Improving target cell specificity using a novel monovalent bispecific IgG design

Abstract

(2015) Improving target cell specificity using a novel monovalent bispecific IgG design, mAbs, 7:2, 377-389, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Monovalent bispecific IgGs cater to a distinct set of mechanisms of action but are difficult to engineer and manufacture because of complexities associated with correct heavy and light chain pairing. We have created a novel design, "DuetMab," for efficient production of these molecules. The platform uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the C H 1-C L interfaces with an engineered disulfide bond. Using two pairs of antibodies, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), and anti-CD40 and anti-CD70 antibodies, we demonstrate that DuetMab antibodies can be produced in a highly purified and active form, and show for the first time that monovalent bispecific IgGs can concurrently bind both antigens on the same cell. This last property compensates for the loss of avidity brought about by monovalency and improves selectivity toward the target cell.

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

“…Bis-A (DuetMab 24,25 ) is an asymmetric bispecific antibody with a KIH Fc design to promote correct heavy-chain heterodimerization. Bis-A contains two light chains, a lambda light chain and a kappa light chain.…”

“…The results of our studies suggest that the hydrophobicity difference between mispaired and correctly paired bispecific antibodies is significant and can be exploited to develop a hydrophobic interaction chromatography (HIC)–based mispairing assay. Here we describe the use of a different DuetMab, 24,25 Bis-B, as an example to illustrate the development of the HIC method.…”

“…The design and production of these bispecific antibodies have been disclosed previously. 24,25 LC and LC-MS grade solvents were purchased from EMD (Billerica, MA). Chemical reagents, including TFA, formic acid, sodium phosphates, sodium sulfate, and TCEP, were purchased from Sigma-Aldrich (St. Louis, MO).…”

“…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.…”

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

“…[26][27][28] Although these approaches generate IgG-Bs, the deviation from the IgG chain sequence and structure remains a substantial issue, [6][7][8][9][10][11][12] and novel antibody engineering solutions are thus still needed.…”