A phase I/IIa study of the mRNA-based cancer immunotherapy CV9201 in patients with stage IIIB/IV non-small cell lung cancer.

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.

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.

Protein expression from exogenous mRNA: uptake by receptor-mediated endocytosis and trafficking via the lysosomal pathway.

Insertional mutagenesis and the inherent risk of malignancy compromise the clinical use of DNA-based therapies. Being a transient copy of genetic material, mRNA is a safe alternative, overcoming this limitation. As a prerequisite for the development of efficient mRNA-based therapies, we investigated the cellular uptake and intracellular fate of mRNA for the first time. To this end we determined cell-type, dose and energy dependence of mRNA internalisation. Moreover, we employed markers for uptake pathways and cellular compartments to analyse the route of mRNA internalisation and its intracellular destination. Finally, we addressed the involvement of receptors and their nature using a competitor-based approach. We found that all cell types tested were amenable to uptake and expression of naked mRNA. Internalisation mainly occurred via caveolae/lipid raft-rich membrane domains and involved scavenger-receptor(s). Following endocytosis, mRNA eventually accumulated in lysosomes, while part of it escaped into the cytosol giving rise to protein synthesis. Taken together, our findings provide unprecedented insights into the internalisation and trafficking of exogenous mRNA, greatly facilitating the development of effective mRNA-based therapies in the future.

Characterization of the ribonuclease activity on the skin surface.

The rapid degradation of ribonucleic acids (RNA) by ubiquitous ribonucleases limits the efficacy of new therapies based on RNA molecules. Therefore, our aim was to characterize the natural ribonuclease activities on the skin and in blood plasma i.e. at sites where many drugs in development are applied. On the skin surfaces of Homo sapiens and Mus musculus we observed dominant pyrimidine-specific ribonuclease activity. This activity is not prevented by a cap structure at the 5'-end of messenger RNA (mRNA) and is not primarily of a 5'- or 3'-exonuclease type. Moreover, the ribonuclease activity on the skin or in blood plasma is not inhibited by chemical modifications introduced at the 2'OH group of cytidine or uridine residues. It is, however, inhibited by the ribonuclease inhibitor RNasin although not by the ribonuclease inhibitor SUPERase* In. The application of our findings in the field of medical science may result in an improved efficiency of RNA-based therapies that are currently in development.

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

BACKGROUND: Anti-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. RESULTS: Random 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 MEL02's library, a large number of tumor associated antigens (TAAs) either patient specific or shared by several melanomas were found. CONCLUSION: Our results show that unlimited amounts of cRNA representing tumor's transcriptome could be obtained and that this cRNA was a reliable source of a large variety of tumor antigens.

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.

Self-adjuvanted mRNA vaccination in advanced prostate cancer patients: a first-in-man phase I/IIa study.

BACKGROUND: 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. METHODS: 44 Patients received up to 5 intra-dermal vaccinations. Three dose levels of total mRNA were tested in Phase I in cohorts of 3-6 patients to determine a recommended dose. In phase II, 32 additional patients were treated at the recommended dose. The primary endpoint was safety and tolerability, the secondary endpoint was induction of antigen specific immune responses monitored at baseline and at weeks 5, 9 and 17. RESULTS: The most frequent adverse events were grade 1/2 injection site erythema, injection site reactions, fatigue, pyrexia, chills and influenza-like illness. Possibly treatment related urinary retention occurred in 3 patients. The recommended dose was 1280 µg. A total of 26/33 evaluable patients treated at 1280 µg developed an immune response, directed against multiple antigens in 15 out of 33 patients. One patient showed a confirmed PSA response. In the subgroup of 36 metastatic patients, the Kaplan-Meier estimate of median overall survival was 31.4 months [95 % CI: 21.2; n.a]. CONCLUSIONS: The self-adjuvanted RNActive® vaccine CV9103 was well tolerated and immunogenic. The technology is a versatile, fast and cost-effective platform allowing for creation of vaccines. The follow-up vaccine CV9104 including the additional antigens prostatic acid phosphatase (PAP) and Muc1 is currently being tested in a randomized phase IIb trial to assess the clinical benefit induced by this new vaccination approach. TRIAL REGISTRATION: EU Clinical Trials Register: EudraCT number 2008-003967-37, registered 27 Jan 2009.

Messenger RNA-based vaccines: progress, challenges, applications.

Twenty years after the demonstration that messenger RNA (mRNA) was expressed and immunogenic upon direct injection in mice, the first successful proof-of-concept of specific protection against viral infection in small and large animals was reported. These data indicate wider applicability to infectious disease and should encourage continued translation of mRNA-based prophylactic vaccines into human clinical trials. At the conceptual level, mRNA-based vaccines-more than other genetic vectors-combine the simplicity, safety, and focused immunogenicity of subunit vaccines with favorable immunological properties of live viral vaccines: (1) mRNA vaccines are molecularly defined and carry no excess information. In the environment and upon physical contact, RNA is rapidly degraded by ubiquitous RNases and cannot persist. These characteristics also guarantee tight control over their immunogenic profile (including avoidance of vector-specific immune responses that could interfere with repeated administration), pharmacokinetics, and dosing. (2) mRNA vaccines are synthetically produced by an enzymatic process, just requiring information about the nucleic acid sequence of the desired antigen. This greatly reduces general complications associated with biological vaccine production, such as handling of infectious agents, genetic variability, environmental risks, or restrictions to vaccine distribution. (3) RNA can be tailored to provide potent adjuvant stimuli to the innate immune system by direct activation of RNA-specific receptors; this may reduce the need for additional adjuvants. The formation of native antigen in situ affords great versatility, including intracellular localization, membrane association, posttranslational modification, supra-molecular assembly, or targeted structural optimization of delivered antigen. Messenger RNA vaccines induce balanced immune responses including B cells, helper T cells, and cytotoxic T lymphocytes, rendering them an extremely adaptable platform. This article surveys the design, mode of action, and capabilities of state-of-the-art mRNA vaccines, focusing on the paradigm of influenza prophylaxis.

Messenger RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity.

Direct vaccination with messenger RNA (mRNA) molecules encoding tumor-associated antigens is a novel and promising approach in cancer immunotherapy. The main advantage of using mRNA for vaccination is that the same molecule not only provides an antigen source for adaptive immunity, but can simultaneously bind to pattern recognition receptors, thus stimulating innate immunity. However, achieving both features remains challenging, as the complexation of mRNA required for immune-stimulating activity may inhibit its translatability. In this study, we present a new and more effective vaccine design: a two-component mRNA-based tumor vaccine that supports both: antigen expression and immune stimulation, mediated by Toll like receptor 7 (TLR7). The two-component mRNA vaccines, containing free and protamine-complexed mRNA, induce balanced adaptive immune responses providing humoral as well as T cell mediated immunity. This balanced immune response is based on the induction of antigen-specific CD4(+) T helper cells and cytotoxic CD8(+) T cells. Once activated, these CD4(+) and CD8(+) T cells secrete a wide set of cytokines, which drive a TH1 response. Immunization with the two-component vaccines induces sustained memory responses, mediated by antigen-specific memory T cells. Moreover, treatment of mice with the two-component mRNA vaccine mediates a strong antitumor response against OVA-expressing tumor cells, not only in a prophylactic but also in a therapeutic setting. In conclusion, two-component mRNA vaccines with self-adjuvanting activity induce balanced adaptive immune responses and mediate sustained antitumor activity.

Delivery of messenger RNA using poly(ethylene imine)-poly(ethylene glycol)-copolymer blends for polyplex formation: biophysical characterization and in vitro transfection properties.

Nucleic acid based therapies have so far mainly been focused on plasmid DNA (pDNA), small interfering RNA (siRNA), antisense and immunostimulatory oligonucleotides. Messenger RNA (mRNA) was the subject of only a few studies. The objective of this investigation was the preparation of new composite polyplexes with mRNA consisting of poly(ethylene imine) (PEI) and poly(ethylene imine)-poly(ethylene glycol)-copolymers (PEI-PEG) as blends. These complexes were designed to increase the stability of mRNA, to improve transfection efficiency and to reduce cytotoxicity. Hydrodynamic diameters of the polyplexes were measured by dynamic light scattering, polyplex stability was analyzed by gel retardation assay and transfection efficiency of luciferase (Luc) encoding mRNA was evaluated under in vitro conditions. Most of the polyplexes generated showed small particle sizes <200 nm and positive zeta-potentials of +20 mV to +30 mV. Stable complexes were formed even at low nitrogen to phosphate ratios. Polyplexes with mRNA Luc and blends of low molecular weight PEI(5 kDa) and PEI(25k Da)-PEG(20 kDa)1-block-copolymer showed protein expression as high as polyplexes with PEI(25 kDa). Moreover, luciferase expression was significantly higher than that obtained with one of the components alone. These results suggest that delivery systems for pulmonary application of mRNA merit further investigation under in vitro and in vivo conditions.

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.

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-alpha and IFN-alpha 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.

The non-classical HLA class I molecule HFE does not influence the NK-like activity contained in fresh human PBMCs and does not interact with NK cells.

In humans, four beta2-microglobulin-associated non-classical class I molecules are encoded in the MHC: HLA-E, -F, -G and -H. Three of them (HLA-E, -F and -G) were shown to inhibit NK activity. On the contrary, the fourth one, HLA-H, named HFE after it was found to be mutated in patients suffering from inherited hemochromatosis, has been shown to be involved only in the regulation of iron uptake. We tested the capacity of HFE to affect (enhance or reduce) specifically the NK activity contained in non-manipulated fresh human PBMCs. We showed that HFE expression by target cells does not affect their killing by the NK-like activity contained in PBMCs. Moreover, using fluorescent HFE tetramers, we could confirm that blood NK cells as well as blood gammadelta T cells do not bind HFE. Altogether, our data indicate that HFE does not affect the NK activity contained in the PBMCs.

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.

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

Although the V-gene segments coding for the TCR alpha and delta chains are mixed together in the alpha delta locus and are recombined by the same processes, some gene segments (TRAV) are rearranged only with TCR Jalpha gene segments, some (TRDV) only with TCR Ddelta gene segments and some (TRADV) with both. To date, no molecular signal is known that can characterize these three different types 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 alpha delta locus.