Apogenix (Germany)

Abstract BACKGROUND There have been few advances in glioblastoma treatment in recent decades. Treatments fail due to tumour heterogeneity (genetic, epigenetic, cell-state) and persistence of GBM stem-like cells (GSCs) within the tumour margin. Sox 2 and Sox 9 are consistently overexpressed in GSCs – an exploitable commonality across diverse tumour subtypes. We designed a synthetic superenhancer (SSE), activated by transcription factors known to be highly expressed in GSCs. TGX-007 delivers dual genetic payloads, via AAV-1 mediated delivery, which are activated only in tumour cells under control of this SSE. In our validated immunocompetent mouse model of GBM, TGX-007 administration cures >85% of animals with no associated toxicity and provides immunological memory, preventing tumour formation on re-exposure. METHODS This is a first-in-human, Phase I/II study designed to establish the safety and OBD of TGX-007, and to assess preliminary efficacy in patients with newly-diagnosed GBM or with first radiological progression of previously treated GBM. Tumours must be suitable for ≥90% debulking. TGX-007 is an AAV-1 capsid delivering a transgene encoding HSV-tk and IL12. HSV-tk is an enzyme that converts the nucleoside analogue valaciclovir to a cytotoxic molecule, resulting in cell death. IL-12 is an immune-activating cytokine. TGX-007 will be delivered with intra-operative MRI-guidance via convection enhanced delivery (Clearpoint Smartflow® cannula). Newly-diagnosed patients will first have intra-operative histological confirmation. Patients then receive 2 weeks valaciclovir prior to surgical debulking, then standard care treatment. The study employs a two-stage BOIN12 design to establish the OBD of TGX-007, based on toxicity and signal of activity (HSVtk mRNA in tumour tissue), followed by an expansion for preliminary clinical efficacy evaluation. Primary endpoints are: Phase I - safety and tolerability, OBD determination; Phase II – OS rate at 12 months (newly-diagnosed population) and 6 months (recurrent population). Secondary and exploratory endpoints include PFS, viral shedding, proof-of-mechanism within tumour tissue.

An alternate mask absorber is a generally applicable approach as a mitigation strategy for Mask 3-D effects (M3D) observed in EUV lithography. It is also an efficient solution in a production-worthy environment compared to multilayer modification or Source Mask Optimization (SMO) techniques. Absorbers with a high EUV extinction coefficient k allow for lower Best Focus Variation (BFV) through pitch and reduced Telecentricity Errors (TCE). This study evaluates Ta-Co alloys as potential high-k mask absorbers. It includes an experimental study of film morphology, surface composition, and stability of Ta-Co alloys as well as a theoretical investigation of the imaging performance. The optical constants were determined from EUV angle-dependent reflectivity measurements for three selected compositions, viz. TaCo, Ta₂Co, and TaCo₃. The Ta-Co alloys exhibit a higher EUV extinction coefficient k compared to the currently used TaBN absorber. TaCo and Ta₂Co demonstrated smooth surfaces, were stable in a hydrogen environment, and in mask cleaning solutions. These qualified compositions of Ta-Co alloys were selected for aerial image simulations and compared with a TaBN absorber. The optimized absorber thickness for Ta-Co alloy absorbers is based upon NILS enhancement, Threshold to Size (TtS) and balancing of diffraction order amplitudes. A 10 nm alternate line-space pattern with a pitch of 20 nm and 14 nm square contact holes with a pitch of 28 nm were considered for the simulation study using High NA 0.55 EUV lithography process settings. The through pitch imaging performance was evaluated using NILS, TCE and BFV as metrics. Ta-Co alloys allow for a reduction in M3D effects at smaller absorber thickness compared to a 60 nm TaBN absorber.

Innovative medicinal products are required for progress in many therapeutic areas, and in particular, oncology. For these products to succeed, pharmaceutical companies must generate the relevant and robust clinical data required to meet the needs of regulators and healthcare providers. In addition, real-world and health economic evidence is increasingly required to support pricing and reimbursement for innovative medicinal products. To incorporate all of these requirements into the innovative medicinal product strategy early in development, the core principles of how product strategies are developed and applied must be revisited and end-to-end strategic planning implemented. This paper reviews the hurdles faced during development of the integrated strategy for innovative medicinal products, particularly in the oncology field, and examines which functions of a pharmaceutical company should play the greatest role in addressing patient and different stakeholders' needs. It will then illustrate how Medical Affairs activities are evolving to take on this strategic leadership role.

Abstract Glucocorticoid-induced TNFR-related protein (GITR, TNFRSF18, CD357), a TNFR-SF member, is a co-stimulatory receptor that increases anti-tumor T cell activation. Based on Apogenix TNFR-SF agonist HERA-technology, we created a fully human hexavalent GITR ligand fusion protein - HERA-GITRL - intended for T cell costimulatory approaches in immuno-oncology (IO) therapy. HERA-GITRL is composed of a trivalent single chain GITRL-receptor-binding-domain fused to an IgG1-derived silenced Fc-domain serving as dimerization scaffold. The unique design that combines a molecular mimic of the endogenous GITRL with a silenced Fc-domain, allows the study of pure GITR agonism in contrast to Fc-mediated mixed modes of action. Here we report in vitro and in vivo properties of our novel HERA-GITRL construct. For functional characterization of HERA-GITRL in vitro, human immune cells isolated from healthy-donor blood were profiled by multicolor flow cytometry and real-time cell analysis. Stimulation of unfractionated human T cells or purified naïve CD4+ T cells by anti-CD3 antibody was further augmented by HERA-GITRL. This effect was accompanied by increased proliferation, differentiation and elevated levels of TNF-α and IFN-γ. Importantly, HERA-GITRL-mediated T cell activation increases tumor cell killing by PBMCs in vitro and showed in vivo anti-tumor efficacy as a single agent in a subcutaneous syngeneic colon cancer model (CT26wt) in mice. This anti-tumor effect is independent of its Fc functionality, as murine HERA-GITR ligands with functional Fc- or silenced Fc-domains show similar tumor growth inhibition. TRX518, an anti-human GITR monoclonal antibody currently investigated in a clinical Phase I study, was used in a direct in vitro comparison with trivalent GITRL and our hexavalent HERA-GITRL. Without crosslinking HERA-GITRL showed superior agonistic activity over trivalent GITRL and TRX518. Crosslinking increased the activity of trivalent GITRL while the residual activity of TRX518 was even decreased. We constructed a CHO cell line stably expressing fully human HERA-GITRL with high purity and yield. The resulting research cell bank is ready to be used for subsequent GMP process development. By clustering the receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional Fc-mediated crosslinking. A comparison of HERA-GITRL with the anti-GITR antibody TRX518 showed superior agonistic activity of our HERA construct in vitro with and without cross-linking. The HERA-ligand concept has also been successfully translated to HERA-TRAIL (now in Phase I), -CD40L, -CD27L,-LIGHT and -4-1BBL. Citation Format: Matthias Schröder, Viola Marshall, Meinolf Thiemann, David M. Richards, Christian Merz, Jaromir Sykora, Julian P. Sefrin, Mauricio Redondo-Müller, Karl Heinonen, Katharina Billian-Frey, Oliver Hill, Christian Gieffers. The novel hexavalent human GITR agonist HERA-GITRL promotes anti-tumor efficacy independent of Fc-functionality and shows superior activity compared with the monoclonal anti-GITR antibody TRX518 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4143.

The protein therapeutic and CD95L inhibitor asunercept is currently under clinical investigation for the treatment of glioblastoma and myelodysplastic syndrome. The purpose of this study was to predict the asunercept pharmacokinetics in children and to give dose recommendations for its first use in pediatric glioblastoma patients. A physiologically-based pharmacokinetic (PBPK) model of asunercept in healthy and diseased adults was successfully developed using the available clinical Phase I and Phase II study data. This model was then extrapolated to different pediatric populations, to predict the asunercept exposure in children and to find equivalent starting doses. Simulation of the asunercept serum concentration-time curves in children between 1–18 years of age shows that a dosing regimen based on body weight results in a similar asunercept steady-state exposure in all patients (pediatric or adult) above 12 years of age. For children between 1–12 years, higher doses per kg body weight are recommended, with the highest dose for the very young patients. Translational PBPK modeling is strongly encouraged by regulatory agencies to help with the initial dose selection for pediatric trials. To our knowledge, this is the first report of pediatric PBPK to support the dose selection of a therapeutic protein before its administration to children.

Abstract The tumor necrosis factor superfamily (TNFSF) member LIGHT (TNFSF14) plays an important role in regulating the activity of immune cells, especially T cells. Several studies have shown that engagement of HVEM (herpesvirus entry mediator), one of the known receptors of LIGHT, can deliver a co-stimulatory signal to support T cell activation and expansion and promote tumor clearance. The HERA technology platform developed by Apogenix generates fully human hexavalent TNFSF fusion proteins that mimic the natural receptor binding mode in order to co-stimulate T cells. HERA ligands are pure agonists whose signaling capacity does not rely on secondary Fcγ-receptor crosslinking. Here we report the in vitro and in vivo properties of a novel HERA-LIGHT construct. Similar to all HERA fusion proteins, HERA-LIGHT has been engineered as a perfect molecular mimic of the natural ligand with high clustering capacity for the cognate receptor. The core unit consists of a single chain polypeptide comprising the three minimal LIGHT-subsequences necessary for folding into a functional trivalent receptor binding domain (RBD). By fusing a silenced IgG1 Fc-domain as a dimerization scaffold to the C-terminus of the RBD we generated HERA-LIGHT, a hexavalent fusion protein. HERA-LIGHT was expressed in CHO suspension cells followed by a lab-scale purification process including AFC- and SEC-based polishing, resulting in homogenous, aggregate-free protein lots. HERA-LIGHT was proven to bind both the human and murine HVEM receptor, as determined by ELISA. Qualitative analytics revealed excellent stability following heat- and pH-stress as well as freeze-thaw cycles. Analyzing serum samples from a PK study in CD1-mice, the terminal half-life of the compound was 36.5 hours. This short half-life, relative to antibodies, allows for fast-in/fast-out dynamics essential for improving combination therapy and reducing serious side effects associated with immune system overstimulation. In order to test biological activity, T cells were isolated by magnetic sorting from human PBMCs and treated with HERA-LIGHT in vitro. Flow cytometric analysis revealed that HERA-LIGHT enhanced activation and proliferation of naïve effector T cells (Teff) following stimulation with anti-CD3 antibody, as determined by CFSE dilution. Importantly, co-stimulation with HERA-LIGHT prevented regulatory T cell (Treg)-mediated suppression of Teff proliferation. In vivo, treatment with a murine surrogate of HERA-LIGHT resulted in significant tumor growth inhibition in a pilot study using the syngeneic CT-26 colorectal cancer model.In summary, the unique hexavalent design of HERA-LIGHT mediates efficient co-stimulation of Teff even in the presence of Treg cells and independent of secondary crosslinking events. Being true agonists, all HERA molecules are unique from current antibody-based concepts rendering them attractive candidates for cancer immunotherapy. Citation Format: Julian P. Sefrin, David M. Richards, Jaromir Sykora, Meinolf Thiemann, Christian Merz, Viola Marschall, Mauricio Redondo Müller, Karl Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. Novel hexavalent HVEM agonist HERA-LIGHT promotes T cell activation and expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 630.

Abstract CD137/4-1BB is an inducible costimulatory receptor mainly expressed on immune cells following activation. Binding of the cognate ligand CD137L/4-1BBL leads to receptor trimerization and activation of signaling cascades involved in expansion and survival of T cells and myeloid cells as well as memory formation and protection against autoimmunity. Apogenix´ HERA technology is based on trivalent single-chain molecular mimics of the TNF-SF receptor binding domains fused to a silenced human IgG1-Fc-domain which serves exclusively as a dimerization scaffold. This results in hexavalent agonists replicating the natural receptor binding mode. Due to their inherent high receptor clustering capacity, HERA compounds are true agonists and their biological activity is, in contrast to bivalent anti-TNFR-SF antibodies, independent of FcR-mediated crosslinking events. HERA-CD137L was produced in CHO-S cells and purified resulting in highly pure non-aggregating protein lots. PK studies in mice demonstrated a terminal half-life of 75 hours indicating excellent in vivo stability. To study the effects on immune cells in vitro, T cells were isolated from healthy-donor buffy coats and stimulated with anti-CD3 antibody alone or in combination with HERA-CD137L. Using multicolor flow cytometry, we confirmed that expression of CD137 increased on CD8+ T cells following stimulation with anti-CD3 antibody. In accord with upregulation of the activation markers CD25 and CD69 and the memory marker CD45RO, treatment with HERA-CD137L enhanced proliferation of both CD4+ and CD8+ T cells, as determined by CFSE analysis. Intracellular accumulation of IFN-γ, TNF-α, Granzyme B and Perforin upon CD137 ligation was observed in CD8+ but not CD4+ T cells. HERA-CD137L treatment of THP-1 monocytes co-cultured with primary T cells also increased their cytotoxic activity against multiple tumor cell lines, including colorectal and mammary, as shown in a real-time live cell analysis (RTCA) assay. Primary human monocytes express low levels of CD137 and differentiation to macrophages in vitro did not increase expression. However, pro-inflammatory cytokines such as TNF-α and the chemokine CCL4 were secreted after stimulation with HERA-CD137L. RTCA assays further demonstrated enhanced antigen-specific killing of MDA-MB231 tumor cells by HERA-CD137L treated T cells. HERA-CD137L conveys its activity through effector T cell proliferation while regulatory T (Treg) cell proliferation or production of anti-inflammatory cytokines are not altered in Treg cell cultures. In contrast, HERA-CD137L prevented Treg-mediated suppression of effector T cells. Based on the in vitro data presented, HERA-CD137L is a promising candidate to promote anti-tumor immune responses either as single agent or in combination with other IO-compounds. Citation Format: Meinolf Thiemann, Jaromir Sykora, David M. Richards, Christian Merz, Viola Marschall, Mauricio Redondo Mueller, Julian P. Sefrin, Karl Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. The novel hexavalent human CD137/4-1BB agonist HERA-CD137L promotes anti-cancer immunity by activating CD8 T cells while regulatory T cells are not affected [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3754.

Abstract Introduction: HERA-CD40L is a novel hexavalent CD40 agonist engineered with the HERA-Technology developed by Apogenix. We have previously shown that the natural binding mode via the receptor/ligand binding domains and the high clustering capacity for the cognate receptor clearly distinguish HERA-CD40L from other, e.g. antibody-based, CD40-targeting compounds. Here, we report on the effects of HERA-CD40L on crosstalk between T cells and antigen presenting cells (APC) and the functional consequences in vitro. Materials & Methods: Biological activity of CD40 agonists was analyzed using co-cultures of primary T cells with B cells or monocytes/macrophages. All primary cells were isolated by negative selection using magnetic sorting from healthy donor buffy coats. Expression of CD markers upon CD40 ligation on B cells and monocytes was analyzed by flow cytometry (FC). Monitoring of T cell-induced killing of tumor cells primed in direct co-cultures with APC was done on a real-time cell analysis system (xCELLigence). For analysis of phagocytosis, we developed an FC-based assay employing primary monocytes/macrophages and Jurkat A3 cells. Results: Treatment of primary B cells and monocytes with HERA-CD40L induced expression of co-stimulatory molecules, like CD86, and promotes M1 maturation of naïve (M0) monocytes. In vitro, treatment of alternatively activated M2 macrophages with HERA-CD40L induced an M2 to M1 phenotype switch (re-programming) which concurs with CD16 downregulation and a dose-dependent decrease of phagocytic activity of re-programmed macrophages compared to M0 or M2 macrophages. Primary B cells and M1 macrophages enhanced the proliferation and cytotoxic activity of naïve T cells in direct co-cultures in the presence of HERA-CD40L. The activating effect on T cells required direct cell-cell contact with APC and was not observed in indirect co-cultures. Functionally, neutralization of either MHC-I or CD80/CD86 in direct co-cultures inhibited full activation of the T cells in vitro as shown by kill assays with various tumor cell lines. Conclusion: The hexavalent CD40 agonist HERA-CD40L produced by the Apogenix HERA-Technology is a potent immune-regulator acting on B cells and myeloid cells. HERA-CD40L promotes activation of B cells, maturation of APC and induces an M2 to M1 phenotype switch which inhibits tolerance-inducing phagocytic activity of the repolarized macrophages in vitro. In response to CD40 ligation on APC, an efficient anti-tumor response is conferred to primary T cells through cell-cell interactions via MHC-I and CD80/CD86. Citation Format: Christian Merz, Jaromir Sykora, Viola Marschall, David M. Richards, Meinolf Thiemann, Harald Fricke, Oliver Hill, Christian Gieffers. The hexavalent CD40 agonist HERA-CD40L augments multi-level crosstalk between T cells and antigen-presenting cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1760.

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.