Christian Merz

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.

Abstract LB-382: Inhibition of CD95 signalling by APG-101 enhances efficacy of radiotherapy (RT) and reduces RT-induced tumor satellite formation

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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

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.

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 A83: Hexavalent agonists targeting co-stimulatory receptors of the tumor necrosis factor superfamily

Tumor necrosis factor receptor superfamily (TNFRSF) proteins are widely expressed by immune and tumor cells highlighting their importance in multiple locations and phases of the anti-tumor immune response. Whereas agonistic stimulation of TRAIL-Receptor-1 or -2 can directly induce apoptosis in tumor cells, signaling through many TNFRSF members, such as CD40, CD27, OX40, HVEM, GITR and 4-1BB, results in co-stimulation of immune cells. Therefore, agonistic stimulation of certain members of the TNFRSF is considered to have a positive impact on immune-based therapeutic concepts in clinical oncology. Apogenix has developed a proprietary technology platform for the construction of novel hexavalent TNFRSF agonists (HERA) for the treatment of cancer. HERA fusion proteins are based on a perfect molecular mimic of the TNFSF cytokine structure. The HERA core unit consists of one single polypeptide chain comprising the three receptor-binding domain-forming subsequences (TNFSF-protomers). These single-chain TNFSF receptor-binding domains (scTNFSF-RBD) preserve the structural organization of the trimeric natural TNFSF cytokine and can be utilized to engineer fully human fusion-proteins in a modular manner. Fusing an IgG1 Fc-domain as a dimerization scaffold to the C-terminus of a scTNFSF-RBD creates a hexavalent agonist from two trivalent scTNFSF-RBDs. As a result of this molecular design, each molecule is capable of clustering six receptors in a spatially well-defined manner in close proximity to each other. Therefore, signaling following treatment with the Apogenix HERA scTNFSF-RBD-Fc fusion proteins in vivo is independent of secondary clustering through Fc-γ receptors that is required for many agonistic anti-TNFRSF antibodies (e.g., anti-TRAIL-R2 or anti-CD40). The described HERA engineering concept has been successfully translated to TRAIL, CD40L, LIGHT and CD27L resulting in hexavalent agonists suitable for further development. CD27-Ligand is a potent co-stimulatory molecule that drives T-cell activation and survival through interaction with its receptor (CD27). Here we show in vitro and in vivo data for APG1293 (scCD27L-RBD-Fc), a hexavalent CD27 agonist. APG1293 was expressed in CHO suspension cells followed by a lab-scale purification process including affinity chromatography and SEC-based polishing, resulting in homogenous aggregate-free protein lots. The purified protein binds its respective target-receptor 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, APG1293 was able to bind CD27 expressed on primary human CD4+ and CD8+ T cells and importantly, binding significantly increased T-cell expansion following activation. In vivo the efficacy of APG1293 was evaluated in the colorectal syngeneic murine tumor models MC38-CEA and CT26. In both models APG1293 treatment resulted in a dose dependent tumor growth inhibition. In summary, the data on the hexavalent APG1293 indicate a potent immune cell driven anti tumor efficacy. Therefore, APG1293 could be applied as a single agent or in combination with check-point inhibitors. Citation Format: Christian Gieffers, Christian Merz, David Richards, Mauricio Redondo, Viola Marschall, Jaromir Sykora, Meinolf Thiemann, Harald Fricke, Oliver Hill. Hexavalent agonists targeting co-stimulatory receptors of the tumor necrosis factor superfamily. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A83.

Abstract B48: Structure to function: comparison of CD40 agonist formats reveals superior immune-modulating properties of hexavalent scCD40L-RBD-Fc fusion protein APG1233

Introduction: Initiation and perpetuation of anti-tumor responses of the immune system are in the focus of current approaches to fight cancer. Manipulation of the tumor microenvironment, e.g. shifting the M1/M2 ratio towards M1 is one goal to support immunologic cancer treatment. Here we examined and compared the functional effects of bivalent agonistic anti-CD40 antibodies, homo-trimeric trivalent CD40-Ligand and the novel hexavalent CD40-agonist APG1233 on the maturation and differentiation of primary monocytes, B cells and T cell mediated tumor cell killing. Materials and Methods: Following treatment with various CD40 agonists, cytokine secretion by monocytes, PBMCs and T cells from blood samples was assessed by ELISA. Monocytes isolated from healthy-donor blood samples were differentiated in vitro into either M1- or M2-type macrophages, or dendritic cells (DC) which was confirmed by multicolor flow-cytometry (MC-FC). Subsequently, we analyzed the respective M1- and M2-type macrophages and DCs regarding their ability to induce proliferation in a direct allogenic co-culture system with naive CD4-positive T cells by a flow cytometry-based CFSE-assay. Macrophage plasticity, e.g. re-polarisation of M2-like to M1-like macrophages upon exposure to CD40 agonists was assessed by MC-FC. A real-time cell analysis system (Roche xCelligence RTCA DP) was used to attest T cell activation by CD40 stimulated B cells, resulting in killing of tumor cells in direct co-cultures. Results: Stimulation of CD40 on PBMCs, T cells and monocytes increased secretion of cytokines (e.g. IL-12, TNFαa, CCL4) dependent on the agonist format and moreover was strictly dependent on Fc-crosslinking when using agonistic anti-CD40 mAb. In vitro, when the hexavalent APG1233 was added to the cytokine cocktail during the in vitro differentiation process, the appearance of M1-type macrophages was substantially increased. Moreover, M2-macrophages underwent conversion and acquired M1-type surface markers after exposure to APG1233. Finally, in direct co-culture of the in vitro differentiated cell populations with naive CD4+ T cells, M1-macrophages induced strong lymphocyte proliferation, while the induction by monocytes and M2-macrophages was low. On a functional level, T lymphocytes co-cultured with M1 or DC acquired direct cytotoxic activity against tumor cells in a real-time cell analysis assay. Similarly, induction of cytolytic activity of purified T cells in vitro required the presence of both CD40 expressing B cells and APG1233. Conclusion: Stimulation of CD40 on immune cells triggers development of anti-tumor responses, but efficacy of various agonist formats dramatically varies. Compared to trimeric CD40L formats and agonistic anti-CD40 mAbs, the novel hexavalent CD40 agonist APG1233 emerging from Apogenix HERA Technology platform excels as a strong inducer of B cell activation, M1-type macrophage differentiation and M2->M1 conversion. M1-macrophages generated in vitro are functional and enhance proliferation of naive CD4+ T cells. CD40 stimulation on a CD40 expressing B cell line enhances its ability to activate T cells and trigger an anti-tumor response. Citation Format: Christian Merz, Jaromir Sykora, Thamara Beyer, Stefanie Knorn, Harald Fricke, Christian Gieffers, Oliver Hill, David M. Richards. Structure to function: comparison of CD40 agonist formats reveals superior immune-modulating properties of hexavalent scCD40L-RBD-Fc fusion protein APG1233. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B48.

Abstract 4690: Hexavalent CD27 agonists show single agent anti-tumor activity and enhanced memory formation in mouse syngeneic tumor models

Tumor necrosis factor receptor superfamily (TNFRSF) proteins are widely expressed by immune and tumor cells highlighting their importance in multiple locations and phases of the anti-tumor immune response. Apogenix has developed a proprietary technology platform for the construction of novel hexavalent TNFRSF agonists (HERA) for the treatment of cancer. HERA fusion proteins comprise a perfect molecular mimic of the TNFSF cytokine structure and are based on dimerization of trivalent single-chain TNFSF receptor-binding domains (scTNFSF-RBD) via a Fc-γ receptor (FcγR) binding deficient immunoglobulin Fc domain. As a result of this molecular design, HERA proteins are capable of clustering six receptors in a spatially well-defined manner. Signaling following treatment with the Apogenix HERA “scTNFSF-RBD-Fc fusion proteins” is entirely independent of secondary crosslinking through FcγRs that is required for many agonistic anti-TNFRSF antibodies. The HERA engineering concept has been successfully translated to TRAIL, GITRL, CD40L, LIGHT and CD27L resulting in agonists that are currently in development. CD27L is a potent co-stimulatory molecule that drives T cell activation and survival through interaction with its receptor (CD27). HERA-CD27L is expressed in CHO suspension cells followed by a lab-scale purification process that results in homogenous aggregate-free protein lots. The purified protein binds its respective target-receptor with high affinity. In vitro, HERA-CD27L was able to bind CD27 expressed on primary human CD4+ and CD8+ T cells. Binding significantly increased T cell expansion following αCD3/αCD28 stimulation and leads to increased expression of OX40 on CD4+ T cells and 4-1BB on CD8+ T cells, respectively. In vivo, a single dose of 10mg/kg HERA-CD27L increases clonal expansion of antigen-specific CD8+ T cells upon immunization with Ovalbumin (Ova) in the mouse OT-1 model with a kinetics leading to peak levels of >25% Ova-specific CD8+ T cells at day 6 after treatment. Anti-tumor efficacy of HERA-CD27 was evaluated in MC38-CEA and CT26 colorectal syngeneic murine tumor models. In both models treatment with HERA-CD27L resulted in a dose dependent inhibition of tumor growth. CT26 tumor bearing mice treated with 1mg/kg HERA-CD27L, twice weekly showed an 85% tumor-growth inhibition (TGI) compared to the control group. A significant TGI of 48% could be observed in the MC38-CEA model upon treatment with 10mg/kg, twice weekly. Analysis of peripheral lymphoid tissues in the MC38-CEA bearing mice could furthermore show that HERA-CD27L treatment is accompanied with enhanced memory formation in both CD4+ & CD8+ T cells. In summary, the data on the hexavalent HERA-CD27L indicate a potent immune cell driven anti tumor efficacy. Therefore, HERA-CD27 agonists could be applied for the treatment of cancer as a single agent or in combination with check-point Inhibitors. Citation Format: Christian Gieffers, David Richards, Jaromir Sykora, Mauricio Redondo-Muller, Meinolf Thiemann, Christian Merz, Karl Heinonen Heinonen, Viola Marschall, Harald Fricke, Oliver Hill. Hexavalent CD27 agonists show single agent anti-tumor activity and enhanced memory formation in mouse syngeneic tumor models [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 4690. doi:10.1158/1538-7445.AM2017-4690

Abstract 4963: Novel hexavalent GITR agonists stimulate T cells and enhance memory formation

Introduction: The co-stimulatory receptor GITR plays an important role in initiating the immune response in the lymph nodes and in maintaining the immune response in the tumor tissue. Binding of GITR to its natural ligand directly leads to increased anti-tumor T cell activation and their survival. It also reduces the suppressive abilities of Treg cells, further increasing the anti-tumor immune response. The HERA-technology developed by Apogenix targets the TNF-receptor superfamily and generates fully human hexavalent fusion proteins with high clustering capacity for the cognate receptor. Hexavalent HERA-ligands are pure agonists whose signaling capacity is independent of secondary Fcγ-receptor crosslinking. Here we report in vitro and in vivo properties of novel hexavalent HERA-GITRL constructs. Experimental procedures: For the assessment of in vivo stability, serum samples from a PK study with three HERA-GITRL constructs in CD1-mice were analyzed with respect to their drug levels employing a specific ELISA assay. For functional characterization of HERA-GITRL in vitro, immune cells were isolated from healthy-donor blood samples and profiled by multicolor flow cytometry (MC-FC). Subsequently, immune cells were cultured in growth media containing different HERA-GITRL constructs and anti-CD3. Changes in activation and memory markers on T cells (e.g. CD25, CD69, CD45RA, CD45RO), their proliferation rate (CFSE assay) and the intracellular staining of cytokines (e.g. TNF-α and IFN-γ) was assessed by MC-FC. Results: Minor modifications led to three HERA-GITRL drug candidates with unique pharmacokinetic properties / in vivo stability as explored in mice. Terminal half-life was between 61.7 and 200.6 hours. Stimulation of pan T cells as well as naive CD4+ T-lymphocytes by anti-CD3 was further augmented by HERA-GITRL as demonstrated by CD69 and CD25 expression. This effect was accompanied by an increased proliferation and an increased memory formation. Furthermore, we observed an increased level of intracellular TNF-α and IFN-γ in naive CD4+ T-lymphocytes incubated with anti-CD3 that could be further raised by the addition of HERA-GITRL. Conclusion: HERA-GITRL demonstrate excellent in vivo stability. Their ability to enhance proliferation and activation of naive CD4+ T cells and to induce memory formation render them as attractive candidates for immunotherapeutic treatments of cancer. Citation Format: Meinolf Thiemann, Christian Gieffers, David M. Richards, Christian Merz, Karl Heinonen, Mauricio Redondo Mueller, Viola Marschall, Jaromir Sykora, Harald Fricke, Oliver Hill. Novel hexavalent GITR agonists stimulate T cells and enhance memory formation [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 4963. doi:10.1158/1538-7445.AM2017-4963

Abstract 1688: HERA-CD40L: A novel hexavalent CD40 agonist with superior biological activity

Introduction: Targeted therapeutics for cancer treatment are mostly developed as antibodies, however, the natural signaling complexes of the members of the TNF superfamily and their receptors consist of clusters of trimers. Consequently, most of these bivalent agonistic antibodies depend on Fc receptor mediated crosslinking for biological activity. The HERA-Technology developed by Apogenix generates hexavalent fusion proteins targeting the TNF-receptor superfamily with high clustering capacity for the cognate receptor, which overcomes this disadvantage of antibody-based drugs. Here we compared the efficacy of different CD40 agonist formats, including the novel HERA-CD40L and the functional consequences of differential receptor clustering. Materials & Methods: Biological activity of CD40 agonists was compared using an engineered reporter cell line and by flow cytometric analysis of CD40-induced IκBα degradation in Ramos B cells. T lymphocytes and monocytes were isolated from buffy coats and expression of CD markers upon CD40 ligation was analysed by multicolor flow cytometry (MC-FC). Secretion of cytokines in response to CD40 ligation was determined by ELISA. Monitoring of T cell-induced killing of tumor cells in direct co-cultures employed a real-time cell analysis system (xCELLigence). Results: Direct comparison of bivalent CD40 antibodies with trivalent CD40L and the hexavalent HERA-CD40L in two independent bioactivity assay formats demonstrated that only the hexavalent agonist was fully active without additional crosslinking. In contrast to HERA-CD40L, neither the bivalent agonistic CD40 antibody nor the trivalent CD40L were able to upregulate expression of activation markers on B cells or to induce secretion of proinflammatory cytokines such as IL-12 and TNFα by PBMCs. In vitro generated M2-macrophages acquired an M1 phenotype and enhanced proliferation of naive CD4+ T cells in direct co-culture. Similarly, direct co-culture of CD4+ T cells with Ramos B cells in the presence of HERA-CD40L induced cytotoxic activity of CD4+ cells against tumor cells. The activating effect was dependent on cell-cell contacts and was not observed in indirect co-cultures. Importantly, only the hexavalent HERA-CD40L showed full biological activity without additional crosslinking. Conclusion: The hexavalent CD40 agonist HERA-CD40L produced by Apogenix HERA-Technology platform triggers CD40 signaling on B cells and cells of the monocytic lineage, leads to direct cytolytic activation and proliferation of CD4+ T cells and shifts the M1/M2 balance towards proinflammatory conditions. Unlike bivalent CD40 antibodies or trivalent CD40L_ based agonists, the hexavalent HERA-CD40L forms highly clustered signaling complexes and thus exhibits superior biological activity over other agonistic formats without the need for Fc receptor mediated crosslinking. Citation Format: Christian Merz, Jaromir Sykora, Meinolf Thiemann, Viola Marschall, Karl H. Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. HERA-CD40L: A novel hexavalent CD40 agonist with superior biological activity [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 1688. doi:10.1158/1538-7445.AM2017-1688