cMAb design and binding characteristics. (A) Crystal structure of CCR5 (PDB entry 4MBS), with the extracellular loop 2 (ECL2) region shown in red and the N-terminal (Nt) region (PDB entry 2L87) superimposed in blue. The orange lines represent the top and bottom layers of the plasma membrane, with the transmembrane region in between. The top view is shown on the right. (B) Cartoon representation of the cMAb, including the T366W change in the RoAb13 CH3 domain to give the knob (red) and the modification of three residues (T366S/L368A/Y407V) to create the hole (blue) in the CH3 domain of the other corresponding heavy chain on PRO 140. The key or knob-in-hole (KIH) design was used to ensure that the two different heavy chains would pair up. The constant domain of the light chain (CL) (gray) and constant domain 1 of the heavy chain (CH1) (black) on the PRO 140 side of the IgG molecule were swapped to ensure correct association of the light chains. The KIH design and the CL/CH1 swap on one of the IgG arms ensure that the IgG molecules are in the bispecific cMAb format, eliminating the undesirable monospecific IgG formats. On the right is an SDS-PAGE gel showing PRO 140 IgG (lanes 1), RoAb13 (IgG) (lanes 2), and the cMAb (lanes 3), run under nonreducing (NR) and reducing (R) conditions. (C) Human (PRO 140, RoAb13, and cMAb) and mouse (CTC8 and 45523) anti-CCR5 antibodies are represented as cartoons. PRO 140 and 45523 are ECL2 specific, while RoAb13 and CTC8 are Nt-specific antibodies. The cMAb is dually specific, recognizing both Nt and ECL2 epitopes. (D) Table summarizing the cross-competition results between the human antibodies (PRO 140, RoAb13, and cMAb) and the mouse antibodies (CTC8 and 45523) to confirm the binding specificity of the cMAb. MFI, mean fluorescence intensity; APC, allophycocyanin; FITC, fluorescein isothiocyanate.