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PDF file - 1926K, Description of effects of Rapamycin on CD8 T cell phenotype.

PDF file - 1350K, Description of effects of Rapamycin on blood hematogram values and on frequencies of antigen specific T cells.

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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.

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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.

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Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B

BNT162b2 is a vaccine developed to prevent coronavirus disease 2019 (COVID-19). BNT162b2 is a lipid nanoparticle formulated nucleoside-modified messenger RNA (mRNA) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein locked in its prefusion conformation. A developmental and reproductive toxicity study was conducted in rats according to international regulatory guidelines. The full human BNT162b2 dose of 30 μg mRNA/dose (>300 times the human dose on a mg/kg basis) was administered intramuscularly to 44 female rats 21 and 14 days prior to mating and on gestation days 9 and 20. Half of the rats were subject to cesarean section and full fetal examination at the end of gestation, and the other half were allowed to deliver and were monitored to the end of lactation. A robust neutralizing antibody response was confirmed prior to mating and at the end of gestation and lactation. The presence of neutralizing antibodies was also confirmed in fetuses and offspring. Nonadverse effects, related to the local injection site reaction, were noted in dams as expected from other animal studies and consistent with observations in humans. There were no effects of BNT162b2 on female mating performance, fertility, or any ovarian or uterine parameters nor on embryo-fetal or postnatal survival, growth, physical development or neurofunctional development in the offspring through the end of lactation. Together with the safety profile in nonpregnant people, this ICH-compliant nonclinical safety data supports study of BNT162b2 in women of childbearing potential and pregnant and lactating women.

The progressive drive train electrification of vehicles also leads to a technological and technical development of battery cells and systems.

We investigate the functional renormalization group (FRG) flow of the two-particle vertex function of a model for X-ray absorption in metals. Concerning the appearance of logarithmic divergences, the model is prototypical for an important class of mostly zero- and one-dimensional systems which includes the Kondo model and the interacting one-dimensional Fermi gas. For our analysis we formulate the FRG in the framework of the real-time zero-temperature formalism, in which the model was studied before with a parquet-based approach. We establish that a reasonably crafted, purely-fermionic one-loop FRG approximation is fully equivalent on a detailed level to the leading-logarithmic parquet approximation. These two approximation schemes are thus found to just represent different perspectives on the same technical steps. This finding also reconfirms the traditional understanding of the capabilities of one-loop RG approximations for such models, which was recently put into question by an investigation of the X-ray-absorption model with multiloop FRG., 22 pages, 6 figures, version essentially as published

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.

Nowadays recycling processes for lithium-ion batteries focus on the recovery of nickel and cobalt. These processes are mainly pyro-metallurgical and as such energy and cost intensive. The project LithoRec, sponsored by the German Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB), aims at a realization of a new ecological recycling process tracing mechanical process-steps and considering recycling of lithium. This work presents the main challenges of the recycling of battery systems of electric and hybrid electric vehicles and the developed process chain, as well as the influence of a second crushing step on the yield and purity of the separated black mass containing the aged active materials.

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.

Lithium-ion batteries will play a crucial role in the development of mobile consumer devices, stationary energy storage systems, and electric mobility. The growth in these fields will bring about a surge in the lithium-ion battery market. This leads experts to agree that more effective recycling processes are needed in conjunction with the recycling of lithium. This calls for an entirely revolutionary recycling process which we here have attempted to develop. Our approach uses thermal decomposition of the polyvinylidene fluoride binder to lessen the cohesion of coated active material particles and weaken the adhesion between coating and foil. Then, an air-jet-separator is able to detach the coating powder from the current collector foils while stressing remaining particulate agglomerates. This separation process named ANVIIL (Adhesion Neutralization via Incineration and Impact Liberation) was tested on a laboratory scale with electrode rejects. We compared this to the widely used mechanical recycling process that utilizes a cutting mill to separate the current collector and coating. Intermediates and products were characterized using thermogravimetric analysis, tape adhesion tests, atomic absorption spectroscopy, particle size analysis, and gravimetric sieve analysis. We found that 97.1% w/w of the electrode coating can be regained with aluminum impurities of only 0.1% w/w, 30 times purer than the comparative process. This demonstrates a more effective recycling process than is currently available that also enables the recapture of lithium from the electrode coating.

Internal short circuit tests of Lithium-Ion Batteries (LIBs) are used to test battery safety behavior in a custom made battery cell stressing chamber. However, systematic investigations regarding the test setup and test procedure are rare. In our research commercially available pouch cells (5 Ah) are employed for the method development and validation of nail penetration tests including measurement of gaseous reaction products. The effects of the thermal insulation material, the nail material (conductive and non-conductive), the influence of the penetration depth and the nail velocity were examined. It was observed that low penetration velocities (1 mm s−1) in combination with a conductive nail and a nail motion control, which is based on monitoring the temporal evaluation of the cell voltage change, provide the most promising results in terms of reproducibility at low standard deviation. By applying this method, only the energy required for a Thermal Runaway (TR) is released, which makes it possible to determine a novel key value for the assessment of battery safety. Based on this, a proposal has been made for a nail penetration test method which would allow the results to be compared between different test facilities.

Due to their high voltage, high stored energy, and reactive components, lithium-ion batteries present a specific and significant hazard potential. This especially comes into play during recycling because nearly every safety precaution of a battery system and battery cell needs to be bypassed. Because the project partners of LithoRec II spared a thermal pre-treatment step to deactivate the batteries, the hazard potential and its handling played a major role. This chapter gives an overview of the hazards associated with lithium-ion batteries and describes their role in every process step.

Crushing is a substantial process step for the following separation, as it transfers the battery cells or modules to a storable and conveyable bulk material. Crushing also leads to the opening of the battery cells and release of valuable materials. Because of the deliberate destruction of the battery cells, this process has a high hazard potential due to the remaining attached energy of the battery cells and the flammable electrolyte components. The project partners evaluated requirements for the design of a crusher and drafted a concept for the realization of a safe and efficient crushing process.

The LithoRec projects were funded by the German Federal Ministry of the Environment, Nature Conservation Building and Nuclear Safety and VDI/VDE Innovation+Technik GmbH. The projects aimed to develop a new recycling process for lithium-ion batteries from electric and hybrid electric vehicles with a focus on energy efficiency and a high material recycling rate. The developed process route combines mechanical, mild thermal and hydrometallurgical treatment to regain nearly all materials of a battery system.

This chapter describes the areas and aggregates, as well as the operating cycle of the realized temporary demonstration plant in Braunschweig. This plant was run by employees of TU Braunschweig and Lion Engineering GmbH. As especially safe processing of entire battery systems had to be assured, the developed safety concept is presented. Overall, the demonstration plant processed 1.4 tons of the battery systems of electric vehicles, reaching a material recycling rate of up to 80%.

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'.

Having a closer look at the details, recycling of scraps from the production of lithium-ion batteries is different from recycling of spent batteries. On the one hand it is less dangerous on the other hand pristine electrodes retain the original non-aged quality and thus the separation process is more difficult. Two different separation mechanisms, one mechanical and one based on the solvent N-methyl-2-pyrrolidone, are examined in this work. The resulting separated coatings are directly re-applied on new electrodes and electrochemically characterized in full cells.

The electrolyte of a used lithium-ion battery from a hybrid electric vehicle (HEV) was investigated. The liquid electrolyte was collected through the pressure valve of these 5 Ah cells. It consists of (29.8 ± 0.2) wt.% dimethyl carbonate (DMC), (21.7 ± 0.1) wt.% ethyl methyl carbonate (EMC), (30.3 ± 0.3) wt.% ethylene carbonate (EC) and (2.2 ± 0.1) wt.% cyclohexyl benzene (CHB) which were identified with GC–MS and quantified with GC–FID. Li + (1.29 ± 0.04) mol L −1 and PF 6 − were determined with IC as the main ionic species in the solution. Furthermore, BF 4 − was clearly identified with IC–ESI–MS, IC–ICP–MS and 11 B NMR and quantified to a concentration of (120.8 ± 8.3) mg L −1 with ICP–OES. The presence of POF 3 (detected with GC–MS), F − , PO 2 F 2 − , HPO 3 F − and H 2 PO 4 − (determined with IC–ESI–MS) can be attributed to the reaction of the conducting salt LiPF 6 via PF 5 with traces of water. HPO 3 F − and H 2 PO 4 − could only be observed in cells which were opened in a laboratory hood under exposure of air humidity. This experiment was done to simulate escaping electrolyte from an HEV battery pack. Furthermore, several alkyl phosphates (identified with GC–MS and IC–ESI–MS) are present in the solution due to further reaction of the different fluorinated phosphates with organic carbonates.

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.

Cancer immunotherapy localized to the tumor microenvironment holds great potential to promote innate and adaptive immune responses against tumors, while avoiding toxicities related to systemic administration of immuno-modulatory therapeutics. Current strategies for tumor-targeted, gene-based delivery of immune therapies face limitations in the clinic due to suboptimal target expression, anti-vector immunity, potential for unwanted genomic rearrangements and other off target effects. We developed a highly potent synthetic mRNA-based platform for in vivo transfection and sustained intratumoral expression of immuno-modulatory molecules that is capable of inducing immunity to tumor specific antigens while avoiding anti-vector immunity and distal tissue expression often associated with viral gene therapy or oncolytic viruses. Using iterative rounds of in vivo screening in murine syngeneic tumor models, we identified a combination of four synthetic mRNAs encoding potent cytokines that mediate high frequency of complete tumor regressions across different tumor models. Mechanistically, anti-tumor activity of cytokine mRNAs relied on the action of T cells as well as NK cells and was accompanied by robust intratumoral induction of interferon gamma, systemic expansion of antigen-specific T cells and increased granzyme B positive CD8+ T cell infiltration. Immunological memory to both dominant and subdominant antigens was formed that protected long-term survivors from re-challenge with autologous tumors. Importantly, although cytokine mRNAs were administered intratumorally, resulting in local target expression, anti-tumor activity extended beyond the injected lesion to effectively control the growth of distant tumors in both a dual tumor model and in an experimental lung metastasis model. Finally, the combination of four cytokine mRNAs with immunomodulatory antibodies further enhanced the abscopal effect leading to improved overall survival and higher incidence of complete tumor regressions across several preclinical models. In summary, the robust and versatile synthetic mRNA platform reported herein was used to identify multi-modal localized cancer immunotherapy with broad anti-tumor activity against treated and untreated lesions in a range of preclinical cancer models. Citation Format: Timothy R. Wagenaar, Christian Hotz, Friederike Gieseke, Hui Cao, Jan Diekmann, Mustafa Diken, Christian Grunwitz, Andy Hebert, Karl Hsu, Marie Jordan, Katalin Kariko, Sebastian Kreiter, Andreas N. Kuhn, Mikhail Levit, Natalia Malkova, Serena Masciari, Jack Pollard, Hui Qu, Abderaouf Selmi, Julia Schlereth, Fangxian Sun, Bodo Tillmann, Tatiana Tolstykh, Lena Wicke, Sonja Witzel, Qunyan Yu, Yu-An Zhang, Gang Zheng, Gary Nabel, Joanne Lager, Ugur Sahin, Dmitri Wiederschain. Combinatorial treatment with intratumoral cytokine mRNAs results in high frequency of tumor rejection and development of anti-tumor immunity across a range of preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-130.

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.

The use of lithium-ion batteries (LIBs) has grown since the market entry of portable power tools and consumer electronic devices. Soon, the need for LIB will rise, when they are used in hybrid and full electric vehicles as well as in energy storage systems to enable the use of renewable energies. To prevent a future shortage of cobalt, nickel, and lithium and to enable a sustainable life cycle of these technologies, new recycling processes for LIBs are needed. These new processes have to regain not only cobalt, nickel, copper, and aluminum from spent battery cells but also a significant share of lithium. Therefore, this article approaches unit operations and their combination to set up for efficient LIB recycling processes, especially considering the task to recover high rates of valuable materials with regard to involved safety issues. Further discussed unit operations are Deactivation/discharging of the battery Disassembly of battery systems (specifically for EV-battery systems) Mechanical processes (including inert crushing, sorting, and sieving processes and a special case: thermomechanical separation) Hydrometallurgical processes Pyrometallurgical processes Specific dangers are associated with LIB recycling processes: electrical dangers, chemical dangers, burning reactions, and potential interactions of the single dangers. Furthermore, industrial process chains, already in use, as well as research approaches are summarized. The processes of the companies Retriev Technologies, Recupyl, Batrec, Inmetco, Xstrata, Umicore, Accurec, AEA Technology, OnTo Technology, and Lion Engineering are discussed and illustrated briefly. A closer look is given to some results of the research project LithoRec. Keywords: recycling; lithium-ion battery; recovery; lithium; cobalt; nickel; electric vehicles

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

The mutant IDH1 protein, which is expressed in a large fraction of human gliomas, is shown to be immunogenic; mutant-specific immune responses can be detected in patients with IDH1 mutated gliomas and generated in mice and are shown to treat established IDH1 mutant tumours in a syngeneic MHC humanized mouse model in a CD4 T-cell-dependent manner. Isocitrate dehydrogenase type 1 (IDH1) point mutations are associated with certain slow-growing gliomas and other tumours. This study in a humanized syngeneic tumour mouse model shows that IDH1(R132H), the mutant IDH1 protein most commonly expressed in gliomas, is immunogenic, capable of inducing a human MHC class II-restricted spontaneous and functionally relevant immune response. These findings suggest that some patients with gliomas with a high prevalence of the IDH1(R132H) mutation may benefit from a tumour vaccine based on the IDH1(R132H) antigen. Monoallelic point mutations of isocitrate dehydrogenase type 1 (IDH1) are an early and defining event in the development of a subgroup of gliomas1,2,3 and other types of tumour4,5,6. They almost uniformly occur in the critical arginine residue (Arg 132) in the catalytic pocket, resulting in a neomorphic enzymatic function, production of the oncometabolite 2-hydroxyglutarate (2-HG)7,8, genomic hypermethylation9,10,11, genetic instability and malignant transformation12. More than 70% of diffuse grade II and grade III gliomas carry the most frequent mutation, IDH1(R132H) (ref. 3). From an immunological perspective, IDH1(R132H) represents a potential target for immunotherapy as it is a tumour-specific potential neoantigen with high uniformity and penetrance expressed in all tumour cells13,14. Here we demonstrate that IDH1(R132H) contains an immunogenic epitope suitable for mutation-specific vaccination. Peptides encompassing the mutated region are presented on major histocompatibility complexes (MHC) class II and induce mutation-specific CD4+ T-helper-1 (TH1) responses. CD4+ TH1 cells and antibodies spontaneously occurring in patients with IDH1(R132H)-mutated gliomas specifically recognize IDH1(R132H). Peptide vaccination of mice devoid of mouse MHC and transgenic for human MHC class I and II with IDH1(R132H) p123-142 results in an effective MHC class II-restricted mutation-specific antitumour immune response and control of pre-established syngeneic IDH1(R132H)-expressing tumours in a CD4+ T-cell-dependent manner. As IDH1(R132H) is present in all tumour cells of these slow-growing gliomas15, a mutation-specific anti-IDH1(R132H) vaccine may represent a viable novel therapeutic strategy for IDH1(R132H)-mutated tumours.

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.

The majority of metastatic cancers remain incurable since the current methods of treatment often fail to target the heterogeneous nature of each individual patient's tumor. Personalized approaches targeting each individual patient's tumor may therefore bring significant improvements. The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically validate a pioneering RNA-based immunotherapy concept for the treatment of triple negative breast cancer (TNBC) by targeting shared tumor antigens and individual neo-antigens in TNBC patients. MERIT combines two personalized treatment concepts: (i) treatment with vaccines containing “off-the-shelf” mRNAs selected from a pre-synthesized mRNA vaccine warehouse (MERIT WAREHOUSE) that encode shared breast cancer tumor antigens expressed in the respective patient's tumor; (ii) treatment with mRNAs engineered on-demand that encode patient-specific mutated neo-antigens identified by next generation sequencing (NGS) and ranked according to the predicted immunogenicity (MERIT MUTANOME). The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation, which specifically targets APCs and consequently induces antigen-specific T cell responses. MERIT is a multi-center phase I trial (NCT02316457) conducted in four European countries to assess the feasibility, safety and biological efficacy of this personalized immunotherapy. TNBC patients (pT1cN0M0 - anyTanyNM0) after surgery and adjuvant chemotherapy will be allocated to one of two study arms. Patients in ARM1 will receive eight vaccination cycles with a personalized set of shared tumor antigens selected from the WAREHOUSE that correspond to the patient tumor's antigen-expression profile. Patients in ARM2 will be first treated with the personalized WAREHOUSE vaccine approach followed by six vaccination cycles of on-demand manufactured MUTANOME vaccine targeting the unique mutation signature of the individual patient. During the clinical trial, patients will receive the individualized combination of the RNAs in parallel to standard radiotherapy. The clinical trial is approved and the study start is planned for Q1 2016. The consortium has built a multi-disciplinary clinical workflow and trial design tailored to this unique therapeutic concept, which covers the whole individualized drug development cycle from target discovery, validation to GMP manufacturing and drug release for each individual patient. We will present the therapeutic concept and study protocol as well as the methodologies required for this highly innovative phase I trial. The personalized immunotherapy overcomes the current limitations of fixed, off-the-shelf therapeutics and thus might increase the clinical benefit for TNBC patients. This project is a collaborative effort of five partners from academia and industry funded by the European Commission's FP7 and led by BioNTech AG. Citation Format: Sandra Heesch, Valesca Bukur, Janina Buck, Jan Diekmann, Mustafa Diken, Kerstin Ewen, Heinrich Haas, Alexandra Kemmer-Brueck, Björn-Philipp Kloke, Sebastian Kreiter, Andreas N. Kuhn, Klaus Kuehlcke, Martin Loewer, Anna Paruzynski, Kathrin Schultheiß, Doreen Schwarck, Marcus Schmidt, Fabrice André, Jacques De Greve, Thomas Kuendig, Henrik Lindman, Steve Pascolo, Tobias Sjöblom, Kris Thielemans, Laurence Zitvogel, Özlem Türeci, Ugur Sahin. MERIT: introducing individualized cancer vaccines for the treatment of TNBC - a phase I trial. [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 CT020.

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.

The increasing demand of lithium-ion batteries requires the recovery of active materials from spent lithium-ion batteries and production rejects in order to prevent a future shortage of lithium and other valuable raw materials. In contrast to existing recycling processes, the main goal of the LithoRec process is to achieve a high recycling yield and a low ecological impact as well as to efficiently recover all battery materials at battery grade quality. The project spams the entire recycling process starting from the discharging and disassembly stages, and following through with mechanical, chemical and thermal processing (e.g. crushing, grinding, classification, sorting, and extraction) used to separate the battery into recyclable fractions. In order to match the high purity standards of hydro-metallurgical processes for the production of battery active materials, the battery active materials have to be separated from the current collector foil very purely. For that purpose, a new patented separation process has been developed which shows a very high separation rate at high purity. The entire process was set up in pilot scale and operated with different spent battery systems. Results of this pilot trials will be discussed in the paper. Subsequently, new battery active materials have been produced and characterized in battery test cells.

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.

Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single-targeted drugs difficult. The multiplicity of gene mutations derived from clonal heterogeneity therefore represents an ideal setting for multiepitope tumor vaccination. Here, we used next generation sequencing exome resequencing to identify 962 nonsynonymous somatic point mutations in B16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration, and apoptosis. Aim1 and Trrap mutations known to be altered in human melanoma were included among those found. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60% in this group eliciting immune responses directed preferentially against the mutated sequence as compared with the wild-type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Together, our findings provide a comprehensive picture of the mutanome of B16F10 melanoma which is used widely in immunotherapy studies. In addition, they offer insight into the extent of the immunogenicity of nonsynonymous base substitution mutations. Lastly, they argue that the use of deep sequencing to systematically analyze immunogenicity mutations may pave the way for individualized immunotherapy of cancer patients. Cancer Res; 72(5); 1081–91. ©2012 AACR.

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.