Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects

Abstract

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications.

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

“…2 Thus, they generally require further engineering, extensive purification, or special production systems imposing specific limitations. 8,[13][14][15][16][17][18][19][20][21][22] Furthermore, their avidity per antigen is reduced compared to the parental antibodies.…”

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

“…A number of alternative approaches for the same purpose have been developed recently. 9,10,11,12,13,14 Upon application of one of these methods alone, however, there still remains a mixture of 4 compounds except for cases where the chain association issue is circumvented by a common LC approach. 15 The LC mispairing problem is more difficult to address because a total of 4 possible pairings of heavy and light chains have to be considered.…”

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

“…20 Similarly, electrostatic interactions have been used to promote heterodimerization between two heavy chains. 21,22 By mutating charged groups in the Fc region or introducing new charged groups in the hinge region, one heavy chain is enriched with positively charged functionality and vice versa. Homodimerization is thus minimized due to electrostatic repulsion, and the desired heterodimerization is maximized due to electrostatic attraction.…”

“…Near-perfect heterodimerization can be achieved by utilizing this electrostatic steering effect. 21 Subsequent efforts to improve fidelity have focused on pairing between cognate heavy and light chains. Technologies introduced to improve the correct heavy chain–light chain pairing include the use of a common light chain 20 (which tends to compromise specificity of each binding arm and limit the diversity of bispecific antibodies); CrossMab, 23 in which the light chain of one antigen-binding fragment (Fab) arm is exchanged for the anti-human IgG (Fd) region of the corresponding heavy chain; and DuetMab, 24,25 in which the correct heavy chain–light chain pairing is achieved by replacing the native disulfide bond in one of the Fab arms with an engineered disulfide bond.…”