Joerg Benz

Structural basis for the cyclophilin A binding affinity and immunosuppressive potency of E-ISA247 (voclosporin)

X-ray crystal structures of the cyclosporin A analogue E-ISA247 (voclosporin) and its stereoisomer Z-ISA247 bound to cyclophilin A suggest the molecular basis for the differences in their binding affinities and immunosuppressive efficacies.

Identification of Potent and Selective Cathepsin S Inhibitors Containing Different Central Cyclic Scaffolds

Starting from the weakly active dual CatS/K inhibitor 5, structure-based design supported by X-ray analysis led to the discovery of the potent and selective (>50,000-fold vs CatK) cyclopentane derivative 22 by exploiting specific ligand-receptor interactions in the S2 pocket of CatS. Changing the central cyclopentane scaffold to the analogous pyrrolidine derivative 57 decreased the enzyme as well as the cell-based activity significantly by 24- and 69-fold, respectively. The most promising scaffold identified was the readily accessible proline derivative (e.g., 79). This compound, with an appealing ligand efficiency (LE) of 0.47, included additional structural modifications binding in the S1 and S3 pockets of CatS, leading to favorable in vitro and in vivo properties. Compound 79 reduced IL-2 production in a transgenic DO10.11 mouse model of antigen presentation in a dose-dependent manner with an ED50 of 5 mg/kg.

Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies

Humanized hapten‐binding IgGs were designed with an accessible cysteine close to their binding pockets, for specific covalent payload attachment. Individual analyses of known structures of digoxigenin (Dig)‐ and fluorescein (Fluo) binding antibodies and a new structure of a biotin (Biot)‐binder, revealed a “universal” coupling position (52+2) in proximity to binding pockets but without contributing to hapten interactions. Payloads that carry a free thiol are positioned on the antibody and covalently linked to it via disulfides. Covalent coupling is achieved and driven toward complete (95‐100%) payload occupancy by spontaneous redox shuffling between antibody and payload. Attachment at the universal position works with different haptens, antibodies, and payloads. Examples are the haptens Fluo, Dig, and Biot combined with various fluorescent or peptidic payloads. Disulfide‐bonded covalent antibody‐payload complexes do not dissociate in vitro and in vivo. Coupling requires the designed cysteine and matching payload thiol because payload or antibody without the Cys/thiol are not linked (<5% nonspecific coupling). Hapten‐mediated positioning is necessary as hapten‐thiol‐payload is only coupled to antibodies that bind matching haptens. Covalent complexes are more stable in vivo than noncovalent counterparts because digoxigeninylated or biotinylated fluorescent payloads without disulfide‐linkage are cleared more rapidly in mice (approximately 50% reduced 48 hour serum levels) compared with their covalently linked counterparts. The coupling technology is applicable to many haptens and hapten binding antibodies (confirmed by automated analyses of the structures of 140 additional hapten binding antibodies) and can be applied to modulate the pharma‐cokinetics of small compounds or peptides. It is also suitable to link payloads in a reduction‐releasable manner to tumor‐ or tissue‐targeting delivery vehicles.—Dengl, S., Hoffmann, E., Grote, M., Wagner, C., Mundigl, O., Georges, G., Thorey, I., Stubenrauch, K.‐G., Bujotzek, A., Josel, H.‐P., Dziadek, S., Benz, J., Brinkmann, U. Hapten‐directed spontaneous disulfide shuffling: a universal technology for site‐directed covalent coupling of payloads to antibodies. FASEB J. 29, 1763‐1779 (2015). www.fasebj.org

Structure of the malaria vaccine candidate antigen CyRPA and its complex with a parasite invasion inhibitory antibody

Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines. DOI: http://dx.doi.org/10.7554/eLife.20383.001

Stepwise Evolution Improves Identification of Diverse Peptides Binding to a Protein Target.

Considerable efforts have been made to develop technologies for selection of peptidic molecules that act as substrates or binders to a protein of interest. Here we demonstrate the combination of rational peptide array library design, parallel screening and stepwise evolution, to discover novel peptide hotspots. These hotspots can be systematically evolved to create high-affinity, high-specificity binding peptides to a protein target in a reproducible and digitally controlled process. The method can be applied to synthesize both linear and cyclic peptides, as well as peptides composed of natural and non-natural amino acid analogs, thereby enabling screens in a much diverse chemical space. We apply this method to stepwise evolve peptide binders to streptavidin, a protein studied for over two decades and report novel peptides that mimic key interactions of biotin to streptavidin.

Variable heavy–variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly

Abstract Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach. There are three different crossovers possible, namely Fab-arm, constant domain and variable domain crossovers. The CrossMabCH1–CL exchange does not lead to the formation of unexpected side products, whereas the CrossMabFab and the CrossMabVH–VL formats result in the formation of typical side products. Thus, CrossMabCH1–CL was initially favored for therapeutic antibody development. Here, we report a novel improved CrossMab design principle making use of site-specific positional exchanges of charged amino acid pairs in the constant domain of these CrossMabs to enable the correct light chain assembly in the CrossMabVH–VL and improvements for the CrossMabFab design.

Abstract PR04: Targeting tumor-asoociated macrophages with a novel anti-CSF1R antibody in cancer patients

Myeloid cells represent the most abundant immune cell type within the tumor microenvironment of certain tumor entities, including tumor associated macrophages (TAMs). Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for TAMs is macrophage colony stimulating factor 1 (CSF1). We generated a monoclonal antibody (RG7155) that binds to the secondary dimerization interface of CSF1 receptor (CSF1R) as a specific and potent allosteric inhibitor. In vitro, RG7155 treatment results in cell death of CSF1-differentiated macrophages. In animal models, CSF1R inhibition reduced the F4/80+ TAMs infiltrate by 90% and was accompanied by an increase of the CD8+/CD4+ T cell ratio. The ability of RG7155 to reduce TAMs is currently evaluated in a first-in-man phase I clinical study in patients suffering either from pigmented villonodular synovitis (PVNS), a neoplastic disorder characterized by CSF1 overexpression, or other tumor entities. The associated biomarker program involves mandatory paired pre- and on-treatment biopsies of tumor and surrogate skin tissue as well as pharmacodynamic marker assessment in circulating blood. In patients treated with RG7155 an increase of CSF1 associated with a sustained decrease of CD14+CD16+ alternatively activated monocytes in peripheral blood was detected. In PVNS patients administration of RG7155 led to striking reductions of CSF1R+ and CD163+ macrophages in tumor tissue resulting in objective clinical responses according to RECIST (Response Evaluation Criteria in Solid Tumors) in 5 out of 6 patients. All six evaluable PVNS patients showed partial metabolic response in FDG-PET imaging and significant symptomatic improvement as early as 4 weeks after treatment initiation. Furthermore, TAM reduction was also observed in paired tumor samples of patients with various advanced solid malignancies, suggesting broad applicability of this therapeutic approach. This abstract is also presented as Poster A50. Citation Format: Michael Cannarile, Sabine Hoves, Ann-Marie Broeske, Joerg Benz, Katharina Wartha, Valeria Runza, Flora Rey-Giraud, Leon P. Pradel, Friedrich Feuerhake, Irina Klaman, Tobin Jones, Ute Jucknischke, Stefan Scheiblich, Ingo H. Gorr, Antje Walz, Keelara Abiraj, Philippe Cassier, Antonio Sica, Carlos Gomez-Roca, Christophe Le Tourneau, Jean-Pierre Delord, Antoine Italiano, Hyam Levitsky, Jean-Yves Blay, Dominik Ruettinger, Carola H. Ries. Targeting tumor-asoociated macrophages with a novel anti-CSF1R antibody in cancer patients. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR04. doi:10.1158/1538-7445.CHTME14-PR04

A novel recombinant human amyloid plaque-specific antibody for the treatment of Alzheimer's disease: Molecular properties and preclinical efficacy

Background: Preclinical profile of Ab binding properties and efficacy of a fully human monoclonal anti-Ab antibody. Methods: Binding properties were assessed by Biacore and X-ray crystallography. In life efficacy for plaque binding and Ab clearance was characterized by immunoassays and quantitative image analysis. Results: Anti Ab antibodies are currently explored as promising therapeutics to treat Alzheimer’s disease (AD). We have generated a new type of a human recombinant antibody with amyloid plaque specificity by phage display and in vitro maturation technologies. Antibody binding properties were optimized to achieve subnanomolar affinity and strong reactivity with human amyloid plaques and maximal target occupancy in vivo. A conformation specific binding mode was indicated by higher binding affinity to fibrillar versus monomeric Ab40/42. Epitope mapping with linear Ab decapeptides showed reactivity against N-terminal (EFRHDSGYE) and central (FFAEDVGS) amino acids, suggesting recognition of a unique structure present on Ab fibrils. X-ray crystallography revealed an interesting binding mode involving multiple amino acids of heavy and light chain CDRs and all residues of Ab 3-11. In vivo, the human antibody entered the brain and efficiently co-localized to all types of plaques in APPswe x PS2N141I (PS2APP) mice. Plaque binding was dose-dependent with a minimal effective dose of 0.1 mg/kg and detectable over 2 months after single administration. Chronic treatment of PS2APP mice revealed a significant decrease of amyloid plaque load by partial clearance of pre-existing plaques and substantial prevention of de novo plaque formation. Efficacy of amyloid lowering was directly dependent on the ability of the antibody to cross the blood-brain-barrier and to bind to amyloid plaques. Remarkably, peripheral Ab40 and Ab42 levels were not increased after administration of the human antibody to PS2APP mice indicating that clearance of peripheral Ab was not affected. The absence of stable antibody-Ab complexes in blood may also have facilitated the pronounced and long lasting target occupancy observed in vivo in PS2APP mice. Conclusions: The fully human antibody described represents a novel, centrally-active anti-Ab antibody with a broad and sensitive Ab-amyloid specificity. Its binding mode and amyloid lowering capacity are promising features supporting further development as an immunotherapeutic treatment for Alzheimer’s Disease.