Design and characterization of the tumor vaccine MGN1601, allogeneic fourfold gene-modified vaccine cells combined with a TLR-9 agonist

The tumor vaccine MGN1601 was designed and developed for treatment of metastatic renal cell carcinoma (mRCC). MGN1601 consists of a combination of fourfold gene-modified cells with the toll-like receptor 9 agonist dSLIM, a powerful connector of innate and adaptive immunity. Vaccine cells originate from a renal cell carcinoma cell line (grown from renal cell carcinoma tissue), express a variety of known tumor-associated antigens (TAA), and are gene modified to transiently express two co-stimulatory molecules, CD80 and CD154, and two cytokines, GM-CSF and IL-7, aimed to support immune response. Proof of concept of the designed vaccine was shown in mice: The murine homologue of the vaccine efficiently (100%) prevented tumor growth when used as prophylactic vaccine in a syngeneic setting. Use of the vaccine in a therapeutic setting showed complete response in 92% of mice as well as synergistic action and necessity of the components. In addition, specific cellular and humoral immune responses in mice were found when used in an allogeneic setting. Immune response to the vaccine was also shown in mRCC patients treated with MGN1601: Peptide array analysis revealed humoral CD4-based immune response to TAA expressed on vaccine cells, including survivin, cyclin D1, and stromelysin

Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

<p>Abstract</p> <p>Background</p> <p>Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as β-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE) vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS)-coupled MIDGE encoding the β-endorphin precursor proopiomelanocortin (POMC) on complete Freund's adjuvant-induced inflammatory pain in rats.</p> <p>Results</p> <p>POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of β-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing β-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of β-endorphin.</p> <p>Conclusion</p> <p>Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or β-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.</p

Ballistomagnetic transfer of nucleic acids into eucaryotic cells

0\. Titelblatt und Inhaltsverzeichnis 1\. Einleitung 1 2\. Material 12 3\. Methoden 16 4\. Ergebnisse 37 5\. Diskussion 60 6\. Zusammenfassung 72 7\. Abbildungs- und Tabellenverzeichnis 73 8\. Abkürzungen 78 9\. Eigene Veröffentlichungen 79 10\. Literatur 84Die Dissertation beschreibt ein Verfahren, mit dessen Hilfe Nukleinsäuren effektiv und einfach in Zellen eingebracht werden können. Dieses Verfahren beruht auf der Kombination von partikelvermittelter Transfektion mit magnetischer Zellsortierung. Im ersten Teil der Arbeit konnte durch Transfektion von kurzen fluoreszenzmarkierten Oligodesoxynukleotiden gezeigt werden, dass es mit dem Verfahren möglich ist, eine Population von Zellen zu isolieren, in der mehr als 90% der Zellen transfiziert sind. Durch Einbringen von "antisense"-ODN in die Zellen konnte gezielt eine G-Protein-Untereinheit ausgeschaltet werden. Am Modellsystem der eGFP-transfizierten Zelle konnte demonstriert werden, dass auch bei nachgewiesener antisense-Wirkung bei stark exprimierten Proteinen mit langer Halblebenszeit in der Zelle keine vollständige Unterdrückung der Genexpression erreichbar war. Im zweiten Teil der Arbeit wurden Experimente mit Plasmid-basierten Genexpressionskonstrukten durchgeführt. Dabei stand die Frage im Vordergrund, wie viele Zellen tatsächlich nach der Transfektion das transfizierte Gen exprimieren. Besondere Gesichtspunkte dabei waren die quantitative Ermittlung der Anzahl der transferierten Plasmide und die Optimierung der Transfektionsmethode in Hinblick auf die Transfektionseffizienz. Dazu wurde eine quantitative PCR- Methode entwickelt, die für diese Fragestellung geeignet war. Anhand der Expression der Reportergene Luciferase und eGFP konnte nachgewiesen werden, dass mit dem Verfahren eine etwa zehnfache Erhöhung der Gesamtmenge an Proteinaktivität in der angereicherten transfizierten Zellpopulation bzw. des Anteils der exprimierenden Zellen an der Gesamtpopulation erreicht wurde. Das Verfahren stellt eine Erweiterung des Methodenspektrums der experimentellen und klinisch angewandten Molekularbiologie dar und wurde in einer klinischen Studie zur immuntherapeutischen Therapie des Nierenzellkarzinoms mit genmodifizierten Tumorzellen zur Anwendung gebracht.The dissertation decribes a method for the efficacious transfection of eucaryotic cells with nucleic acids. This method is based on the combination of ballistic transfer and magnetic cell sorting. In the first part the enrichment of transfected cells was demonstrated by transfection of FITC coupled oligodeoxyribonucleotides (ODN). More than 90% of the enriched cell were transfected cells. The transfection of antisense ODN resulted in the complete supression of gene expression. In the second part of the dissertation cells were transfected with plasmid based expression vectors. The focus in this part was to determine parameters like amount of transfected vectors or optimization of the transfection method regarding transfection efficacy. For this purpose a quantitative PCR method was developed. The increase of the gene expression in the enriched fraction was determined with reporter genes like GFP or Luciferase and was in a range of 10. The transfection method is an improvement compared to the existing ballistic transfer and was used in clinical gene therapy trials against cancer

Genuine Immunomodulation With dSLIM

Toll-like receptors are sensing modulators of the innate immune system. One member of this protein family, Toll-like receptor (TLR)-9, is increasingly being investigated as therapeutic target for infectious diseases and cancer. Double-Stem Loop ImmunoModulator (dSLIM) is a new TLR-9 agonist in clinical development for patients with metastatic colorectal carcinoma. Compared with other TLR-9 ligands developed as immunomodulators, dSLIM comprises single- and double-stranded DNA, is covalently closed, and consists of natural nucleotide components only. All investigated biologic effects of dSLIM are strongly dependent on CG motifs, and the relevant cellular activation profile of dSLIM is distinct to that of other TLR-9 agonists. Here we describe the structure and biologic profile of dSLIM: in isolated human peripheral blood mononuclear cells (PBMCs), dSLIM induced a unique pattern of cytokine secretion, activated within the PBMC pool particular cell subpopulations, and exhibited specific cytotoxicity on target cells. Using cellular isolation and depletion setups, the mechanism of immunoactivation by dSLIM was deduced to be dependent on, but not restricted to, TLR-9-bearing plasmacytoid dendritic cells. The dSLIM-promoted cellular stimulation directs systemic activation of the immune response as revealed in cancer patients. The observed cellular activation cascades are discussed in the context of cancer therapy

Combination of MIDGE-Th1 DNA vaccines with the cationic lipid SAINT-18: Studies on formulation, biodistribution and vector clearance

We have previously shown that the combination of MIDGE-Th1 DNA vectors with the cationic lipid SAINT-18 increases the immune response to the encoded antigen in mice. Here, we report on experiments to further optimize and characterize this approach. We evaluated different formulations of MIDGE-Th1 vectors with SAINT-18 by assessing their influence on the transfection efficiency in cell culture and on the immune response in mice. We found that high amounts of SAINT-18 in formulations with a w/w ratio MIDGE Th1/SAINT-18 of 1:4.8 are beneficial for cell transfection in vitro. In contrast, the formulation of HBsAg-encoding MIDGE-Th1 DNA vectors with the lowest amount of SAINT-18 (w/w ratio MIDGE Thl/SAINT-18 of 1:0.5) resulted in the highest serum IgG1 and IgG2a levels after intradermal immunization of mice. Consequently, latter formulation was selected for a comparative biodistribution study in rats. Following intradermal administration of both naked and formulated MIDGE-Th1 DNA, the vectors localized primarily at the site of injection. Vector DNA levels decreased substantially over the two months duration of the study. When administered in combination with SAINT-18, the vectors were found in significantly higher amounts in draining lymph nodes in comparison to administration of naked MIDGE-Th1 DNA. We propose that the high immune responses induced by MIDGE-Th1/SAINT-18 lipoplexes are mediated by enhanced transfection of cells in vivo, resulting in stronger antigen expression and presentation. Importantly, the combination of MIDGE-Thl vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications. (C) 2014 Elsevier Ltd. All rights reserved

Cationic lipid-formulated DNA vaccine against hepatitis B virus: immunogenicity of MIDGE-Th1 vectors encoding small and large surface antigen in comparison to a licensed protein vaccine.

Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals