Binding of type I and II anti-CD20 mAbs. In the top panels the binding sites of the clinically relevant type I (rituximab, ofatumumab) and type II (obinutuzumab, tositumomab) mAbs are shown. As can be seen, the fully human ofatumumab binds to a discontinuous region of CD20 apparently composed of both the smaller CD20 loop and the N-terminal region of the larger loop, unlike the other mAbs, which cluster around the ANP motif. At the bottom is a hypothesis of how these mAbs might bind to CD20 in the plasma membrane that may serve to explain their differing effects, such as the 2:1 binding ratio, raft accumulation, and subsequent effector functions. Accordingly, type I mAbs bind and interact with closed conformations of CD20 (red loops, labeled a-d) and are able to occupy all available binding sites. If these mAbs are favorably orientated to bind between tetramers, this would facilitate clustering and aggregation, suitable for raft accumulation, optimal C1q binding, and efficient CDC. In contrast, type II mAbs bind the first molecule of CD20 and are subsequently constrained for binding to an adjacent molecule, possibly within the same tetramer, potentially through the interaction with Asn 171 and opening of CD20 molecules (red and green loops). Intra-tetrameric binding will not facilitate large-scale aggregation and will preclude further binding to the same CD20 tetramer, thus causing half-maximal binding in comparison with type I mAbs. Both the reduced binding and lack of raft accumulation serve to limit C1q binding and impotent CDC. However, binding to the open CD20 may be the trigger for signaling to actin, resulting in homotypic adhesion and lysosomal cell death.