Detlef Oswald

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

BackgroundLeukocytes 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 Freunds adjuvant-induced inflammatory pain in rats.ResultsPOMC-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.ConclusionAlthough 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.

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.

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 Th1/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-Th1 vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications.

Local and systemic effect of transfection-reagent formulated DNA vectors on equine melanoma

BackgroundEquine melanoma has a high incidence in grey horses. Xenogenic DNA vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. In a clinical study, twenty-seven, grey, melanoma-bearing, horses were assigned to three groups (n = 9) and vaccinated on days 1, 22, and 78 with DNA vectors encoding for equine (eq) IL-12 and IL-18 alone or in combination with either human glycoprotein (hgp) 100 or human tyrosinase (htyr). Horses were vaccinated intramuscularly, and one selected melanoma was locally treated by intradermal peritumoral injection. Prior to each injection and on day 120, the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. Specific serum antibodies against hgp100 and htyr were measured using cell based flow-cytometric assays. An Analysis of Variance (ANOVA) for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. For post-hoc testing a Tukey-Kramer Multiple-Comparison Test was performed to compare the relative volumes on the different examination days. An ANOVA for repeated measurements was performed to analyse changes in body temperature over time. A one-way ANOVA was used to evaluate differences in body temperature between the groups. A p–value < 0.05 was considered significant for all statistical tests applied.ResultsIn all groups, the relative tumor volume decreased significantly to 79.1 ± 26.91% by day 120 (p < 0.0001, Tukey-Kramer Multiple-Comparison Test). Affiliation to treatment group, local treatment and examination modality had no significant influence on the results (ANOVA for repeated measurements). Neither a cellular nor a humoral immune response directed against htyr or hgp100 was detected. Horses had an increased body temperature on the day after vaccination.ConclusionsThis is the first clinical report on a systemic effect against equine melanoma following treatment with DNA vectors encoding eqIL12 and eqIL18 and formulated with a transfection reagent. Addition of DNA vectors encoding hgp100 respectively htyr did not potentiate this effect.

Abstract 2622: Two new TLR9 agonists for cancer immunotherapy: Combination with checkpoint inhibitors

Introduction TLR9 agonists are developed as anti-cancer therapies based on their broad activation of the innate and adaptive immune system. Single-stranded oligodeoxynucleotides (ODN) containing non-methylated CG-motifs activate TLR9. Previously, chemical modification was used to prevent their degradation by exonucleases. To avoid the off-target effects observed with chemical modifications, new TLR9 agonists containing only natural DNA were stabilized by structural components. The dSLIM® family of TLR9 agonists is protected from exonucleolytic degradation by its covalently-closed dumbbell-shaped structure. It contains an immunomodulatory sequence with CG-motifs in its loops. The linear single-stranded EnanDIM® family of TLR9 agonists utilizes L-deoxyribonucleotides (natural enantiomers of D-deoxyribonucleotides) at their 3’-ends to prevent degradation. Since the mode-of-action of TLR9 agonists starts upstream of the targets of checkpoint inhibitors anti-PD-1/anti-PD-L1 a combinatory approach may support synergistic immune activation and thus enhanced anti-tumor effects. Methods The impact of dSLIM2006 and EnanDIM-1 on T cell responses was analyzed employing an in vitro assay using human peripheral blood mononuclear cells (PBMC). PBMC were treated with peptides selected from HLA class I-restricted T-cell epitopes of recall-antigens (CMV, EBV, Flu = CEF), TLR9 agonists and anti-PD-1 as checkpoint inhibitor. In addition, in vivo studies were used to investigate the anti-tumor effect of dSLIM2006 and EnanDIM-1 in combination with anti-PD-1 in a syngeneic murine CT26 tumor model. Results The IFN-gamma secretion of human PBMC after stimulation of CEF peptides was roughly 5-fold increased by EnanDIM-1 and dSLIM2006, whereas treatment with anti-PD-1 resulted barely in a two-fold increase. The combination of the TLR9 agonists and anti-PD-1 further enforced IFN-gamma secretion by about 7-fold. In the murine colon cancer model CT26 the subcutaneous injection of EnanDIM-1 or intraperitoneal injection of anti-PD-1 had a moderate effect on the tumor growth when used in monotherapy (28.3% or 57.0% tumor growth inhibition, TGI). Notably, a combination of EnanDIM-1 and anti-PD-1 further reduced tumor growth (74.7% TGI). Intratumoral injection of dSLIM2006 in combination with intraperitoneal injection of anti-PD-1 reduced tumor growth (54.2% TGI) whereas the single components had more limited effects (dSLIM2006: 18.7%, anti-PD-1: no inhibition). Combined treatment with TLR9 agonists and anti-PD-1 prolonged survival of the mice in comparison to single treatments. Conclusions The TLR9 agonists and immune surveillance reactivators (ISR) EnanDIM-1 and dSLIM2006 enhance T cell responses and anti-tumor effects of the anti-PD-1 checkpoint inhibitor. These data show their promising potential not only for monotherapeutic but also combinatory approaches. Citation Format: Kerstin Kapp, Barbara Volz, Detlef Oswald, Burghardt Wittig, Manuel Schmidt. Two new TLR9 agonists for cancer immunotherapy: Combination with checkpoint inhibitors [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 2622. doi:10.1158/1538-7445.AM2017-2622

Abstract B067: Preclinical data of novel enantiomeric oligonucleotides for cancer immunotherapy: The TLR9 agonist EnanDIM

Introduction: The use of TLR9 agonists as immunomodulators is supported by preclinical and clinical studies, showing their anti-tumor effect by enhancing both cellular and humoral responses. Two different families of DNA molecules containing non-methylated CG-motifs for TLR9 activation have been established so far: Dumbbell-shaped dSLIM molecules are protected against exonucleolytic degradation by their covalently-closed, natural phosphodiester (PO) backbone. In contrast, single-stranded, oligodeoxynucleotides (CpG-ODN) are most commonly chemically-stabilized by phosphorothioates (PTO) in their phosphate moieties. However, PTO modifications produce off-target effects in immune cell populations and have resulted in an unfavorable risk-to-benefit ratio. Methods: Linear single-stranded ODN were synthesized using L-deoxyribonucleotides at their 39-ends, which are the natural enantiomers of D-deoxyribonucleotides, to ensure protection against exonucleases and avoid the off-target effects of PTO-modified CpG-ODN. The vast majority of deoxyribose in present organisms are D-deoxyribose, thus co-evolved nucleases are blind for L-deoxyribose thereby leaving L-protected ODN intact. Using high secretion of IFN-alpha and IP-10 from human peripheral blood mononuclear cells as marker, we selected nucleotide sequences of such L-protected, CG-motif containing, single-stranded ODN, EnanDIM. First, we employed a maximum feasible dose (MFD) approach. Mice received subcutaneous injection of single doses of 10 to 50 mg EnanDIM to evaluate its acute toxicity. Immunomodulatory properties were evaluated as well. Finally, a pilot tumor model in mice was used to investigate the anti-tumor effect of EnanDIM. Results: EnanDIM581 and EnanDIM532 were selected due to their pronounced activation of immune cells (e.g. monocytes, NK cells and plasmacytoid dendritic cells) and their prominent induction of IFN-alpha and IP-10 secretion in vitro. EnanDIM744, comprising EnanDIM581 with additional 59-end L-nucleotide protection and exhibiting an activation pattern similar to EnanDIM581, was also used for MFD studies. Safety assessments were performed throughout the study period and no mortality, clinical signs and body weight changes were observed, despite the extremely high doses of app. 300 to 1700 mg/kg. A gross necropsy consisting of a macroscopic organ evaluation at day 15 revealed also no toxicity. Dose-dependent increase of IP-10 levels in serum was observed between 6 and 24 hours after injection (maximum at 6h) but after 15 days IP-10 levels were undetectable, confirming that L-nucleotides in EnanDIM do not change the kinetic profile known from other DNA-based TLR9 agonists. Preliminary data from a murine tumor model suggest that multiple doses of EnanDIM can reduce tumor growth. Conclusions: EnanDIM, a new family of TLR9 agonists, broadly activates the immune system in vitro and in vivo. While maximal feasible doses of EnanDIM resulted in no signs of toxicity, an indication of reduced tumor growth was observed in a murine tumor model in vivo. Therefore EnanDIM has the potential for clinical development in the treatment of cancer. Citation Format: Kerstin Kapp, Barbara Volz, Detlef Oswald, Burghardt Wittig, Manuel Schmidt. Preclinical data of novel enantiomeric oligonucleotides for cancer immunotherapy: The TLR9 agonist EnanDIM [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 B067.

Abstract A035: Pharmacokinetics and pharmacodynamics of the immunotherapeutic TLR-9 agonist MGN1703 – conclusions by comparison of data from clinical trials with healthy volunteers and cancer patients

Background: The synthetic DNA-based immunomodulator MGN1703 is a member of the new dSLIM® family of TLR-9 agonists, comprising covalently closed dumbbell-like DNA molecules which consist entirely of natural DNA with two single-stranded CG-containing loops separated by a double-stranded stem. MGN1703 exerts its function by activating the innate and both arms of the adaptive immune system. MGN1703 has already shown a good safety profile and benefit for patients compared to placebo in maintenance therapy of metastatic colorectal carcinoma (mCRC) after fist-line induction therapy in a phase 2 clinical trial (MGN1703-C02, IMPACT). Methods: Following preclinical studies in mice and monkeys, a human clinical program was performed, including a phase 1 trial (MGN1703-C01), on a variety of solid tumors, and the double-blinded, placebo-controlled phase 2 IMPACT trial with mCRC patients. Additionally, a single dose crossover, placebo-controlled phase 1 cardiac safety trial, MGN1703-C04, was conducted to assess cardiac and general safety, pharmacokinetics (PK) and pharmacodynamics (PD) in healthy volunteers (HV). Fourteen HV were randomized and 13 subjects completed the study receiving a subcutaneous dose of 60 mg MGN1703 during one treatment period and a single dose of placebo during the other. Results: After single dose administration of MGN1703 standard PK parameters were determined in the MGN1703-C04 trial: mean Tmax (14h), AUC0-t (5000 ng•hr/mL) and shape of the curve of MGN1703 concentration in serum. These were similar to single dose data obtained from cancer patients, where a mean Tmax of 10h and a mean AUC0-t of 5340 ng•hr/mL have been observed. Notably, multiple doses resulted in similar levels in serum of cancer patients, without accumulation of MGN1703. PD was mainly evaluated by analyzing levels of the chemokine IP-10 in serum of the HV as an equivalent of innate immune activation. Elevated IP-10 levels in the serum of HV were shown peaking at 24-48 hours after dosing with MGN1703. This is clearly in line with expected time frame of immune activation subsequent to MGN1703 appearance in the serum of the HV. Timing and levels of IP-10 elevation are in keeping with data from the peripheral blood of cancer patients, where in addition to IP-10 also the subsequent activation of innate immune cells – especially the up-regulation of CD169 on monocytes – as a consequence of MGN1703 administration was observed. Conclusions: The data show obvious similarities between healthy volunteers and cancer patients with respect to PK and PD and also confirm the appearance of the appropriate immunomodulatory signature downstream of MGN1703 appearance. This supports the twice weekly application scheme of current immunotherapy studies with MGN1703, e.g. the ongoing phase 3 IMPALA trial in mCRC patients. Further data will allow for evaluation of a possible association with clinical parameters in cancer patients. Citation Format: Manuel Schmidt, Kerstin Kapp, Detlef Oswald, Matthias Schroff, Burghardt Wittig, Alfredo Zurlo. Pharmacokinetics and pharmacodynamics of the immunotherapeutic TLR-9 agonist MGN1703 – conclusions by comparison of data from clinical trials with healthy volunteers and cancer patients. [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 A035.