Methods behind neoantigen prediction for personalized anticancer vaccines.

Next to conventional cancer therapies, immunotherapies such as immune checkpoint inhibitors have broadened the cancer treatment landscape over the past decades. Recent advances in next generation sequencing and bioinformatics technologies have made it possible to identify a patient's own immunogenic neoantigens. These cancer neoantigens serve as important targets for personalized immunotherapy which has the benefit of being more active and effective in targeting cancer cells. This paper is a step-by-step guide discussing the different analyses and challenges encountered during in-silico neoantigen prediction. The protocol describes all the tools and steps required for the identification of immunogenic neoantigens.

Neoantigen-directed therapeutics in the clinic: where are we?

In the past decade, immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) T cell therapy have brought immunotherapy to the forefront of cancer treatment; however, only subsets of patients benefit from current approaches. Neoantigen-driven therapeutics specifically redirect the immune system of the patient to enable or reinduce its ability to recognize and eliminate cancer cells. The tumor specificity of this strategy spares healthy and normal cells from being attacked. Consistent with this concept, initial clinical trials have demonstrated the feasibility, safety, and immunogenicity of neoantigen-directed personalized vaccines. We review neoantigen-driven therapy strategies as well as their promise and clinical successes to date.

Challenges in neoantigen-directed therapeutics.

A fundamental prerequisite for the efficacy of cancer immunotherapy is the presence of functional, antigen-specific T cells within the tumor. Neoantigen-directed therapy is a promising strategy that aims at targeting the host's immune response against tumor-specific antigens, thereby eradicating cancer cells. Initial forays have been made in clinical environments utilizing vaccines and adoptive cell therapy; however, many challenges lie ahead. We provide an in-depth overview of the current state of the field with an emphasis on in silico neoantigen discovery and the clinical aspects that need to be addressed to unlock the full potential of this therapy.

A synthetic DNA template for fast manufacturing of versatile single epitope mRNA.

A flexible, affordable, and rapid vaccine platform is necessary to unlock the potential of personalized cancer vaccines in order to achieve full clinical efficiency. mRNA cancer vaccine manufacture relies on the rigid sequence design of multiepitope constructs produced by laborious bacterial cloning and time-consuming plasmid preparation. Here, we introduce a synthetic DNA template (SDT) assembly process, which allows cost- and time-efficient manufacturing of single (neo)epitope mRNA. We benchmarked SDT-derived mRNA against mRNA derived from a plasmid DNA template (PDT), showing that monocyte-derived dendritic cells (moDCs) electroporated with SDT-mRNA or PDT-mRNA, encoding HLA-I- or HLA-II-restricted (neo)epitopes, equally activated T cells that were modified to express the cognate T cell receptors. Furthermore, we validated the SDT-mRNA platform for neoepitope immunogenicity screening using the characterized HLA-A2-restricted neoepitope DHX40B and four new candidate HLA-A2-restricted melanoma neoepitopes. Finally, we compared SDT-mRNA with PDT-mRNA for vaccine development purposes. moDCs electroporated with mRNA encoding the HLA-A2-restricted, mutated Melan-A/Mart-1 epitope together with TriMix mRNA-generated high levels of functional Melan-A/Mart-1-specific CD8+ T cells. In conclusion, SDT single epitope mRNA can be manufactured in a more flexible, cost-efficient, and time-efficient way compared with PDT-mRNA, allowing prompt neoepitope immunogenicity screening, and might be exploited for the development of personalized cancer vaccines.