Although the use of checkpoint inhibiting antibodies has proven its merit in the treatment of patients suffering from nonsmall cell lung cancer (NSCLC), the limited response rate (20%) to these therapeutics remains a major issue. To increase the number of responders, it is of the essence to increase the infiltration of effector T cells into a patient’s tumor lesions, to provide a point of engagement for these therapeutic moieties. To increase immune infiltration, we will use mRNA vaccination as a novel approach to immunize NSCLC patients against antigens expressed by malignant cells. Delivery of the tumor antigen-encoding mRNAs to the patient’s antigen-presenting cells is ensured by intravenous injection of mRNA packaged into lipid nanoparticles together with a-galactosylceramide (aGC), which acts as a potent immune adjuvant by activating natural killer T (NKT) cells. Importantly, the mRNA is synthesized using modified nucleosides to prevent toxic type I interferon production. In this phase I trial, mRNA encoding a defined combination of 4 cancer testis antigens (CTAs), covering >90% of NSCLC patients, will be encapsulated. We showed in a murine melanoma model that these mRNA liposome complexes or Galsomes induced a very broad, but controllable immune response with a high intratumoral influx of CTLs, NKT and NK cells. Furthermore, Galsomes significantly enhanced survival in combination with checkpoint inhibitors compared to either treatment alone. As such, the proposed strategy is the use of a broadly applicable, off-the-shelf mRNA vaccine for use in NSCLC, in combination with checkpoint inhibiting antibodies. In this proposal, we will set up an early phase clinical trial in late stage NSCLC patients with Galsomes containing mRNA encoding 4 CTAs (4CTA-Galsomes). In order to ensure the safety of the patients, a number of essential preclinical studies are included in this project. Firstly, we will optimize the composition of the Galsomes for use in humans. In addition, we will set up the manufacture of the 4CTA-Galsomes according to Good Manufacturing Practices (GMP). Subsequently, we will extensively test toxicity in mice and in a highly relevant pig model. Finally, after obtaining the required licenses from FAMHP (FAGG), we will use these 4CTA-Galsomes in a first-in-human, phase I clinical trial in NSCLC to evaluate safety, as well as specific immune activation and clinical responses. This new cell-free vaccination approach is expected to have several advantages compared to the current state-of-the-art cellular vaccinations (e.g. dendritic cell vaccines) with regard to production, costs, administration and applicability. In addition, as intravenously administered Galsomes directly target lung dendritic cells, this approach may be optimal for the treatment of lung carcinomas.