'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization

‘Knobs-into-holes’ was originally proposed by Crickin 1952 as a model for the packing of amino acid side chainsbetween adjacent -helices. ‘Knobs-into-holes’ isdemonstrated here as a novel and effective design strategy forengineering antibody heavy chain homodimers for heterodimerization.In this approach a ‘knob’ variant was first obtainedby replacement of a small amino acid with a larger one in theC_H3 domain of a CD4-IgG immuno-adhesin: T366Y. The knob wasdesigned to insert into a ‘hole’ in the C_H3 domainof a humanized anti-CD3 antibody created by judicious replacementof a large residue with a smaller one: Y407T. The anti-CD3/CD4-IgGhybrid represents up to 92% of the protein A purified proteinpool following co-expression of these two different heavy chainstogether with the anti-CD3 light chain. In contrast, only upto 57% of the anti-CD3/CD4-IgG hybrid is recovered followingco-expression in which heavy chains contained wild-type C_H3domains. Thus knobs-into-holes engineering facilitates the constructionof an antibody/ immunoadhesin hybrid and likely other Fc-containingbifunctional therapeutics including bispecific immuno-adhesinsand bispecific antibodies.