The prospect of stimulating immune responses to specifically kill cancer cells, instigated research
into cancer vaccines. The positive results on systemic delivery of mRNA packaged in lipid
nanoparticles (LNPs) in patients are very exciting, since LNPs avoid the complex logistics associated
with ex vivo strategies. We recently developed LNPs containing tumor antigen mRNA and the
adjuvant α−galactosylceramide (α-GC-LNPs). These are able to induce a high tumor influx of
cytotoxic T lymphocytes (CTLs) and invariant natural killer T (iNKT) cells, thereby tackling cancer
from two angles. However, recently we discovered that these cells receive inhibitory signals via the
PD-1 receptor, which binds PD-L1 expressed on antigen-presenting cells (APCs) and cancer cells
resulting in iNKT-cell anergy and CTL-paralysis. Therefore, we aim to boost the efficiency of the
developed α-GC-LNPs by developing a dual treatment regimen, based on the spatio-temporal
expression PD-L1 after vaccination, in which α-GC-LNPs are paired with PD-1/PD-L1 inhibition at
these two fronts. We will study the treatment regimen in vivo, evaluating its effect on APCs, CTLs,
iNKT cells, and its ability to mediate tumor regression. As most experiments are performed in
laboratory animals, and therefore designed to accrue to the principle of reduction, we included the
development of a novel non-parametric statistical toolbox with hypothesis tests suitable for small
datasets with high power.