Lena M. Kranz

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.

mRNA Cancer Vaccines—Messages that Prevail

During the last decade, mRNA became increasingly recognized as a versatile tool for the development of new innovative therapeutics. Especially for vaccine development, mRNA is of outstanding interest and numerous clinical trials have been initiated. Strikingly, all of these studies have proven that large-scale GMP production of mRNA is feasible and concordantly report a favorable safety profile of mRNA vaccines. Induction of T-cell immunity is a multi-faceted process comprising antigen acquisition, antigen processing and presentation, as well as immune stimulation. The effectiveness of mRNA vaccines is critically dependent on making the antigen(s) of interest available to professional antigen-presenting cells, especially DCs. Efficient delivery of mRNA into DCs in vivo remains a major challenge in the mRNA vaccine field. This review summarizes the principles of mRNA vaccines and highlights the importance of in vivo mRNA delivery and recent advances in harnessing their therapeutic potential.

Abstract CT034: A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent melanoma immunotherapy

Therapeutic vaccination with tumor antigen-encoding RNAs by local administration is currently being successfully employed in various clinical trials. Advancing from local to more efficient systemic targeting of antigen-presenting cells (APCs), we have developed pioneering RNA-lipoplex (RNA(LIP)) immunotherapeutics for intravenous application based on the employment of well-known lipid carriers without the need for functionalization of particles with molecular ligands. The novel RNA(LIP) formulation has been engineered to preserve RNA integrity after intravenous injection and physicochemically optimized for efficient uptake and expression of the encoded antigen by APCs in various lymphoid compartments, resulting in the synchronized induction of both potent adaptive as well as type-I-IFN-mediated innate immune responses. The first-in-human phase I/II dose escalation Lipo-MERIT trial (NCT02410733) assesses the safety, tolerability, and biological efficacy of the innovative RNA(LIP) immunotherapy in four study centers in Germany. This is the first example of a clinically applicable and systemic RNA-based cancer vaccine. Following selective antigen stratification on routinely collected tumor samples, eligible patients with malignant melanoma are treated with increasing doses of the tetravalent Lipo-MERIT vaccine - a fixed set of four RNA(LIP) products, each encoding one shared melanoma-associated antigen, i.e. NY-ESO-1, tyrosinase, MAGE-A3, and TPTE, that are administered successively within one treatment cycle. Accompanying correlative biomarker studies and concerted immunological assessments evaluate the pharmacodynamic activity and immunogenicity upon multiple vaccination cycles with the Lipo-MERIT vaccine. As of January 2017, 15 patients have been treated within five dose escalation cohorts thoroughly guided by an independent data safety and monitoring board. Multiple dosing with the Lipo-MERIT vaccine was generally well-tolerated and no dose-limiting toxicities (DLTs) were observed so far. Further patient enrollment is continuing. Detailed information on the ongoing trial, the recruitment and treatment status as well as preliminary data on the assessment of vaccine-induced immune responses from the first patients treated will be presented. Citation Format: Robert A. Jabulowsky, Carmen Loquai, Jochen Utikal, Jessica Hassel, Roland Kaufmann, Evelyna Derhovanessian, Mustafa Diken, Lena M. Kranz, Heinrich Haas, Sebastian Attig, Christine Anft, Janina Buck, Jan Diekmann, Daniel Fritz, Kerstin Hartmann, Alexandra Kemmer-Brueck, Klaus Kuehlcke, Andreas N. Kuhn, Peter Langguth, Ulrich Luxemburger, Martin Meng, Richard Rae, Fatih Sari, Doreen Schwarck-Kokarakis, Malte Stein, Stephan Grabbe, Sebastian Kreiter, Oezlem Tuereci, Christoph Huber, Ugur Sahin. A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent melanoma immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT034. doi:10.1158/1538-7445.AM2017-CT034

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.”

Abstract B041: A novel nanoparticular formulated tetravalent RNA cancer vaccine for treatment of patients with malignant melanoma

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.

Abstract CT032: A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent cancer immunotherapy in patients with malignant melanoma

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 Spies, 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.

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.

CIMT 2017: Anniversary symposium - Report on the 15th CIMT Annual Meeting of the Association for Cancer Immunotherapy

The 15th Annual Meeting of the Association for Cancer Immunotherapy (CIMT) took place May 10–11, 2017, Mainz, Germany during which scientists and CIMT members from all over the world not only celebrated CIMT`s 15th Anniversary but also had the chance to present and discuss the past and current status, and the future of cancer immunotherapy. This annual symposium report highlights and summarizes the sessions held in various fields of this promising cancer therapy.

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 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.