2021

Costa B, Fletcher MNC, Boskovic P, Ivanova EL, Eisemann T, Lohr S, Bunse L, Löwer M, Burchard S, Korshunov A, Coltella N, Cusimano M, Naldini L, Liu HK, Platten M, Radlwimmer B, Angel P, Peterziel H. (2021) A Set of Cell Lines Derived from a Genetic Murine Glioblastoma Model Recapitulates Molecular and Morphological Characteristics of Human Tumors. Cancers (Basel).13(2):230.

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Glioblastomas (GBM) are the most aggressive tumors affecting the central nervous system in adults, causing death within, on average, 15 months after diagnosis. Immunocompetent in-vivo models that closely mirror human GBM are urgently needed for deciphering glioma biology and for the development of effective treatment options. The murine GBM cell lines currently available for engraftment in immunocompetent mice are not only exiguous but also inadequate in representing prominent characteristics of human GBM such as infiltrative behavior, necrotic areas, and pronounced tumor heterogeneity. Therefore, we generated a set of glioblastoma cell lines by repeated in vivo passaging of cells isolated from a neural stem cell-specific Pten/p53 double-knockout genetic mouse brain tumor model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts, they formed high-grade gliomas that faithfully recapitulated the histopathological features, invasiveness and immune cell infiltration characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioblastoma pathomechanism and to test novel treatments in an intact immune microenvironment.

Aravamudhan S, Türk C, Bock T, Keufgens L, Nolte H, Lang F, Krishnan RK, König T, Hammerschmidt P, Schindler N, Brodesser S, Rozsivalova DH, Rugarli E, Trifunovic A, Brüning J, Langer T, Braun T, Krüger M. (2021) Phosphoproteomics of the developing heart identifies PERM1 – An outer mitochondrial membrane protein. J Mol Cell Cardiol. 154:41-59.

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Heart development relies on PTMs that control cardiomyocyte proliferation, differentiation and cardiac morphogenesis. We generated a map of phosphorylation sites during the early stages of cardiac postnatal development in mice; we quantified over 10,000 phosphorylation sites and 5000 proteins that were assigned to different pathways. Analysis of mitochondrial proteins led to the identification of PGC-1- and ERR-induced regulator in muscle 1 (PERM1), which is specifically expressed in skeletal muscle and heart tissue and associates with the outer mitochondrial membrane. We demonstrate PERM1 is subject to rapid changes mediated by the UPS through phosphorylation of its PEST motif by casein kinase 2. Ablation of Perm1 in mice results in reduced protein expression of lipin-1 accompanied by accumulation of specific phospholipid species. Isolation of Perm1-deficient mitochondria revealed significant downregulation of mitochondrial transport proteins for amino acids and carnitines, including SLC25A12/13/29/34 and CPT2. Consistently, we observed altered levels of various lipid species, amino acids, and acylcarnitines in Perm1-/- mitochondria. We conclude that the outer mitochondrial membrane protein PERM1 regulates homeostasis of lipid and amino acid metabolites in mitochondria.

Dölen Y, Gileadi U, Chen JL, Valente M, Creemers JHA, Van Dinther EAW, van Riessen NK, Jäger E, Hruby M, Cerundolo V, Diken M, Figdor CG, de Vries IJM. (2021) PLGA Nanoparticles Co-encapsulating NY-ESO-1 Peptides and IMM60 Induce Robust CD8 and CD4 T Cell and B Cell Responses. Front Immunol. 12:641703.

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Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85-111, 117-143, and 157-165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells via various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo. Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.

Beck JD, Reidenbach D, Salomon N, Sahin U, Türeci Ö, Vormehr M, Kranz LM. (2021) mRNA therapeutics in cancer immunotherapy. Mol Cancer. 20(1):69. doi: 10.1186/s12943-021-01348-0.

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Synthetic mRNA provides a template for the synthesis of any given protein, protein fragment or peptide and lends itself to a broad range of pharmaceutical applications, including different modalities of cancer immunotherapy. With the ease of rapid, large scale Good Manufacturing Practice-grade mRNA production, mRNA is ideally poised not only for off-the shelf cancer vaccines but also for personalized neoantigen vaccination. The ability to stimulate pattern recognition receptors and thus an anti-viral type of innate immune response equips mRNA-based vaccines with inherent adjuvanticity. Nucleoside modification and elimination of double-stranded RNA can reduce the immunomodulatory activity of mRNA and increase and prolong protein production. In combination with nanoparticle-based formulations that increase transfection efficiency and facilitate lymphatic system targeting, nucleoside-modified mRNA enables efficient delivery of cytokines, costimulatory receptors, or therapeutic antibodies. Steady but transient production of the encoded bioactive molecule from the mRNA template can improve the pharmacokinetic, pharmacodynamic and safety properties as compared to the respective recombinant proteins. This may be harnessed for applications that benefit from a higher level of expression control, such as chimeric antigen receptor (CAR)-modified adoptive T-cell therapies. This review highlights the advancements in the field of mRNA-based cancer therapeutics, providing insights into key preclinical developments and the evolving clinical landscape.

Lang F, Ferreiro PR, Löwer M, Sahin U, Schrörs B. (2021) NeoFox: annotating neoantigen candidates with neoantigen features. Bioinformatics. 2021 May 10:btab344. doi: 10.1093/bioinformatics/btab344.

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The detection and prediction of true neoantigens is of great importance for the field of cancer immunotherapy. We searched the literature for proposed neoantigen features and integrated them into a toolbox called NeoFox (NEOantigen Feature toolbOX). NeoFox is an easy-to-use Python package that enables the annotation of neoantigen candidates with 16 neoantigen features.

Bresadola L, Weber D, Ritzel C, Löwer M, Bukur V, Akilli-Öztürk Ö, Becker J, Mehanna H, Schrörs B, Vascotto F, Sahin U, Kong A. Comprehensive Genomic and Transcriptomic Analysis of Three Synchronous Primary Tumours and a Recurrence from a Head and Neck Cancer Patient. Int J Mol Sci. 2021 Jul 15;22(14):7583.

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Synchronous primary malignancies occur in a small proportion of head and neck squamous cell carcinoma (HNSCC) patients. Here, we analysed three synchronous primaries and a recurrence from one patient by comparing the genomic and transcriptomic profiles among the tumour samples and determining the recurrence origin. We found remarkable levels of heterogeneity among the primary tumours, and through the patterns of shared mutations, we traced the origin of the recurrence. Interestingly, the patient carried germline variants that might have predisposed him to carcinogenesis, together with a history of alcohol and tobacco consumption. The mutational signature analysis confirmed the impact of alcohol exposure, with Signature 16 present in all tumour samples. Characterisation of immune cell infiltration highlighted an immunosuppressive environment in all samples, which exceeded the potential activity of T cells. Studies such as the one described here have important clinical value and contribute to personalised treatment decisions for patients with synchronous primaries and matched recurrences.

Kappel C, Seidl C, Medina-Montano C, Schinnerer M, Alberg I, Leps C, Sohl J, Hartmann AK, Fichter M, Kuske M, Schunke J, Kuhn G, Tubbe I, Paßlick D, Hobernik D, Bent R, Haas K, Montermann E, Walzer K, Diken M, Schmidt M, Zentel R, Nuhn L, Schild H, Tenzer S, Mailänder V, Barz M, Bros M, Grabbe S. (2021) Density of Conjugated Antibody Determines the Extent of Fc Receptor Dependent Capture of Nanoparticles by Liver Sinusoidal Endothelial Cells. ACS Nano 15(9):15191-15209

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Despite considerable progress in the design of multifunctionalized nanoparticles (NPs) that selectively target specific cell types, their systemic application often results in unwanted liver accumulation. The exact mechanisms for this general observation are still unclear. Here we asked whether the number of cell-targeting antibodies per NP determines the extent of NP liver accumulation and also addressed the mechanisms by which antibody-coated NPs are retained in the liver. We used polysarcosine-based peptobrushes (PBs), which in an unmodified form remain in the circulation for >24 h due to the absence of a protein corona formation and low unspecific cell binding, and conjugated them with specific average numbers (2, 6, and 12) of antibodies specific for the dendritic cell (DC) surface receptor, DEC205. We assessed the time-dependent biodistribution of PB–antibody conjugates by in vivo imaging and flow cytometry. We observed that PB–antibody conjugates were trapped in the liver and that the extent of liver accumulation strongly increased with the number of attached antibodies. PB–antibody conjugates were selectively captured in the liver via Fc receptors (FcR) on liver sinusoidal endothelial cells, since systemic administration of FcR-blocking agents or the use of F(ab′)2 fragments prevented liver accumulation. Cumulatively, our study demonstrates that liver endothelial cells play a yet scarcely acknowledged role in liver entrapment of antibody-coated NPs and that low antibody numbers on NPs and the use of F(ab′)2 antibody fragments are both sufficient for cell type-specific targeting of secondary lymphoid organs and necessary to minimize unwanted liver accumulation.

Hotz C, Wagenaar TR, Gieseke F, Bangari DS, Callahan M, Cao H, Diekmann J, Diken M, Grunwitz C, Hebert A, Hsu K, Bernardo M, Karikó K, Kreiter S, Kuhn AN, Levit M, Malkova N, Masciari S, Pollard J, Qu H, Ryan S, Selmi A, Schlereth J, Singh K, Sun F, Tillmann B, Tolstykh T, Weber W, Wicke L, Witzel S, Yu Q, Zhang YA, Zheng G, Lager J, Nabel GJ, Sahin U, Wiederschain D. (2021) Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across multiple preclinical tumor models. Sci Transl Med. 13(610):eabc7804.

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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+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.

Schrörs B, Riesgo-Ferreiro P, Sorn P, Gudimella R, Bukur T, Rösler T, Löwer M, Sahin U. (2021) Large-scale analysis of SARS-CoV-2 spike-glycoprotein mutants demonstrates the need for continuous screening of virus isolates. PLoS One. 16(9):e0249254

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Due to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it. We analyzed 1,036,030 SARS-CoV-2 genome assemblies and 30,806 NGS datasets from GISAID and European Nucleotide Archive (ENA) focusing on non-synonymous mutations in the spike protein. Only around 2.5% of the samples contained the wild-type spike protein with no variation from the reference. Among the spike protein mutants, we confirmed a low mutation rate exhibiting less than 10 non-synonymous mutations in 99.6% of the analyzed sequences, but the mean and median number of spike protein mutations per sample increased over time. 5,472 distinct variants were found in total. The majority of the observed variants were recurrent, but only 21 and 14 recurrent variants were found in at least 1% of the mutant genome assemblies and NGS samples, respectively. Further, we found high-confidence subclonal variants in about 2.6% of the NGS data sets with mutant spike protein, which might indicate co-infection with various SARS-CoV-2 strains and/or intra-host evolution. Lastly, some variants might have an effect on antibody binding or T-cell recognition. These findings demonstrate the continuous importance of monitoring SARS-CoV-2 sequences for an early detection of variants that require adaptations in preventive and therapeutic strategies.

Tadmor AD, Phillips R. (2021) MCRL: using a reference library to compress a metagenome into a nonredundant list of sequences, considering viruses as a case study. Bioinformatics, btab703

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Metagenomes offer a glimpse into the total genomic diversity contained within a sample. Currently, however, there is no straightforward way to obtain a nonredundant list of all putative homologs of a set of reference sequences present in a metagenome.

To address this problem, we developed a novel clustering approach called “metagenomic clustering by reference library” (MCRL), where a reference library containing a set of reference genes is clustered with respect to an assembled metagenome. According to our proposed approach, reference genes homologous to similar sets of metagenomic sequences, termed “signatures”, are iteratively clustered in a greedy fashion, retaining at each step the reference genes yielding the lowest E values, and terminating when signatures of remaining reference genes have a minimal overlap. The outcome of this computation is a nonredundant list of reference genes homologous to minimally overlapping sets of contigs, representing potential candidates for gene families present in the metagenome. Unlike metagenomic clustering methods, there is no need for contigs to overlap to be associated with a cluster, enabling MCRL to draw on more information encoded in the metagenome when computing tentative gene families. We demonstrate how MCRL can be used to extract candidate viral gene families from an oral metagenome and an oral virome that otherwise could not be determined using standard approaches. We evaluate the sensitivity, accuracy, and robustness of our proposed method for the viral case study and compare it with existing analysis approaches.

Formes H, Bernardes JP, Mann A, Bayer F, Pontarollo G, Kiouptsi K, Schäfer K, Attig S, Nikolova T, Hofmann TG, Schattenberg JM, Todorov H, Gerber S, Rosenstiel P, Bopp T, Sommer F, Reinhardt C. (2021) The gut microbiota instructs the hepatic endothelial cell transcriptome. iScience. 24(10):103092.

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The gut microbiota affects remote organ functions but its impact on organotypic endothelial cell (EC) transcriptomes remains unexplored. The liver endothelium encounters microbiota-derived signals and metabolites via the portal circulation. To pinpoint how gut commensals affect the hepatic sinusoidal endothelium, a magnetic cell sorting protocol, combined with fluorescence-activated cell sorting, was used to isolate hepatic sinusoidal ECs from germ-free (GF) and conventionally raised (CONV-R) mice for transcriptome analysis by RNA sequencing. This resulted in a comprehensive map of microbiota-regulated hepatic EC-specific transcriptome profiles. Gene Ontology analysis revealed that several functional processes in the hepatic endothelium were affected. The absence of microbiota influenced the expression of genes involved in cholesterol flux and angiogenesis. Specifically, genes functioning in hepatic endothelial sphingosine metabolism and the sphingosine-1-phosphate pathway showed drastically increased expression in the GF state. Our analyses reveal a prominent role for the microbiota in shaping the transcriptional landscape of the hepatic endothelium.

Weiden J, Schluck M, Ioannidis M, van Dinther EAW, Rezaeeyazdi M, Omar F, Steuten J, Voerman D, Tel J, Diken M, Bencherif SA, Figdor CG, Verdoes M. (2021) Robust Antigen-Specific T Cell Activation within Injectable 3D Synthetic Nanovaccine Depots. ACS Biomater. Sci. Eng. https://pubs.acs.org/doi/10.1021/acsbiomaterials.0c01648

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Synthetic cancer vaccines may boost anticancer immune responses by co-delivering tumor antigens and adjuvants to dendritic cells (DCs). The accessibility of cancer vaccines to DCs and thereby the delivery efficiency of antigenic material greatly depends on the vaccine platform that is used. Three-dimensional scaffolds have been developed to deliver antigens and adjuvants locally in an immunostimulatory environment to DCs to enable sustained availability. However, current systems have little control over the release profiles of the cargo that is incorporated and are often characterized by an initial high-burst release. Here, an alternative system is designed that co-delivers antigens and adjuvants to DCs through cargo-loaded nanoparticles (NPs) incorporated within biomaterial-based scaffolds. This creates a programmable system with the potential for controlled delivery of their cargo to DCs. Cargo-loaded poly(d,l-lactic-co-glycolic acid) NPs are entrapped within the polymer walls of alginate cryogels with high efficiency while retaining the favorable physical properties of cryogels, including syringe injection. DCs cultured within these NP-loaded scaffolds acquire strong antigen-specific T cell-activating capabilities. These findings demonstrate that introduction of NPs into the walls of macroporous alginate cryogels creates a fully synthetic immunostimulatory niche that stimulates DCs and evokes strong antigen-specific T cell responses.

Krienke C, Kolb L, Diken E, Streuber M, Kirchhoff S, Bukur T, Akilli-Öztürk Ö, Kranz LM, Berger H, Petschenka J, Diken M, Kreiter S, Yogev N, Waisman A, Karikó K, Türeci Ö, Sahin U. (2021) A noninflammatory mRNA vaccine for treatment of experimental autoimmune encephalomyelitis. Science. 371(6525):145-153.

DOI, PMID

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The ability to control autoreactive T cells without inducing systemic immune suppression is the major goal for treatment of autoimmune diseases. The key challenge is the safe and efficient delivery of pharmaceutically well-defined antigens in a noninflammatory context. Here, we show that systemic delivery of nanoparticle-formulated 1 methylpseudouridine-modified messenger RNA (m1Ψ mRNA) coding for disease-related autoantigens results in antigen presentation on splenic CD11c+ antigen-presenting cells in the absence of costimulatory signals. In several mouse models of multiple sclerosis, the disease is suppressed by treatment with such m1Ψ mRNA. The treatment effect is associated with a reduction of effector T cells and the development of regulatory T cell (Treg cell) populations. Notably, these Treg cells execute strong bystander immunosuppression and thus improve disease induced by cognate and noncognate autoantigens.

Vogel AB, Kanevsky I, Che Y, et al. (2021) Immunogenic BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature. https://doi.org/10.1038/s41586-021-03275-y.

DOI, PMID

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A safe and effective vaccine against COVID-19 is urgently needed in quantities sufficient to immunise large populations. We report the preclinical development of two BNT162b vaccine candidates, which contain lipid-nanoparticle (LNP) formulated nucleoside-modified mRNA encoding SARS-CoV-2 spike glycoprotein-derived immunogens. BNT162b1 encodes a soluble, secreted, trimerised receptor-binding domain (RBD-foldon). BNT162b2 encodes the full-length transmembrane spike glycoprotein, locked in its prefusion conformation (P2 S). The flexibly tethered RBDs of the RBD-foldon bind ACE2 with high avidity. Approximately 20% of the P 2S trimers are in the two-RBD ‘down,’ one-RBD ‘up’ state. In mice, one intramuscular dose of either candidate elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong TH1 CD4+ and IFNγ+ CD8+ T-cell responses. Prime/boost vaccination of rhesus macaques with BNT162b candidates elicits SARS-CoV-2 neutralising geometric mean titres 8.2 to 18.2 times that of a SARS-CoV-2 convalescent human serum panel. The vaccine candidates protect macaques from SARS-CoV-2 challenge, with BNT162b2 protecting the lower respiratory tract from the presence of viral RNA and with no evidence of disease enhancement. Both candidates are being evaluated in phase 1 trials in Germany and the United States1–3. BNT162b2 is being evaluated in an ongoing global, pivotal Phase 2/3 trial (NCT04380701, NCT04368728).

Miederer M, Pektor S, Miederer I , Bausbacher N, Keil IS, Hefesha H, Haas H, Sahin U, Diken M. (2021) Iodine-124 PET quantification of organ-specific delivery and expression of NIS-encoding RNA. EJNMMI Res 11, 14

DOI, PMID

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Background

RNA-based vaccination strategies tailoring immune response to specific reactions have become an important pillar for a broad range of applications. Recently, the use of lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. Here, we have investigated whether small animal PET/MRI can be employed to image the biodistribution of RNA-encoded protein. For this purpose, a reporter RNA coding for the sodium-iodide-symporter (NIS) was in vitro transcribed in cell lines and evaluated for expression. RNA-lipoplex nanoparticles were then assembled by complexing RNA with liposomes at different charge ratios, and functional NIS protein translation was imaged and quantified in vivo and ex vivo by Iodine-124 PET upon intravenous administration in mice.

Results

NIS expression was detected on the membrane of two cell lines as early as 6 h after transfection and gradually decreased over 48 h. In vivo and ex vivo PET/MRI of anionic spleen-targeting or cationic lung-targeting NIS-RNA lipoplexes revealed a visually detectable rapid increase of Iodine-124 uptake in the spleen or lung compared to control-RNA-lipoplexes, respectively, with minimal background in other organs except from thyroid, stomach and salivary gland.

Conclusions

The strong organ selectivity and high target-to-background acquisition of NIS-RNA lipoplexes indicate the feasibility of small animal PET/MRI to quantify organ-specific delivery of RNA.