Karen Silence

Antithrombotic drug candidate ALX-0081 shows superior preclinical efficacy and safety compared with currently marketed antiplatelet drugs

Neutralizing the interaction of the platelet receptor gpIb with VWF is an attractive strategy to treat and prevent thrombotic complications. ALX-0081 is a bivalent Nanobody which specifically targets the gpIb-binding site of VWF and interacts avidly with VWF. Nanobodies are therapeutic proteins derived from naturally occurring heavy-chain-only Abs and combine a small molecular size with a high inherent stability. ALX-0081 exerts potent activity in vitro and in vivo. Perfusion experiments with blood from patients with acute coronary syndrome on standard antithrombotics demonstrated complete inhibition of platelet adhesion after addition of ALX-0081, while in the absence of ALX-0081 residual adhesion was observed. In a baboon efficacy and safety model measuring acute thrombosis and surgical bleeding, ALX-0081 showed a superior therapeutic window compared with marketed antithrombotics. Pharmacokinetic and biodistribution experiments demonstrated target-mediated clearance of ALX-0081, which leads to a self-regulating disposition behavior. In conclusion, these preclinical data demonstrate that ALX-0081 combines a high efficacy with an improved safety profile compared with currently marketed antithrombotics. ALX-0081 has entered clinical development.

Acute activation of the endothelium results in increased levels of active von Willebrand factor in hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome

Summary.  Background: HELLP (hemolysis, elevated liver enzymes and low platelets) syndrome is a severe complication of pre‐eclampsia in pregnancy, characterized by microvascular platelet thrombi. Activation of the endothelium is thought to play a key role in pre‐eclampsia and HELLP syndrome. Activation of endothelial cells may lead to release of von Willebrand factor (VWF) multimers, which are highly reactive with platelets. Normally, newly released multimers are cleaved by ADAMTS13, resulting in less reactive derivatives. Objective: We hypothesized that HELLP syndrome is characterized by increased amounts of active VWF compared with healthy pregnancy and pre‐eclampsia, due to acute activation of endothelial cells. This might contribute to thrombocytopenia and thrombotic microangiopathy. Methods: Active VWF and ADAMTS13 activity were measured in healthy pregnant volunteers (n = 9), patients with pre‐eclampsia (n = 6) and patients with HELLP syndrome (n = 14) at similar gestational ages. To study the role of endothelial cell activation, the propeptide/mature VWF ratio was determined, and VWF released by cultured endothelial cells was analyzed. Results: Active VWF levels were increased 2.1‐fold in HELLP syndrome compared with healthy pregnant volunteers (P < 0.001) and 1.6‐fold compared with patients with pre‐eclampsia (P = 0.001). ADAMTS13 activity was moderately decreased in patients with HELLP syndrome compared with healthy pregnant volunteers (P < 0.004), but not compared with patients with pre‐eclampsia. The propeptide/mature VWF ratio was increased 1.7‐fold compared with healthy pregnant volunteers (P < 0.001) and 1.5‐fold compared with patients with pre‐eclampsia (P < 0.05). A significant correlation was found between this ratio and the activation factor of VWF (r = 0.68, P < 0.001). The amount of active VWF was increased 1.4‐fold in medium of stimulated endothelial cells when compared with non‐stimulated cells (P < 0.05). Conclusion: Acute endothelial cell activation in HELLP syndrome and decreased ADAMTS13 activity result in increased amounts of active VWF. This might explain the consumptive thrombocytopenia and thrombotic microangiopathy associated with HELLP syndrome. Inhibition of circulating active VWF could be a potential new approach in the treatment of patients with HELLP syndrome.

Strong in vivo maturation compensates for structurally restricted H3 loops in antibody repertoires.

A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites. Much of the diversity of this repertoire is provided by varying one antigen binding loop, created by inserting randomly a D (diversity) gene out of a small pool between the V and J genes. It is therefore assumed that any particular D-encoded region surrounded by different V and J regions adopts a different conformation. We have solved the structure of two lysozyme-specific variable domains of heavy-chain antibodies isolated from two strictly unrelated dromedaries. These antibodies recombined identical D gene sequences to different V and J precursors with significant variance in their V(D)J junctions. Despite these large differences, the D-encoded loop segments adopt remarkably identical architectures, thus directing the antibodies toward identical epitopes. Furthermore, a striking convergent maturation process occurred in the V region, adapting both binders for their sub-nanomolar affinity association with lysozyme. Hence, on a structural level, humoral immunity may rely more on well developed maturation and selection systems than on the acquisition of large primary repertoires.

CD70: An emerging target in cancer immunotherapy

Over the last decades, advances in the knowledge of immunology have led to the identification of immune checkpoints, reinvigorating cancer immunotherapy. Although normally restricted to activated T and B cells, constitutive expression of CD70 in tumor cells has been described. Moreover, CD70 is implicated in tumor cell and regulatory T cell survival through interaction with its ligand, CD27. In this review, we summarize the targetable expression patterns of CD70 in a wide range of malignancies and the promising mechanism of anti-CD70 therapy in stimulating the anti-tumor immune response. In addition, we will discuss clinical data and future combination strategies.

ARGX-110, a highly potent antibody targeting CD70, eliminates tumors via both enhanced ADCC and immune checkpoint blockade

Overexpression of CD70 has been documented in a variety of solid and hematological tumors, where it is thought to play a role in tumor proliferation and evasion of immune surveillance. Here, we describe ARGX-110, a defucosylated IgG1 monoclonal antibody (mAb) that selectively targets and neutralizes CD70, the ligand of CD27. ARGX-110 was generated by immunization of outbred llamas. The antibody was germlined to 95% human identity, and its anti-tumor efficacy was tested in several in vitro assays. ARGX-110 binds CD70 with picomolar affinity. In depletion studies, ARGX-110 lyses tumor cells with greater efficacy than its fucosylated version. In addition, ARGX-110 demonstrates strong complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis activity. ARGX-110 inhibits signaling of CD27, which results in blocking of the activation and proliferation of Tregs. In a Raji xenograft model, administration of the fucosylated version of ARGX-110 resulted in a prolonged survival at doses of 0.1 mg/kg and above. The pharmacokinetics of ARGX-110 was tested in cynomolgus monkeys; the calculated half-life is 12 days. In conclusion, ARGX-110 is a potent blocking mAb with a dual mode of action against both CD70-bearing tumor cells and CD70-dependent Tregs. This antibody is now in a Phase 1 study in patients with advanced malignancies expressing CD70 (NCT01813539).

Depleting MET-Expressing Tumor Cells by ADCC Provides a Therapeutic Advantage over Inhibiting HGF/MET Signaling

Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies.

A novel llama antibody targeting Fn14 exhibits anti-metastatic activity in vivo

Expression of fibroblast growth factor (FGF)-inducible 14 (Fn14), a member of the tumor necrosis factor receptor superfamily, is typically low in healthy adult organisms, but strong Fn14 expression is induced in tissue injury and tissue remodeling. High Fn14 expression is also observed in solid tumors, which is why this receptor is under consideration as a therapeutic target in oncology. Here, we describe various novel mouse-human cross-reactive llama-derived recombinant Fn14-specific antibodies (5B6, 18D1, 4G5) harboring the human IgG1 Fc domain. In contrast to recombinant variants of the established Fn14-specific antibodies PDL192 and P4A8, all three llama-derived antibodies efficiently bound to the W42A and R56P mutants of human Fn14. 18D1 and 4G5, but not 5B6, efficiently blocked TNF-like weak inducer of apoptosis (TWEAK) binding at low concentrations (0.2–2 µg/ml). Oligomerization and Fcγ receptor (FcγR) binding converted all antibodies into strong Fn14 agonists. Variants of 18D1 with enhanced and reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity were further analyzed in vivo with respect to their effect on metastasis. In a xenogeneic model using human colon carcinoma cancer cells, both antibody variants were effective in reducing metastasis to the liver. In contrast, only the 18D1 variant with enhanced ADCC activity, but not its ADCC-defective counterpart, suppressed lung metastasis in the RENCA model. In sum, this suggests that Fn14 targeting might primarily act by triggering of antibody effector functions, but also by blockade of TWEAK-Fn14 interaction in some cases.

CD70 and PD-L1 in anaplastic thyroid cancer : promising targets for immunotherapy

During recent years, immune checkpoint inhibition has proved to be effective in several solid malignancies. The aim of this study was to identify novel targets for immunotherapy in anaplastic thyroid cancer by analysis of the expression of tumour antigens for which therapeutic agents are available.

Bivalent Llama Single-Domain Antibody Fragments against Tumor Necrosis Factor Have Picomolar Potencies due to Intramolecular Interactions

The activity of tumor necrosis factor (TNF), a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody (VHH, also called Nanobodies™) were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs (VHH#1, VHH#2, and VHH#3) were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF–VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled via linkers of different lengths. Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.

Phase I dose-escalation study of the anti-CD70 antibody ARGX-110 in advanced malignancies

Purpose: The purpose of this study was to evaluate safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor efficacy of ARGX-110, a glyco-engineered monoclonal antibody, targeting CD70, in patients with CD70 expressing advanced malignancies. Experimental Design: Dose escalation with a sequential 3+3 design was performed in five steps at the 0.1, 1, 2, 5, and 10 mg/kg dose levels (N = 26). ARGX-110 was administered intravenously every 3 weeks until progression or intolerable toxicity. Dose-limiting toxicity was evaluated in the 21 days following the first ARGX-110 administration (Cycle 1). Samples for pharmacokinetics and pharmacodynamics were collected. Results: Dose-limiting toxicity was not observed and the maximum tolerated dose was not reached. ARGX-110 was generally well tolerated, with no dose-related increase in treatment-emergent adverse events (TEAE). The most common TEAE were fatigue and drug related infusion-related reactions (IRR). Of the 20 SAEs reported, five events, all IRRs, were considered related to ARGX-110. ARGX-110 demonstrates dose proportionality over the dose range 1 to 10 mg/kg, but not at 0.1 mg/kg and a terminal half-life of 10 to 13 days. The best overall response was stable disease (14/26) in all 26 evaluable patients with various malignancies and the mean duration of treatment was 15 weeks. No dose–response related antitumor activity was observed, but biomarker readouts provided signs of biological activity, particularly in patients with hematologic malignancies. Conclusions: This dose-escalation phase I trial provides evidence of good tolerability of ARGX-110, pharmacokinetics, and preliminary antitumor activity at all dose levels in generally heavily pretreated patients with advanced CD70-positive malignancies. Clin Cancer Res; 23(21); 6411–20. ©2017 AACR.