Fulvia Vascotto

Mutant MHC class II epitopes drive therapeutic immune responses to cancer

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’.

Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy

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.

mTOR Inhibition Improves Antitumor Effects of Vaccination with Antigen-Encoding RNA

Diken, Kreiter, and colleagues report that treatment of mice in the B16 melanoma model with rapamycin at the effector-to-memory transition phase skews the vaccine-induced immune response toward the memory pool, with alterations of the tumor microenvironment, and prolongs survival. 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.

Uptake of synthetic naked RNA by skin-resident dendritic cells via macropinocytosis allows antigen expression and induction of T-cell responses in mice

Intradermal administration of antigen-encoding RNA has entered clinical testing for cancer vaccination. However, insight into the underlying mechanism of RNA uptake, translation and antigen presentation is still limited. Utilizing pharmacologically optimized naked RNA, the dose–response kinetics revealed a rise in reporter signal with increasing RNA amounts and a prolonged RNA translation of reporter protein up to 30xa0days after intradermal injection. Dendritic cells (DCs) in the dermis were shown to engulf RNA, and the signal arising from the reporter RNA was significantly diminished after DC depletion. Macropinocytosis was relevant for intradermal RNA uptake and translation in vitro and in vivo. By combining intradermal RNA vaccination and inhibition of macropinocytosis, we show that effective priming of antigen-specific CD8+ T-cells also relies on this uptake mechanism. This report demonstrates that direct antigen translation by dermal DCs after intradermal naked RNA vaccination is relevant for efficient priming of antigen-specific T-cells.

CIMT 2015: The right patient for the right therapy - Report on the 13th annual meeting of the Association for Cancer Immunotherapy.

The 13th Annual Meeting of the Association for Cancer Immunotherapy (CIMT) brought together more than 800 scientists in Mainz, Germany, from May 11–13, 2015, to present and discuss current research on various fields of cancer immunotherapy. Special focus was set on personalized approaches, and independent of the specific therapeutic strategy, the exploitation of mutated neoantigens predominated all sessions - in line with the motto of this years meeting, “The right patient for the right therapy.”

CIMT 2013: advancing targeted therapies--report on the 11th Annual Meeting of the Association for Cancer Immunotherapy, May 14-16 2013, Mainz, Germany.

The 11th Annual Meeting of Association for Cancer Immunotherapy (CIMT) welcomed more than 700 scientists around the world to Mainz, Germany and continued to be the largest immunotherapy meeting in Europe. Renowned speakers from various fields of cancer immunotherapy gave lectures under CIMT2013’s tag: “Advancing targeted therapies” the highlights of which are summarized in this meeting report.

Monitoring Translation Activity of mRNA-Loaded Nanoparticles in Mice

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.

CIMT 2016: Mechanisms of efficacy in cancer immunotherapy — Report on the 14th Annual Meeting of the Association for Cancer Immunotherapy May 10–12 2016, Mainz, Germany

Lena M. Kranz, Matthias Birtel, Lina Hilscher, Christian Grunwitz, Jutta Petschenka, Fulvia Vascotto, Mathias Vormehr, Ralf-Holger Voss, Sebastian Kreiter, and Mustafa Diken TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz gGmbH, Mainz, Germany; Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University, Mainz, Germany; BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany

Abstract A110: Mutant MHC class II epitopes drive therapeutic immune responses to cancer

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 Lower, Fulvia Vascotto, Jan Diekmann, Sebastian Boegel, Barbara Schroers, Arbel D. Tadmor, Ozlem Tureci, 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.

Abstract A004: Systemic RNA vaccines: Connecting effective cancer immunotherapy with antiviral defense mechanisms

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 Husemann, 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, Ozlem Tureci, 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.