Hans-Peter Josel

Bispecific digoxigenin-binding antibodies for targeted payload delivery

Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 2∶1 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.

Competitive homogeneous digoxigenin immunoassay based on fluorescence quenching by gold nanoparticles

We report on a competitive, homogeneous immunoassay for the detection of the hapten digoxigenin. The assay is based on competitive fluorescence quenching by gold nanoparticles. Digoxigenin is indirectly labeled with the fluorophore Cy3B through bovine serum albumin and used as a marker. Gold nanoparticles functionalized with anti-digoxigenin antibodies serve as fluorescence quenchers. Free digoxigenin molecules in the analyte solution compete with the labeled markers for antibodies on the gold nanoparticles. The fluorescence signal depends linearly on the free digoxigenin concentration within a range of concentration from 0.5 to 3 ng mL(-1). The limit of detection is estimated as 0.2 ng mL(-1) and the limit of quantitation is estimated as 0.6 ng mL(-1). The method can be used to detect digoxin, a drug used to cure cardiac arrhythmia.

Photophysics and Electrochemiluminescence of Bright Cyclometalated Ir(III) Complexes in Aqueous Solutions

A family of neutral bis-cyclometalated iridium complexes [Ir(C^N)2(LX)] has been investigated as ECL labels under immunoassay conditions. Among them, the complex based on phenylphenanthridine (pphent) as the C^N ligand, exhibits outstanding performance and it is a candidate to substitute the commercially available Ru-based label in diagnostics.

PK modulation of haptenylated peptides via non-covalent antibody complexation

We applied noncovalent complexes of digoxigenin (Dig) binding antibodies with digoxigeninylated peptide derivatives to modulate their pharmacokinetic properties. A peptide derivative which activates the Y2R receptor was selectively mono-digoxigeninylated by reacting a NHS-Dig derivative with an ε-amino group of lysine 2. This position tolerates modifications without destroying receptor binding and functionality of the peptide. Dig-peptide derivatives can be loaded onto Dig-binding IgGs in a simple and robust reaction, thereby generating peptide-IgG complexes in a defined two to one molar ratio. This indicates that each antibody arm becomes occupied by one haptenylated peptide. In vitro receptor binding and signaling assays showed that Dig-peptides as well as the peptide-antibody complexes retain better potency than the corresponding pegylated peptides. In vivo analyses revealed prolonged serum half-life of antibody-complexed peptides compared to unmodified peptides. Thus, complexes are of sufficient stability for PK modulation. We observed more prolonged weight reduction in a murine diet-induced obesity (DIO) model with antibody-complexed peptides compared to unmodified peptides. We conclude that antibody-hapten complexation can be applied to modulate the PK of haptenylated peptides and in consequence improve the therapeutic efficacy of therapeutic peptides.

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

Overview of Colorimetric, Chemiluminometric, and Fluorimetric Detection Systems

A large number of detection systems for biomolecules have been described in the literature; therefore, only a brief overview of the most important nonradioactive methods will be given here. More details can be found in the other chapters of this book or in the cited literature.

Chemiluminescent Detection with Horseradish Peroxidase and luminol

Principle and The chemiluminescence (CL) of luminol and related compounds is a well-applications known reaction and has been studied intensively (Gundermann, 1974). The mechanism of the CL reaction of the so-called diacyl hydrazides is very complex and depends on several conditions (Gundermann and McCapra, 1987), e.g., whether the reaction is carried out in water or in aprotic solvents. In DMSO luminol exists as an activated aminophthalic acid, which gives off a bright blue-green light in the presence of a strong base together with hydrogen peroxide.

Labeling of Biomolecules with Fluorophores

Coumarins, fluorescein, and resorufin derivatives are three important fluorescence labels which can be used for labeling of biomolecules. During the last years a wide range of new fluorescence labels have been published and are commercially available, especially covering the long wavelength/ NIR range. Among these are new rhodamine and oxazine derivatives (Arden-Jakob et al., 1997) cyanine derivatives (Thompson, 1994) and also BODIPY fluorophores (Haugland, 1996). The development was especially driven by the upcoming cheap laser diodes (Wersig et al., 1993) and the demand to reduce background fluorescence.