Design and evaluation of immunotherapeutic imaging for cancer vaccination

Cancer immunotherapy, where a patient's immune system is harnessed in the battle against a tumor, is currently on the rise. One possible approach comprises the in vitro modulation of dendritic cells with tumor antigens, after which the cells are injected as a therapeutic cancer vaccine. Despite extensive progress and promising (pre)clinical results, such cancer vaccines often remain patient-specific, laborious in production and therefore very expensive. In this thesis, these issues are addressed by developing imageable biomaterials as cancer immunotherapeutics. Firstly, we highlight the use of antigen-loaded perfluorocarbon particles for 19F MRI tracking of dendritic cells is explored. Such imageable cellular vaccines could provide information on the fate and migratory potential of the cells after their injection, and therefore give important initial feedback on the vaccine efficacy. Secondly, we investigated an alternative strategy that makes use of mRNA-loaded microbubbles for ultrasound-guided and ultrasound-triggered immune activation. We demonstrated that this principle could indeed be used to induce antigen expression in dendritic cells in vitro. Moreover, by simultaneously delivering both antigen mRNA and adjuvant mRNA (TriMix), highly immunogenic dendritic cell vaccines could be produced, allowing complete tumor regression in 30% of the vaccinated animals, which were protected against tumor recurrence. Interestingly, these mRNA-loaded microbubbles show potential for the immediate in vivo delivery of mRNA to dendritic cells as they spontaneously migrate to the lymph nodes upon subcutaneous injection. What is more, the image feedback information provided by these contrast agents on the lymphatic anatomy underpins their interesting role as immunotheranostic agents

Choose your models wisely: how different murine bone marrow-derived dendritic cell protocols influence the success of nanoparticulate vaccines in vitro

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Hitchhiking nanoparticles: reversible coupling of lipid-based nanoparticles to cytotoxic T lymphocytes

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mRNA in cancer immunotherapy : beyond a source of antigen

mRNA therapeutics have become the focus of molecular medicine research. Various mRNA applications have reached major milestones at high speed in the immuno-oncology field. This can be attributed to the knowledge that mRNA is one of nature's core building blocks carrying important information and can be considered as a powerful vector for delivery of therapeutic proteins to the patient.For a long time, the major focus in the use of in vitro transcribed mRNA was on development of cancer vaccines, using mRNA encoding tumor antigens to modify dendritic cells ex vivo. However, the versatility of mRNA and its many advantages have paved the path beyond this application. In addition, due to smart design of both the structural properties of the mRNA molecule as well as pharmaceutical formulations that improve its in vivo stability and selective targeting, the therapeutic potential of mRNA can be considered as endless.As a consequence, many novel immunotherapeutic strategies focus on the use of mRNA beyond its use as the source of tumor antigens. This review aims to summarize the state-of-the-art on these applications and to provide a rationale for their clinical application

Broadening the message : a nanovaccine co-loaded with messenger RNA and α-GalCer induces antitumor immunity through conventional and natural killer T cells

Messenger RNA encoding tumor antigens has the potential to evoke effective antitumor immunity. This study reports on a nanoparticle platform, named mRNA Galsomes, that successfully co-delivers nucleoside-modified antigen-encoding mRNA and the glycolipid antigen and immunopotentiator α-galactosylceramide (α-GC) to antigen-presenting cells after intravenous administration. By co-formulating low doses of α-GC, mRNA Galsomes induce a pluripotent innate and adaptive tumor-specific immune response in mice, with invariant natural killer T cells (iNKT) as a driving force. In comparison, mRNA Galsomes exhibit advantages over the state-of-the-art cancer vaccines using unmodified ovalbumin (OVA)-encoding mRNA, as we observed up to seven times more tumor-infiltrating antigen-specific cytotoxic T cells, combined with a strong iNKT cell and NK cell activation. In addition, the presence of suppressive myeloid cells (myeloid-derived suppressor cells and tumor-associated macrophages) in the tumor microenvironment was significantly lowered. Owing to these antitumor effects, OVA mRNA Galsomes significantly reduced tumor growth in established E.G7-OVA lymphoma, with a complete tumor rejection in 40% of the animals. Moreover, therapeutic vaccination with mRNA Galsomes enhanced the responsiveness to treatment with a PD-L1 checkpoint inhibitor in B16-OVA melanoma, as evidenced by a synergistic reduction of tumor outgrowth and a significantly prolonged median survival. Taken together, these data show that intravenously administered mRNA Galsomes can provide controllable, multifaceted, and effective antitumor immunity, especially when combined with checkpoint inhibition

Theranostic mRNA-loaded microbubbles in the lymphatics of dogs: implications for drug delivery

Microbubbles have shown potential as intralymphatic ultrasound contrast agents while nanoparticle-loaded microbubbles are increasingly investigated for ultrasound-triggered drug and gene delivery. To explore whether mRNA-nanoparticle loaded microbubbles could serve as theranostics for detection of and mRNA transfer to the lymph nodes, we investigate the behavior of unloaded and mRNA-loaded microbubbles using contrast-enhanced ultrasound imaging after subcutaneous injection in dogs. Our results indicate that both types of microbubbles are equally capable of rapidly entering the lymph vessels and nodes upon injection, and novel, valuable and detailed information on the lymphatic structure in the animals could be obtained. Furthermore, additional observations were made regarding the dynamics of microbubble lymph node uptake. Importantly, neither the microbubble migration distance within the lymphatics, nor the observed contrast signal intensity was influenced by mRNA-loading. Although further optimization of acoustic parameters will be needed, this could represent a first step towards ultrasound-guided, ultrasound-triggered intranodal mRNA delivery using these theranostic microbubbles

Soil texture can predominantly control organic matter mineralization in temperate climates by regulating soil moisture rather than through direct stabilization

Soil organic carbon (OC) levels generally increase with increasing clay and silt content under a similar climatic zone because of increased association of OC to clay minerals and stronger occlusion inside aggregates. Surprisingly though, in Western Europe many silt loam soils actually bear low topsoil OC levels compared to lighter textured soils. Soil texture obviously also strongly controls moisture availability with consequent indirect impact on heterotrophic activity. We hypothesized that with increasingly frequent summer drought: 1) soil microbial activity in sandy soils is more likely impeded due to their limited water holding capacity retention during droughts, while soil OC mineralization in silty soils remain be less drought-limited; 2) capillary rise from sufficiently shallow groundwater would, on the other hand, alleviate the water stress in lighter textures. To test these hypotheses, we established a one-year field trial with manipulation of soil texture, monitoring of soil moisture and maize-C decomposition via 13/12C-CO2 emissions. The upper 0.5 m soil layer was replaced by sand, sandy loam and silt loam soil with low soil OC. Another sandy soil treatment with a gravel layer was also included beneath the sand layer to exclude capillary rise. Soil texture did not affect maize-C mineralization (Cmaize-min) until April 2019 and thereafter Cmaize-min rates were higher in the silt loam than in the sandy soils (P=0.01). θv correlated positively with the Cmaize-min rate for the sand-textured soils only but not for the finer textures. These results clearly highlight that soil texture controlled Cmaize-min indirectly through regulating moisture under the field conditions starting from about May, when soils faced a period of drought. By the end of the experiment, more added Cmaize was mineralized in the silt loam soil (81%) (P<0.05) than in the sandy soil (56%). Capillary rise did not result in a significant increase in cumulative Cmaize-min in the sandy soil, seemingly because the capillary fringe did not reach the sandy topsoil layer. These results imply that, under future climate scenarios the frequency of drought is expected to increase, the largely unimpeded microbial activity in silty soils might lead to a further stronger difference in soil OC with coarser textured soils under similar management