Engineered nanovaccines for anti-tumour immuno-therapy

The overall goal of the project was to develop a new light-activated nanovaccine against cancer. For this purpose, nanoparticles based on biodegradable and biocompatible hyaluronic acid have been used, which act as the backbone of tumor-associated antigens. To deliver tumor-associated antigens to dendritic cells (DCs), the most potent antigen-presenting cells, the nanovaccines were surface-treated with ligands for DC targeting and activation. These nanovaccines are combined with photosensitizing compounds (PSs) that enable light-activated induction of inflammatory signals. In vitro and in vivo immunobiological evaluation has been performed in preclinical melanoma mouse models, on the skin of tumor-free mice on DC cells in vitro. The results may lead to a more effective or pre- or postoperative treatment of cancer patients and reduce the risk of recurrence. The Norwegian part of the project started in February 2019 and has had the task of optimizing and mapping the photochemically induced local inflammation to recruit DCs before immunization with nanovaccines. The photochemical treatment was performed by injecting PS subcutaneously and then activating the PS with light that will induce inflammation. The induced local inflammation condition depends on both the light dose, the drug-light enterval and the dose and type of photosensitizer injected into the skin. In the first part of the project, the optimal doses that are sufficient for the induction of moderate inflammation without causing undesirable effects, such as skin irritation or wounds of any kind, were mapped. Obtained inflammation was measured by determining increase in levels of various proinflammatory cytokines. After finding the optimal doses, the time aspect of the inflammation was described. Such an acutely induced inflammatory condition will be quickly cleared up by the body's immune system and go away after a short time. It is therefore necessary to know when to achieve optimal conditions for injection of a nanovaccine. Samples of the skin were taken at several times after treatment. The samples were analyzed for proinflammatory cytokines and digital pathology was developed to quantify the infiltration of immune cells into the skin tissue. With the developed digital pathology method, it is possible to map at which times you have the largest immune cell infiltration and thus which times are most suitable for injection of nanovaccine. With digital pathology, we followed the infiltration of immune cells into blood vessels and then over the blood vessel wall and into the various parts of the skin tissue. In these studies, 2 different clinically relevant PSers were used with different physicochemical properties that results in different intracellular and extracellular localization in the skin. These analyzes have provided important understanding of the differences between photosensitizers that bind to different parts of the cell and how this affects the inflammation in different parts of the skin. The photochemical treatment induces a significant accumulation of immune cells, including antigen presenting cells, in the irradiated area of the skin. Dosages and time of light treatment after PS administration in the skin for optimal accumulation of immune cells before vaccination are documented. These are important parameters for the optimal vaccination effect of the nanovaccine particles that will be administered to the photochemically treated area.

Resultatene gir en grunnleggende forståelse av kinetikken og mekanismene rundt fotokjemisk indusert inflammasjon i huden og kan dermed være svært viktige for hvordan man setter opp optimale eksperimenter for å forsterke den immunologiske effekten av vaksiner. Utviklingen av metodene som er utviklet i dette prosjektet vil også være nyttig i andre forskningsprosjekter. Spesielt digital patologi som muliggjør kvantifisering av immunceller i vev kan ha stor nytteverdi i studier av immunbehandling av kreft. Det er flere selskaper i næringslivet som jobber med å bruke lysindusert inflammasjon i vaksineteknologi og denne studien kan gi informasjon som øker sannsynligheten for å lykkes med denne utviklingen. I dagens samfunn er man nå helt avhengig av å utvikle effektive vaksiner og dette prosjektet er med å skaffe informasjon om hvordan man kan optimalisere vaksiner for størst mulig immunologisk effekt.

In addition to more established treatments such as chemotherapy and radiation therapy, delivering tumour associated antigens to dendritic cells (DCs), the most potent class of antigen presenting cells of our immune system, has emerged as a promising approach by harnessing patients? own immune system at recognizing and eliminating metastatic growth. However, one of the major bottlenecks in this approach is the inability, upon sub-cutaneous or intra muscular injection, of tumour associated peptide antigens to target and activate DCs because of their small size and low immunogenicity. Indeed, peptides rapidly diffuse in the body, instead of reaching the immune-inducing sites in the lymph nodes, and by consequence are incapable of inducing potent anti-tumour immunity. Therefore, there is a clear medical need for a targeted approach that delivers tumour associated antigens and activation stimuli to DCs. In this project we aim at priming the immune system through the development of a novel class of potent but safe nanovaccines based on degradable nanoparticles that target tumour associated antigens and activation stimuli to DCs. For this purpose, we will use the functional and biodegradable/biocompatible properties of the natural polysaccharide hyaluronic acid as backbone for subsequent ligation of DC targeting and DC activating ligands together with lymph node homing cues and tumour associated antigen. These nanovaccines will be combined with photosensitizers for photo-activated engineering of the inflammatory environment at the injection site to promote DC recruitment and to improve antigen presentation in the draining lymph nodes through photochemical internalization. In vitro and in vivo immuno-biological evaluation will be performed in pre-clinical mouse models for melanoma. Promising results obtained in this would lead to a more efficient pre- or post-operative treatment of cancer patients to reduce the risk of recurrence.