Jochen Probst

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 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.

A conserved sequence in the mouse variable T cell receptor α recombination signal sequence 23-bp spacer can affect recombination

Although the V‐gene segments coding for the TCR α and δ chains are mixed together in the α δ locus and are recombined by the same processes, some gene segments (TRAV) are rearranged only with TCR Jα gene segments, some (TRDV) only with TCR Dδ gene segments and some (TRADV) with both. To date, no molecular signal is known that can characterize these three differenttypes of gene segments. Studying the recombination signal sequences (RSS) of all mouse TCR V‐gene segments we observed that 80% of the TRAV contain a palindrome sequence (CTGCAG) or its related variant CTGTAG in their 23‐bp spacer. Using gel‐shift assays we show that these sequences are specifically recognized by some nuclear proteins that are expressed by fresh thymocytes, fresh lymphocytes and tumor cells. Recombination assays on plasmid substrates in a pre‐B cell line showed that RSS containing the CTGCAG sequence can impair recombination. From the protein fractions containing the CTGCAG‐binding activity, three proteins were identified: G3BP1 (a nucleic‐acid‐binding protein with a proposed helicase activity) and two proteins from the high‐mobility group (HMG) family — HMGB2 and HMGB3. We hypothesize that these proteins can affect recombination at the TCR α δ locus.

Messenger RNA Vaccines

Twenty years after the seminal observation of Wolff et al. that injection of naked RNA and DNA vectors results in protein expression in vivo, messenger RNA (mRNA) vaccines have found entry into clinical development. Through improved vector design, formulation, and delivery, mRNA, initially perceived as unstable and difficult to manipulate, has been developed into a convenient, efficacious, and flexible vaccine platform. Importantly, the same production process can be used to produce a variety of different vaccines, independent of the specifics of particular constructs, which ultimately decreases costs and development time.

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.