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.

Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties, mAbs, 5:1, 22-33,

CD20 is a cell-surface marker of normal and malignant B cells. Rituximab, a monoclonal antibody targeting CD20, has improved the treatment of malignant lymphomas. Therapeutic CD20 antibodies are classified as either type I or II based on different mechanisms of killing malignant B cells. To reveal the molecular basis of this distinction, we fine-mapped the epitopes recognized by both types. We also determined the first X-ray structure of a type II antibody by crystallizing the obinutuzumab (GA101) Fab fragment alone and in complex with a CD20 cyclopeptide. Despite recognizing an overlapping epitope, GA101 binds CD20 in a completely different orientation than type I antibodies. Moreover, the elbow angle of GA101 is almost 30°wider than in type I antibodies, potentially resulting in different spatial arrangements of 2 CD20 molecules bound to a single GA101 or rituximab molecule. Using protein tomography, different CD20 complexes were found to be associated with the 2 antibodies, and confocal microscopy showed different membrane compartmentalization of these subpopulations of the cellular CD20 pool. Our findings offer a possible molecular explanation for the different cellular responses elicited by type I and II antibodies. (Blood. 2011;118(2):358-367)

The p53 protein is a transcription factor that acts as the major tumor suppressor in mammals. The core DNAbinding domain is mutated in about 50% of all human tumors. The crystal structure of the core domain in complex with DNA illustrated how a single core domain specifically interacts with its DNA consensus site and how it is inactivated by mutation. However, no structural information for the tetrameric full-length p53-DNA complex is available. Here, we present novel experimental insight into the dimerization of two p53 core domains upon cooperative binding to consensus DNA in solution obtained by NMR. The NMR data show that the p53 core domain itself does not appear to undergo major conformational changes upon addition of DNA and elucidate the dimerization interface between two DNA-bound core domains, which includes the short H1 helix. A NMR-based model for the dimeric p53 core-DNA complex incorporates these data and allows the conclusion that the dimerization interface also forms the actual interface in the tetrameric p53-DNA complex. The significance of this interface is further corroborated by the finding that hot spot mutations map to the H1 helix, and by the binding of the putative p53 inhibitor 53BP2 to this region via one of its ankyrin repeats. Based on symmetry considerations it is proposed that tetrameric p53 might link non-contigous DNA consensus sites in a sandwich-like manner generating DNA loops as observed for transcriptionally active p53 complexes.The tumor suppressor gene p53 is the most frequent site of genetic alterations found in human tumors (1) and acts as the major tumor suppressor in mammals. In addition to non-transcriptional functions, p53 acts primarily as a transcriptional activator, that regulates the expression of several genes involved in cell cycle arrest, cellular senescence, anti-angiogenesis, and apoptosis (reviewed in Refs. 2-4). Recently, two homologues of p53, p63 and p73, were discovered, coding for a variety of different isoforms. These three p53 family members play distinct roles in differentiation, development, and tumor suppression (reviewed in Ref. 5). p53 possesses a modular architecture with an N-terminal transactivation domain (TAD), 1 a strongly conserved core DNA-binding domain (DBD), a tetramerization domain (TD), and a regulatory C terminus (6, 7). Tetrameric p53 binds specifically to a DNA consensus sequence consisting of two consecutive palindromic 10-bp halfsites, where each half-site is formed by two head-to-head quarter-sites (8 -12). The isolated TD forms a symmetric dimer of dimers (13-15), and contrasting models have been proposed that describe how the DBDs of each dimer are attached to DNA, namely with either consecutive or alternating arrangements (16). The p53 DBD comprises several hot spot regions for mutation that inactivate p53 in more than half of all human tumors (1). Therefore, wild-type and mutant p53 DBDs have been the focus of various studies (17-21). The crystal structure of the p53 DBD in complex with DNA (10) showed that almost all known...

EmrE is an Escherichia coli multidrug transport protein that confers resistance to a wide range of toxicants by active transport across the bacterial cell membrane. The highly hydrophobic polytopic integral membrane protein has been purified and studied in its full-length form by high-resolution NMR spectroscopy in a mixture of chloroform/methanol/water (6 :6:1, by vol.). Full activity is maintained after reconstitution of the protein into proteoliposomes from this solvent mixture. A series of heteronuclear ( 1 H-15 N) two-and three-dimensional experiments, as well as triple resonance experiments, were applied to the 110-residue protein and led to the assignment of the 1 H, 15 N and a large part of the 13 C backbone resonances as well as many of the sidechain resonances. A preliminary analysis of the secondary structure, based on sequential NOE connectivities, deviation of chemical shifts from random coil values and 3 JNH-HA coupling constants supports a model where the protein forms four A-helices between residues 4Ϫ26 (TM1), 32Ϫ53 (TM2), 58Ϫ76 (TM3) and 85Ϫ106 (TM4). For the residues of helices TM2 and TM3 a significant line broadening occurs due to slow conformational processes.