Scientific excellence is not an end in itself, but is a crucial tool for efficient translation of new ideas into viable solutions.

Genetic diseases are driven by aberrations of the human genome. Identification of such aberrations including structural variations (SVs) is key to our understanding. Conventional short-reads whole genome sequencing (cWGS) can identify SVs to base-pair resolution, but utilizes only short-range information and suffers from high false discovery rate (FDR). Linked-reads sequencing (10XWGS) utilizes long-range information by linkage of short-reads originating from the same large DNA molecule. This can mitigate alignment-based artefacts especially in repetitive regions and should enable better prediction of SVs. However, an unbiased evaluation of this technology is not available. In this study, we performed a comprehensive analysis of different types and sizes of SVs predicted by both the technologies and validated with an independent PCR based approach. The SVs commonly identified by both the technologies were highly specific, while validation rate dropped for uncommon events. A particularly high FDR was observed for SVs only found by 10XWGS. To improve FDR and sensitivity, statistical models for both the technologies were trained. Using our approach, we characterized SVs from the MCF7 cell line and a primary breast cancer tumor with high precision. This approach improves SV prediction and can therefore help in understanding the underlying genetics in various diseases.

Treating patients who have cancer with vaccines that stimulate a targeted immune response is conceptually appealing, but cancer vaccine trials have not been successful in late-stage patients with treatment-refractory tumours1,2. We are testing melanoma FixVac (BNT111)—an intravenously administered liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma—in an ongoing, first-in-human, dose-escalation phase I trial in patients with advanced melanoma (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733). We report here data from an exploratory interim analysis that show that melanoma FixVac, alone or in combination with blockade of the checkpoint inhibitor PD1, mediates durable objective responses in checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. Clinical responses are accompanied by the induction of strong CD4+ and CD8+ T cell immunity against the vaccine antigens. The antigen-specific cytotoxic T-cell responses in some responders reach magnitudes typically reported for adoptive T-cell therapy, and are durable. Our findings indicate that RNA-LPX vaccination is a potent immunotherapy in patients with CPI-experienced melanoma, and suggest the general utility of non-mutant shared tumour antigens as targets for cancer vaccination.

Background: Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. The 4T1 murine mammary cancer cell line is one of the most widely used breast cancer models. Here, we present an integrated map of the genome, transcriptome, and immunome of 4T1.Results: We found Trp53 (Tp53) and Pik3g to be mutated. Other frequently mutated genes in breast cancer, including Brca1 and Brca2, are not mutated. For cancer related genes, Nav3, Cenpf, Muc5Ac, Mpp7, Gas1, MageD2, Dusp1, Ros, Polr2a, Rragd, Ros1, and Hoxa9 are mutated. Markers for cell proliferation like Top2a, Birc5, and Mki67 are highly expressed, so are markers for metastasis like Msln, Ect2, and Plk1, which are known to be overexpressed in triple-negative breast cancer (TNBC). TNBC markers are, compared to a mammary gland control sample, lower (Esr1), comparably low (Erbb2), or not expressed at all (Pgr). We also found testis cancer antigen Pbk as well as colon/gastrointestinal cancer antigens Gpa33 and Epcam to be highly expressed. Major histocompatibility complex (MHC) class I is expressed, while MHC class II is not. We identified 505 single nucleotide variations (SNVs) and 20 insertions and deletions (indels). Neoantigens derived from 22 SNVs and one deletion elicited CD8+ or CD4+ T cell responses in IFNγ-ELISpot assays. Twelve high-confidence fusion genes were observed. We did not observe significant downregulation of mismatch repair (MMR) genes or SNVs/indels impairing their function, providing evidence for 6-thioguanine resistance. Effects of the integration of the murine mammary tumor virus were observed at the genome and transcriptome level.Conclusions: 4T1 cells share substantial molecular features with human TNBC. As 4T1 is a common model for metastatic tumors, our data supports the rational design of mode-of-action studies for pre-clinical evaluation of targeted immunotherapies.

Antigen-encoding, lipoplex-formulated RNA (RNA-LPX) enables systemic delivery to lymphoid compartments and selective expression in resident antigen-presenting cells.  We report here that the rejection of CT26 tumors, mediated by local radiotherapy (LRT), is further augmented in a CD8+ T cell-dependent manner by an RNA-LPX vaccine that encodes CD4+ T cell-recognized neoantigens (CD4 neoantigen vaccine).  Whereas CD8+ T cells induced by LRT alone were primarily directed against the immunodominant gp70 antigen, mice treated with LRT plus the CD4 neoantigen vaccine rejected gp70-negative tumors and were protected from rechallenge with these tumors, indicating a potent poly-antigenic CD8+ T cell response and T cell memory.  In the spleens of CD4 neoantigen-vaccinated mice, we found a high number of activated, poly-functional, Th1-like CD4+ T cells against ME1, the immunodominant CD4 neoantigen within the poly-neoantigen vaccine.  LRT itself strongly increased CD8+ T cell numbers and clonal expansion.  However, tumor infiltrates of mice treated with CD4 neoantigen vaccine/LRT, as compared to LRT alone, displayed a higher fraction of activated gp70-specific CD8+ T cells, lower PD-1/LAG-3 expression and contained ME1-specific IFNγ+ CD4+ T cells capable of providing cognate help.  CD4 neoantigen vaccine/LRT treatment followed by anti-CTLA-4 antibody therapy further enhanced the efficacy with complete remission of gp70-negative CT26 tumors and survival of all mice.  Our data highlight the power of combining synergistic modes of action and warrants further exploration of the presented treatment schema.

TRON is delighted to announce the publication of a scientific article in OncoImmunology.In this article, TRON authors Nadja Salomon et al. investigated combination therapy in CT26 tumors. CT26 rejection using local radiotherapy (LRT) is augmented by a lipoplex-formulated RNA vaccine that encodes CD4+ T cell-recognized neoantigens. CD4 neoantigen vaccine/LRT treatment followed by anti-CTLA-4 antibody therapy further enhanced the efficacy with complete remission of gp70-negative CT26 tumors.

PLAC1 (placenta enriched 1) is a mammalian trophoblast-specific protein. Aberrant expression of PLAC1 is observed in various human cancers, where it is involved in the motility, migration, and invasion of tumor cells, which are associated with the phosphoinositide 3-kinase (PI3K)/AKT pathway. We previously demonstrated that AKT activation mediates the downstream effects of PLAC1; however, the molecular mechanisms of PLAC1-induced AKT-mediated tumor-related processes are unclear. We studied human choriocarcinoma and breast cancer cell lines to explore the localization and receptor-ligand interactions, as well as the downstream effects of PLAC1. We show secretion and adherence of PLAC1 to the extracellular matrix, where it forms a trimeric complex with fibroblast growth factor 7 (FGF7) and its receptor, FGF receptor 2 IIIb (FGFR2IIIb). We further show that PLAC1 signaling via FGFR2IIIb activates AKT phosphorylation in cancer cell lines. As the FGF pathway is of major interest in anticancer therapeutic strategies, these data further promote PLAC1 as a promising anticancer drug target.

Rewiring of energy metabolism and adaptation of mitochondria are considered to impact on prostate cancer development and progression. Here, we report on mitochondrial respiration, DNA mutations and gene expression in paired benign/malignant human prostate tissue samples. Results reveal reduced respiratory capacities with NADH-pathway substrates glutamate and malate in malignant tissue and a significant metabolic shift towards higher succinate oxidation, particularly in high-grade tumors. The load of potentially deleterious mitochondrial-DNA mutations is higher in tumors and associated with unfavorable risk factors. High levels of potentially deleterious mutations in mitochondrial Complex I-encoding genes are associated with a 70% reduction in NADH-pathway capacity and compensation by increased succinate-pathway capacity. Structural analyses of these mutations reveal amino acid alterations leading to potentially deleterious effects on Complex I, supporting a causal relationship. A metagene signature extracted from the transcriptome of tumor samples exhibiting a severe mitochondrial phenotype enables identification of tumors with shorter survival times.

Our immune system plays a key role in health and disease as it is capable of responding to foreign antigens as well as acquired antigens from cancer cells. Latter are caused by somatic mutations, the so-called neoepitopes, and might be recognized by T cells if they are presented by HLA molecules on the surface of cancer cells. Personalized mutanome vaccines are a class of customized immunotherapies, which is dependent on the detection of individual cancer-specific tumor mutations and neoepitope (i.e., prediction, followed by a rational vaccine design, before on-demand production. The development of next generation sequencing (NGS) technologies and bioinformatic tools allows a large-scale analysis of each parameter involved in this process. Here, we provide an overview of the bioinformatic aspects involved in the design of personalized, neoantigen-based vaccines, including the detection of mutations and the subsequent prediction of potential epitopes, as well as methods for associated biomarker research, such as high-throughput sequencing of T-cell receptors (TCRs), followed by data analysis and the bioinformatics quantification of immune cell infiltration in cancer samples.

Chimeric antigen receptor (CAR)-T cells have shown efficacy in patients with B cell malignancies. Yet their application for solid tumors has challenges that include limited cancer-specific targets and non-persistence of adoptively transferred CAR-T cells. Here we introduce the developmentally regulated tight junction protein claudin 6 (CLDN6) as a CAR target in solid tumors, and a strategy to overcome inefficient CAR-T cell stimulation in vivo. We demonstrate that a nanoparticulate RNA vaccine, designed for body-wide delivery of the CAR antigen into lymphoid compartments, stimulates adoptively transferred CAR-T cells. Presentation of the natively folded target on resident dendritic cells promotes cognate and selective expansion of CAR-T cells. Improved engraftment of CAR-T cells and regression of large tumors in difficult-to-treat mouse models was achieved at sub-therapeutic CAR-T cell doses.