Drug delivery for cancer immunotherapy : The development and evaluation of a versatile platform for delivery of oligodeoxynucleotides for cancer immunotherapy

Abstract

Cancer remains one of the leading causes of death worldwide, with approximately 40% of all people experiencing cancer at some point during their lives. By now, it is acknowledged that immunological recognition of cancer is a multifaceted interplay, in which the elicited immune response can both promote and reject tumor growth depending on the inflammatory state of the tumor microenvironment. Accordingly, the eradication of established cancer requires more cunning strategies than currently available. Innate immunomodulators are capable of increasing the inflammatory state within the body, and thereby, enhancing immune cell recognition of cancer. The toll-like receptor (TLR) 9 agonist CpG (Cytosine-phosphate-Guanine) oligodeoxynucleotide (ODN) has existed for over 30 years, and we're just now grasping the potential of it. CpG ODNs are synthetic TLR9 agonists that exhibit great immunostimulatory capacity and are capable of linking innate and adaptive immune responses, thus making them clinically relevant. Even though nucleotide-based therapy is capable of mounting immune responses, it still faces the challenge of specificity, which is an obstacle that can be tackled through targeted delivery, immunization with antigens of either tumor-specific or other origin, or through radiation-mediated antigen release. In this thesis, Manuscript I investigates and describes the development of a lipoplex based delivery platform, which enables co-delivery of CpG and immunogenic proteins for direct intratumoral injection to enhance the therapeutic outcome of radiotherapy. Particles were either functionalized by engraftment of polyethylene glycol (PEG) bioconjugated protein ovalbumin (OVA) or OVA adsorption onto the particle surface. We demonstrate that the method used for protein functionalization greatly impacts the immunological capacity of the particles. Functionalized particles exhibited low toxicity and similar immune activity independent of the functionalization method in vitro, but PEG bioconjugated proteins post-inserted into the lipoplexes exhibited superior potential in terms of in vivo efficacy compared to protein adsorbed lipoplexes in combination with radiotherapy. Manuscript I thereby highlights that the developed protein-CpG-lipoplexes can potentially enhance the specificity of CpG and the therapeutic outcome of radiation therapy, mediating a new strategy to promote anti-cancer immunity. Through interaction with the TLR9, CpG promotes dendritic cell maturation, antigen presentation, and production of proinflammatory cytokines, which leads to enhanced T cell-mediated anti-tumor immune responses. Once we established that the developed delivery platform of CpG in conjunction with proteins tended to exhibit enhanced synergistic effects with radiotherapy, we further investigated the impact of the lipoplex platform on adoptive T cell transfer (ACT). ACT is a form of cancer immunotherapy in which the number of T cells capable of recognizing the cancer cells are enlarged. For this purpose, the T cells are isolated, expanded to large numbers, and reinjected into the patient. Besides the fact that this process is both costly and time consumptions, the outcome of the therapy highly depends on the T cell's ability to survive and persist within the biological system. As such, there is excellent room for improvements. As such, in the second manuscript ( Manuscript II ) of this thesis, we investigate the potential of the developed lipoplex-based platform in combination with ACT. We demonstrate that a single administration of the protein-CpG-platform causes priming and proliferation of adoptively transferred non-activated T cells in vivo, leading to cancer eradication. This is important because it proves that upon correct stimulation, minimally-activated adoptively transferred T cells can be expanded in vivo, which in turn can minimize the waiting time for critically ill patients. Thereby, Manuscript II highlights that the treatment with the lipoplex platform might be a way to enhance ACT efficacy and bypass ex vivo expansion and ultimately broaden the scope of the therapy. Collectively, the studies reported in this thesis provide documentation for the development of a new cancer immunotherapy platform with versatile functions for more efficacious combinatorial cancer immunotherapy.