer vaccine in association with a tumour associated carbohydrate antigen, Globo-H to determine whether the novel formulation could improve immune response and trigger antigen presentation in vitro and in vivo. In an in vivo setting, these were also investigated against lung cancer to determine their potential in preventing tumour occurrence. Within this study RAW 264.7 mouse macrophages were initially exposed to varying concentrations of CpG 7909, RNA:DNA hybrid, and Globo-H to determine the optimum dose concentration for the activation of antigen presenting cells by measuring the level of TNF-α secreted in cell culture supernatant. Following dose optimization, the individual agents were combined in a vaccine formulation, sharing the same antigen, where two cell lines, mouse RAW 264.7 macrophages and human monocytic cell line, THP-1 that was differentiated into macrophages and dendritic cells were exposed to the vaccines. TNF-α, IL-12 and IL-6 cytokine response was analysed as measured by ELISA and real-time PCR that were determined to be partially significant in response to the vaccines. The degree of immune response demonstrated by RNA:DNA hybrid-based vaccine (HG-Vax) confirmed that HG-Vax is more effective than CpG 7909-based vaccine (CG-Vax). This response was further analysed in an in vivo model where C57/BL mice were exposed to immune stimulators and vaccines separately for 14- and 28-days before end point termination. Serum was analysed for IL-12 and TNF-α to determine the level of inflammatory response. Even though statistical significance was not achieved in vivo, contrasting observations compared to the in vitro model were exhibited. Secretion of IL-12 in animals confirmed the induction of an anti-tumour immune response. Interestingly, cytokines induced by single dose CG-Vax persisted for a longer period of time in blood in comparison to HG-Vax. However, opposite was true with the booster immunization. In animal model of lung cancer, statistical significance validates that booster immunization with HG-Vax triggers an immune response and upregulates MHC II expression. Unfortunately, CG-Vax did not induce a significant immune response and failed to induce MHC II expression in an in vivo model of lung cancer. Although these findings present RNA:DNA hybrid to be a novel immune stimulator that may have the potential to be used in future cancer vaccinations, further investigation on its immunological potential and mechanism of action remains of need.