Barbara Volz

Carbohydrate structures of soluble human L-selectin recombinantly expressed in baby-hamster kidney cells.

A soluble form of L‐selectin was recombinantly produced, which might be an effective therapeutic agent in inflammatory disorders, acting as an inhibitor for leucocyte endothelium adhesion. In the present study the oligosaccharide structures of soluble human L‐selectin, recombinantly expressed in baby‐hamster kidney cells, were determined. The N‐linked glycans were enzymically released and fluorescently labelled with 2‐aminobenzamide. Sialylation of the N‐glycans was analysed by anion‐exchange chromatography followed by rechromatography of the resulting fractions on amino‐phase HPLC after release of the sialic acid residues. Desialylated oligosaccharides were separated using two‐dimensional HPLC and characterized by digestion with exoglycosidases and MS. More than 30 oligosaccharide structures representing at least 95% of the overall glycosylation of this protein were determined. The results revealed that recombinant soluble human L‐selectin carries bi‐, tri‐ and tetra‐antennary sugar chains, which are fucosylated on the innermost residue of N‐acetylglucosamine. The number of sialic acid residues linked to these glycans ranges from 0 (neutral glycans) to 4 (tetrasialylated oligosaccharides). The sialic acid is found exclusively in the α2–3 linkage to galactose. In addition to the main glycans, different minor structures containing terminal N‐acetylgalactosamine, or the H (O) blood‐group determinant were also identified. O‐Glycosylation of mucin‐type sugar chains was not detected in recombinant soluble human L‐selectin.

H (0) blood group determinant is present on soluble human L‐selectin expressed in BHK‐cells

In the present study we show that the H (0) blood group determinant Fucα1‐2Galβ1‐4GlcNAcβ1‐R is present on N‐linked glycans of soluble human L‐selectin recombinantly expressed in baby hamster kidney (BHK) cells. The glycans were isolated using complementary HPLC techniques and characterized by a combination of exoglycosidase digestion and mass spectrometry. The linkage of the fucose residues was determined by incubation of the glycans with specific fucosidases. The H blood determinant Fucα1‐2Galβ1‐4GlcNAcβ1 was detected for bi‐, 2,4 branched tri‐ and tetraantennary structures. To our knowledge, the proposed oligosaccharide structures represent a new glycosylation motif for recombinant glycoproteins expressed on BHK cells.

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

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.

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.

213. Treatment with MGN1601 Induces Immune Responses Against Tumor Associated Antigens in Patients with Renal Cell Carcinoma

The cell-based RCC tumor vaccine MGN1601 consists of two active pharmaceutical ingredients: allogeneic (human) cells gene-modified with four different MIDGE vectors encoding IL-7, GM-CSF, CD80 and CD154 combined with the synthetic DNA-based immunomodulator dSLIM®, a TLR-9 agonist. In a first in men, proof-of-principle, multi-center, open-label, single-arm, non-randomized phase 1/2 clinical trial (ClinicalTrials.gov: NCT01265368; EudraCT No.: 2009-016853-16) in patients with advanced RCC MGN1601 has shown a good safety profile and improved overall survival in the per-protocol (PP) treated patient population.

MIDGE Technology for the Production of a Fourfold Gene-Modified, Allogenic Cell-Based Vaccine for Cancer Therapy

Gene modification of eukaryotic cells by electroporation is a widely used method to express selected genes in a defined cell population for various purposes, like gene correction or production of therapeutics. Here, we describe the generation of a cell-based tumor vaccine via fourfold transient gene modification of a human renal cell carcinoma (RCC) cell line for high expression of CD80, CD154, GM-CSF, and IL-7 by use of MIDGE(®) vectors. The two co-stimulatory molecules CD80 and CD154 are expressed at the cell surface, whereas the two cytokines GM-CSF and IL-7 are secreted yielding cells with enhanced immunological properties. These fourfold gene-modified cells have been used as a cell-based tumor vaccine for the treatment of RCC.