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

Area of Science:

• Protein Engineering
• Immunology
• Biochemistry

Background:

• The 'knobs-into-holes' model, originally described by Crick in 1952, explains amino acid side chain packing in alpha-helices.
• This model has been adapted as a protein engineering strategy to control the assembly of protein subunits.

Purpose of the Study:

• To demonstrate the 'knobs-into-holes' strategy as an effective method for engineering antibody heavy chain homodimers for heterodimerization.
• To create a hybrid immunoadhesin by combining an anti-CD3 antibody with a CD4-IgG immunoadhesin.

Main Methods:

• Engineered a 'knob' variant in the CH3 domain of CD4-IgG by replacing threonine 366 with tyrosine (T366Y).
• Created a complementary 'hole' in the CH3 domain of a humanized anti-CD3 antibody by replacing tyrosine 407 with threonine (Y407T).
• Co-expressed the engineered heavy chains with the anti-CD3 light chain and purified the resulting hybrid protein using Protein A chromatography.

Main Results:

• The 'knobs-into-holes' engineered hybrid anti-CD3/CD4-IgG immunoadhesin reached up to 92% of the purified protein pool.
• Co-expression with wild-type CH3 domains yielded only up to 57% of the hybrid protein.
• Demonstrated successful heterodimerization of distinct antibody heavy chains.

Conclusions:

• The 'knobs-into-holes' engineering approach is a powerful tool for constructing antibody-immunoadhesin hybrids.
• This strategy is applicable for developing other Fc-containing bifunctional therapeutics, including bispecific antibodies and immunoadhesins.