Herein, we produced and characterized cavrotolimod (formerly AST-008), a 20-30 nanometer-diameter immunostimulatory spherical nucleic acid targeting Toll-like receptor 9 (TLR9), for the treatment of solid tumors such as advanced Merkel cell carcinoma (MCC) and cutaneous squamous cell carcinoma (CSCC). Cavrotolimod agonizes TLR9 to produce T-helper 1 (Th1)-type immune responses. We studied cavrotolimod in two mouse models: in an MCC model (using cavrotolimod monotherapy) and in a CSCC model (monotherapy and combination therapy). In the MCC model, a 7.5 mg/kg intratumoral (IT) dose of cavrotolimod elicited tumor growth inhibition (TGI) of up to 68%. In the CSCC model, treatment with 5.0 and 7.5 mg/kg cavrotolimod alone and in combination with an anti-PD-1 antibody produced tumor growth inhibition (TGI = 33- 42%). Cavrotolimod was well-tolerated, induced immune cell population changes in the tumors and blood consistent with TGI, and reduced tumor burden in MCC and CSCC mouse models. Cavrotolimod is a promising nanoparticle therapy for these advanced solid tumors.

From the immune reaction cascade explained with the nonscientist reader in mind to illustrate existing and eventually unexplored therapeutic options at multiple levels of the immune reaction, the therapeutic potential of dendritic cells will be explored in oncology via a description of their basic biological attributes and the latest advances achieved in developing novel therapies based on these cells, either minimally manipulated or engineered. A Perspectives section at the end of this chapter will summarize key learnings, hypotheses and objectives highlighted in this chapter, combining as much as possible business and scientific insights.

Abstract Significant progress has been achieved recently in cancer vaccines that aim at engaging or reengaging the tumor immunity, in particular in the rapidly growing field of personalized immunotherapy. Yet novel immunization solutions to deliver efficiently tumor-associated (neo)antigens to professional antigen-presenting cells, and to overcome the peripheral tolerance and the immunosuppressive tumor microenvironment that prevent the eradication of cancer in most patients, are urgently needed. VAXIMM is developing a unique and versatile oral T-cell vaccination platform based on the FDA-approved live-attenuated Salmonella Typhi strain Ty21a vaccine Vivotif®, capable of delivering tumor-associated antigens encoded in DNA expression construct to the gut-associated lymphoid tissue, breaking immune tolerance and inducing antitumor immunity. This study summarizes the immunogenicity and antileukemia efficacy of VXM10 vaccines based on the live-attenuated Salmonella Typhimurium strain SL7207, transformed with a eukaryotic expression plasmid encoding the murine programmed death-ligand 1 (PD-L1) protein. It also describes for the first time the systemic immunogenicity of Salmonella Typhimurium-based polyepitope oral vaccines in healthy animals. The antileukemia activity of VXM10 was evaluated in the FBL-3 disseminated model of leukemia, in which the tumor cells express high levels of PD-L1. Multiple oral administrations of VXM10 used at 1010 colony-forming units (CFU) were generally well tolerated, and neither sign of toxicity nor body weight loss were observed throughout the study. Oral administration of VXM10 produced a strong antitumor effect in the FBL-3 leukemia model, with 100% of surviving animals 80 days after leukemia challenge in the highest-dose groups. In contrast, administration of the empty vector control did not show any anticancer effect. Moreover, 100% of long-term surviving mice resisted rechallenge with FBL-3 cells, demonstrating that vaccination with VXM10 generated a potent memory T-cell response against the leukemia. Importantly, full leukemia control was achieved in both prophylactic and therapeutic settings. Immunogenicity and antibody response towards PD-L1 are being evaluated to dissect the exact mechanism of action of these novel vaccines. Finally, different polyepitope vaccines encoding dominant epitopes from VEGFR2, Mesothelin, WT1, CEA, and Ovalbumin induced a significant systemic immunogenicity for up to 6 out of 9 epitopes, 10 days after vaccination of healthy C57BL/6J mice via the oral route, with doses up to 1010 CFU, as measured in the spleen by flow cytometry using peptide-MHC class I pentamers. This study provides further evidence that VAXIMM’s oral T-cell vaccination platform can be employed to stimulate antitumor immunity against antigens of the immune checkpoint regulatory protein PD-L1, and also against T-cell antigens encoded by polyepitope constructs. These data pave the way for advancing the development of VXM10 and neoantigen-based vaccines into clinical development. Citation Format: Sébastien Wieckowski, Lilli Podola, Marco Springer, Iris Kobl, Heiko Smetak, Anne-Lucie Nugues, Philippe Slos, Amine Adda Berkane, Ming Wei, Klaus Breiner, Albrecht Meichle, Beckhove Philipp, Marc Mansour, Matthias Schroff, Heinz Lubenau. Live attenuated oral Salmonella platform for effective targeting of multiple tumor-associated epitopes and PD-L1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B196.

74 Background: Significant progresses have been recently achieved in cancer vaccines, yet novel immunization solutions to deliver efficiently tumor-associated antigens to professional antigen-presenting cells, and to overcome the peripheral tolerance and the immunosuppressive tumor microenvironment that prevent the eradication of cancer in most of patients, are urgently needed. VAXIMM is developing a unique and versatile oral T-cell vaccination platform based on the FDA-approved live-attenuated Salmonella Typhi strain Ty21a vaccine Vivotif, capable of delivering tumor-associated antigens encoded in DNA expression construct to the gut-associated lymphoid tissue, breaking immune tolerance and inducing effective anti-tumor immunity. Methods: This study summarizes the immunogenicity and antileukemia efficacy of VXM10 vaccines based on the live-attenuated Salmonella Typhimurium strain SL7207, transformed with a eukaryotic expression plasmid encoding different variants of the murine PD-L1 protein. Results: The antileukemia activity of VXM10 was evaluated in the FBL-3 disseminated model of leukemia, in which the tumor cells strongly express PD-L1. Multiple oral administrations of VXM10 vaccines produced a strong anti-tumor effect, with 100% of surviving animals 80 days after challenge with FBL3 leukemia in the highest dose groups. Moreover, 100% of long-term surviving mice resisted re-challenge with FBL-3 cells, demonstrating that vaccination with VXM10 generated a potent memory T cell response against the leukemia. Importantly, full leukemia control was achieved in both prophylactic and therapeutic settings. Upon immunization with VXM10 vaccines, T cell response was raised against PD-L1 epitopes after in vitro restimulation of the splenocytes, and anti-PD-L1 antibodies were detected in the serum. The precise mechanism of action of VXM10 vaccines is currently being investigated. Conclusions: This study provides further evidence that VAXIMM’s oral T-cell vaccination platform can be employed to stimulate anti-tumor immunity against antigens of the immune checkpoint regulatory protein PD-L1. These data paved the way for advancing the development of VXM10 into clinical development.

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 Background: In a first in men, proof-of-principle, multi-center, open-label, single-arm, non-randomized phase 1/2 clinical ASET trial (ClinicalTrials.gov: NCT01265368; EudraCT No.: 2009-016853-16) in patients with advanced RCC the tumor vaccine MGN1601 has shown a good safety profile and improved overall survival in the per-protocol (PP) treated patient population. MGN1601 is a cell-based tumor vaccine consisting of two active pharmaceutical ingredients: Allogeneic (human) cells originating from renal cell carcinoma were gene-modified with four different MIDGE vectors encoding IL-7, GM-CSF, CD80 and CD154. These vaccine cells were combined with the synthetic DNA-based immunomodulator dSLIM®, a potent TLR-9 agonist. Methods: The tumor vaccine MGN1601 – each dose consists of 107 vaccine cells and 5 mg dSLIM® – was administered 8 times over 12 weeks with 3 weekly and 5 bi-weekly administrations (PP population) in the ASET trial. Specific humoral immune responses in sera from patients before and after treatment with MGN1601 against a natural set of 17 known tumor associated antigens (TAA) expressed by the vaccine cells were evaluated ex-vivo in a translational research program using peptide arrays. On the array each TAA was represented by peptides of 15 amino acids length with an overlap of 13 amino acids. Results: Nineteen heavily pre-treated patients with advanced RCC have been included into the trial. In the PP population – comprising 10 of these 19 patients – the humoral immune response against TAA expressed on vaccine cells was evaluated. Within each antigen subsets of overlapping peptides were recognized by patient's sera with an abundant or clearly increased intensity after MGN1601 treatment indicating favoured immunogenic regions within a TAA for an individual immune response. Such sets of peptides were identified within each antigen showing individual responses by sera from 2 to eight patients. Remarkably, some of these overlapping peptides were recognized by sera of more than one patient (“shared immunogenic regions”): In particular, 50 to 70 % of treated patients shared response to immunogenic regions within Survivin, Stromelysin, c-Myc, Histone H1.2 and G1/S-specific Cyclin-D1 indicating their comprehensive immunogenic potential. Within other TAA (e.g. p53, telomerase) shared immune responses to peptides were observed at lower rates (20 to 40% of patients), whereas for some of the antigens (e.g. MUC1, Her2/neu) only individual immune responses to the peptides were identified. Conclusions: The humoral immune response – assessed in patients treated with the tumor vaccine MGN1601 – revealed overlapping peptides within various TAA that induced an immune response shared by up to 7 of 10 patients, indicating comprehensive immunogenic regions within those TAA and their potential use as vaccine epitopes. The specific evaluation of immune response to those TAA remains to further studies. Citation Format: Manuel Schmidt, Barbara Volz, Kerstin Kapp, Viktor Gruenwald, Matthias Schroff, Burghardt Wittig. Immune responses against tumor associated antigens are induced by treatment with the cell-based tumor vaccine MGN1601 in patients with renal cell carcinoma. [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 A034.

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 immune responses. So far, two different families of DNA molecules containing non-methylated CG-motifs for TLR9 activation have been established: 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. PTO modification, however, produce off-target effects in immune cell populations and have resulted in an unfavorable risk-to-benefit ratio. Methods To avoid the off-target effects of PTO-modified CpG-ODN, linear single-stranded ODN were synthezised using L-deoxyribonucleotides at their 3'-ends, which are the natural enantiomers of Ddeoxyribonucleotides. 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. We selected nucleotide sequences of such L-protected, CG-motif containing, single-stranded ODN, EnanDIM(R), for high secretion of IFN-alpha and IP-10 from human peripheral blood mononuclear cells (PBMC). In a maximum feasible dose approach in CD-1 mice EnanDIM(R) doses of 10 to 50 mg per mice were injected subcutaneously to evaluate the acute toxicity and immunomodulatory properties of EnanDIM(R) molecules in vivo. Results EnanDIM581 and EnanDIM532 were chosen since they caused high secretion of IFN-alpha and IP-10 from human PBMC and resulted in a strong activation of monocytes, NK cells and plasmacytoid dendritic cells (pDC) in vitro. Notably, both showed a distinct immune activation pattern, with the highest secretion of IFN-alpha by EnanDIM581 and the strongest maturation of TLR9-bearing pDC by EnanDIM532. EnanDIM744, comprising EnanDIM581 with additional 5'-end L-nucleotide protection and exhibiting an immune activation pattern similar to EnanDIM581, was selected as third EnanDIM(R) for in vivo studies. In the maximum feasible dose approach, safety assessments were performed throughout the study period and no mortality, clinical signs and body weight changes were observed, despite the fact that extremely high doses of app. 300 to 1700 mg/kg were used. A gross necropsy consisting of a macroscopic organ evaluation at day 15 revealed also no toxicity. Regarding immune activation, increased levels of IP-10 in serum were observed 24 hours after injection but not after 15 days confirming that L-nucleotides in EnanDIM(R) do not change the kinetic profile known from other DNA-based TLR9 agonists. Conclusions EnanDIM®, a new family of TLR9 agonists with conformation-mediated nuclease-resistance, broadly activates the immune system in vitro. Maximal feasible doses of EnanDIM(R) resulted in no signs of toxicity and confirmed immunomodulatory effects in vivo. Therefore EnanDIM(R) has the potential for clinical development in the treatment of cancer.

INTRODUCTION:DNA-based TLR9 agonists are potent activators of the immune system. ProMune® and dSLIM® belong to different families of TLR9 agonists and both have been established as cancer immunotherapeutics in clinical proof-of-concept studies. Unfortunately, ProMune® failed in pivotal oncological trials. dSLIM®, the active ingredient of Lefitolimod (MGN1703), successfully finished a double-blinded, placebo-controlled phase II study in patients with advanced colorectal cancer, exhibiting improved progression-free survival and durable disease control. METHODS:To explain the different systemic efficacies of dSLIM® and ProMune®, both TLR9 agonists and chimeric molecules thereof are analyzed side-by-side in a panel of in vitro assays for immune activation. RESULTS AND CONCLUSIONS:Indeed, dSLIM® exposure results in an IFN-α dependent broad activation of immune cells whereas ProMune® strongly stimulates B cells. Moreover, all functional effects of dSLIM® strictly depend on the presence of CG-motifs within its dumbbell-shaped, covalently closed structural context. Conversely, several immunological effects of ProMune® like IL-8 secretion are independent of CG-motifs and could be ascribed to the phosphorothioate-modifications of its DNA backbone, which may have caused the side effects of ProMune® in clinical trials. Finally, we showed that the implementation of ProMune® (ODN2006) base sequence into the characteristic dSLIM® dumbbell form resulted in dSLIM2006 with all beneficial effects for immunostimulation combined from both TLR9 classes without any CG-independent effects.

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.

Single-stranded oligodeoxynucleotides (ODN), containing nonmethylated cytosine-guanine motifs (CpG ODN), are recognized by the innate immune system as "danger signals." CpG ODN are efficacious immunomodulators but require phosphorothioate (PT) or other backbone modifications for metabolic stability, which cause toxicities in mice and primates. We therefore designed a covalently closed DNA molecule (dSLIM(®)) where two single-stranded loops containing CG motifs are connected through a double-stranded stem in the absence of any nonnatural DNA component. The most promising immunomodulator, MGN1703, comprises two loops of 30 nucleotides containing three CG motifs each, and a connecting stem stem of 28 base pairs. MGN1703 stimulates cytokine secretion [interferon (IFN)-α, IFN-γ, interleukin (IL)-12, IL-6, and IL-2] and activates immune cells by increased expression of CD80, CD40, human leukocyte antigen (HLA)-DR and ICAM-1. Efficacy of immunomodulation strictly depends on the descriptive dumbbell shape and size of the molecule. Variations in stem length and loop size lead to reduced potency of the respective members of the dSLIM(®) class. In a representative mouse model, toxicities from injections of high amounts of a CpG ODN-PT and of MGN1703 were evaluated. The CpG ODN-PT group showed severe organ damage, whereas no such or other pathologies were found in the MGN1703 group. Oncological clinical trials of MGN1703 already confirmed our design.

Aim: The synthetic DNA-based immunomodulator MGN1703 is a potent TLR-9 agonist, activating the innate and adaptive immune system. MGN1703 has already shown a good safety profile and efficacy compared to placebo in patients receiving maintenance therapy for metastatic colorectal carcinoma (mCRC) in a randomised trial (MGN1703-C02, IMPACT). Methods: A single dose crossover, placebo-controlled phase 1 trial (MGN1703-C04) to assess cardiac and general safety, pharmacokinetics (PK) and pharmacodynamics (PD) was conducted in healthy volunteers (HV). Fourteen HV were randomized to 2 groups and 13 subjects completed the study receiving a single subcutaneous (sc) dose of 60 mg MGN1703 during one treatment period and a single sc dose of placebo during the other. Results: Seven of 13 HV experienced 21 adverse events (AE) after receiving MGN1703 (6 with 16 AE possibly or probably related); 6 of 14 HV experienced 11 AE after receiving placebo (3 with 5 AE possibly or probably related). All AE were mild or moderate in severity. No SAE was reported and no subject discontinued the study due to an AE. Changes in ECG numerical data were small and similar in both MGN1703 and placebo groups. The changes observed were not clinically significant and consistent with spontaneous variability and circadian change. Coagulation test results were within normal levels from screening throughout the study. There were no laboratory abnormalities reported as AE. The PK analysis of MGN1703 determined a mean Cmax of 189 ng/mL, a mean AUC0-t of 5001 ng•hr/mL, a median Tmax of 14 hours and a median t1/2 of 12.7 hours, confirming single dose data from cancer patients. All healthy volunteers had an increase in IP-10 concentration in serum app. 24 hours after injection of MGN1703. This is in line with expected time frame of immune activation subsequent to MGN1703 administration and with data from cancer patients. Conclusions: The data confirm the good safety profile of MGN1703 and the PK/PD data from cancer patients. This supports the twice weekly application scheme of immunotherapy studies with MGN1703, e.g. the ongoing phase 3 IMPALA trial in mCRC patients. Clinical trial identification: NCT01982747 Disclosure: A. Zurlo and M. Schroff: Are board members of Mologen AG. M. Schmidt, A. Dax and K. Kapp: Are employees of Mologen AG. B. Wittig: Is an advisor and shareholder of Mologen AG.

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. In the ASET trial, MGN1601 – containing 10e7 gene-modified vaccine cells and 5 mg dSLIM® – was administered 8 times over 12 weeks with 3 weekly and 5 bi-weekly administrations (PP population). Specific immune responses of sera from patients before and after treatment with MGN1601 against a natural set of 17 known tumor associated antigens (TAA) expressed by the vaccine cells were evaluated ex-vivo in a translational research program using peptide arrays. Each TAA was represented on the array by overlapping peptides of 15 amino acids length with an overlap of 13 amino acids. The PP population comprised 10 of 19 heavily pre-treated patients with advanced RCC and of these the humoral immune response against TAA expressed on vaccine cells was evaluated. Within each antigen subsets of overlapping peptides were recognized by patient's sera with an abundant or clearly increased intensity after MGN1601 treatment indicating favoured immunogenic regions for an individual immune response. Such sets of peptides were identified within each antigen showing individual responses by sera from 2 to eight patients. Remarkably, some of these overlapping peptides were recognized by sera of more than one patient ("shared immunogenic regions"): In particular, 50 to 70% of treated patients shared response to immunogenic regions within survivin, stromelysin, c-myc, histone H1.2 and G1/S-specific cyclin-D1 indicating their comprehensive immunogenic potential. Within other TAA (e.g. p53, telomerase) shared immune responses to peptides were observed at lower rates (20 to 40% of patients), whereas within some of the antigens (e.g. MUC1, WT-1) no overlapping peptides recognized by sera of more than one patient were identified. The humoral immune response – assessed in patients treated with the tumor vaccine MGN1601 – revealed overlapping peptides within various TAA that induced an immune response shared by up to 7 of 10 patients, indicating comprehensive immunogenic regions within those TAA and their potential use as vaccine epitopes. The specific evaluation of immune response to those TAA remains to further studies.

Background MGN1703 is a synthetic DNA-based TLR-9 agonist which has shown a good safety profile and a benefit compared to placebo in maintenance therapy of patients with metastatic colorectal carcinoma. Methods In addition to the phase 1 and 2 trials with cancer patients a single dose crossover placebo-controlled phase 1 trial, MGN1703-C04, was performed to assess safety, pharmacokinetics (PK) and pharmacodynamics in healthy volunteers (HV). Results Standard PK parameters like mean Tmax (14 h), AUC0-t (5001 ng h/mL) and shape of the curve in the MGN1703-C04 trial were similar to single dose data obtained from cancer patients, with mean Tmax of 10 h and AUC0-t of 5336 ng h/mL. Notably, multiple doses resulted in similar levels without accumulation of MGN1703. In MGN1703-C04 elevated IP-10 levels were detected in the serum of HV peaking at 24–48 h after dosing with MGN1703. The elevation of IP-10 is in keeping with data from cancer patients, which additionally showed activation of innate immune cells after dosing with MGN1703, e.g. up-regulation of CD169 on blood monocytes. Conclusions The data show similarities between HV and cancer patients with respect to PK and – following-up downstream – the immunomodulatory profile, supporting the twice weekly application scheme of ongoing cancer studies. Further data will allow evaluation of possible association with clinical parameters. MGN1703 is a synthetic DNA-based TLR-9 agonist which has shown a good safety profile and a benefit compared to placebo in maintenance therapy of patients with metastatic colorectal carcinoma. In addition to the phase 1 and 2 trials with cancer patients a single dose crossover placebo-controlled phase 1 trial, MGN1703-C04, was performed to assess safety, pharmacokinetics (PK) and pharmacodynamics in healthy volunteers (HV). Standard PK parameters like mean Tmax (14 h), AUC0-t (5001 ng h/mL) and shape of the curve in the MGN1703-C04 trial were similar to single dose data obtained from cancer patients, with mean Tmax of 10 h and AUC0-t of 5336 ng h/mL. Notably, multiple doses resulted in similar levels without accumulation of MGN1703. In MGN1703-C04 elevated IP-10 levels were detected in the serum of HV peaking at 24–48 h after dosing with MGN1703. The elevation of IP-10 is in keeping with data from cancer patients, which additionally showed activation of innate immune cells after dosing with MGN1703, e.g. up-regulation of CD169 on blood monocytes. The data show similarities between HV and cancer patients with respect to PK and – following-up downstream – the immunomodulatory profile, supporting the twice weekly application scheme of ongoing cancer studies. Further data will allow evaluation of possible association with clinical parameters.

e14015 Background: The synthetic DNA-based immunomodulator MGN1703 acts as TLR-9 agonist by activating the innate and adaptive immune system. MGN1703 has already shown a good safety profile and a benefit compared to placebo in maintenance therapy of patients with metastatic colorectal carcinoma (mCRC) in a phase 2 clinical trial (MGN1703-C02, IMPACT). Methods: In addition to preclinical studies, a phase 1 trial (MGN1703-C01) including patients with solid tumors and the phase 2 IMPACT trial with mCRC patients, a single dose crossover placebo-controlled phase 1 trial, MGN1703-C04, was performed 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: Standard PK parameters like mean Tmax (14h), AUC0-t (5001 ng•hr/mL) and shape of the curve in the MGN1703-C04 trial were similar to single dose data obtained from cancer patients, with a mean Tmax of 10h and a mean AUC0-t of 5336 ng•hr/mL. Notably, multiple doses resulted in similar levels, but no accumulation of MGN1703. PD was mainly evaluated analyzing levels of the chemokine IP-10 in serum of the HV representing activation of the immune system. In MGN1703-C04 elevated IP-10 levels were detected in the serum of HV peaking at 24-48 hours after dosing with MGN1703. This clearly shows an expected delayed immune activation with respect to appearance of MGN1703 in the serum of the HV. The elevation of IP-10 is in keeping with data from cancer patients, in which an additional activation of innate immune cells – especially the up-regulation of CD169 on monocytes – after dosing with MGN1703 was observed. Conclusions: The data show obvious similarities between HV and cancer patients with respect to PK and PD – with the appearance of the immunomodulatory profile following-up downstream. This supports the twice weekly application scheme of ongoing cancer studies with MGN1703, e.g. the phase 3 IMPALA trial in mCRC patients. Further data will allow for evaluation of a possible association with clinical parameters. Clinical trial information: NCT01982747.

The leishmaniases are a complex of vector-borne diseases caused by protozoan parasites of the genus Leishmania. LEISHDNAVAX is a multi-antigen, T-cell epitope-enriched DNA vaccine candidate against human leishmaniasis. The vaccine candidate has been proven immunogenic and showed prophylactic efficacy in preclinical studies. Here, we describe the safety testing of LEISHDNAVAX in naive mice and rats, complemented by the demonstration of tolerability in Leishmania-infected mice. Biodistribution and persistence were examined following single and repeated intradermal (i.d.) administration to rats. DNA vectors were distributed systemically but did not accumulate upon repeated injections. Although vector DNA was cleared from most other tissues within 60 days after the last injection, it persisted in skin at the site of injection and in draining lymph nodes. Evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after i. d. administration to naive, non-infected mice did not reveal any safety concerns. LEISHDNAVAX was also well tolerated in Leishmania-infected mice. Taken together, our results substantiate a favorable safety profile of LEISHDNAVAX in both naive and infected animals and thus, support the initiation of clinical trials for both preventive and therapeutic applications of the vaccine.

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.

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.

Aim: Allogeneic tumor cells gene-modified via MIDGE® vectors to express IL-7, GM-CSF, CD80, and CD154, immunomodulated with dSLIM® constitute the therapeutic cancer vaccine MGN1601, whose safety, immunological effects, and clinical efficacy were assessed in heavily pre-treated patients with RCC.