S4C)

S4C). Comparison and docking with previously determined structures The insight gained from 2D and 3D EM reconstruction of the DVD-Ig? molecule alone and bound to antigen was corroborated by high resolution X-ray crystallography data. domain name molecules, structure, crystallography Introduction Monoclonal antibodies (mAbs) have emerged as an important class of therapeutics in oncology, cardiovascular disease, inflammation, transplantation and infectious disease.1-6 This class of therapeutics has proven successful in the medical center due to their high specificity, good security profiles, and overall tolerability. Opportunities for innovation exist, however, in the areas of fulfilling currently unmet medical needs, as well as delivering molecules with superior efficacy and physicochemical properties. A new class of molecules, bispecific mAbs, has thus emerged, including the dual-variable-domain immunoglobulin (DVD-Ig?) molecules currently being developed at AbbVie, two-in-one molecules (Genentech), dual affinity retargeting molecules (MacroGenics), bispecific T-cell engager (Micromet), kappa-lambda antibodies (NovImmune), chemical generation (CovX/Pfizer) and tetravalent bispecific antibodies (Merrimack).7,8 The bispecific molecules offer potential economic and therapeutic superiority in blocking two soluble ligands or cell surface receptors, cross-linking of two receptor molecules or recruiting T-cells to aid in tumor killing. Most of these molecules are in early clinical development, although one (catuxomab) that simultaneously targets EpCAM and CD3 has been approved in the European Union. Simultaneous targeting of multiple disease mediators using the DVD-Ig? format was first explained in 20079 and at least two are currently in clinical trials, a DVD-Ig? molecule targeting tumor necrosis factor and interleukin IL-17, and a DVD-Ig? molecule targeting IL-1 alpha and beta.10-13 These molecules, as well as a host of others in preclinical development, have demonstrated the potential of this class of molecules to retain affinity and potency for both intended targets. The molecules have good physicochemical properties and pharmacokinetics; the ability of the DVD-Ig? molecules to simultaneously bind both antigens was shown by Biacore analysis.9 The asymmetry and flexibility of IgG Mulberroside C molecules have been suggested from your few existing solved crystal structures of intact antibodies.14,15 This flexibility enables IgG molecules to bind antigens of a variety of shapes and sizes and also allows the effector Mulberroside C domain (Fc) to bind the Fc receptor or complement.15,16 Flexibility is mediated through the hinge region and human IgG isotypes have different amino acid composition and inter-chain disulfide bridges in their hinge regions due to the number and position of cysteine residues. Human IgG1 and IgG4 have two disulfide linkages; whereas, IgG2 and IgG3 have four and 11 respectively.1 The hinge region has three structural components, i.e., the upper, core and lower hinge segments (Fig. 1). The core segment (CPPC) contains cysteine residues that connect the two heavy chains and paired poly (L)-proline helices that make this segment inflexible. The upper hinge region (DKTHT) of human IgG1 connects Gdf7 the Fab arms to the core segment and influences Fab-Fab flexibility; N-terminal residues from your upper hinge region are reported to interact with the CH1 domain name.17-19 The lower hinge region (PAPELLGG) connects the Fc to the core region and plays a role in Fab/Fc flexibility and Fc tail wagging.16,20 The length of the hinge region also plays a role in flexibility; mouse IgG1, which has a shorter hinge region (12 residues and 3 inter-chain cysteines), is usually more compact than either IgG2a (19 residues with 3 cysteines) Mulberroside C or human IgG1 (17 residues with 2 cysteines).15 The inherent flexibility in IgG molecules has hampered crystallization efforts and only a few crystal structures of intact IgG molecules have been solved.15,21-27 The majority of structures reported in the literature are either Fab or Fab fragments. Open in a separate window Physique 1. (A) Schematic depiction of the overall architecture of a DVD-IgTM molecule showing the outer and inner antigen binding domains in the Fab region, the hinge region and the Fc region. Class averages showing (B) an isolated DVD-Ig? molecule (n = 761), (C) inner antigen (n Mulberroside C = 127), (D) outer antigen (n = 234); complexes of.