title: 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization.
abstract: 'Knobs-into-holes' was originally proposed by Crick in 1952 as a model for the packing of amino acid side chains between adjacent alpha-helices. 'Knobs-into-holes' is demonstrated here as a novel and effective design strategy for engineering antibody heavy chain homodimers for heterodimerization. In this approach a 'knob' variant was first obtained by replacement of a small amino acid with a larger one in the CH3 domain of a CD4-IgG immunoadhesin: T366Y. The knob was designed to insert into a 'hole' in the CH3 domain of a humanized anti-CD3 antibody created by judicious replacement of a large residue with a smaller one: Y407T. The anti-CD3/CD4-IgG hybrid represents up to 92% of the protein A purified protein pool following co-expression of these two different heavy chains together with the anti-CD3 light chain. In contrast, only up to 57% of the anti-CD3/CD4-IgG hybrid is recovered following co-expression in which heavy chains contained wild-type CH3 domains. Thus knobs-into-holes engineering facilitates the construction of an antibody/immunoadehsin hybrid and likely other Fc-containing bifunctional therapeutics including bispecific immunoadhesins and bispecific antibodies.
authors.affiliation: Department of Molecular Oncology, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: J B
authors.initials: JB
authors.lastname: Ridgway
authors.forename: L G
authors.initials: LG
authors.lastname: Presta
authors.forename: P
authors.initials: P
authors.lastname: Carter
mesh_terms: D000906:Antibodies / Q000235:genetics / Q000378:metabolism*; D015703:Antigens, CD / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology; D015704:CD4 Antigens / Q000276:immunology; D019281:Dimerization; D014644:Genetic Variation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D017252:CD3 Complex; D015704:CD4 Antigens; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains