Refined Structure of an Intact IgG2a Monoclonal Antibody^,

Abstract

The structure of an intact, anti-canine lymphoma monoclonal antibody (Mab231) was determined by molecular replacement and refined in a triclinic cell to an R-value of 20.9%, using synchrotron diffraction data from 2.8 to 20 A resolution. All segments of the antibody, including the hinge region and carbohydrate component, are visible in electron density maps. There is no overall symmetry to the antibody, as the Fc is disposed in an entirely oblique manner with respect to the Fabs. The CH2 and CH3 domains do, however, possess a nearly exact, local 2-fold relationship. The Fab segments are related by a second, independent, local dyad axis, exact only with respect to constant domains. Variable domains exhibit no symmetry relationship as a consequence of the 16 degrees difference in Fab elbow angles. Variable domain pair associations VL:VH for the Fabs are virtually the same, and corresponding CDRs of the two Fabs also are nearly identical in structure. CDR-H3 displays the greatest difference. Hypervariable loops of both Fabs are involved in contacts with symmetry-related Fc segments at the CH2-CH3 switch junction, suggesting a "complex" structure. The hinge segment connecting Fabs with the Fc is quite extended and exhibits thermal factors indicative of a high degree of mobility. It consists of a well-defined upper hinge that partially maintains dyad symmetry and a fairly rigid core bounded above and below by fluid polypeptides that provide segmental flexibility. This structure represents the first visualization by X-ray analysis of a murine Fc segment, and its CH2 domains exhibit substantial rigid body conformational changes with respect to the human Fc used as an initial molecular replacement model. The oligosaccharides were found by difference Fourier syntheses to be very similar to those of the free human Fc fragment, although differences are present in the terminal residues. The detailed structure of the IgG presented here, and the distribution of effector binding sites, appears consistent with effector activation mechanisms involving translocation and/or aggregation of the Fc following antigen binding by the Fabs.

Molecular modeling of antibodies for the treatment of TNFα‐related immunological diseases

“…2011), and the complete structure of a murine immunoglobulin (1IGT) used either as a protein template (Harris et al. 1997) to drive the 3D modeling or to extract its Fc portion for building one of our chimera models. First, the infliximab Fab fragment was duplicated by using PyMOL and the two Fab fragments obtained were superimposed on the Fab domains of the 1IGT protein template (Fig.…”

“…Notably, thanks to the crystallization of three complete antibodies, it is now possible to build a complete mAb model based on the available three mAb templates 1IGT (Harris et al. 1997), 1IGY (Harris et al. 1998), 1HZH (Saphire et al.…”

“…2013) with the heavy chains of the Fab fragment of the murine immunoglobulin 1IGT (Harris et al. 1997). The murine Ig heavy chain (from 1IGT) is 223 amino acids long, whereas the infliximab heavy chain (from 4G3Y) is 226 amino acids long.…”

“…2011) with the Fc portion of the murine Ig (1IGT.pdb) (Harris et al. 1997). The Fc portions of the two Ig (1IGT, 3SGK) were both 225 amino acids long and share the 64.71% of identical amino acids.…”

Oligomerisation and thermal stability of polyvalent integrin α5β1 ligands

“…2). The IgG-derived spacer between FIII9'-10 and the coiled coil was modelled as a rod with the centre of rotation arbitrarily chosen in the middle of the spacer; this was considered reasonable since the IgG hinge is predicted to be highly flexible [17].…”