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.

Preclinical Development of U3-1784, a Novel FGFR4 Antibody Against Cancer, and Avoidance of Its On-target Toxicity.

The FGFR4/FGF19 signaling axis is overactivated in 20% of liver tumors and currently represents a promising targetable signaling mechanism in this cancer type. However, blocking FGFR4 or FGF19 has proven challenging due to its physiological role in suppressing bile acid synthesis which leads to increased toxic bile acid plasma levels upon FGFR4 inhibition. An FGFR4-targeting antibody, U3-1784, was generated in order to investigate its suitability as a cancer treatment without major side effects.U3-1784 is a high-affinity fully human antibody that was obtained by phage display technology and specifically binds to FGFR4. The antibody inhibits cell signaling by competing with various FGFs for their FGFR4 binding site thereby inhibiting receptor activation and downstream signaling via FRS2 and Erk. The inhibitory effect on tumor growth was investigated in 10 different liver cancer models in vivo The antibody specifically slowed tumor growth of models overexpressing FGF19 by up to 90% whereas tumor growth of models not expressing FGF19 was unaffected. In cynomolgus monkeys, intravenous injection of U3-1784 caused elevated serum bile acid and liver enzyme levels indicating potential liver damage. These effects could be completely prevented by the concomitant oral treatment with the bile acid sequestrant colestyramine, which binds and eliminates bile acids in the gut. These results offer a new biomarker-driven treatment modality in liver cancer without toxicity and they suggest a general strategy for avoiding adverse events with FGFR4 inhibitors.

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.

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.