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Glioblastoma is the most common and aggressive malignant tumor of the central nervous system. Despite the existing high unmet medical needs, the past few decades have seen no notable improvement in overall survival for glioblastoma patients. One active area of research to develop new therapeutic options for this disease is focusing on the CD95/Fas receptor and its ligand CD95L/FasL. It is now recognized that in addition to its role in programmed cell death, CD95/CD95L signaling is involved in a wide range of other apoptotic and non-apoptotic pathways directed toward T-effector cells and cells in the tumor microenvironment involved in tumor progression and invasiveness. Asunercept is a first-in-class recombinant glycosylated fusion protein, which has been designed to selectively bind to CD95L and therefore disrupt CD95/CD95L signaling. The current report provides a brief overview of the role of the CD95/CD95L signaling pathway in cancer pathogenesis and discusses how asunercept was designed to bind and neutralize CD95L and disrupt signaling thereby potentially improving outcomes in glioblastoma and other malignancies.

Background Patients with glioblastoma without O6-methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation are unlikely to benefit from alkylating chemotherapy with temozolomide (TMZ). Trials aiming at replacing TMZ with targeted agents in unselected patient populations have failed to demonstrate any improvement of survival. Advances in molecular understanding and diagnostic precision enable identification of key genetic alterations in a timely manner and in principle allow treatments with targeted compounds based on molecular markers. Methods The NCT Neuro Master Match (N2M2) trial is an open-label, multicenter, phase I/IIa umbrella trial for patients with newly diagnosed isocitrate dehydrogenase (IDH) wildtype glioblastoma without MGMT promoter hypermethylation to show safety, feasibility, and preliminary efficacy of treatment with targeted compounds in addition to standard radiotherapy based on molecular characterization. N2M2 is formally divided into a Discovery and a Treatment part. Discovery includes broad molecular neuropathological diagnostics to detect predefined biomarkers for targeted treatments. Molecular diagnostics and bioinformatic evaluation are performed within 4 weeks, allowing a timely initiation of postoperative treatment. Stratification for Treatment takes place in 5 subtrials, including alectinib, idasanutlin, palbociclib, vismodegib, and temsirolimus as targeted therapies, according to the best matching molecular alteration. Patients without matching alterations are randomized between subtrials without strong biomarkers using atezolizumab and asinercept (APG101) and the standard of care, TMZ. For the phase I parts, a Bayesian criterion is used for continuous monitoring of toxicity. In the phase II trials, progression-free survival at 6 months is used as endpoint for efficacy. Results Molecular diagnostics and bioinformatic evaluation are performed within 4 weeks, allowing a timely initiation of postoperative treatment. Stratification for Treatment takes place in 5 subtrials, including alectinib, idasanutlin, palbociclib, vismodegib, and temsirolimus as targeted therapies, according to the best matching molecular alteration. Patients without matching alterations are randomized between subtrials without strong biomarkers using atezolizumab and asinercept (APG101) and the standard of care, TMZ. For the phase I parts, a Bayesian criterion is used for continuous monitoring of toxicity. In the phase II trials, progression-free survival at 6 months is used as endpoint for efficacy. Discussion Molecularly informed trials may provide the basis for the development of predictive biomarkers and help to understand and select patient subgroups who will benefit.

In low risk MDS, increased apoptosis of erythroid progenitors mediated via CD95 (Fas) activation has been described to result in peripheral cytopenia. Blockade of the CD95 system can improve erythropoiesis in MDS. Asunercept (APG101) is a fusion protein consisting of the extracellular domain of human CD95 and the Fc domain of human IgG1 blocking the interaction between CD95 and its ligand. Here we report on results from a phase I study in 20 transfusion-dependent low and intermediate risk MDS patients treated with intravenous asunercept (EudraCT 2012-003027-37). Primary objectives were safety and tolerability as well as pharmacodynamic effects. Secondary objectives were hematologic improvement, incidence and time to leukemic progression as well as overall survival. Frequency and severity of adverse events were in range of what could be expected in a patient cohort comprising of elderly MDS patients. Two patients experienced a serious adverse event with a suspected relationship to asunercept. The incidence of disease progression was low. In the 20 patients a decrease of the transfusion need from a mean of 10,8 (±5,1) pRBCs during the 12 weeks treatment phase to a mean of 10,0 (±4,2) pRBCs at the end of the study was observed. In conclusion, asunercept was well tolerated and showed efficacy in transfusion-dependent low and intermediate risk MDS patients. Further clinical investigation is warranted, particularly in combination with erythropoiesis stimulating agents (ESAs).

CD95 (Fas/APO-1), a death receptor family member, activity has been linked to tumorigenicity in multiple cancers, including glioblastoma multiforme (GBM). A phase II clinical trial on relapsed glioblastoma patients demonstrated that targeted inhibition of CD95 signaling via the CD95 ligand (CD95L) binding and neutralizing Fc-fusion protein APG101 (asunercept) prolonged patient survival. Although CD95 signaling may be relevant for multiple aspects of tumor growth, the mechanism of action of APG101 in glioblastoma is not clear. APG101 action was examined by in vitro proliferation, apoptosis, and invasion assays with human and murine glioma and human microglial cells, as well as in vivo therapy studies with orthotopic gliomas and clinical data. APG101 inhibits CD95L-mediated invasion of glioma cells. APG101 treatment was effective in glioma-bearing mice, independently of the presence or absence of CD4 and CD8 T lymphocytes, which should be sensitive to CD95L. Combined with radiotherapy, APG101 demonstrated a reduction of tumor growth, fewer tumor satellites, reduced activity of matrix metalloproteinases (MMP) as well as prolonged survival of tumor-bearing mice compared with radiotherapy alone. Inhibiting rather than inducing CD95 activity is a break-of-paradigm therapeutic approach for malignant gliomas. Evidence, both in vitro and in vivo, is provided that CD95L-binding fusion protein treatment enhanced the efficacy of radiotherapy and reduced unwanted proinfiltrative effects by reducing metalloproteinase activity by directly affecting the tumor cells. Implications: APG101 (asunercept) successfully used in a controlled phase II glioblastoma trial (NCT01071837) acts anti-invasively by inhibiting matrix metalloproteinase signaling, resulting in additive effects together with radiotherapy and helping to further develop a treatment for this devastating disease. Mol Cancer Res; 16(5); 767–76. ©2018 AACR.

Asunercept (APG101) is a well-tolerated CD95-ligand inhibitor that showed promising efficacy in a prospective, single-arm phase I study in anemic, transfusion-dependent patients with low and intermediate risk myelodysplastic syndrome (MDS). In this retrospective post hoc analysis, serum levels of biomarkers were measured in study patients focusing on cytokines associated with erythropoiesis, inflammation, apoptosis, bone marrow fibrosis, and inflammasome activity. Baseline serum biomarkers were correlated with treatment response, in order to propose a hypothetical responder serum profile. After an updated median follow-up of 54 months (range 7&ndash, 65), response to asunercept was associated with improved overall survival (at 3-years: 67% [95%CI 36&ndash, 97] versus 13% [95%CI 0&ndash, 36] in responders versus non-responders, respectively). Higher baseline values of interleukin-18 (IL-18), S100 calcium-binding protein A9 (S100A9) and soluble p53 were predictive of non-response to asunercept (area under the receiver operating characteristic curve 0.79&ndash, 0.82). Furthermore, non-responding patients showed a distinct, pro-inflammatory serum cytokine profile which was persistent throughout the first half of the treatment phase and appeared unaffected by asunercept. Although prospective validation is required, our post hoc analysis suggests that serum cytokine profiling based on IL-18, S100A9 and soluble p53 may represent an approach to identify and select low-risk MDS patients most likely to benefit from asunercept treatment.

The CD95 ligand (CD95L) is frequently overexpressed in cancers and tumor-associated endothelia, but also other immune cells like MDSCs and Tregs. Binding of CD95L expressed on tumors or regulatory immune cells to activated CD95-expressing effector cells triggers activation-induced apoptosis (AICD) or impairs their proliferation. In contrast, most tumors do also express CD95, but are intrinsically resistant to CD95-induced apoptosis. Thus, CD95L in the tumor microenvironment greatly contributes to the observed immunosuppression and escape from tumor surveillance by the immune system, making CD95 a potential immune checkpoint. Asunercept (APG101) is a fully human fusion protein consisting of the extracellular domain of CD95 and the Fc-domain of human IgG1, which efficiently inhibits CD95/CD95L signaling. Clinical efficacy has been demonstrated in a controlled randomized phase 2 study in patients with recurrent glioblastoma. Here we examined the effects of APG101 on innate and adaptive immune cells and subsequent effects on tumor cell killing. Subtypes of in vitro differentiated macrophages generated with and without exposure to APG101 were functionally and phenotypically analyzed by ELISA and multi-color flow cytometry following various stimuli. APG101 did not alter differentiation patterns and response of M1- and M2-like macrophages in vitro. Direct co-culture of monocytes with tumor cells resulted in an M2/TAM-like phenotype which was not influenced by APG101, but re-programming to an M1-like state was achieved by addition of a CD40 agonist. Effects of APG101 on the proliferation and activation of CD8+ T cells in the presence of autologous CD4+ T(reg) cells and allogeneic APC was assessed by CFSE-dilution and multi-color flow cytometry, respectively. The proliferation rate of CD8 T cells in co-cultures with CD4 T(reg) cells in response to stimulation with APCs was increased in the presence of APG101. Real-time cell analysis was performed employing direct co-cultures of activated T cells and tumor cell lines. Tumor killing assays using direct co-cultures of in vitro activated T cells with and without APG101 demonstrate that tumor killing was not impaired by APG101. Conclusion: Asunercept (APG101) is a potent inhibitor of pro-apoptotic/anti-proliferative CD95/CD95L signaling in immune cells and protects activated immune cells from activation induced cell death (AICD). Our results suggest that APG101 does not impair CD8 T cell activation, but rather supports their proliferation by disrupting CD95/CD95L interaction with regulatory T cells. Importantly, the primary anti-tumor killing mechanisms is most likely CD95L-independent and remains unaffected by the presence of APG101. The inhibition of CD95 signaling as an immune checkpoint represents an attractive and novel concept for immunologic treatment of tumors and the combination of Asunercept/APG101 with co-stimulatory TNFR-SF agonists is currently being investigated. Citation Format: Christian Merz, Jaromir Sykora, Rebecca Hussong, Meinolf Thiemann, Oliver Hill, Christian Gieffers. Neutralization of pro-apoptotic CD95L by Asunercept/APG101 does not impair anti-tumor immune responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4529.

Supplemental Digital Content is available in the text., Glioblastoma is a disease characterized by rapid invasive tumour growth. Studies on the proapoptotic CD95/CD95L signalling pathway recently suggested a significant contribution of CD95 signalling towards the high degree of motility in glioma cells. Apogenix has developed APG101, a clinical phase II compound designed to bind and neutralize CD95L, and thus to interfere with CD95/CD95L-based signalling. APG101 has shown clinical efficacy in a controlled randomized phase II trial in patients with recurrent glioma. Because APG101 is not cytotoxic to tumour cells in vitro, we postulated that the anti-invasive function of APG101 is the main mechanism of action for this compound. Using three-dimensional spheroid invasion assays in vitro and in murine brain tissue cultures, we found that knockdown of endogenous CD95L reduced the invasive phenotype in our two glioblastoma model cell lines U87-MG and U251-MG. Invasion was restored in CD95L knockdown cells upon the addition of soluble recombinant CD95L and this effect was inhibited by APG101. We conclude that CD95L from autocrine and paracrine sources contributes towards the invasive phenotype of glioblastoma cells and that APG101 acts as a suppressor of proinvasive signalling by the CD95/CD95L pathway in glioblastoma.

Insights into the molecular pathogenesis of glioblastoma have not yet resulted in relevant clinical improvement. With standard therapy, which consists of surgical resection with concomitant temozolomide in addition to radiotherapy followed by adjuvant temozolomide, the median duration of survival is 12–14 months. Therefore, the identification of novel molecular targets and inhibitory agents has become a focus of research for glioblastoma treatment. Recent results of bevacizumab may represent a proof of principle that treatment with targeted agents can result in clinical benefits for patients with glioblastoma. This review discusses limitations in the existing therapy for glioblastoma and provides an overview of current efforts to identify molecular targets using large-scale screening of glioblastoma cell lines and tumor samples. We discuss preclinical and clinical data for several novel molecular targets, including growth factor receptors, phosphatidylinositol-3 kinase, SRC-family kinases, integrins, and CD95 ligand and agents that inhibit these targets, including erlotinib, enzastaurin, dasatinib, sorafenib, cilengitide, AMG102, and APG101. By combining advances in tumor screening with novel targeted therapies, it is hoped that new treatment options will emerge for this challenging tumor type.

// Anna Raimbault 1, 2, 3, 4, 5, * , Cecile Pierre-Eugene 1, * , Alexandra Rouquette 6 , Celine Deudon 1 , Lise Willems 7 , Nicolas Chapuis 1, 2, 3, 4, 5 , Stephanie Mathis 1, 2, 3, 4, 5 , Claudia Kunz 8 , Harald Fricke 8 , Olivier Kosmider 1, 2, 3, 4, 5 , Valerie Bardet 1, 2, 3, 4, 5 , Michaela Fontenay 1, 2, 3, 4, 5 , on behalf of the Groupe Francophone des Myelodysplasies 1 Assistance Publique-Hopitaux de Paris, Service d’Hematologie Biologique, Hopitaux Universitaires Paris Centre, Hopital Cochin, Paris, France 2 Universite Paris Descartes, Faculte de Medecine, Paris, France 3 Institut National de la Sante et de la Recherche Medicale (INSERM) U1016, Paris, France 4 Centre National de la Recherche Scientifique, Unite Mixte de Recherche 8104, Paris, France 5 Institut Cochin, Department of Development, Reproduction and Cancer, Paris, France 6 Departement d’Epidemiologie et de Biostatistiques, Hopitaux Universitaires Paris Centre, Paris, France 7 Assistance Publique-Hopitaux de Paris, Service d’Hematologie Clinique, Hopitaux Universitaires Paris Centre, Hopital Cochin, Paris, France 8 APOGENIX, GmbH, Heidelberg, Germany * These authors have contributed equally to this work Correspondence to: Michaela Fontenay, e-mail: michaela.fontenay@inserm.fr Keywords: myelodysplastic syndromes, erythropoiesis, CD95, CD95 ligand, anemia Received: September 07, 2015 Accepted: January 02, 2016 Published: February 18, 2016 ABSTRACT CD95, a member of the death receptor family initiates a caspase-dependent apoptosis, when activated by its ligand CD95L, thought to negatively regulate erythrocyte production in the bone marrow. We have previously shown that CD95 is overexpressed in two thirds of patients with a lower risk myelodysplastic syndrome (MDS) and that resistance to erythropoiesis-stimulating agents (ESA) is linked to poor residual erythropoiesis. In the present study, we show that CD95 overexpression and previous transfusion are independent predictive factors of ESA resistance. To investigate an alternative therapeutic strategy of anemia in ESA-resistant patients, we have conducted a preclinical study of the effects of APG101, a fusion protein consisting of the extracellular domain of human CD95 and the Fc region of human IgG1 on MDS erythropoiesis in vitro . APG101 increases the number of burst-forming unit-erythroid (BFU-E) progenitors derived from CD34 + progenitors in liquid culture and improves overall proliferation rate of erythroid precursors by inhibiting apoptosis. APG101 rescues BFU-E growth in MDS patients presenting with attrition of erythroid progenitors at baseline, independently of CD95 or CD95L expression level. Our data show that overexpression of CD95 at diagnosis is a hallmark of ESA resistance and that severe impairment of erythropoiesis is predictive of erythroid response to APG101 in vitro . These data provide a rationale for further clinical investigation of APG101 in an attempt to treat anemia in lower risk MDS patients.

Purpose: Preclinical data indicate anti-invasive activity of APG101, a CD95 ligand (CD95L)–binding fusion protein, in glioblastoma. Experimental Design: Patients (N = 91) with glioblastoma at first or second progression were randomized 1:2 between second radiotherapy (rRT; 36 Gy; five times 2 Gy per week) or rRT+APG101 (400 mg weekly i.v.). Patient characteristics [N = 84 (26 patients rRT, 58 patients rRT+APG101)] were balanced. Results: Progression-free survival at 6 months (PFS-6) rates were 3.8% [95% confidence interval (CI), 0.1–19.6] for rRT and 20.7% (95% CI, 11.2–33.4) for rRT+APG101 (P = 0.048). Median PFS was 2.5 (95% CI, 2.3–3.8) months and 4.5 (95% CI, 3.7–5.4) months with a hazard ratio (HR) of 0.49 (95% CI, 0.27–0.88; P = 0.0162) adjusted for tumor size. Cox regression analysis adjusted for tumor size revealed a HR of 0.60 (95% CI, 0.36–1.01; P = 0.0559) for rRT+APG101 for death of any cause. Lower methylation levels at CpG2 in the CD95L promoter in the tumor conferred a stronger risk reduction (HR, 0.19; 95% CI, 0.06–0.58) for treatment with APG101, suggesting a potential biomarker. Conclusions: CD95 pathway inhibition in combination with rRT is an innovative concept with clinical efficacy. It warrants further clinical development. CD95L promoter methylation in the tumor may be developed as a biomarker. Clin Cancer Res; 20(24); 6304–13. ©2014 AACR.

Introduction:Particularly in low risk MDS, a pro-apoptotic milieu has been described with increased levels of apoptosis-promoting factors such as tumor necrosis factor and fas ligand (CD95 ligand). Evidence exists that in low risk MDS increased apoptosis of erythroid progenitors mediated via CD95 activation results in peripheral cytopenia. APG101 is a fusion protein consisting of the extracellular domain of human CD95 and the Fc domain of human IgG1. APG101 binds to CD95 ligand on effector cells as well as to soluble ligand, thus blocking the interaction between CD95 and its ligand. Here we report on results of a phase I study in transfusion-dependent low risk MDS patients treated with APG101. Primary objectives were safety and tolerability of APG101. Secondary objectives were hematologic, cytologic and cytogenetic response rates using modified International Working Group (IWG) response criteria (Cheson et al., 2006), incidence and time to leukemic progression as well as overall survival (OS) at 37 weeks. Furthermore, this study was focused on the depiction of molecular and functional changes underlying the stem cell defect in MDS including age related changes and high-throughput molecular profiling to evaluate the biological effect of APG101 in patients with lower risk MDS. Methods: The study was performed according to ICH-GCP guidelines and the Declaration of Helsinki as an open-label, non-randomized study for up to 37 weeks including an initial 12 weeks treatment phase with APG101 and a follow-up period of 25 weeks. 400 mg of APG101 were given every week as an intravenous (i.v.) infusion to the first 6 patients, 100 mg APG101 were given to all further patients. Bone marrow aspirates were obtained during the screening period (baseline), at the end of treatment (EOT, week 13), at week 25 (follow up) and at week 37 (End of Study) with subsequent comprehensive cytologic, cytogenetic, molecular and immunophenotypic analyses. In order to track clonal development and heterogeneity, methylcellulose assays on isolated CD34+cells from bone marrow aspirates at the different time points were performed and subjected to amplicon deep sequencing to detect gene mutations prior to APG101 dosing and to follow the mutational allele burden during and after treatment. Results: A total of 29 patients were screened for this study. Twenty patients received at least one dose of APG101, 17 patients completed the treatment phase and 15 patients completed the entire study including the follow-up phase. Median age was 74.5 years (range 56-82). WPSS categories were low in 19 patients and intermediate in 1 patient, respectively. Except for two patients with RCMD-RS and RARS, all others were classified as RCMD (WHO 2008). According to the exclusion criteria, none of the patients showed a medullary blast count ≥ 5%. Quantification of the allele burden of pre-existing mutations (mutations of ASXL1, SF3B1, TTBK, del(ETV6) and IDH2, SRSF2, SPEG2, BRCC3, NF1) showed no expansion of the mutated clones during the course of treatment. One patient, however, was reported with leukemic progression and AML transformation. One patient died from sepsis due to pre-existing neutropenia. Ex vivo differentiation analyses revealed an increase in CFU-E/BFU-E forming capacity. With regard to efficacy, an improvement in Hb level was observed in 6 patients at various assessment periods in this study. In 8 patients, a decrease in transfusion frequency from treatment until EOS was observed. Conclusions: APG101 is a very well tolerable compound in transfusion-dependent lower risk MDS patients comprising mostly of elderly patients. Risk of AML progression was not elevated. Deep amplicon sequencing did not show any signs of expansion of preexisting malignant clones during therapy, i.e. APG101 did not provide these clones with a survival benefit. Analysis of efficacy as secondary objective revealed that APG101 has the potential to improve hematopoiesis in MDS patients. Therefore, APG101 is an interesting compound for further clinical studies. Disclosures Fricke: Apogenix AG: Employment. Kunz:Apogenix AG: Employment.

CD95 (APO-1/Fas) is a member of the Tumor Necrosis Factor Receptor Super Family. Binding of CD95-ligand (CD95L) to CD95 triggers intracellular signal transduction that is critically involved in the invasive growth of glioblastoma cells. Invasion of malignant glioblastoma cells into the brain parenchyma is responsible for poor therapeutic outcome of currently available treatments. The inhibition of CD95/CD95L mediated invasive growth of glioblastoma cells represents an attractive novel therapeutic concept. Apogenix has developed APG101, a fully human fusion protein consisting of the extracellular domain of CD95 and the Fc-domain of an IgG. APG101 has been confirmed as a potent inhibitor of CD95L induced invasion of glioblastoma cells in vitro. In a randomized phase 2 study in glioblastoma patients with 1st or 2nd relapse the combined therapy of APG101 plus radiotherapy (RT) was found to be superior to RT alone in a clinically relevant order of magnitude in all efficacy endpoints (i.e. PFS-6, PFS and OS). At the same time APG101 exhibited an excellent safety profile and was well tolerated. To identify potential biomarkers we used available tissue sections originating from archived primary tumor of the study patients and analyzed them for the expression of CD95L as well as for the DNA methylation status. A genome-wide assessment of DNA methylation identified a single CpG-site (CpG2) upstream of the CD95L-promotor that showed differential methylation between APG101 responders (PFS>5 months) and non-responders (PFS < 2 months). Available patient DNAs were in addition analyzed by MassARRAY and Pyro-sequencing to confirm differential CpG2 methylation. Based on this data we used the median of the CpG2 methylation level as a threshold to analyze for a correlation of CpG2 methylation and response to APG101 therapy. Patients showing a low level of CpG2 methylation responded best to therapy with APG101 whereas patients with a high level of CpG2 methylation did not show a relevant benefit when treated with APG101 compared to the control RT-group. The analysis shows a significant survival benefit achieved in patients with low CpG2 methylation (median OS: 16.1 vs 7.3 months, p = 0.029). Hence, the level of CpG2 methylation in the CD95L promoter in the patients' glioblastoma tissue is a prognostic biomarker predicting response to therapy with APG101. Based on the observations described, Apogenix currently develops a qPCR-based assay to quantify CpG2 methylation. This assay is intended as companion diagnostic to identify patients that may respond best to APG101 treatment. Citation Format: Christian Gieffers, Claudia Kunz, Jaromir Sykora, Christian Merz, Meinolf Thiemann, Harald Fricke, Benedikt Wiestler, Wolfgang Wick. Methylation of a single CpG site in the CD95-ligand promoter is a biomarker predicting the response to therapy with APG101 in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 464.

e18552Background: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis resulting in peripheral cytopenia. In low risk MDS, a pro-apoptotic milieu was found with increased ...

Since the first description of CD95/Fas this member of the death receptor superfamily has been regarded as paradigmatic for death receptors, capable of induction of caspase-driven apoptosis. More recently, CD95 has also emerged as an inducer of other signaling pathways including pro-inflammatory and migratory mechanisms (for an overview see Roeder C. et al., Eur J Cell Biol. 2011). Previously, we demonstrated a strong induction of NFkB activity after CD95 stimulation of pancreatic cancer cells resulting in increased invasiveness under in vitro conditions. Inhibition of CD95 mediated mechanisms in tumor gains importance since malignant cells may misuse this system for progression as demonstrated by others eg. for ovarian cancer and glioblastoma. From a clinical point of view, several observations also point into the same direction. Studies on colorectal cancer, head and neck, hepatocellular carcinoma for example suggest CD95 ligand expression to correlate negatively with patients prognosis. APG101 is an inhibitor of CD95 ligand consisting of the CD95 receptor extracellular domain fused to the Fc domain of IgG. A randomized controlled phase II ‘proof-of-concept’ study in patients with recurrent glioblastoma was successfully completed recently (NCT01071837). Up to date there exist no in vivo studies concerning the influence of blocking the interaction between CD95 and its ligand using this recombinant inhibitor in pancreatic cancer. Thus we tested this novel tool in a palliative and adjuvant orthotopic xenotransplantation model of human pancreatic cancer. In this study PancTuI Luc cells were used, which establish a highly infiltrating primary tumor with very low/no metastatic capacity in the liver. All mice (n=8/group) survived the operative procedures for the palliative treatment and were randomly assigned into five groups: control group; treatment with different concentrations of APG101: 12.5, 25, 50, and 100 µg/g bw. (i.p. injection, twice per week). Upon tumor resection (2/3rd pancreatectomy 10 days after tumor cell inoculation) local as well as distant recurrent disease (primarily in the liver) is regularly observed. All mice surviving the operative procedures for the adjuvant treatment (n=9/group) were randomly assigned into two groups: control group with 200 µl saline 0.9 % i.p. and the APG 101 group with a dose of 25 µg/g bw i.p (twice a week). Treatment was started three days after subtotal pancreatectomy and lasted for 28 days until sacrifice of the animals. Palliative treatment: mean tumor weight was 353 ± 179 mg for animals in the control group compared to 210.4 ± 86.5 mg to the mice treated with 25 µg APG101/g bw (p = 0.09) and 221.4 ± 94 mg (p = 0.021) in the group treated with 50 µg APG101/g bw Tumor volume was significantly reduced in all treatment groups, again with 25 µg APG101/g bw achieving maximal growth inhibition. Within the 4 weeks no metastases occurred in any group as previously observed within this model. Adjuvant treatment: All mice developed recurrent tumors. The mean weight of locally recurrent tumor was 887.5 ± 467.3 mg in the control group (treated with 0.9% saline). The other group treated with APG101 (25 µg/g bw) showed a local tumor recurrence with a significant lower tumor weight (p=0.027). The average number of metastases per mouse in the therapy group was 2.3 versus 4 metastases / mouse in the control group. Reduced tumor growth in both therapeutic settings was reflected by a reduced Ki67 proliferation index. Our data underline the importance of the CD95 antagonist APG101 as a new option in pancreatic cancer therapy that might be further evaluated also in combination with chemotherapies. Note: These results were published in the International Journal of Cancer as a full-length article before the online publication of this abstract. Citation information: Inhibition of IL-6 signaling significantly reduces primary tumor growth and recurrencies in orthotopic xenograft models of pancreatic cancer. Goumas FA, Holmer R, Egberts JH, Gontarewicz A, Heneweer C, Geisen U, Hauser C, Mende MM, Legler K, Röcken C, Becker T, Waetzig GH, Rose-John S, Kalthoff H. Int J Cancer. 2015 Jan 21. doi: 10.1002/ijc.29445. [Epub ahead of print] PMID: 25604508. Citation Format: Freya Goumas, Khalid Rhashid, Anna Trauzold, Harald Fricke, Michael Kluge, Thomas Becker, Jan Egberts, Holger Kalthoff. The CD95 ligand inhibitor APG 101 reduces tumor recurrence and metastasis in an adjuvant orthotopic mouse model of pancreatic cancer as well as primary tumor load in a palliative setting. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A115.

2006^ Background: Preclinical data indicate antiinvasive activity of APG101, a CD95-ligand (CD95L)-binding fusion protein, and synergistic activity with radiotherapy in glioblastoma. In healthy vol...

CD95 is a prototype death receptor that regulates the induction of apoptosis, upon binding of its ligand CD95L. Therefore, activation of the CD95L/CD95 system has been regarded an excellent target for treatment of various malignancies. In contrast to the aim of promoting CD95 activation, there is compelling evidence that CD95 is able to promote tumor growth through intracellular non-apoptotic signalling mechanisms. This mechanism has recently been described for glioblastoma, in which activation of CD95 by CD95L leads to invasive growth and glioma cell migration. This is signalled through increased activity of pivotal glioblastoma invasion-related proteases, that is matrix metalloproteinases (MMP). With this shift of paradigms, blocking of the CD95L mediated invasion of tumor cells and subsequent tumor progression may therefore be a promising approach in anti-glioma therapy. APG101 is a fusion protein, consisting of the extracellular domain of human CD95 and the Fc-region of human immunoglobulin G. Hence, it acts as a soluble CD95 receptor trapping the CD95 ligand. The presented project aims at analyzing the clinical relevance of blocking CD95 signalling, given the fact that glioma patients with upregulated CD95 expression have worse survival rates than those with intermediate expression levels. In proof-of principle experiments APG101 inhibits CD95L-mediated invasion of glioma cells. More importantly, APG101-treatment (100 mg/kg body weight) resulted in significantly prolonged survival of SMA560-tumor bearing Vm/Dk mice, less glioma cell satellites in the surrounding tissue and reduced activity of MMP. APG101 in combination with focal irradiation at 6 Gy demonstrated a remarkable reduction of tumor growth with a significantly prolonged survival compared with irradiation treatment alone and inhibition of the proinvasive properties of radiotherapy as demonstrated by magnetic resonance imaging and histology. Surprisingly, APG-101 added to the vascular endothelial growth factor receptor (VEGFR) inhibitor cediranib (AZD2171) did not increase the survival of SMA-560-tumor bearing mice as compared to cediranib alone nor did it impair the proinvasive consequences of cediranib. Our data strongly support the potential use and clinical evaluation of APG101 in combination with radiotherapy in the treatment of malignant glioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-382. doi:10.1158/1538-7445.AM2011-LB-382

CD95 (APO-1/FAS) is a receptor that belongs to the Tumor Necrosis Factor Super Family (TNFSF). Binding of the cognate ligand (CD95L) to CD95 triggers intracellular signaling, ultimately leading to apoptotic cell death. Recent research indicates that CD95 depending on the tissue and the conditions also mediates diverse non-apoptotic functions (e.g. liver regeneration, neuronal development, inflammation and cellular migration/invasion of tumor cells). The potential impact on multiple cellular processes classifies the CD95 pathway as an attractive target for pharmacological interference. Apogenix developed APG101 a human fusion protein consisting of the extracellular domain of the CD95 receptor and the Fc-domain of an IgG antibody. APG101 can be used for the treatment of patho-physiological conditions showing an excess of CD95 induced apoptosis (e.g. acute Graft versus Host Disease, [GvHD]) and for malignancies that depend on CD95 regulated cell migration/invasion (e.g. cancer). Comprehensive, preclinical studies have demonstrated the therapeutic potential of APG101 in Glioblastoma Multiforme (GBM) and aGvHD. GBM is a malignant astrocytic tumor that is highly resistant to radiation and chemotherapy induced apoptosis. A major reason for the particular poor prognosis of GBM is the diffuse invasive growth of infiltrating tumor cell into the surrounding brain. These infiltrating tumor cells evade surgery and are a major cause for tumor relapse. For this reason treatment regimens inhibiting the invasive phenotype should be beneficial for the treatment of GBM. It was shown recently that binding of CD95L to its cognate receptor is an important trigger for the invasive growth of glioblastoma cells and that inhibition of CD95 signaling with neutralizing antibodies abolished the invasion of glioblastoma in vitro and in vivo. Data presented here indicate that APG101 is capable to interfere with the invasive phenotype of glioblastoma cell lines in vitro. Furthermore APG101 effectively reduced the migration of invading tumor cells in a syngeneic mouse model of intracranial GBM (SMA-model). In a modified experimental setup the SMA-mouse model was additionally tested for the efficacy of a combinatorial treatment of APG101 and irradiation. These experiments revealed that a combination of APG101 and sub-lethal irradiation inhibits the invasive growth of GBM cells in vivo, whereas radiation alone had no effect on invasive growth. Detailed analysis of the treatment groups indicated that the combination of APG101 and sub-lethal irradiation prevents formation of satellite tumors, an effect that could not be observed in the control groups. Based on the preclinical data set presented, APG101 is currently tested in a clinical Phase II for the treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-273.