Meinolf Thiemann

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.

The design of potent and selective inhibitors of DPP-4: optimization of ADME properties by amide replacements.

For a series of beta-homophenylalanine based inhibitors of dipeptidyl peptidase IV ADME properties were improved by the incorporation of amide replacements. These efforts led to a novel series of potent and selective inhibitors of DPP-4 that exhibit an attractive pharmacokinetic profile and show excellent efficacy in an animal model of diabetes.

Discovery of β-homophenylalanine based pyrrolidin-2-ylmethyl amides and sulfonamides as highly potent and selective inhibitors of dipeptidyl peptidase IV

Modifications of DPP-4 inhibitor 5, that was discovered by structure based design, are described and structure-activity relationships discussed. With analogue 7k one of the most potent non-covalent inhibitors of DPP-4 reported to date (IC(50)=0.38nM) was discovered. X-ray structure of inhibitor 7k bound to DPP-4 revealed a hydrogen bonding interaction with Q553. First successful efforts in balancing overall properties, as demonstrated by improved metabolic stability, highlight the potential of this series.

From lead to preclinical candidate: Optimization of β-homophenylalanine based inhibitors of dipeptidyl peptidase IV

A series of highly potent and selective inhibitors of DPP-4 was optimized for ADMET properties. The effort resulted in the discovery of inhibitor 1g, that exhibits excellent efficacy in an oral glucose tolerance test and an attractive pharmacokinetic profile.

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 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