ABSTRACT
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.
Subject(s)
B-Lymphocytes/immunology , Cancer Vaccines/immunology , Carcinoma, Non-Small-Cell Lung/therapy , Immunotherapy/methods , Lung Neoplasms/therapy , RNA, Messenger/therapeutic use , T-Lymphocytes/immunology , Aged , Aged, 80 and over , Antigens, Neoplasm/genetics , Cancer Vaccines/genetics , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/mortality , Cells, Cultured , Female , Humans , Immunotherapy/adverse effects , Injection Site Reaction/etiology , Lung Neoplasms/immunology , Lung Neoplasms/mortality , Lymphocyte Activation , Male , Middle Aged , Neoplasm Staging , RNA, Messenger/administration & dosage , RNA, Messenger/genetics , RNA, Messenger/immunology , Survival AnalysisABSTRACT
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.
Subject(s)
Adjuvants, Immunologic , Cancer Vaccines/immunology , RNA, Long Noncoding/immunology , Uterine Cervical Neoplasms/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cancer Vaccines/pharmacology , Cell Line, Transformed , Cytokines/immunology , Cytokines/metabolism , Disease Models, Animal , Female , Flow Cytometry , Humans , Immunologic Memory/immunology , Mice, Inbred BALB C , Mice, Inbred C57BL , Papillomavirus E7 Proteins/immunology , Peptides/immunology , Peptides/pharmacology , Poly I-C/immunology , Poly I-C/pharmacology , RNA, Long Noncoding/genetics , Treatment Outcome , Uterine Cervical Neoplasms/drug therapyABSTRACT
BACKGROUND: Advanced non-small cell lung cancer (NSCLC) represents a significant unmet medical need. Despite advances with targeted therapies in a small subset of patients, fewer than 20% of patients survive for more than two years after diagnosis. Cancer vaccines are a promising therapeutic approach that offers the potential for durable responses through the engagement of the patient's own immune system. CV9202 is a self-adjuvanting mRNA vaccine that targets six antigens commonly expressed in NSCLC (NY-ESO-1, MAGEC1, MAGEC2, 5 T4, survivin, and MUC1). METHODS/DESIGN: The trial will assess the safety and tolerability of CV9202 vaccination combined with local radiation designed to enhance immune responses and will include patients with stage IV NSCLC and a response or stable disease after first-line chemotherapy or therapy with an EGFR tyrosine kinase inhibitor. Three histological and molecular subtypes of NSCLC will be investigated (squamous and non-squamous cell with/without EGFR mutations). All patients will receive two initial vaccinations with CV9202 prior to local radiotherapy (5 GY per day for four successive days) followed by further vaccinations until disease progression. The primary endpoint of the study is the number of patients experiencing Grade >3 treatment-related adverse events. Pharmacodynamic analyses include the assessment of immune responses to the antigens encoded by CV9202 and others not included in the panel (antigen spreading) and standard efficacy assessments. DISCUSSION: RNActive self-adjuvanted mRNA vaccines offer the potential for simultaneously inducing immune responses to a wide panel of antigens commonly expressed in tumors. This trial will assess the feasibility of this approach in combination with local radiotherapy in NSCLC patients. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01915524/EudraCT No.: 2012-004230-41.
Subject(s)
Cancer Vaccines/genetics , Carcinoma, Non-Small-Cell Lung/therapy , Lung Neoplasms/therapy , RNA, Messenger/administration & dosage , Cancer Vaccines/administration & dosage , Cancer Vaccines/adverse effects , Cancer Vaccines/therapeutic use , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/pathology , Chemotherapy, Adjuvant , Combined Modality Therapy , Humans , Lung Neoplasms/immunology , Lung Neoplasms/pathology , RNA, Messenger/adverse effects , RNA, Messenger/therapeutic use , Radiation Dosage , Radiotherapy , Treatment OutcomeABSTRACT
BACKGROUND: Preclinical studies demonstrate synergism between cancer immunotherapy and local radiation, enhancing anti-tumor effects and promoting immune responses. BI1361849 (CV9202) is an active cancer immunotherapeutic comprising protamine-formulated, sequence-optimized mRNA encoding six non-small cell lung cancer (NSCLC)-associated antigens (NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1), intended to induce targeted immune responses. METHODS: We describe a phase Ib clinical trial evaluating treatment with BI1361849 combined with local radiation in 26 stage IV NSCLC patients with partial response (PR)/stable disease (SD) after standard first-line therapy. Patients were stratified into three strata (1: non-squamous NSCLC, no epidermal growth factor receptor (EGFR) mutation, PR/SD after ≥4 cycles of platinum- and pemetrexed-based treatment [n = 16]; 2: squamous NSCLC, PR/SD after ≥4 cycles of platinum-based and non-platinum compound treatment [n = 8]; 3: non-squamous NSCLC, EGFR mutation, PR/SD after ≥3 and ≤ 6 months EGFR-tyrosine kinase inhibitor (TKI) treatment [n = 2]). Patients received intradermal BI1361849, local radiation (4 × 5 Gy), then BI1361849 until disease progression. Strata 1 and 3 also had maintenance pemetrexed or continued EGFR-TKI therapy, respectively. The primary endpoint was evaluation of safety; secondary objectives included assessment of clinical efficacy (every 6 weeks during treatment) and of immune response (on Days 1 [baseline], 19 and 61). RESULTS: Study treatment was well tolerated; injection site reactions and flu-like symptoms were the most common BI1361849-related adverse events. Three patients had grade 3 BI1361849-related adverse events (fatigue, pyrexia); there was one grade 3 radiation-related event (dysphagia). In comparison to baseline, immunomonitoring revealed increased BI1361849 antigen-specific immune responses in the majority of patients (84%), whereby antigen-specific antibody levels were increased in 80% and functional T cells in 40% of patients, and involvement of multiple antigen specificities was evident in 52% of patients. One patient had a partial response in combination with pemetrexed maintenance, and 46.2% achieved stable disease as best overall response. Best overall response was SD in 57.7% for target lesions. CONCLUSION: The results support further investigation of mRNA-based immunotherapy in NSCLC including combinations with immune checkpoint inhibitors. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01915524 .
Subject(s)
Adjuvants, Immunologic/therapeutic use , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/therapy , Immunotherapy , Lung Neoplasms/radiotherapy , Lung Neoplasms/therapy , Pemetrexed/therapeutic use , Protamines/therapeutic use , RNA, Messenger/therapeutic use , Adult , Aged , Aged, 80 and over , Antigens, Neoplasm/genetics , Carcinoma, Non-Small-Cell Lung/immunology , Combined Modality Therapy , Female , Humans , Lung Neoplasms/immunology , Male , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Middle Aged , Mucin-1/genetics , Neoplasm Proteins/genetics , Survivin/geneticsABSTRACT
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.
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
RNA Interference , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Animals , Cells, Cultured , Dendritic Cells , Electroporation , Gene Expression , Mice , TransfectionABSTRACT
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.
ABSTRACT
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.
Subject(s)
Cancer Vaccines/administration & dosage , Cancer Vaccines/immunology , RNA/administration & dosage , RNA/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/immunology , Humans , Toll-Like Receptor 7/immunology , Vaccination/methodsABSTRACT
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.
Subject(s)
Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Bone Neoplasms/immunology , Bone Neoplasms/therapy , Cancer Vaccines , Immunotherapy, Active , Lung Neoplasms/immunology , Lung Neoplasms/therapy , Melanoma/immunology , Melanoma/therapy , Myeloid Cells/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , T-Lymphocytes, Regulatory/metabolism , Adolescent , Adult , Aged , Antigens, Neoplasm/metabolism , Antigens, Neoplasm/therapeutic use , Bone Neoplasms/metabolism , Bone Neoplasms/secondary , CD11b Antigen , CD4 Antigens , Cell Proliferation , Feasibility Studies , Female , Forkhead Transcription Factors , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , HLA-DR Antigens , Hemocyanins/metabolism , Humans , Injections, Intradermal , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Male , Melanoma/metabolism , Melanoma/pathology , Melanoma-Specific Antigens , Middle Aged , Myeloid Cells/immunology , Myeloid Cells/pathology , Neoplasm Proteins/metabolism , Neoplasm Proteins/therapeutic use , Neoplasm Staging , Protamines/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Messenger/therapeutic use , Remission Induction , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathologyABSTRACT
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.
Subject(s)
Cancer Vaccines/therapeutic use , Melanoma/therapy , RNA, Messenger/therapeutic use , Adult , Aged , Antibody Formation/immunology , Antigens, Neoplasm/genetics , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cancer Vaccines/adverse effects , Cell Line, Tumor , Female , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use , Humans , Immunity, Cellular/immunology , Injections, Intradermal , Interferon-gamma/metabolism , Male , Melanoma/immunology , Melanoma/pathology , Middle Aged , Monitoring, Immunologic/methods , Neoplasm Metastasis , Neoplasm Staging , RNA, Messenger/administration & dosage , RNA, Messenger/chemical synthesis , Treatment OutcomeABSTRACT
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.
Subject(s)
Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/immunology , RNA, Messenger/immunology , RNA, Messenger/therapeutic use , Animals , Cell Line, Tumor , CpG Islands/immunology , DNA/immunology , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Mice , Protamines/immunologyABSTRACT
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.
Subject(s)
Leukocytes, Mononuclear/immunology , Membrane Glycoproteins/immunology , Protamines/immunology , RNA, Messenger/immunology , Receptors, Cell Surface/immunology , Adaptor Proteins, Signal Transducing , Animals , Antigens, Differentiation/immunology , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/immunology , Enzyme-Linked Immunosorbent Assay , Granulocytes/immunology , Humans , Mice , Myeloid Differentiation Factor 88 , Neutrophil Activation/immunology , Protamines/metabolism , RNA, Messenger/metabolism , Receptors, Immunologic/immunology , Toll-Like Receptor 7 , Toll-Like Receptor 8 , Toll-Like ReceptorsABSTRACT
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.