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J Mol Biol. 2014 May 1;426(9):1947-57. doi: 10.1016/j.jmb.2014.02.015. Epub 2014 Feb 24.

Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction.

Author information

1
Department of Protein Chemistry, Genentech Research and Early Development, 1 DNA Way, South San Francisco, CA 94080, USA.
2
Department of Structural Biology, Genentech Research and Early Development, 1 DNA Way, South San Francisco, CA 94080, USA.
3
Department of Structural Biology, Genentech Research and Early Development, 1 DNA Way, South San Francisco, CA 94080, USA; Department of Antibody Engineering, Genentech Research and Early Development, 1 DNA Way, South San Francisco, CA 94080, USA.
4
Department of Protein Chemistry, Genentech Research and Early Development, 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: scheer.justin@gene.com.

Abstract

Bispecific antibody and antibody-like molecules are of wide interest as potential therapeutics that can recognize two distinct targets. Among the variety of ways such molecules have been engineered is by creating "knob" and "hole" heterodimerization sites in the CH3 domains of two antibody heavy chains. The molecules produced in this manner maintain their biological activities while differing very little from the native human IgG sequence. To better understand the knob-into-hole interface, the molecular mechanism of heterodimerization, and to engineer Fc domains that could improve the assembly and purity of heterodimeric reaction products, we sought crystal structures of aglycosylated heterodimeric and homodimeric "knob" and "hole" Fc fragments derived from bacterial expression. The structure of the knob-into-hole Fc was determined at 2.64 Å. Except for the sites of mutation, the structure is very similar to that of the native human IgG1 Fc, consistent with a heterodimer interaction kinetic K(D) of <1 nM. Homodimers of the "knob" and "hole" mutants were also obtained, and their X-ray structures were determined at resolutions 2.5 Å and 2.1 Å, respectively. Both kinds of homodimers adopt a head-to-tail quaternary structure and thus do not contain direct knob/knob or hole/hole CH3 interactions. The head-to-tail arrangement was disfavored by adding site-directed mutations at F241 and F243 in the CH2 domains, leading to increases in both rate and efficiency of bispecific (heterodimer) assembly.

KEYWORDS:

CH3 domain; antibody structure; bispecific antibody; heterodimer interface; hydrophobic interface

PMID:
24576605
DOI:
10.1016/j.jmb.2014.02.015
[Indexed for MEDLINE]

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