Josefin-Beate Holz

Evaluation of efficacy and safety of the anti-vWF Nanobody ALX-0681 in a preclinical baboon model of acquired thrombotic thrombocytopenic purpura

ALX-0681 is a therapeutic Nanobody targeting the A1-domain of VWF. It inhibits the interaction between ultra-large VWF and platelet GpIb-IX-V, which plays a crucial role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In the present study, we report the efficacy and safety profile of ALX-0681 in a baboon model of acquired TTP. In this model, acute episodes of TTP are induced by administration of an ADAMTS13-inhibiting mAb. ALX-0681 completely prevented the rapid onset of severe thrombocytopenia and schistocytic hemolytic anemia. After induction of TTP, platelet counts also rapidly recovered on administration of ALX-0681. This effect was corroborated by the full neutralization of VWF activity. The schistocytic hemolytic anemia was also halted and partially reversed by ALX-0681 treatment. Brain CT scans and post mortem analysis did not reveal any sign of bleeding, suggesting that complete neutralization of VWF by ALX-0681 under conditions of thrombocytopenia was not linked with an excessive bleeding risk. The results obtained in this study demonstrate that ALX-0681 can successfully treat and prevent the most important hallmarks of acquired TTP without evidence of a severe bleeding risk. Therefore, ALX-0681 offers an attractive new therapeutic option for acquired TTP in the clinical setting.

The TITAN trial – Assessing the efficacy and safety of an anti-von Willebrand factor Nanobody in patients with acquired thrombotic thrombocytopenic purpura

The Phase II TITAN trial is designed to assess the efficacy and safety of an anti-von Willebrand factor (vWF) Nanobody in patients with acquired thrombotic thrombocytopenic purpura (TTP). Nanobodies are a novel class of therapeutic proteins and are based on the smallest functional fragments of single-chain antibodies that occur naturally in the Camelidae family (Nanobody® and Nanobodies® are registered trademarks of Ablynx NV). With vWF implicated in the thrombotic process underlying TTP, an anti-vWF Nanobody may hold significant promise as adjunctive therapy to plasma exchange. Recruitment is currently ongoing, and aims to include a total of 110 patients from countries in Europe, the Middle East, Australia and Northern America.

Novel antiplatelet agents: ALX-0081, a Nanobody directed towards von Willebrand factor.

This manuscript reviews the studies performed with ALX-0081 (INN: caplacizumab), a Nanobody targeting von Willebrand factor, in the context of current antithrombotic therapy in coronary artery disease. ALX-0081 specifically inhibits platelet adhesion to the vessel wall, and may control platelet aggregation and subsequent clot formation without increasing bleeding risk. A substantial number of antithrombotics are aimed at this cascade; however, their generally indiscriminative mode of action can result in a narrow therapeutic window, defined by the risk for bleeding complications, and thrombotic events. Nonclinically, ALX-0081 compared favorably to several antithrombotics. In Phase I studies in healthy subjects and stable angina patients undergoing percutaneous coronary intervention (PCI), ALX-0081 was well tolerated, and effectively inhibited pharmacodynamic markers. Following these results, a phase II study was initiated in high-risk acute coronary syndrome patients undergoing PCI. Based on its mechanism of action, ALX-0081 is also being developed for acquired thrombotic thrombocytopenic purpura.

FRI0021 Alx-0061, an anti-IL-6r nanobody® for therapeutic use in rheumatoid arthritis, demonstrates in vitro a differential biological activity profile as compared to tocilizumab

Background Interleukin-6 (IL-6) is a pleiotropic cytokine inducing a wide range of biological activities via its receptor, which can either be soluble (sIL-6R) or membrane-bound (mIL-6R). Blocking of IL-6R results in clinical benefit in rheumatoid arthritis as demonstrated by the marketed IL-6R inhibitor tocilizumab (TCZ). Signalling via the mIL-6R (“classical pathway”) is confined to selected cell types due to the restricted expression of mIL-6R. However, IL-6 can also activate cells through sIL-6R in a process known as trans-signalling. Unwanted pharmacology associated with IL-6 pathway inhibition has been linked to inhibition of mIL-6R. Preferential inhibition of sIL-6R could therefore provide higher therapeutic efficacy with a better side effect profile compared to equivalent inhibition of both IL-6R forms (1). Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain-only antibodies, naturally occurring in the Camelidae family. ALX-0061 is a bispecific anti-IL-6R Nanobody engineered to have an extended half-life in vivo by targeting human serum albumin (HSA), in combination with a high target affinity and potency using a single anti-IL-6R building block. Objectives ALX-0061 was extensively characterised using in vitro systems: biological activity and affinity for both sIL-6R and mIL-6R were assessed and compared to TCZ. Methods Biological activity of ALX-0061 and TCZ was analysed in a cell-based assay for mIL-6R, ELISA-based neutralisation assays for sIL-6R, and cell-binding and cell-signalling (mIL-6R) experiments in whole blood from human donors using flow cytometry. Due to very tight target binding, the affinity of ALX-0061 for sIL-6R could not be accurately determined via surface plasmon resonance. Consequently, the more sensitive GyrolabTM platform was used to assess affinity for both receptors. For the KD determination on mIL-6R, free compound concentrations were measured in the supernatant, after pre-incubation of mIL-6R-transfected cells with a constant compound concentration. Results Flow cytometry experiments demonstrated that ALX-0061 binds to mIL-6R expressed on peripheral blood leukocyte populations with expected pharmacology. ALX-0061 specifically neutralised sIL-6R with a 10-fold higher in vitro potency compared to TCZ, while the (apparent) affinity of ALX-0061 for sIL-6R (0.20 pM) was 2000-fold superior compared to TCZ (462 pM). In the mIL-6R-driven cell-based assay, however, in vitro potencies were similar for ALX-0061 and TCZ, with the latter one showing avid binding due to its bivalency. In addition, TCZ showed a 3-fold higher affinity for mIL-6R (160 pM) compared to sIL-6R, while the affinity of ALX-0061 was 45-fold lower for mIL-6R (9 pM) compared to sIL-6R. Conclusions ALX-0061 demonstrates in vitro a preferential biological activity profile for sIL-6R with a lesser activity for mIL-6R, while TCZ has a higher preference for mIL-6R. Preferential inhibition of sIL-6R trans-signalling by ALX-0061 could provide superior therapeutic efficacy with a better side effect profile than TCZ. ALX-0061 is currently in clinical development. Analysis of a phase I/II study demonstrated a strong efficacy and an attractive safety profile. References Waetzig G.H. & Rose-John S., Expert Opin Ther Targets (2012) 16(2) Disclosure of Interest None Declared

Abstract LB-316: The anti-c-Met Nanobody®, a novel and promising anti-cancer therapeutic.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The hepatocyte growth factor (HGF) and its receptor, c-Met, are implicated in many human cancers to promote tumor proliferation, migration, invasion and angiogenesis. Dysregulation of the HGF/c-Met pathway is known to correlate with poor prognosis. In recent years, multiple anti-cancer agents have been developed interfering at different levels in the HGF/c-Met pathway. Here, we investigated the in vitro and in vivo efficacy of a novel Nanobody effectively antagonizing c-Met. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies, naturally occurring in Camelidae. The Nanobody platform allows designing modular drugs, including multivalent, biparatopic and bifunctional molecules. The anti-c-Met Nanobody is a bispecific Nanobody that comprises two building blocks, one targeting c-Met and one binding human serum albumin for half-life extension purposes. In vitro characterization revealed that the Nanobody is able to specifically and completely inhibit HGF/c-Met interaction with high potency in an ELISA-based assay. In addition, the anti-c-Met Nanobody was able to completely block the HGF-dependent c-Met-phosphorylation in the A549 non-small cell lung cancer cell line. Importantly, no agonistic activity was observed in absence of HGF, as the Nanobody was designed to have a monovalent interaction with c-Met. Next, the in vivo effect was translated towards a potent in vivo effect. The anti-tumor efficacy of the anti-c-Met Nanobody was assessed in xenograft mouse models in which autocrine U87MG (HGF+, c-Met+) glioblastoma cells, or autocrine KP4 (HGF+, c-Met+) pancreatic carcinoma cells were subcutaneously injected. In the U87MG xenograft model, Nanobody treatment (10 mg/kg i.p 3x/week) resulted in significant tumor growth inhibition compared to vehicle and sustained tumor growth suppression after cessation of the treatment. Treatment with the anti-c-Met Nanobody in the KP4 xenograft model (10 mg/kg i.p 3x/week) led to a significant tumor growth inhibition and even tumor regression. In the latter model, serum levels of soluble c-Met, IL-8 and HGF were shown to be significantly reduced upon Nanobody treatment, which could make these markers attractive as potential translational pharmacodynamics biomarkers. Furthermore, the ratio of tyrosine phosphorylated c-Met over total c-Met levels was significantly lower in KP4 tumors treated with Nanobody versus vehicle. It could be postulated based on literature data that the anti-c-Met Nanobody, by possessing an anti-albumin Nanobody building block, has the potential to show superior tumor penetration than c-Met targeting antibodies. Hence, the anti-c-Met Nanobody might be a valuable novel biological for the treatment of cancer driven by HGF-c-Met signaling. Citation Format: Tinneke Denayer, Thomas Stohr, Gerald Beste, Ann Brige, Cedric Ververken, Josefin-Beate Holz. The anti-c-Met Nanobody®, a novel and promising anti-cancer therapeutic. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-316. doi:10.1158/1538-7445.AM2013-LB-316