Michael Grote

Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies

Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.

Bispecific antibody derivatives based on full-length IgG formats.

Monoclonal antibodies have emerged as an effective therapeutic modality, and numerous antibodies have been approved for the treatment of several severe diseases or are currently in clinical development. To improve their therapeutic potential, monoclonal antibodies are constantly evolved by protein engineering. Particularly, the generation of bispecific antibodies raised special interest because of their ability to bind two different antigens at the same time, and the efficiency of these formats has been demonstrated in several clinical and preclinical studies. Up to now, the major drawbacks in using bispecific antibodies as a therapeutic agent have been difficult design and low-yield expression of homogeneous antibody populations. However, major technological improvements were made in protein engineering during the last years. This allows the design of several new IgG-based bispecific antibody formats that can be prepared in high yields and high homogeneity using conventional expression and purification techniques. Especially, recent development of IgG-fusions with disulfide-stabilized Fv fragments and of CrossMab-technologies facilitates the generation of bispecific antibodies with IgG-like architectures. Here we describe design principles and methods to express and purify different bispecific antibody formats derived from full-length IgGs.

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

Hapten-Binding Bispecific Antibodies for the Targeted Delivery of SiRNA and SiRNA-Containing Nanoparticles

Hapten-binding bispecific antibodies (bsAbs) are effective and versatile tools for targeting diverse payloads, including siRNAs, to specific cells and tissues. In this chapter, we provide examples for successful SiRNA delivery using this powerful targeting platform. We further provide protocols for designing and producing bsAbs, for combining bsAbs with SiRNA into functional complexes, and achieving specific mRNA knockdown in cells by using these functional complexes.

TriFabs--Trivalent IgG-Shaped Bispecific Antibody Derivatives: Design, Generation, Characterization and Application for Targeted Payload Delivery.

TriFabs are IgG-shaped bispecific antibodies (bsAbs) composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain (VH) fused to CH3 with “knob”-mutations, and the variable region of the light chain (VL) fused to CH3 with matching “holes”. The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent “in between” these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic) payloads.