Comparison of the three-dimensional structures of a humanized and a chimeric Fab of an anti-γ-interferon antibody

Abstract

The objective of this work is to compare the three-dimensional structures of "humanized" and mouse-human chimeric forms of a murine monoclonal antibody elicited against human gamma-interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen-binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8,000 by nearly identical microseeding procedures. Their structures were solved by X-ray analyses at 2.9 A resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity-determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized VH, the substitution of serine for proline in position 7 allowed the N-terminal segment (designated strand 4-1) to be closely juxtaposed to an adjacent strand (4-2) and form hydrogen bonds typical of an antiparallel beta-pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an alpha-helix involving residues 25-32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 A to a random coil which terminated in a single turn of 310 helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space-saving alpha-helix, contrasting with a more extended 310 helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use.

Aggregation risk prediction for antibodies and its application to biotherapeutic development

“…We will refer to the parental chimeric antibody and a variant designed by Fan and co-workers 23 as the WT and the reference (REF), respectively. www.tandfonline.com…”

The immunoglobulin constant region contributes to affinity and specificity

“…For example, Ab responses to polysaccharide Ags are notoriously restricted in both V-and C-region usage [65], and they preferentially use 5 0 -V regions attached to IgM and IgG3 C regions. Perhaps this restriction is a reflection of structural constraints that allow certain V regions [66,67] to bind antigen preferentially in the context of certain C regions. Similarly, the predominance of the IgG2 isotype among viral-neutralizing Abs could reflect structural constraints imposed by the C region.…”

Conformational Dynamics of Complementarity-determining Region H3 of an Anti-dansyl Fv Fragment in the Presence of its Hapten

“…Thus, it is difficult to classify the backbone conformations of CDRH3 of this Fv fragment into the known categories, and to specify the causes for the conformational variety. However, because the CDRH3s of the humanized anti-IFN-g Fab fragments 21,22 have partially homologous sequences (Pro-Trp-Ala-X-Trp) and structures similar to the C-terminal region of CDRH3 of the Fv fragment, structural comparisons of them provide clues to understanding the causes for the structural variation of CDRH3 ( Figure 6). The backbone conformations of the homologous region resemble that of the corresponding region in the Fv fragment in the complex with DNS-lys rather than in the unliganded state.…”