Birgit Scheel

Direct Injection of Protamine-protected mRNA: Results of a Phase 1/2 Vaccination Trial in Metastatic Melanoma Patients

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-DRlo 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. This trial was registered at www.clinicaltrials.gov as ♯NCT00204607.

Results of the first phase I/II clinical vaccination trial with direct injection of mRNA.

Vaccination against tumor antigens has been shown to be a safe and efficacious prophylactic and therapeutic antitumor treatment in many animal models. Clinical studies in humans indicate that specific immunotherapy can also result in clinical benefits. The active pharmaceutical ingredient in such vaccines can be DNA, RNA, protein, or peptide and can be administered naked, encapsulated, or after delivery in vitro into cells that are then adoptively transferred. One of the easiest, most versatile and theoretically safest technologies relies on the direct injection of naked messenger RNA (mRNA) that code for tumor antigens. We and others have shown in mice that intradermal application of naked mRNA results in protein expression and the development of an immune response. We used this protocol to vaccinate 15 melanoma patients. For each patient a growing metastasis was removed, total RNA was extracted, reverse-transcribed, amplified, and cloned. Libraries of cDNA were transcribed to produce unlimited amounts of copy mRNA. Autologous preparations were applied intradermally in combination with granulocyte macrophage colony-stimulating factor as adjuvant. We demonstrate here that such treatment is feasible and safe (phase 1 criteria). Furthermore, an increase in antitumor humoral immune response was seen in some patients. However, a demonstration of clinical effectiveness of direct injection of copy mRNA for antitumor immunotherapy was not shown in this study and must be evaluated in subsequent trials.

Immunostimulating capacities of stabilized RNA molecules.

Since direct injection of naked mRNA induces an immune response, we tested the capacity of RNA to signal danger. We show here that mRNA molecules that are protected from immediate degradation either through interaction with cationic proteins (trans protection) or through chemical modification of the phosphodiester backbone (phosphorothioate RNA; cis protection) act as sequence‐independent danger signals on mouse DC. As opposed to CpG DNA, the cis‐stabilized RNA is degraded in a few minutes, does not activate B cells and, in contrast to double‐stranded RNA, requires MyD88 for activation of the DC. We postulate that phosphorothioate RNA, which mimics trans‐stabilized RNA, is a new PAMP.

Toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mRNA

We reported that RNA condensed on protamine is protected from RNase‐mediated degradation and can be used for vaccination. Here, we show that such complexes are also danger signals that activate mouse cells through a MyD88‐dependent pathway. Moreover, mRNA‐protamine complexes stimulate human blood cells. They strongly activate DC and monocytes, leading to TNF‐α and IFN‐α secretion. In addition, protamine‐RNA complexes directly activate B cells, NK cells and granulocytes. The detailed analysis of the activated cell types, the study of the cytokines released from PBMC cultured with protamine‐RNA complexes and recently published results suggest that TLR‐7 and TLR‐8 may be involved in the recognition of protamine‐stabilized RNA. Our data indicate that protamine‐stabilized RNA, which may be similar to RNA condensed in the nucleocapsids of RNA viruses, is a strong danger signal. Thus, similarly to plasmid DNA, protamine‐RNA combines antigen production and non‐specific immunostimulation. The studies presented here explain the capacity of protamine‐RNA to act as a vaccine, and pave the way towards the development of safe and efficient mRNA‐based immunotherapies.

Therapeutic anti-tumor immunity triggered by injections of immunostimulating single-stranded RNA

Stabilized synthetic RNA oligonucleotides (ORN) and protected messenger RNA (mRNA) were recently discovered to possess an immunostimulatory capacity through their recognition by TLR 7 and 8. We wanted to find out whether this danger signal is capable of triggering anti‐tumor immunity when injected locally into an established tumor. Using the mouse glioma tumor cell line SMA‐560 in syngenic VM/Dk mice, we were able to show that intra‐tumor injections of protamine‐stabilized mRNA do indeed induce tumor regression and long‐term anti‐tumor immunity. Residual RNA‐injected tumors show CD8 infiltration. Distant injections of protamine‐protected mRNA and intra‐tumor injection of naked mRNA also result in anti‐tumor immunity. Although they are strong danger signals, RNA are labile molecules with a short half‐life: they do not trigger side effects such as long‐term, uncontrolled immunostimulation evidenced by splenomegaly in CpG DNA‐treated mice. In conclusion, RNA molecules are potent and safe danger signals that are relevant for active immunotherapy strategies aimed at the eradication of solid tumors.

Production and characterization of amplified tumor-derived cRNA libraries to be used as vaccines against metastatic melanomas

BackgroundAnti-tumor vaccines targeting the entire tumor antigen repertoire represent an attractive immunotherapeutic approach. In the context of a phase I/II clinical trial, we vaccinated metastatic melanoma patients with autologous amplified tumor mRNA. In order to provide the large quantities of mRNA needed for each patient, the Stratagene Creator™ SMART™ cDNA library construction method was modified and applied to produce libraries derived from the tumors of 15 patients. The quality of those mRNA library vaccines was evaluated through sequencing and microarray analysis.ResultsRandom analysis of bacterial clones of the library showed a rate of 95% of recombinant plasmids among which a minimum of 51% of the clones contained a full-Open Reading Frame. In addition, despite a biased amplification toward small abundant transcripts compared to large rare fragments, we could document a relatively conserved gene expression profile between the total RNA of the tumor of origin and the corresponding in vitro transcribed complementary RNA (cRNA). Finally, listing the 30 most abundant transcripts of patient MEL02s library, a large number of tumor associated antigens (TAAs) either patient specific or shared by several melanomas were found.ConclusionOur results show that unlimited amounts of cRNA representing tumors transcriptome could be obtained and that this cRNA was a reliable source of a large variety of tumor antigens.

A novel, disruptive vaccination technology: Self-adjuvanted RNActive® vaccines

Nucleotide based vaccines represent an enticing, novel approach to vaccination. We have developed a novel immunization technology, RNActive® vaccines, that have two important characteristics: mRNA molecules are used whose protein expression capacity has been enhanced by 4 to 5 orders of magnitude by modifications of the nucleotide sequence with the naturally occurring nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) that do not affect the primary amino acid sequence. Second, they are complexed with protamine and thus activate the immune system by involvement of toll-like receptor (TLR) 7. Essentially, this bestows self-adjuvant activity on RNActive® vaccines. RNActive® vaccines induce strong, balanced immune responses comprising humoral and cellular responses, effector and memory responses as well as activation of important subpopulations of immune cells, such as Th1 and Th2 cells. Pre-germinal center and germinal center B cells were detected in human patients upon vaccination. RNActive® vaccines successfully protect against lethal challenges with a variety of different influenza strains in preclinical models. Anti-tumor activity was observed preclinically under therapeutic as well as prophylactic conditions. Initial clinical experiences suggest that the preclinical immunogenicity of RNActive® could be successfully translated to humans.

A novel RNA-based adjuvant combines strong immunostimulatory capacities with a favorable safety profile

Protein‐ and peptide‐based tumor vaccines depend on strong adjuvants to induce potent immune responses. Here, we demonstrated that a recently developed novel adjuvant based on a non‐coding, long‐chain RNA molecule, termed RNAdjuvant®, profoundly increased immunogenicity of both antigen formats. RNAdjuvant® induced balanced, long‐lasting immune responses that resulted in a strong anti‐tumor activity. A direct comparison to Poly(I:C) showed superior efficacy of our adjuvant to enhance antigen‐specific multifunctional CD8+ T‐cell responses and mediate anti‐tumor responses induced by peptide derived from HPV‐16 E7 protein in the syngeneic TC‐1 tumor, a murine model of human HPV‐induced cervical cancer. Moreover, the adjuvant was able to induce functional memory responses that mediated complete tumor remission. Despite its remarkable immunostimulatory activity, our RNA‐based adjuvant exhibited an excellent pre‐clinical safety profile. It acted only locally at the injection site where it elicited a transient but strong up‐regulation of pro‐inflammatory and anti‐viral cytokines as well as cytoplasmic RNA sensors without systemic cytokine release. This was followed by the activation of immune cells in the draining lymph nodes. Our data indicate that our RNA‐based adjuvant is a safe and potent immunostimulator that may profoundly improve the efficacy of a variety of cancer vaccines.

Distinct transcriptional changes in non-small cell lung cancer patients associated with multi-antigenic RNActive® CV9201 immunotherapy

ABSTRACT 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.

Co-transfection of messenger RNA and siRNA as a method to study the efficiency of siRNA.

The definition of an optimal siRNA results from the in vitro testing of several siRNA designed to specifically target a gene. Usually, such in vitro tests consist in the transfection of the several siRNA duplexes in a cell expressing stably the gene of interest. When a siRNA specific for a mRNA coding toxic proteins (certain transcription factors, transporters, toxins, cell cycle controlling proteins, etc.) must be tested, the generation of a target cell is difficult. Here we report a quick method to test the efficiency of a siRNA through its co-transfection with the targeted mRNA. This technique can be used as a fast method to test siRNA even when they target genes that cannot be stably expressed in the cells of interest. *Both authors contributed equally to the work. This work was supported by “Förderprogramm Biotechnologie,” a grant from the government of Baden Württemberg, Germany and by a “IZKF-Verbundprojekt,” a grant from the University of Tübingen. JP is supported by the DFG: Graduiertenkolleg “Infektionsbiologie” in Tübingen and JPC is supported by a “Fortüne” grant from the University of Tübingen.