Sebastian Breuer

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.

Impact of selective anti‐BMP9 treatment on tumor cells and tumor angiogenesis

The role of bone morphogenic protein 9 (BMP9) signaling in angiogenesis has been controversial, with a number of studies showing that it acts either as a pro‐angiogenic or, conversely, as an anti‐angiogenic factor in a context‐dependent manner. Notably, BMP9 was also reported to function in both pro‐ or anti‐tumorigenic roles during tumor progression. It has therefore remained unclear, whether selective BMP9 inhibition is a useful target for antibody therapy of cancer. To shed light on these questions, we characterized BMP9 expression in plasma of patients with different cancer indications and found elevated levels of pro‐domains and precursor BMP9 with a strong response in renal cell carcinoma (RCC). These studies prompted us to evaluate the potential of selective anti‐BMP9 cancer therapy in RCC. We generated a novel monoclonal therapeutic antibody candidate, mAb BMP9‐0093, that selectively targets all different BMP9 variants but does not bind to the closest homolog BMP10. In vitro, mAb BMP9‐0093 treatment inhibited signaling, endothelin‐1 (ET‐1) production and spreading of endothelial cells and restored BMP9‐induced decrease in pericyte migration and attachment. Furthermore, BMP9‐mediated epithelial–mesenchymal transition of renal cell carcinoma cells was reversed by mAb BMP9‐0093 treatment in vitro. In vivo, mAb BMP9‐0093 showed significant anti‐tumor activity that was associated with an increase in apoptosis as well as a decrease in tumor cell proliferation and ET‐1 release. Furthermore, mAb BMP9‐0093 induced mural cell coverage of endothelial cells, which was corroborated by a reduction in vascular permeability, demonstrated by a diminished penetration of omalizumab‐Alexa 647 into tumor tissue. Our findings provide new evidence for a better understanding of BMP9 contribution in tumor progression and angiogenesis that may result in the development of effective targeted therapeutic interventions.