RanBP2 is a nucleoporin with SUMO E3 ligase activity that functions in both nucleocytoplasmic transport and mitosis. However, the biological relevance of RanBP2 and the in vivo targets of its E3 ligase activity are unknown. Here we show that animals with low amounts of RanBP2 develop severe aneuploidy in the absence of overt transport defects. The main chromosome segregation defect in cells from these mice is anaphase-bridge formation. Topoisomerase IIalpha (Topo IIalpha), which decatenates sister centromeres prior to anaphase onset to prevent bridges, fails to accumulate at inner centromeres when RanBP2 levels are low. We find that RanBP2 sumoylates Topo IIalpha in mitosis and that this modification is required for its proper localization to inner centromeres. Furthermore, mice with low amounts of RanBP2 are highly sensitive to tumor formation. Together, these data identify RanBP2 as a chromosomal instability gene that regulates Topo IIalpha by sumoylation and suppresses tumorigenesis.

We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible “CrossMab” formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab CH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.

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The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.

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