One hundred fifty years ago, Friedrich Miescher discovered DNA when he isolated "Nuclein"-as he named it-from nuclei of human pus cells. Miescher recognized his isolate as a new type of molecule equal in importance to proteins. He realised that it is an acid of large molecular weight and high phosphorus content. Subsequently, he discovered Nuclein also in the nuclei of other cell types, realised that it chemically defines the nucleus, and speculated on its role in proliferation, heredity and fertilisation. While now universally recognised as the discoverer of DNA, whether Miescher also discovered RNA has not yet been addressed. To determine whether his isolation also yielded RNA, we first reproduced his historic protocols. Our resulting modern Nuclein contained a significant percentage of RNA. Encouraged by this result, we then analysed a sample of Nuclein isolated by Miescher from salmon sperm. Assuming that the RNA present in this sample had degraded to nucleobases, we tested for the presence of uracil in the historic Nuclein. Detection of significant levels of uracil by LC-UV-MS demonstrates that Miescher isolated both forms of nucleic acid-DNA and RNA-and underlines the fundamental nature of his discovery for the field of molecular genetics.

CV9201 is an RNActive®-based cancer immunotherapy encoding five non-small cell lung cancer-antigens: New York esophageal squamous cell carcinoma-1, melanoma antigen family C1/C2, survivin, and trophoblast glycoprotein. In a phase I/IIa dose-escalation trial, 46 patients with locally advanced (n = 7) or metastatic (n = 39) NSCLC and at least stable disease after first-line treatment received five intradermal CV9201 injections (400-1600 µg of mRNA). The primary objective of the trial was to assess safety. Secondary objectives included assessment of antibody and ex vivo T cell responses against the five antigens, and changes in immune cell populations. All CV9201 dose levels were well-tolerated and the recommended dose for phase IIa was 1600 µg. Most AEs were mild-to-moderate injection site reactions and flu-like symptoms. Three (7%) patients had grade 3 related AEs. No related grade 4/5 or related serious AEs occurred. In phase IIa, antigen-specific immune responses against ≥ 1 antigen were detected in 63% of evaluable patients after treatment. The frequency of activated IgD+CD38hi B cells increased > twofold in 18/30 (60%) evaluable patients. 9/29 (31%) evaluable patients in phase IIa had stable disease and 20/29 (69%) had progressive disease. Median progression-free and overall survival were 5.0 months (95% CI 1.8-6.3) and 10.8 months (8.1-16.7) from first administration, respectively. Two- and 3-year survival rates were 26.7% and 20.7%, respectively. CV9201 was well-tolerated and immune responses could be detected after treatment supporting further clinical investigation.

CV9201 is an RNActive®-based cancer immunotherapy encoding five non-small cell lung cancer-antigens: New York esophageal squamous cell carcinoma-1, melanoma antigen family C1/C2, survivin, and trophoblast glycoprotein. In a phase I/IIa dose-escalation trial, 46 patients with locally advanced (n = 7) or metastatic (n = 39) NSCLC and at least stable disease after first-line treatment received five intradermal CV9201 injections (400–1600 µg of mRNA). The primary objective of the trial was to assess safety. Secondary objectives included assessment of antibody and ex vivo T cell responses against the five antigens, and changes in immune cell populations. All CV9201 dose levels were well-tolerated and the recommended dose for phase IIa was 1600 µg. Most AEs were mild-to-moderate injection site reactions and flu-like symptoms. Three (7%) patients had grade 3 related AEs. No related grade 4/5 or related serious AEs occurred. In phase IIa, antigen-specific immune responses against ≥ 1 antigen were detected in 63% of evaluable patients after treatment. The frequency of activated IgD+CD38hi B cells increased > twofold in 18/30 (60%) evaluable patients. 9/29 (31%) evaluable patients in phase IIa had stable disease and 20/29 (69%) had progressive disease. Median progression-free and overall survival were 5.0 months (95% CI 1.8–6.3) and 10.8 months (8.1–16.7) from first administration, respectively. Two- and 3-year survival rates were 26.7% and 20.7%, respectively. CV9201 was well-tolerated and immune responses could be detected after treatment supporting further clinical investigation.

Background Vaccines based on mRNA coding for antigens have been shown to be safe and immunogenic in preclinical models. We aimed to report results of the first-in-human proof-of-concept clinical trial in healthy adults of a prophylactic mRNA-based vaccine encoding rabies virus glycoprotein (CV7201).Methods We did an open-label, uncontrolled, prospective, phase 1 clinical trial at one centre in Munich, Germany. Healthy male and female volunteers (aged 18-40 years) with no history of rabies vaccination were sequentially enrolled. They received three doses of CV7201 intradermally or intramuscularly by needle-syringe or one of three needle-free devices. Escalating doses were given to subsequent cohorts, and one cohort received a booster dose after 1 year. The primary endpoint was safety and tolerability. The secondary endpoint was to determine the lowest dose of CV7201 to elicit rabies virus neutralising titres equal to or greater than the WHO-specified protective antibody titre of 0.5 IU/mL. The study is continuing for long-term safety and immunogenicity follow-up. This trial is registered with ClinicalTrials.gov, number NCT02241135.Findings Between Oct 21, 2013, and Jan 11, 2016, we enrolled and vaccinated 101 participants with 306 doses of mRNA (80-640 mu g) by needle-syringe (18 intradermally and 24 intramuscularly) or needle-free devices (46 intradermally and 13 intramuscularly). In the 7 days post vaccination, 60 (94%) of 64 intradermally vaccinated participants and 36 (97%) of 37 intramuscularly vaccinated participants reported solicited injection site reactions, and 50 (78%) of 64 intradermally vaccinated participants and 29 (78%) of 37 intramuscularly vaccinated participants reported solicited systemic adverse events, including ten grade 3 events. One unexpected, possibly related, serious adverse reaction that occurred 7 days after a 640 mu g intramuscular dose resolved without sequelae. mRNA vaccination by needle-free intradermal or intramuscular device injection induced virus neutralising antibody titres of 0.5 IU/mL or more across dose levels and schedules in 32 (71%) of 45 participants given 80 mu g or 160 mu g CV7201 doses intradermally and six (46%) of 13 participants given 200 mu g or 400 mu g CV7201 doses intramuscularly. 1 year later, eight (57%) of 14 participants boosted with an 80 mu g needle-free intradermal dose of CV7201 achieved titres of 0.5 IU/mL or more. Conversely, intradermal or intramuscular needlesyringe injection was ineffective, with only one participant (who received 320 mu g intradermally) showing a detectable immune response.Interpretation This first-ever demonstration in human beings shows that a prophylactic mRNA-based candidate vaccine can induce boostable functional antibodies against a viral antigen when administered with a needle-free device, although not when injected by a needle-syringe. The vaccine was generally safe with a reasonable tolerability profile.

We recently completed a phase I/IIa trial of RNActive® CV9201, a novel mRNA-based therapeutic vaccine targeting five tumor-associated antigens in non-small cell lung cancer (NSCLC) patients. The aim of the study presented here was to comprehensively analyze changes in peripheral blood during the vaccination period and to generate hypotheses facilitating the identification of potential biomarkers correlating with differential clinical outcomes post RNActive® immunotherapy. We performed whole-genome expression profiling in a subgroup of 22 stage IV NSCLC patients before and after initiation of treatment with CV9201. Utilizing an analytic approach based on blood transcriptional modules (BTMs), a previously described, sensitive tool for blood transcriptome data analysis, patients segregated into two major clusters based on transcriptional changes post RNActive® treatment. The first group of patients was characterized by the upregulation of an expression signature associated with myeloid cells and inflammation, whereas the other group exhibited an expression signature associated with T and NK cells. Patients with an enrichment of T and NK cell modules after treatment compared to baseline exhibited significantly longer progression-free and overall survival compared to patients with an upregulation of myeloid cell and inflammatory modules. Notably, these gene expression signatures were mutually exclusive and inversely correlated. Furthermore, our findings correlated with phenotypic data derived by flow cytometry as well as the neutrophil-to-lymphocyte ratio. Our study thus demonstrates non-overlapping, distinct transcriptional profiles correlating with survival warranting further validation for the development of biomarker candidates for mRNA-based immunotherapy.

Renal cell carcinoma (RCC) is an immunogenic tumor for which immunotherapeutic approaches could be associated with clinically relevant responses. It was recently shown, that induction of T-cell responses against multiple tumor-associated antigen (TAA) epitopes results in prolonged overall survival in RCC patients. In 2003-2005, we performed a phase I/II trial testing an mRNA-based vaccine formulation consisting of a mixture of in vitro transcribed RNA coding for six different TAAs (MUC1, CEA, Her2/neu, telomerase, survivin, MAGE-A1) in 30 metastatic RCC (mRCC) patients. In the first 14 patients, vaccinations were applied i.d. on days 0, 14, 28, and 42. In the consecutive 16 patients, an intensified protocol consisting of i.d. injections (daily on days 0-3, 7-10, 28, and 42) was used. After the respective induction periods, patients in both cohorts were vaccinated monthly until tumor progression. At survival update performed in July 2015, one of the 30 patients was still alive. One patient was lost to follow-up. Median survival of 24.5 mo (all patients) and 89 mo (favorable risk patients) exceeded predicted survival according to Memorial Sloan Kettering Cancer Center (MSKCC) risk score. Impressively, long-term survivors displayed immunological responses to the applied antigens while vice versa no patient without detectable immune response had survived more than 33 mo. The current survival update shows a clear correlation between survival and immunological responses to TAAs encoded by the naked mRNA vaccine. This is one of the first vaccination studies and the only RNA trial that reports on safety and efficacy after a follow-up of more than 10 y.

CV9103 is a prostate-cancer vaccine containing self-adjuvanted mRNA (RNActive®) encoding the antigens PSA, PSCA, PSMA, and STEAP1. This phase I/IIa study evaluated safety and immunogenicity of CV9103 in patients with advanced castration-resistant prostate-cancer.

Introduction Recently Walter et al. (Nature med 2012) demonstrated that induction of T cell responses to tumor-associated antigens (TAA) after peptide vaccination (IMA901) is associated with significantly prolonged overall survival in patients with advanced renal cell carcinoma (RCC). Between August 2003 and November 2005 we performed a phase I/II non-randomized study to analyze the feasibility and safety of an mRNA-based vaccination as well as immunological and clinical responses in patients with stage IV RCC (Rittig et al., Mol Ther. 2011). Here we provide an update on survival and correlative analyses of immunological responses and survival. Methods: In vitro transcribed naked mRNA coding for the TAAs mucin 1, carcinoembryonic antigen (CEA), human epidermal growth factor receptor (EGFR) 2, telomerase, survivin, and melanoma-associated antigen 1 was administered to a total of 30 patients. In the first 14 patients (cohort A), vaccinations were applied on days 0, 14, 28, and 42 (20 µg of each antigen). In the consecutive 16 patients (cohort B), an intensified protocol consisting of injections at days 0-3, 7-10, 28, and 42 with 50 µg/antigen was used. After the respective induction periods, patients in both cohorts received monthly vaccinations until tumor progression according to RECIST. Results: In October 2010 monitoring had revealed a survival in cohort A ranging from 3 to 85 months with 4/14 patients (29%) being alive, while survival in cohort B ranged from 2 to 71 months with 4/16 (25%) patients being alive. Follow up was re-assessed in March 2013. In cohort A, 3 patients were still alive (21%) with survival ranging from 105 to 115 months. Two of these surviving patients had intermediate and one had favourable risk according to Motzer score at enrolment. These respective patients had received between 13 and 32 doses of RNA. In cohort B, three patients (19%) were found to be alive, of which two had favourable and one had intermediate risk according to Motzer score at enrolment. Survival in cohort B ranged from 95 to 97 months, and between 10 and 22 doses of the vaccine had been applied to the patients. Impressively and similar to the findings of Walter et al. with the IMA901 peptide vaccine, all six patients that currently are alive displayed immunological responses to the applied antigens in cytotoxic T cell or ELISpot assays, while vice versa no patient without detectable immune response had survived more than 33 months. Conclusion: The current survival update revealed long term survival (>8 years) in about 20% of patients in this small cohort suffering from stage IV RCC. Notably, in line with data obtained after peptide vaccination, prolonged survival after treatment with our RNA vaccine also showed a clear correlation with in vitro detectable immunological responses to the applied TAA. Disclosures: No relevant conflicts of interest to declare.

The aim of this phase I/II nonrandomized trial was to assess feasibility, safety as well as immunological and clinical responses of a mRNA-based vaccination in patients with stage IV renal cell cancer using granulocyte-macrophage colony stimulating factor (GM-CSF) as adjuvant. Intradermal injections of in vitro transcribed naked mRNA, which was generated using plasmids coding for the tumor-associated antigens mucin 1(MUC1), carcinoembryonic (CEA), human epidermal growth factor receptor 2 (Her-2/neu), telomerase, survivin, and melanoma-associated antigen 1 (MAGE-A1) were performed in 30 enrolled patients. In the first 14 patients (cohort A) vaccinations were administered on days 0, 14, 28, and 42 (20 µg/antigen) while in the consecutive 16 patients (cohort B) an intensified protocol consisting of injections at days 0-3, 7-10, 28, and 42 (50 µg/antigen) was used. In both cohorts, after this induction period, vaccinations were repeated monthly until tumor progression analyzed by Response Evaluation Criteria In Solid Tumors criteria (RECIST). Vaccinations were well tolerated with no severe side effects and induced clinical responses [six stable diseases (SD) and one partial response in cohort A and nine SD in cohort B]. In cohort A, 35.7% survived 4 years (median survival 24 months) compared to 31.25% in cohort B (median survival 29 months). Induction of CD4(+) and CD8(+) T cell responses was shown for several tumor-associated antigens (TAA) using interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and Cr-release assays.

The development of new treatments in the post-genomic era requires methods for safe delivery of foreign genetic information in vivo. As a transient, natural and controllable alternative to recombinant viruses or plasmid DNA (pDNA), purified or in vitro transcribed messenger RNA (mRNA) can be used for the expression of any therapeutic protein in vitro and in vivo. As it has been shown previously, the simple injection of naked mRNA results in local uptake and expression. We show here that this process, in the skin, can greatly be modulated according to the injection solution composition and blocked by an excess of competing nucleic acids or a drug affecting cytosolic mobility. Different cell types at the site of injection can take up the foreign nucleic acid molecules and the protein translated from this is detected for no more than a few days. To test this gene transfer method in humans, we produced in vitro transcribed mRNA under good manufacturing practice (GMP) conditions in a dedicated facility. After injection into the human dermis, we could document the translation of the exogenous mRNA. Our results pave the way toward the use of mRNA as a vehicle for transient gene delivery in humans.

In mice, injection of messenger RNA (mRNA) coding for tumor-associated antigens can induce antitumor immune responses and therefore offers a broadly applicable immunotherapy approach. We injected intradermally protamine-stabilized mRNAs coding for Melan-A, Tyrosinase, gp100, Mage-A1, Mage-A3, and Survivin in 21 metastatic melanoma patients. In 10 patients keyhole limpet hemocyanin (KLH) was added to the vaccine. Granulocyte macrophage colony-stimulating factor was applied as an adjuvant. Endpoints were toxicity and immune responses. No adverse events more than grade II have been observed. During treatment the frequency of Foxp3+/CD4+ regulatory T cells was significantly decreased upon mRNA vaccination in peripheral blood of the patients in the KLH arm, whereas myeloid suppressor cells (CD11b+HLA-DR lo monocytes) were reduced in the patients not receiving KLH. A reproducible increase of vaccine-directed T cells was observed in 2 of 4 immunologically evaluable patients. One of 7 patients with measurable disease showed a complete response. In conclusion, we show here that direct injection of protamine-protected mRNA is feasible and safe. The significant influence of the treatment on the frequency of immunosuppressive cells, the increase of vaccine-directed T cells upon treatment in a subset of patients together with the demonstration of a complete clinical response encourage further clinical investigation of the protamine-mRNA vaccine.