The CD40 agonist HERA-CD40L results in enhanced activation of antigen presenting cells, promoting an anti-tumor effect alone and in combination with radiotherapy.

Introduction: The ability to modulate and enhance the anti-tumor immune responses is critical in developing novel therapies in cancer. The Tumor Necrosis Factor (TNF) Receptor Super Family (TNFRSF) are potentially excellent targets for modulation which result in specific anti-tumor immune responses. CD40 is a member of the TNFRSF and several clinical therapies are under development. CD40 signaling plays a pivotal role in regulating the immune system from B cell responses to myeloid cell driven activation of T cells. The CD40 signaling axis is well characterized and here we compare next generation HERA-Ligands to conventional monoclonal antibody based immune modulation for the treatment of cancer. Methods & results: HERA-CD40L is a novel molecule that targets CD40 mediated signal transduction and demonstrates a clear mode of action in generating an activated receptor complex via recruitment of TRAFs, cIAP1, and HOIP, leading to TRAF2 phosphorylation and ultimately resulting in the enhanced activation of key inflammatory/survival pathway and transcription factors such asNFkB, AKT, p38, ERK1/2, JNK, and STAT1 in dendritic cells. Furthermore, HERA-CD40L demonstrated a strong modulation of the tumor microenvironment (TME) via the increase in intratumoral CD8+ T cells and the functional switch from pro-tumor macrophages (TAMs) to anti-tumor macrophages that together results in a significant reduction of tumor growth in a CT26 mouse model. Furthermore, radiotherapy which may have an immunosuppressive modulation of the TME, was shown to have an immunostimulatory effect in combination with HERA-CD40L. Radiotherapy in combination with HERA-CD40L treatment resulted in an increase in detected intratumoral CD4+/8+ T cells compared to RT alone and, additionally, the repolarization of TAMs was also observed, resulting in an inhibition of tumor growth in a TRAMP-C1 mouse model. Discussion: Taken together, HERA-CD40L resulted in activating signal transduction mechanisms in dendritic cells, resulting in an increase in intratumoral T cells and manipulation of the TME to be pro-inflammatory, repolarizing M2 macrophages to M1, enhancing tumor control.

3D Cellular Architecture Modulates Tyrosine Kinase Activity, Thereby Switching CD95-Mediated Apoptosis to Survival.

The death receptor CD95 is expressed in every cancer cell, thus providing a promising tool to target cancer. Activation of CD95 can, however, lead to apoptosis or proliferation. Yet the molecular determinants of CD95's mode of action remain unclear. Here, we identify an optimal distance between CD95Ligand molecules that enables specific clustering of receptor-ligand pairs, leading to efficient CD95 activation. Surprisingly, efficient CD95 activation leads to apoptosis in cancer cells in vitro and increased tumor growth in vivo. We show that allowing a 3D aggregation of cancer cells in vitro switches the apoptotic response to proliferation. Indeed, we demonstrate that the absence or presence of cell-cell contacts dictates the cell response to CD95. Cell contacts increase global levels of phosphorylated tyrosines, including CD95's tyrosine. A tyrosine-to-alanine CD95 mutant blocks proliferation in cells in contact. Our study sheds light into the regulatory mechanism of CD95 activation that can be further explored for anti-cancer therapies.

HERA-GITRL activates T cells and promotes anti-tumor efficacy independent of FcγR-binding functionality.

BACKGROUND: Glucocorticoid-induced TNFR-related protein (TNFRSF18, GITR, CD357), expressed by T cells, and its ligand (TNFSF18, GITRL), expressed by myeloid populations, provide co-stimulatory signals that boost T cell activity. Due to the important role that GITR plays in regulating immune functions, agonistic stimulation of GITR is a promising therapeutic concept. Multiple strategies to induce GITR signaling have been investigated. The limited clinical efficacy of antibody-based GITR agonists results from structural and functional characteristics of antibodies that are unsuitable for stimulating the well-defined trimeric members of the TNFRSF. METHODS: To overcome limitations of antibody-based TNFRSF agonists, we have developed HERA-GITRL, a fully human hexavalent TNF receptor agonist (HERA) targeting GITR and mimicking the natural signaling concept. HERA-GITRL is composed of a trivalent but single-chain GITRL-receptor-binding-domain (scGITRL-RBD) unit fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. A specific mouse surrogate, mmHERA-GITRL, was also generated to examine in vivo activity in respective mouse tumor models. RESULTS: For functional characterization of HERA-GITRL in vitro, human immune cells were isolated from healthy-donor blood and stimulated with anti-CD3 antibody in the presence of HERA-GITRL. Consistently, HERA-GITRL increased the activity of T cells, including proliferation and differentiation, even in the presence of regulatory T cells. In line with these findings, mmHERA-GITRL enhanced antigen-specific clonal expansion of both CD4+ (OT-II) and CD8+ (OT-I) T cells in vivo while having no effect on non-specific T cells. In addition, mmHERA-GITRL showed single-agent anti-tumor activity in two subcutaneous syngeneic colon cancer models (CT26wt and MC38-CEA). Importantly, this activity is independent of its FcγR-binding functionality, as both mmHERA-GITRL with a functional Fc- and a silenced Fc-domain showed similar tumor growth inhibition. Finally, in a direct in vitro comparison to a bivalent clinical benchmark anti-GITR antibody and a trivalent GITRL, only the hexavalent HERA-GITRL showed full biological activity independent of additional crosslinking. CONCLUSION: In this manuscript, we describe the development of HERA-GITRL, a true GITR agonist with a clearly defined mechanism of action. By clustering six receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional FcγR-mediated crosslinking.

Translational PBPK Modeling of the Protein Therapeutic and CD95L Inhibitor Asunercept to Develop Dose Recommendations for Its First Use in Pediatric Glioblastoma Patients.

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.

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.

The Hexavalent CD40 Agonist HERA-CD40L Induces T-Cell-mediated Antitumor Immune Response Through Activation of Antigen-presenting Cells.

CD40 ligand (TNFSF5/CD154/CD40L), a member of the tumor necrosis factor (TNF) superfamily is a key regulator of the immune system. The cognate receptor CD40 (TNFRSF5) is expressed broadly on antigen-presenting cells and many tumor types, and has emerged as an attractive target for immunologic cancer treatment. Most of the CD40 targeting drugs in clinical development are antibodies which display some disadvantages: their activity typically depends on Fcγ receptor-mediated crosslinking, and depletion of CD40-expressing immune cells by antibody-dependent cellular cytotoxicity compromises an efficient antitumor response. To overcome the inadequacies of antibodies, we have developed the hexavalent receptor agonist (HERA) Technology. HERA compounds are fusion proteins composed of 3 receptor binding domains in a single chain arrangement, linked to an Fc-silenced human IgG1 thereby generating a hexavalent molecule. HERA-CD40L provides efficient receptor agonism on CD40-expressing cells and, importantly, does not require FcγR-mediated crosslinking. Strong activation of NFκB signaling was observed upon treatment of B cells with HERA-CD40L. Monocyte treatment with HERA-CD40L promoted differentiation towards the M1 spectrum and repolarization of M2 spectrum macrophages towards the M1 spectrum phenotype. Treatment of in vitro co-cultures of T and B cells with HERA-CD40L-triggered robust antitumor activation of T cells, which depended upon direct interaction with B cells. In contrast, bivalent anti-CD40 antibodies and trivalent soluble CD40L displayed weak activity which critically depended on crosslinking. In vivo, a murine surrogate of HERA-CD40L-stimulated clonal expansion of OT-I-specific murine CD8 T cells and showed single agent antitumor activity in the CD40 syngeneic MC38-CEA mouse model of colorectal cancer, suggesting an involvement of the immune system in controlling tumor growth. We conclude that HERA-CD40L is able to establish robust antitumor immune responses both in vitro and in vivo.

Infiltration of circulating myeloid cells through CD95L contributes to neurodegeneration in mice.

Neuroinflammation is increasingly recognized as a hallmark of neurodegeneration. Activated central nervous system-resident microglia and infiltrating immune cells contribute to the degeneration of dopaminergic neurons (DNs). However, how the inflammatory process leads to neuron loss and whether blocking this response would be beneficial to disease progression remains largely unknown. CD95 is a mediator of inflammation that has also been proposed as an apoptosis inducer in DNs, but previous studies using ubiquitous deletion of CD95 or CD95L in mouse models of neurodegeneration have generated conflicting results. Here we examine the role of CD95 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP)-induced neurodegeneration using tissue-specific deletion of CD95 or CD95L. We show that DN death is not mediated by CD95-induced apoptosis because deletion of CD95 in DNs does not influence MPTP-induced neurodegeneration. In contrast, deletion of CD95L in peripheral myeloid cells significantly protects against MPTP neurotoxicity and preserves striatal dopamine levels. Systemic pharmacological inhibition of CD95L dampens the peripheral innate response, reduces the accumulation of infiltrating myeloid cells, and efficiently prevents MPTP-induced DN death. Altogether, this study emphasizes the role of the peripheral innate immune response in neurodegeneration and identifies CD95 as potential pharmacological target for neurodegenerative disease.

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.

Pharmacokinetics, pharmacodynamics, safety and tolerability of APG101, a CD95-Fc fusion protein, in healthy volunteers and two glioma patients.

APG101 is a glycosylated fusion protein consisting of the extracellular domain of human CD95 (APO-1/Fas) and the Fc domain of human IgG1. Administration of APG101 blocks the interaction between CD95 and its cognate ligand CD95L, thereby inhibiting various pathways involved in e.g. proliferation, migration, differentiation and apoptosis induction. The safety and tolerability of ascending single doses of intravenously applied APG101 was examined in a randomized, double-blind, placebo-controlled, mono-centre "first in man" dose escalation study in 34 healthy male volunteers. Pharmacokinetics and pharmacodynamics were also assessed. The maximum serum concentration of 460 µg/ml was achieved following 1h infusion of the highest dose of 20 mg/kg. The systemic clearance was low (0.4 to 0.5 ml/hkg). Mean terminal elimination half-life was 12 to 15 days. Two patients suffering from malignant glioma received APG101 intravenously under compassionate use conditions. They received doses ranging from 5mg to 600 mg APG101. No adverse events and no clinical significant changes in laboratory parameters related to APG101 were reported. The presence of anti-drug-antibodies (ADA) was investigated and revealed no detectable levels of ADA. Overall, single ascending doses of APG101 up to 20 mg/kgbody weight (bw) administered as infusion over 1h were considered as safe and well tolerated in healthy volunteers. After the application of multiple doses of 400 mg in two glioma patients, steady state for APG101 seemed to be reached. These results support further clinical evaluation of APG101 at a dose of 400 mg per week in glioblastoma patients.

CD95-ligand on peripheral myeloid cells activates Syk kinase to trigger their recruitment to the inflammatory site.

Injury to the central nervous system initiates an uncontrolled inflammatory response that results in both tissue repair and destruction. Here, we showed that, in rodents and humans, injury to the spinal cord triggered surface expression of CD95 ligand (CD95L, FasL) on peripheral blood myeloid cells. CD95L stimulation of CD95 on these cells activated phosphoinositide 3-kinase (PI3K) and metalloproteinase-9 (MMP-9) via recruitment and activation of Syk kinase, ultimately leading to increased migration. Exclusive CD95L deletion in myeloid cells greatly decreased the number of neutrophils and macrophages infiltrating the injured spinal cord or the inflamed peritoneum after thioglycollate injection. Importantly, deletion of myeloid CD95L, but not of CD95 on neural cells, led to functional recovery of spinal injured animals. Our results indicate that CD95L acts on peripheral myeloid cells to induce tissue damage. Thus, neutralization of CD95L should be considered as a means to create a controlled beneficial inflammatory response.

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.

The death receptor CD95 activates adult neural stem cells for working memory formation and brain repair.

Adult neurogenesis persists in the subventricular zone and the dentate gyrus and can be induced upon central nervous system injury. However, the final contribution of newborn neurons to neuronal networks is limited. Here we show that in neural stem cells, stimulation of the "death receptor" CD95 does not trigger apoptosis but unexpectedly leads to increased stem cell survival and neuronal specification. These effects are mediated via activation of the Src/PI3K/AKT/mTOR signaling pathway, ultimately leading to a global increase in protein translation. Induction of neurogenesis by CD95 was further confirmed in the ischemic CA1 region, in the naive dentate gyrus, and after forced expression of CD95L in the adult subventricular zone. Lack of hippocampal CD95 resulted in a reduction in neurogenesis and working memory deficits. Following global ischemia, CD95-mediated brain repair rescued behavioral impairment. Thus, we identify the CD95/CD95L system as an instructive signal for ongoing and injury-induced neurogenesis.

From lead to preclinical candidate: optimization of beta-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.

Yes and PI3K bind CD95 to signal invasion of glioblastoma.

Invasion of surrounding brain tissue by isolated tumor cells represents one of the main obstacles to a curative therapy of glioblastoma multiforme. Here we unravel a mechanism regulating glioma infiltration. Tumor interaction with the surrounding brain tissue induces CD95 Ligand expression. Binding of CD95 Ligand to CD95 on glioblastoma cells recruits the Src family member Yes and the p85 subunit of phosphatidylinositol 3-kinase to CD95, which signal invasion via the glycogen synthase kinase 3-beta pathway and subsequent expression of matrix metalloproteinases. In a murine syngeneic model of intracranial GBM, neutralization of CD95 activity dramatically reduced the number of invading cells. Our results uncover CD95 as an activator of PI3K and, most importantly, as a crucial trigger of basal invasion of glioblastoma in vivo.

The reversed binding of beta-phenethylamine inhibitors of DPP-IV: X-ray structures and properties of novel fragment and elaborated inhibitors.

The co-crystal structure of beta-phenethylamine fragment inhibitor 5 bound to DPP-IV revealed that the phenyl ring occupied the proline pocket of the enzyme. This finding provided the basis for a general hypothesis of a reverse binding mode for beta-phenethylamine-based DPP-IV inhibitors. Novel inhibitor design concepts that obviate substrate-like structure-activity relationships (SAR) were thereby enabled, and novel, potent inhibitors were discovered.

In silico fragment-based discovery of DPP-IV S1 pocket binders.

Dipeptidyl peptidase IV is a clinically validated target for type-2 diabetes and belongs to a family of peptidases with a quite unique post-proline cleavage specificity. Known inhibitors contain a limited number of molecular anchors occupying the small prototypical S1 pocket. A virtual screening approach for such S1-binding fragments was carried out using FlexX docking to evaluate its potential to confirm known and find novel compounds. Several low molecular weight inhibitors exhibiting activities in the micromolar range could be identified as starting points for structure-based design.