Michael Kluge

APG350 Induces Superior Clustering of TRAIL Receptors and Shows Therapeutic Antitumor Efficacy Independent of Cross-Linking via Fcγ Receptors

Cancer cells can be specifically driven into apoptosis by activating Death-receptor-4 (DR4; TRAIL-R1) and/or Death-receptor-5 (DR5; TRAIL-R2). Albeit showing promising preclinical efficacy, first-generation protein therapeutics addressing this pathway, especially agonistic anti-DR4/DR5-monoclonal antibodies, have not been clinically successful to date. Due to their bivalent binding mode, effective apoptosis induction by agonistic TRAIL-R antibodies is achieved only upon additional events leading to antibody-multimer formation. The binding of these multimers to their target subsequently leads to effective receptor-clustering on cancer cells. The research results presented here report on a new class of TRAIL-receptor agonists overcoming this intrinsic limitation observed for antibodies in general. The main feature of these agonists is a TRAIL-mimic consisting of three TRAIL-protomer subsequences combined in one polypeptide chain, termed the single-chain TRAIL-receptor–binding domain (scTRAIL-RBD). In the active compounds, two scTRAIL-RBDs with three receptor binding sites each are brought molecularly in close proximity resulting in a fusion protein with a hexavalent binding mode. In the case of APG350—the prototype of this engineering concept—this is achieved by fusing the Fc-part of a human immunoglobulin G1 (IgG1)-mutein C-terminally to the scTRAIL–RBD polypeptide, thereby creating six receptor binding sites per drug molecule. In vitro, APG350 is a potent inducer of apoptosis on human tumor cell lines and primary tumor cells. In vivo, treatment of mice bearing Colo205-xenograft tumors with APG350 showed a dose-dependent antitumor efficacy. By dedicated muteins, we confirmed that the observed in vivo efficacy of the hexavalent scTRAIL–RBD fusion proteins is—in contrast to agonistic antibodies—independent of FcγR-based cross-linking events. Mol Cancer Ther; 12(12); 2735–47. ©2013 AACR.

Peripheral monocytes are functionally altered and invade the CNS in ALS patients.

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease affecting primarily the upper and lower motor neurons. A common feature of all ALS cases is a well-characterized neuroinflammatory reaction within the central nervous system (CNS). However, much less is known about the role of the peripheral immune system and its interplay with CNS resident immune cells in motor neuron degeneration. Here, we characterized peripheral monocytes in both temporal and spatial dimensions of ALS pathogenesis. We found the circulating monocytes to be deregulated in ALS regarding subtype constitution, function and gene expression. Moreover, we show that CNS infiltration of peripheral monocytes correlates with improved motor neuron survival in a genetic ALS mouse model. Furthermore, application of human immunoglobulins or fusion proteins containing only the human Fc, but not the Fab antibody fragment, increased CNS invasion of peripheral monocytes and delayed the disease onset. Our results underline the importance of peripheral monocytes in ALS pathogenesis and are in agreement with a protective role of monocytes in the early phase of the disease. The possibility to boost this beneficial function of peripheral monocytes by application of human immunoglobulins should be evaluated in clinical trials.

Inhibition of CD95/CD95L (FAS/FASLG) Signaling with APG101 Prevents Invasion and Enhances Radiation Therapy for Glioblastoma

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.

The novel TRAIL-receptor agonist APG350 exerts superior therapeutic activity in pancreatic cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has raised attention as a novel anticancer therapeutic as it induces apoptosis preferentially in tumor cells. However, first-generation TRAIL-receptor agonists (TRAs), comprising recombinant TRAIL and agonistic receptor-specific antibodies, have not demonstrated anticancer activity in clinical studies. In fact, cancer cells are often resistant to conventional TRAs. Therefore, in addition to TRAIL-sensitizing strategies, next-generation TRAs with superior apoptotic activity are warranted. APG350 is a novel, highly potent TRAIL-receptor agonist with a hexavalent binding mode allowing the clustering of six TRAIL-receptors per drug molecule. Here we report on preclinical in vitro and in vivo studies testing the activity of APG350 on pancreatic ductal adenocarcinoma (PDAC) cells. We found that APG350 potently induced apoptosis of Colo357, PancTuI and Panc89 cells in vitro. In addition, APG350 treatment activated non-canonical TRAIL signaling pathways (MAPK, p38, JNK, ERK1/ERK2 and NF-κB) and induced the secretion of IL-8. Stable overexpression of Bcl-xL inhibited APG350-induced cell death and augmented activation of non-canonical pathways. Intriguingly, pre-treatment of Bcl-xL-overexpressing cells with the BH3-mimic Navitoclax restored their sensitivity to APG350. To study the effects of APG350 on PDAC cells in vivo, we applied two different orthotopic xenotransplantation mouse models, with and without primary tumor resection, representing adjuvant and palliative treatment regimes, respectively. APG350 treatment of established tumors (palliative treatment) significantly reduced tumor burden. These effects, however, were not seen in tumors with enforced overexpression of Bcl-xL. Upon primary tumor resection and subsequent APG350 treatment (adjuvant therapy), APG350 limited recurrent tumor growth and metastases. Importantly, therapeutic efficacy of APG350 treatment was more effective compared with treatment with soluble TRAIL in both models. In conclusion, APG350 represents a promising next-generation TRA for the treatment of PDAC. Moreover, our results suggest that combining APG350 with Navitoclax might be a succesfull strategy for cancers harboring mitochondrial apoptosis resistance.

Abstract LB-232: APG350, a dimerized single chain TRAIL receptor agonist with enhanced functional properties

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL TRAIL (Apo2L) is a member of the Tumor Necrosis Factor Superfamily (TNF-SF) that induces apoptosis through binding to two closely related receptors, TRAIL-R1 and TRAIL-R2. Its unique ability of selective apoptosis induction in tumor cells makes TRAIL an attractive molecule for tumor therapy. APG350 is a newly designed TRAIL-receptor agonist comprising two single-chain TRAIL-receptor binding domains (each consisting of three covalently linked TRAIL protomers) that are dimerized via the Fc-part of human IgG1. APG350 has six receptor binding sites per molecule and binds to both death inducing TRAIL receptors. In vitro , APG350 shows potent apoptosis induction on a wide subset of human tumor cell-lines, on cancer stem cells and on primary tumor cells. Mechanistically its improved ability to form clusters on target cells composed of six TRAIL-receptors each, distinguishes APG350 from current clinical development candidates. Agonistic antibodies like Conatumumab or Apomab are capable to bind two receptor chains per molecule, while recombinant TRAIL (e.g. Dulanermin) binds three receptor chains per molecule. Direct in vitro comparison of APG350 with recombinant TRAIL (APG400) and a TRAIL-R2 specific agonistic monoclonal antibody revealed superior apoptosis induction capacity for APG350. Analysis of the PK-parameters in mice showed a half life of 23.1h for APG350 (1.04h for APG400) indicating a significantly improved PK in comparison to APG400. The half life of APG350 in a Cynomolgus monkey was 26.7h. Comparative treatment of mice bearing Colo205 xenograft tumors with APG350 and APG400 employing one treatment cycle with five consecutive daily intravenous injections showed a superior efficacy of APG350 with respect to tumor volume reduction and number of tumor free animals. Even mice with initially large tumors (∼600 mm3) were treated effectively with APG350. Remarkably, APG350 also showed pronounced dose dependent anti tumor efficacy on xenograft-tumors derived from colon cancer stem cells (CSC). Furthermore large CSC-derived tumors could be treated effectively and successful re-treatment of previously responding tumors demonstrated that tumors did not gain drug resistance. In a pilot experiment APG350 treatment of mice bearing a slowly growing primary rectum tumor xenograft also showed a significant tumor volume reduction. In all in vivo studies APG350 was well tolerated at doses between 0.3–100mg/kg bw in mice and doses up to 10mg/kg bw in a Cynomolgus monkey. General tolerability and potential effects on liver toxicity were assessed by co-application of APG350 together with a crosslinking antibody in mice. This treatment evoked only minor clinical signs at high doses with no relevant increase in liver enzymes. Currently two non-GLP toxicology studies, a 4-week study in mice and a dose escalation study in Cynomolgus monkeys are ongoing to confirm tolerability in vivo. 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-232. doi:10.1158/1538-7445.AM2011-LB-232

A Single-Chain-Based Hexavalent CD27 Agonist Enhances T Cell Activation and Induces Anti-Tumor Immunity

Tumor necrosis factor receptor superfamily member 7 (TNFRSF7, CD27), expressed primarily by T cells, and its ligand CD27L (TNFSF7, CD70) provide co-stimulatory signals that boost T cell activation, differentiation, and survival. Agonistic stimulation of CD27 is therefore a promising therapeutic concept in immuno-oncology intended to boost and sustain T cell driven anti-tumor responses. Endogenous TNFSF/TNFRSF-based signal transmission is a structurally well-defined event that takes place during cell-to-cell-based contacts. It is well-established that the trimeric-trivalent TNFSF-receptor binding domain (TNFSF-RBD) exposed by the conducting cell and the resulting multi-trimer-based receptor clustering on the receiving cell are essential for agonistic signaling. Therefore, we have developed HERA-CD27L, a novel hexavalent TNF receptor agonist (HERA) targeting CD27 and mimicking the natural signaling concept. HERA-CD27L is composed of a trivalent but single-chain CD27L-receptor-binding-domain (scCD27L-RBD) fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. The hexavalent agonist significantly boosted antigen-specific T cell responses while having no effect on non-specific T cells and was superior over stabilized recombinant trivalent CD27L. In addition, HERA-CD27L demonstrated potent single-agent anti-tumor efficacy in two different syngeneic tumor models, MC38-CEA and CT26wt. Furthermore, the combination of HERA-CD27L and an anti-PD-1 antibody showed additive anti-tumor effects highlighting the importance of both T cell activation and checkpoint inhibition in anti-tumor immunity. In this manuscript, we describe the development of HERA-CD27L, a true CD27 agonist with a clearly defined forward-signaling mechanism of action.

Abstract 3641: EGFR/CD16A TandAbs are efficacious NK-cell engagers with favorable biological properties which potently kill EGFR+tumors with and without Ras mutation

Constitutive EGFR activation plays an important role in the pathophysiology of various solid cancers, such as colorectal cancer, non-small cell lung cancer or squamous cell carcinomas of the head and neck. Tyrosine kinase inhibitors (TKI) and monoclonal antibodies (mAbs), which interfere with signal transduction and activation of EGFR, are approved for treatment of such cancers. However, intrinsic or acquired resistance to these treatments has been described for many patients. Natural killer cells (NK-cells) are important effectors of innate immunity and NK-cell engagers have shown evidence of improved safety in patients compared to T-cell engagers. To specifically utilize the cytotoxic potential of NK-cells to eliminate EGFR-expressing tumors, we developed tetravalent bispecific EGFR/CD16A TandAbs comprising fully human Fv domains recognizing human and cynomolgus EGFR and CD16A. TandAbs recognizing epitopes in the extracellular domain of EGFR differing from epitopes targeted by other mAbs were characterized. Lead candidate AFM24 shows superior cytotoxicity in terms of ADCC (main mode of action) and reduced inhibition of EGFR-mediated phosphorylation compared to cetuximab. Importantly, inhibition of EGFR-signaling is believed to contribute to skin toxicity caused by therapeutic mAbs and TKI’s. AFM24’s cytotoxic activity was tested against EGFR+ tumor cell lines including some carrying a Ras mutation, which is a negative prognostic biomarker and renders cells less susceptible to cetuximab or panitumumab. The cetuximab-resistant CRC cell line HCT-116 or the NSCLC cell line A549 (both with Ras mutations) were efficiently killed with EGFR/CD16A TandAbs in vitro. In vivo data in the HCT-116 model indicate anti-tumor efficacy of AFM24, while no efficacy of cetuximab was seen. Importantly, AFM24 does not activate NK-cells without target cell binding and does not bind to any other members of the EGFR family. While binding and cytotoxic efficacy of many therapeutic mAbs are impaired by serum IgG, no substantial change in AFM24’s binding affinity to NK-cells was observed in the presence of high concentrations of human IgG. In calcein-release cytotoxicity assays with NK-cells as effectors, we showed that the presence of IgG had only little inhibitory effect on AFM24 efficacy compared to cetuximab. In addition, competition of an anti-CD16 mAb with AFM24 in cytotoxicity assays was substantially lower than with cetuximab. Taken together our data demonstrate that AFM24 is a highly potent human antibody displaying favorable biological properties over existing mAbs. This human/cynomolgus cross-reactive agent is currently in preclinical development to treat EGFR+ malignancies and has the potential to exhibit a favorable side effect profile and reduced toxicity and to overcome resistance to other targeted anti-EGFR therapeutic agents. Citation Format: Michael Kluge, Kristina Ellwanger, Uwe Reusch, Ivica Fucek, Michael Weichel, Torsten Haneke, Stefan Knackmus, Joachim Koch, Martin Treder. EGFR/CD16A TandAbs are efficacious NK-cell engagers with favorable biological properties which potently kill EGFR+ tumors with and without Ras mutation [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 3641. doi:10.1158/1538-7445.AM2017-3641

Abstract 1204: A construction platform for hexavalent agonists targeting receptors of the tumor necrosis factor superfamily: Where death meets co-stimulation

Tumor necrosis factor receptor superfamily (TNFRSF) proteins are widely expressed by immune and tumor cells. Their importance in many locations and phases of the anti-tumor immune response is now broadly appreciated and several TNFR agonists are currently in preclinical and clinical development. Importantly, signaling through many TNFRSF members, such as CD40, CD27, OX40, 4-1BB, HVEM and GITR, is potentially associated with an enhanced anti tumor response via co-stimulation of immune cells. Apogenix has established a development platform for a novel class of TNFRSF-agonists for the treatment of cancer. Unlike their natural homotrimeric counterparts, the Apogenix recombinant TNFSF proteins consist of one single polypeptide chain composed of three receptor-binding domain-forming protomers. These single-chain TNFSF receptor-binding domains (scTNFSF-RBD) are mimics of the three-dimensional organization of the natural TNFSF-cytokine and can be used to engineer fully human fusion-proteins from a modular toolbox. For example, fusing an IgG1 Fc-domain to the C-terminus of a scTNFSF-RBD creates a hexavalent agonist as the Fc-domain acts as a dimerization scaffold for two trivalent scTNFSF-RBDs. As a result of this molecular design, each drug molecule is capable of clustering six receptors in a spatially well-defined manner. Consequently, TNFSF receptor signaling following treatment with the Apogenix scTNFSF-RBD-Fc in vivo is independent of secondary clustering through Fc-γ receptors that is required for many anti-TNFRSF agonistic antibodies (e.g., anti-TRAILR2 or -CD40). Following up the scTRAIL-RBD-Fc prototype, this engineering concept has now been successfully translated to CD40L and CD27L resulting in hexavalent agonists suitable for further development. Expression of the drug candidates in CHO suspension cells followed by an AFC and SEC-based lab-scale purification process resulted in homogenous aggregate-free protein lots. The purified proteins bind their respective target-receptors with high affinity. In vivo stability/PK studies have been performed in addition to in vitro experiments with primary human and mouse lymphoid and myeloid cell populations. Specifically, it was shown that scCD27L-RBD-Fc was able to bind CD27 expressed on primary human CD4+ and CD8+ T cells. Importantly, binding significantly increased T cell expansion following activation. Treatment with scCD40L-RBD-Fc induced differentiation of B cells and enhanced primary human monocyte differentiation into DCs or M1 macrophages. Encouraged by the promising results obtained with TRAIL, CD40L, and CD27L, Apogenix is currently expanding the TNFRSF-agonist pipeline to target additional cell populations, locations and phases of the immune response in order to develop novel therapies to treat cancer and other conditions. Citation Format: Christian Merz, Christian Gieffers, Michael Kluge, David M. Richards, Tim Schnyder, Jaromir Sykora, Meinolf Thiemann, Harald Fricke, Oliver Hill. A construction platform for hexavalent agonists targeting receptors of the tumor necrosis factor superfamily: Where death meets co-stimulation. [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 1204.

Abstract A115: 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

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, Rocken 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.

Abstract 3856: Dimerized single chain TRAIL-receptor agonists do not depend on Fc-gamma-receptor cross-linking for anti-tumor efficacy in vivo

APG350 is a TRAIL-receptor (TRAIL-R) agonist comprising two single-chain TRAIL-R binding domains (scTRAIL-RBD) that are dimerized via the Fc-part of a human IgG1-mutein thereby creating six receptor binding sites per molecule. This improved ability to form clusters on target cells composed of six TRAIL-Rs each, distinguishes APG350 from current clinical development candidates. In vitro, comparison of APG350 with recombinant APO2L/TRAIL and a TRAIL-R2 specific agonistic antibody revealed superior apoptosis induction for APG350 on primary and established human tumor cell-lines. Treatment of mice bearing Colo205 xenograft tumors with APG350, APO2L/TRAIL or an agonistic TRAIL-R2 specific antibody showed superior anti-tumor efficacy for APG350. Pronounced anti tumor efficacy was also shown on colon cancer stem cell (CSC) derived xenografts and successful APG350 re-treatment of relapsed CSC derived tumors demonstrated that tumors did not develop drug resistance. For most agonistic TRAIL-R antibodies effective apoptosis induction is achieved only upon additional cross-linking. SEC-based fractionation of a TRAIL-R2 specific agonistic antibody indicates a small amount of multimerized antibody that showed efficient apoptosis induction in vitro. However, the respective monomeric antibody showed poor apoptosis induction in vitro that could be enhanced upon cross-linking. In contrast apoptosis induction by APG350 was only marginally enhanced by cross-linking. Although monomeric agonistic TRAIL-R antibodies are poor apoptosis inducers in vitro, they show efficient apoptosis induction on xenograft tumors in vivo. A likely explanation for this difference is given by a recent publication showing that anti-tumor efficacy of an agonistic TRAIL-R2 specific antibody (Drozitumab) depends on cross-linking by Fcα-receptors (FcαR) in vivo. These data suggest that FcαR cross-linking might be a common requirement for the anti-tumor efficacy of agonistic TRAIL-R antibodies. To exclude that in vivo efficacy of APG350 depends on cross-linking by FcαRs we designed APG350-muteins with strongly reduced (APG808) or lacking FcαR binding (APG780). Side by side comparison of APG808, APG780 and APG350 in mice bearing Colo205-derived xenograft tumors showed identical anti-tumor efficacy for all respective proteins. Given that APG780 cannot bind to FcαRs these results suggest that the anti-tumor efficacy of APG808, APG780 and APG350 is solely based on the unique construction principle of the dimerized scTRAIL-RBD. APG350 induces superior clustering of TRAIL-Rs that in contrast to agonistic TRAIL-R antibodies does not require cross-linking via FcαRs for its potent anti-tumor efficacy. APG350 based dimerized scTRAIL-RBD formats may therefore have the capacity to bridge the current gap seen between preclinical and clinical efficacy for agonistic TRAIL-R specific antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3856. doi:1538-7445.AM2012-3856