T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.

Adoptive transfer of dendritic cells (DCs) transfected with in vitro-transcribed, RNA-encoding, tumor-associated antigens has recently entered clinical testing as a promising approach for cancer immunotherapy. However, pharmacokinetic exploration of RNA as a potential drug compound and a key aspect of clinical development is still pending. While investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we identified components located 3 of the coding region that contributed to a higher transcript stability and translational efficiency. With the use of quantitative reverse transcription-polymerase chain reaction (RT-PCR) and eGFP variants to measure transcript amounts and protein yield, we showed that a poly(A) tail measuring 120 nucleotides compared with a shorter one, an unmasked poly(A) tail with a free 3 end rather than one extended with unrelated nucleotides, and 2 sequential ␤-globin 3 untranslated regions cloned head to tail between the coding region and the poly(A) tail each independently enhanced RNA stability and translational efficiency. Consecutively, the density of antigen-specific peptide/MHC complexes on the transfected cells and their potency to stimulate and expand antigen-specific CD4 ؉ and CD8 ؉ T cells were also increased. In summary, our data provide a strategy for optimizing RNA-transfected DC vaccines and a basis for defining release criteria for such vaccine preparations. IntroductionAntigen-encoding RNA 1,2 has the advantages of a genetic vaccine (delivery of all epitopes of the whole antigen, easy manufacturing, standardized purification) and the added value of a safe pharmaceutical characterized by transient expression and lack of integration into the genome of the treated host. The combination of this versatile antigen delivery molecule with dendritic cells (DCs) as the most potent antigen-presenting cells is regarded as an attractive approach to induce cellular and potentially therapeutic immune responses in patients with cancer. Reports demonstrated convincingly that the use of RNA results in efficient induction of antigen-specific immune responses in vitro and in animal models 1,[3][4][5][6][7] and paved the way for trials in humans. Early clinical trials showed feasibility, lack of toxicity, and promising efficacy based on immunologic and clinical read-outs. [8][9][10][11][12] At this early phase of clinical development, antigen-specific RNA has the status of a drug compound requiring detailed exploration.Basic pharmacologic issues that must be addressed in drug development include the pharmacokinetics of the compound of interest within the system of its physiological activity after administration. In the quoted clinical trials, this system would be represented by immature or mature DCs. A key objective of such investigations is better understanding of the impact of the structural features of the drug formulation on its pharmacologic properties. Neither of these questions has thus far been addressed for antigen-e...

Insights into the early infection events of the human hepatitis B (HBV) and hepatitis delta virus (HDV) have been limited because of the lack of a cell culture system supporting the full replication cycle for these important pathogens. The human hepatoma cell line HepaRG allows the experimental induction of a differentiated state, thereby gaining susceptibility toward HBV and HDV infection. We recently identified HBV envelope protein-derived lipopeptides comprising amino acids 2 though 48 of the preS-domain of the L-surface protein, which block infection already at picomolar concentrations. To map the responsible sequence for the peptides' activity we describe an Escherichia coli expression system that permits myristoylation and investigated recombinant HBVpreS-GST fusion proteins with deletion-and point-mutations for their ability to prevent HBV and HDV infection. We found that (1) H epadna-or hepatitis B viruses (HBV) are small enveloped DNA viruses that cause acute and chronic liver infections in mammals and birds. In the case of HBV, progression to the chronic state significantly increases the risk of developing liver cirrhosis and hepatocellular carcinoma. Because 400 million people worldwide suffer from chronic HBV infection, HBV is one of the most important human pathogens. Hepadnaviruses possess remarkable host specificities and preferentially replicate in hepatocytes of their respective hosts. 1 Until recently, in vitro HBV infections were only successful in primary human hepatocytes (PHH) or hepatocytes of related primates. 2,3 Systematic investigations of HBV early infection events were thus difficult and varied with the quality of the hepatocyte preparation. The observation that hepatocytes of Tupaia belangeri are susceptible for HBV 4,5 and the recent establishment of the HepaRG cell line, which also supports the full HBV replication cycle, 6 resolved this limitation and facilitated detailed studies on the contribution of HBV envelope protein domains in virus attachment and entry.The HBV envelope consists of three membrane proteins termed large (L-), middle (M-), and small (S-) proteins. They are encoded in one open reading frame with three in-phase start codons. 1 L-and M-share the S-domain, which serves as a membrane anchor but accom-

Triple-negative breast cancer (TNBC) is a high medical need disease with limited treatment options. CD8+ T cell-mediated immunotherapy may represent an attractive approach to address TNBC. The objectives of this study were to assess the expression of CXorf61 in TNBCs and healthy tissues and to evaluate its capability to induce T cell responses.We show by transcriptional profiling of a broad comprehensive set of normal human tissue that CXorf61 expression is strictly restricted to testis. 53% of TNBC patients express this antigen in at least 30% of their tumor cells. In CXorf61-negative breast cancer cell lines CXorf61 expression is activated by treatment with the hypomethylating agent 5-aza-2′-deoxycytidine.By vaccination of HLA-A*02-transgenic mice with CXorf61 encoding RNA we obtained high frequencies of CXorf61-specific T cells. Cloning and characterization of T cell receptors (TCRs) from responding T cells resulted in the identification of the two HLA-A*0201-restricted T cell epitopes CXorf6166–74 and CXorf6179–87. Furthermore, by in vitro priming of human CD8+ T cells derived from a healthy donor recognizing CXorf6166–74 we were able to induce a strong antigen-specific immune response and clone a human TCR recognizing this epitope.In summary, our data confirms this antigen as promising target for T cell based therapies.

Several viral and non-viral vectors have been developed for exogenous protein expression in specific cells. Conventionally, this purpose is achieved through the use of recombinant DNA. But mainly due to the risks associated with permanent genetic alteration of cells, safety and ethical concerns have been raised for the use of DNA-based vectors in human clinical therapy. In the last years, synthetic messenger RNA has emerged as powerful tool to deliver genetic information. RNA vectors exhibit several advantages compared to DNA and are particularly interesting for applications that require transient gene expression. RNA stability and translation efficiency can be increased by cis-acting structural elements in the RNA such as the 5'-cap, the poly(A)-tail, untranslated regions and the sequence of the coding region. Here we review recent developments in the optimization of messenger RNA as vector for modulation of protein expression emphasizing on stability, transfection and immunogenicity. In addition, we summarize current pre-clinical and clinical studies using RNA-based vectors for immunotherapy, T cell, stem cell as well as gene therapy.