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The emergence of SARS-CoV-2 at the end of 2019 required the swift development of a vaccine to address the pandemic. Nonclinical GLP-compliant studies in Wistar Han rats were initiated to assess the local tolerance, systemic toxicity, and immune response to four mRNA vaccine candidates encoding immunogens derived from the spike (S) glycoprotein of SARS-CoV-2, encapsulated in lipid nanoparticles (LNPs). Vaccine candidates were administered intramuscularly once weekly for three doses at 30 and/or 100 µg followed by a 3-week recovery period. Clinical pathology findings included higher white blood cell counts and acute phase reactant concentrations, lower platelet and reticulocyte counts, and lower RBC parameters. Microscopically, there was increased cellularity (lymphocytes) in the lymph nodes and spleen, increased hematopoiesis in the bone marrow and spleen, acute inflammation and edema at the injection site, and minimal hepatocellular vacuolation. These findings were generally attributed to the anticipated immune and inflammatory responses to the vaccines, except for hepatocyte vacuolation, which was interpreted to reflect hepatocyte LNP lipid uptake, was similar between candidates and resolved or partially recovered at the end of the recovery phase. These studies demonstrated safety and tolerability in rats, supporting SARS-CoV-2 mRNA-LNP vaccine clinical development.

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient's tumour possesses a unique set of mutations ('the mutanome') that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient's individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4(+) T cells. Vaccination with such CD4(+) immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4(+) T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient's tumour with vaccines produced 'just in time'.

Cancer is a disease caused by DNA mutations. Cancer therapies targeting defined functional mutations have shown clinical benefit. However, as 95% of the mutations in a tumour are unique to that single patient and only a small number of mutations are shared between patients, the addressed medical need is modest. A rapidly determined patient-specific tumour mutation pattern combined with a flexible mutation-targeting drug platform could generate a mutation-targeting individualised therapy, which would benefit each single patient. Next-generation sequencing enables the rapid identification of somatic mutations in individual tumours (the mutanome). Immunoinformatics enables predictions of mutation immunogenicity. Mutation-targeting RNA-based vaccines can be rapidly and affordably synthesised as custom GMP drug products. Integration of these cutting-edge technologies into a clinically applicable process holds the promise of a disruptive innovation benefiting cancer patients. Here, we describe our translation of the individualised RNA-based cancer vaccine concept into clinic trials.

Vaccination with in vitro transcribed RNA encoding tumor antigens is an emerging approach in cancer immunotherapy. Attempting to further improve RNA vaccine efficacy, we have explored combining RNA with immunomodulators such as rapamycin. Rapamycin, the inhibitor of mTOR, was used originally for immunosuppression. Recent reports in mouse systems, however, suggest that mTOR inhibition may enhance the formation and differentiation of the memory CD8+ T-cell pool. Because memory T-cell formation is critical to the outcome of vaccination aproaches, we studied the impact of rapamycin on the in vivo primed RNA vaccine-induced immune response using the chicken ovalbumin-expressing B16 melanoma model in C57BL/6 mice. Our data show that treatment with rapamycin at the effector-to-memory transition phase skews the vaccine-induced immune response toward the formation of a quantitatively and qualitatively superior memory pool and results in a better recall response. Tumor-infiltrating immune cells from these mice display a favorable ratio of effector versus suppressor cell populations. Survival of mice treated with the combined regimen of RNA vaccination with rapamycin is significantly longer (91.5 days) than that in the control groups receiving only one of these compounds (32 and 46 days, respectively). Our findings indicate that rapamycin enhances therapeutic efficacy of antigen-specific CD8+ T cells induced by RNA vaccination, and we propose further clinical exploration of rapamycin as a component of immunotherapeutic regimens. Cancer Immunol Res; 1(6); 386–92. ©2013 AACR.

The Ag-specific cellular recall response to herpes virus infections is characterized by a swift recruitment of virus-specific memory T cells. Rapid activation is achieved through formation of the immunological synapse and supramolecular clustering of signal molecules at the site of contact. During the formation of the immunological synapse, epitope-loaded MHC molecules are transferred via trogocytosis from APCs to T cells, enabling the latter to function as Ag-presenting T cells (T-APCs). The contribution of viral epitope expressing T-APCs in the regulation of the herpes virus-specific CD8+ T cell memory response remains unclear. Comparison of CD4+ T-APCs with professional APCs such as Ag-presenting CD40L-activated B cells (CD40B-APCs) demonstrated reduced levels of costimulatory ligands. Despite the observed differences, CD4+ T-APCs are as potent as CD40B-APCs in stimulating herpes virus-specific CD8+ T cells resulting in a greater than 35-fold expansion of CD8+ T cells specific for dominant and subdominant viral epitopes. Virus-specific CD8+ T cells generated by CD4+ T-APCs or CD40B-APCs showed both comparable effector function such as specific lysis of targets and cytokine production and also did not differ in their phenotype after expansion. These results indicate that viral epitope presentation by Ag-specific CD4+ T cells may contribute to the rapid recruitment of virus-specific memory CD8+ T cells during a viral recall response.

Mechanisms of antiviral host defense are important for survival and evolutionarily optimized for high sensitivity and potency. Intending to harvest the multitude of highly specialized and intertwined pathogen immune defense programs for cancer immunotherapy, we simulated a systemic pathogen intrusion into the blood stream by intravenous injection of lipid-formulated, tumor antigen-encoding mRNA nanoparticles. These RNA-lipoplexes (RNA-LPX) were directed to various lymphoid tissues, including the spleen, lymph nodes and bone marrow, which provide the ideal microenvironment for efficient priming and amplification of T cell responses. Solely the RNA-to-lipid ratio was discovered to determine the biodistribution of RNA-LPX, irrespective of the types of lipids used, and a slightly negative particle net charge was able to specifically transfect lymphoid-resident antigen presenting cells (APCs). Following uptake by CD11c+ DCs, pDCs and macrophages in the marginal zone of the spleen and in other lymphoid organs, predominantly by macropinocytosis, RNA recognition via TLR7 triggered two transient waves of type I IFN production by pDCs (early response) and macrophages (delayed response), which established an inflammatory, lymphocyte-activating milieu reminiscent of that initiated during the early systemic phase of viral infection. These IFNα receptor (IFNAR)-dependent immune mechanisms were required for DCs to mature, migrate into the T cell zones and express RNA-encoded tumor antigens. Presentation on MHC class I and II in the context of upregulated CD40, CD69 and CD86 elicited strong effector and memory CD8 and CD4 T cell immunity against viral, mutant neo-antigens or self-antigens, which was able to reject progressive tumors in therapeutic mouse models of melanoma, colon carcinoma and human papilloma virus (HPV)-associated cancer. In an ongoing phase I dose escalation study, the first cohort of three patients with advanced melanoma received RNA-LPX encoding four shared tumor antigens at doses lower than those used in the mouse studies. All patients showed a dose-dependent IFNα- and IP-10-dominated cytokine response, developed de novo CD4 and CD8 T cell responses or enhanced pre-existing immunity against the encoded self-antigens NY-ESO-I, Tyrosinase and MAGE-A3, and have stable disease to date. These results support the preclinically identified mode of action and strong potency of this approach in the clinical setting. Our study presents a novel class of systemically administered nanoparticulate RNA vaccines acting by body-wide delivery of encoded antigens to APCs and simultaneous initiation of a strong type I IFN-driven immunostimulatory program. Precise DC targeting in lymphoid compartments is accomplished using well-known lipid carriers and only by manipulating the net charge of the nanoparticles. RNA-LPX vaccines appear to mimic infectious non-self and thus mobilize both adaptive and innate immune mechanisms, connecting effective cancer immunotherapy with host pathogen-defense mechanisms. The simple but highly versatile design allows vaccine preparation with any type of RNA-encoded antigen and may thus be regarded as a universally applicable, first-in-class vaccine platform for cancer immunotherapy. Citation Format: Lena M. Kranz, Mustafa Diken, Heinrich Haas, Sebastian Kreiter, Carmen Loquai, Kerstin C. Reuter, Martin Meng, Daniel Fritz, Fulvia Vascotto, Hossam Hefesha, Christian Grunwitz, Mathias Vormehr, Yves Hüsemann, Abderraouf Selmi, Andreas N. Kuhn, Janina Buck, Evelyna Derhovanessian, Richard Rae, Sebastian Attig, Jan Diekmann, Robert A. Jabulowsky, Sandra Heesch, Jessica Hassel, Peter Langguth, Stephan Grabbe, Christoph Huber, Özlem Türeci, Ugur Sahin. Systemic RNA vaccines: Connecting effective cancer immunotherapy with antiviral defense mechanisms [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A004.

One of the hallmarks of cancer is the inherent instability of the genome leading to multiple genomic alterations and epigenetic changes that ultimately drive carcinogenesis. These processes lead to a unique molecular profile of every given tumor and to substantial intratumoral heterogeneity of cancer tissues. Recently, a series of independent reports revealed that pre-formed neoantigen specific T-cell responses are of crucial relevance for the clinical efficacy of immune checkpoint inhibitors. However, spontaneous immune recognition of neoantigens seems to be a rare event with only less than 1% of mutations inducing a T-cell response in the tumor-bearing patient. Accordingly, only patients with a high burden of mutations profit from currently approved therapies. To overcome this restriction, the IVAC® MUTANOME-project harnesses the individual patient's mutation profile by manufacturing highly potent neoantigen-coding RNA vaccines. To this end, the individual mutation repertoire is identified by next-generation-sequencing, potentially immunogenic mutations are selected and incorporated into a poly-epitopic RNA vaccine that is tailored to activate and expand the individual patient's neoantigen-specific CD4+ and CD8+ T cells. A phase I study to test this novel concept of an active individualized cancer vaccine for the treatment of malignant melanoma was initiated in 2013 (NCT02035956). Notably, BioNTech RNA Pharmaceutical's IVAC® MUTANOME trial is the first-in-human trial that introduces a fully personalized RNA vaccine for the treatment of malignant melanoma. The objective of this clinical trial is to study the feasibility, safety, tolerability, immunogenicity and the potential clinical activity of the IVAC® MUTANOME approach. The recruitment of a patient into the trial triggers the multi-step IVAC® MUTANOME process covering (i) the receipt and processing of tumor and blood sample specimens, (ii) the identification, prioritization and confirmation of mutations, (iii) testing of pre-existing immunity against identified tumor mutations, (iv) the selection of mutant neoantigen sequences as vaccine targets, (v) design, production of a DNA lead structure, (vi) GMP manufacturing and release of the patient-specific mRNA, (vii) shipment to the clinical trial site and (viii) the administration of the IMP to patients. Detailed information on the trial, the recruitment and treatment status as well as data on the assessment of vaccine induced immune responses will be presented. Citation Format: Matthias Miller, Carmen Loquai, Björn-Philipp Kloke, Sebastian Attig, Nicole Bidmon, Stefanie Bolte, Valesca Bukur, Evelyna Derhovanessian, Jan Diekmann, Angela Filbry, Sandra Heesch, Christoph Hoeller, Klaus Kuehlcke, David Langer, Martin Loewer, Felicitas Mueller, Inga Ortseifer, Burkhard Otte, Anna Paruzynski, Richard Rae, Barbara Schroers, Christine Seck, Kristina Spiess, Arbel D. Tadmor, Isabel Vogler, Mathias Vormehr, Alexandra Kemmer-Brueck, Andreas N. Kuhn, Ulrich Luxemburger, Sebastian Kreiter, Jochen Utikal, Stephan Grabbe, Oezlem Tuereci, Ugur Sahin. IVAC® MUTANOME - A first-in-human phase I clinical trial targeting individual mutant neoantigens for the treatment of melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT022.

Immunotherapeutic approaches have evolved as promising and valid alternatives to available conventional cancer treatments. Amongst others, vaccination with tumor antigen-encoding RNAs by local administration is currently successfully employed in various clinical trials. To allow for a more efficient targeting of antigen-presenting cells (APCs) and to overcome potential technical challenges associated with local administration, we have developed a novel RNA immunotherapeutic for systemic application based on a fixed set of four liposome complexed RNA drug products (RNA(LIP)), each encoding one shared melanoma-associated antigen. The novel RNA(LIP) formulation was engineered (i) to protect RNA from degradation by plasma RNases and (ii) to enable directed in vivo targeting of APCs in lymphoid compartments, thus (iii) allowing for intravenous administration of multiple RNA products advancing from local to systemic targeting of APCs. Here, RNA(LIP) products trigger a Toll-like receptor (TLR)-mediated Interferon-α (IFN-α) release from plasmacytoid dendritic cells (DCs) and macrophages stimulating DC maturation and hence inducing innate immune mechanisms as well as potent vaccine antigen-specific immune responses. Notably, BioNTech RNA Pharmaceuticals′ RNA(LIP) formulation is a universally applicable potent novel vaccine class for intravenous APC targeting and the induction of potent synchronized adaptive and type-I interferon-mediated innate immune responses for cancer immunotherapy. Similar to other liposomal drugs, the ready-to-use RNA(LIP) products are prepared individually in a straight-forward manner directly prior to use from three components, namely solutions containing RNA drug product, NaCl diluent, and liposome excipient, that are provided as a kit. A multi-center phase I/II trial to clinically validate this pioneering RNA(LIP) formulation for the treatment of malignant melanoma was initiated in 2015 (NCT02410733). The objective of the clinical trial is to study the feasibility, safety, tolerability, immunogenicity and evaluate potential clinical activity of the RNA(LIP) immunotherapy concept. Detailed information on the ongoing trial, the recruitment and treatment status as well as data on the assessment of vaccine-induced immune responses will be presented. Citation Format: Robert A. Jabulowsky, Carmen Loquai, Mustafa Diken, Lena M. Kranz, Heinrich Haas, Sebastian Attig, Nicole Bidmon, Janina Buck, Evelyna Derhovanessian, Jan Diekmann, Daniel Fritz, Veronika Jahndel, Alexandra Kemmer-Brueck, Klaus Kuehlcke, Andreas N. Kuhn, Peter Langguth, Ulrich Luxemburger, Martin Meng, Felicitas Mueller, Richard Rae, Fatih Sari, Doreen Schwarck-Kokarakis, Christine Seck, Kristina Spieß, Meike Witt, Jessica C. Hassel, Jochen Utikal, Roland Kaufmann, Sebastian Kreiter, Christoph Huber, Oezlem Tuereci, Ugur Sahin. A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent cancer immunotherapy in patients with malignant melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT032.

Mutations are regarded as ideal targets for cancer immunotherapy. As neoepitopes with strict lack of expression in any healthy tissue, they are expected to be safe and could bypass the central tolerance mechanisms. Recent advances in nucleic acid sequencing technologies have revolutionized the field of genomics, allowing the readily targeting of mutated neoantigens for personalized cancer vaccination. We demonstrated in three independent murine tumor models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that unexpectedly the immunogenic mutanome is pre-dominantly recognized by CD4+ T cells. RNA vaccination with such MHC class II restricted immunogenic mutations leads to infiltration of CD4+ and CD8+ T cells into the tumor, reduces intratumoral regulatory T cells and ultimately confers strong anti-tumor activity. Encouraged by these findings we set up a process comprising mutation detection by exome sequencing, selection of vaccine targets by solely bioinformatical prioritization of mutated epitopes predicted to be abundantly expressed and presented on MHC class II molecules. Synthetic mRNA vaccines encoding multiple of these prioritized mutated epitopes induce potent tumor control and complete rejection of established aggressively growing tumors in mice. Moreover, we demonstrate that CD4+ T cell neoepitope vaccination primes CTL responses against an independent immunodominant antigen in tumor bearing mice indicating orchestration of antigen spread. Our findings reveal that cancer mutation based MHC class II restricted epitopes are attractive vaccination targets and provide the preclinical proof of concept for an integrated process from tumor sample to a cancer vaccine customized to the unique repertoire of each patient`s tumor. Citation Format: Mathias Vormehr, Sebastian Kreiter, Niels van de Roemer, Mustafa Diken, Martin Löwer, Fulvia Vascotto, Jan Diekmann, Sebastian Boegel, Barbara Schroers, Arbel D. Tadmor, Özlem Türeci, Ugur Sahin. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A110.

Immunotherapeutic approaches have evolved as promising and valid alternatives to available conventional cancer treatments. Amongst others, vaccination with tumor antigen-encoding RNAs by local administration is currently successfully employed in various clinical trials. To allow for a more efficient targeting of antigen-presenting cells (APCs) we have developed a novel RNA immunotherapeutic for systemic application based on a fixed set of four liposome complexed RNA drug products (RNA(LIP)) each encoding one shared melanoma-associated antigen. Similar to other liposomal drugs, the four injectable RNA(LIP) products constituting the investigational medicinal product will be prepared individually in a straight-forward manner directly prior to use from three components, namely solutions containing RNA drug product, NaCl diluent, and liposome excipient, that are provided as a kit. The novel lipoplex formulation was engineered (i) to protect RNA from degradation by plasma RNases and (ii) to enable directed in vivo targeting of APCs in lymphoid compartments, thus (iii) allowing for intravenous administration of multiple RNA products advancing from local to systemic targeting of APCs. The improved selective delivery of the RNA(LIP) products into APCs has further been shown to lead to an enhanced induction of vaccine-induced T-cell responses. Extensive pharmacological characterization of the RNA(LIP) platform revealed that upon cellular uptake the encoded antigens will be translated into proteins that will be rapidly processed into peptide fragments, which after presentation by MHC class I and II molecules on the surface of APCs induce tumor antigen-specific CD8+ and CD4+ T-cell responses that spread systemically. These vaccine-induced T cells have been shown to specifically recognize and kill antigen-positive tumor cells eliciting potent anti-tumoral activity in vivo. The potent vaccination effects are additionally enhanced by further immunomodulatory effects based on the transient release of pro-inflammatory cytokines such as IFN-α, IP-10, and IL-6 due to binding of the administered RNA drug products to Toll-like receptors (TLRs). The clinical translation of this pioneering therapeutic concept is currently being realized in a multi-center, first-in-human phase I trial in patients with malignant melanoma. Main objectives of the clinical trial are to study safety, tolerability, and immunogenicity of this innovative immunotherapy approach. The novel lipoplex formulation, RNA(LIP) mechanism of action, study design and clinical workflow, as well as recruitment and treatment status of the ongoing clinical trial will be presented. Citation Format: Robert A. Jabulowsky, Carmen Loquai, Mustafa Diken, Lena M. Kranz, Heinrich Haas, Sebastian Attig, Cedrik M. Britten, Janina Buck, Evelyna Derhovanessian, Jan Diekmann, Isaac Esparza, Daniel Fritz, Yves Huesemann, Veronika Jahndel, Klaus Kuehlcke, Andreas N. Kuhn, Peter Langguth, Ulrich Luxemburger, Martin Meng, Felicitas Mueller, Kerstin C. Reuter, Doreen Schwarck, Kristina Spiess, Meike Witt, Jessica C. Hassel, Jochen Utikal, Roland Kaufmann, Marc Schrott, Sebastian Kreiter, Oezlem Tuereci, Christoph Huber, Ugur Sahin. A novel nanoparticular formulated tetravalent RNA cancer vaccine for treatment of patients with malignant melanoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B041.

Intranodal immunization with antigen-encoding naked RNA may offer a simple and safe approach to induce antitumor immunity. RNA taken up by nodal dendritic cells (DC) coactivates toll-like receptor (TLR) signaling that will prime and expand antigen-specific T cells. In this study, we show that RNA vaccination can be optimized by coadministration of the DC-activating Fms-like tyrosine kinase 3 (FLT3) ligand as an effective adjuvant. Systemic administration of FLT3 ligand prior to immunization enhanced priming and expansion of antigen-specific CD8+ T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodal RNA. Unexpectedly, plasmacytoid DCs (pDC) were found to be essential for the adjuvant effect of FLT3 ligand and they were systemically expanded together with conventional DCs after treatment. In response to FLT3 ligand, pDCs maintained an immature phenotype, internalized RNA, and presented the RNA-encoded antigen for efficient induction of antigen-specific CD8+ T-cell responses. Coadministration of FLT3 ligand with RNA vaccination achieved remarkable cure rates and survival of mice with advanced melanoma. Our findings show how to improve the simple and safe strategy offered by RNA vaccines for cancer immunotherapy. Cancer Res; 71(19); 6132–42. ©2011 AACR.

The EBV Ag latent membrane protein 1 (LMP1) has been described as a potential target for T cell immunotherapy in EBV-related malignancies. However, only a few CD8+ T cell epitopes are known, and the benefit of LMP1-specific T cell immunotherapy has not yet been proven. In this work, we studied the processing of the two LMP1 HLA-A02-restricted epitopes, YLLEMLRWL and YLQQNWWTL. We found that target cells endogenously expressing the native LMP1 are not recognized by CTLs specific for these epitopes because the N-terminal part of LMP1 limits the efficiency of epitope generation. We further observed that the proteasome is not required for the generation of both epitopes and that the YLLEMLRWL epitope seems to be destroyed by the proteasome, because blocking of proteasomal activities enhanced specific CTL activation. Activation of LMP1-specific CTLs could be significantly reduced after inhibition of the tripeptidyl peptidase II, suggesting a role for this peptidase in the processing of both epitopes. Taken together, our results demonstrate that the MHC class I-restricted LMP1 epitopes studied in this work are two of very few epitopes known to date to be processed proteasome independently by tripeptidyl peptidase II.