Efficient production of bispecific antibody by FAST-Ig ^TM and its application to NXT007 for the treatment of hemophilia A

Abstract

Efficient production of bispecific antibodies (BsAbs) in single mammalian cells is essential for basic research and industrial manufacturing. However, preventing unwanted pairing of heavy chains (HCs) and light chains (LCs) is a challenging task. To address this, we created an engineering technology for preferential cognate HC/LC and HC/HC paring called FAST-Ig (Four-chain Assembly by electrostatic Steering Technology – Immunoglobulin), and applied it to NXT007, a BsAb for the treatment of hemophilia A. We introduced charged amino-acid substitutions at the HC/LC interface to facilitate the proper assembly for manufacturing a standard IgG-type BsAb. We generated CH1/CL interface-engineered antibody variants that achieved > 95% correct HC/LC pairing efficiency with favorable pharmacological properties and developability. Among these, we selected a design (C3) that allowed us to separate the mis-paired species with an unintended pharmacological profile using ion-exchange chromatography. Crystal structure analysis demonstrated that the C3 design did not affect the overall structure of both Fabs. To determine the final design for HCs-heterodimerization, we compared the stability of charge-based and knobs into hole-based Fc formats in acidic conditions and selected the more stable charge-based format. FAST-Ig was also applicable to stable CHO cell lines for industrial production and demonstrated robust chain pairing with different subclasses of parent BsAbs. Thus, it can be applied to a wide variety of BsAbs both preclinically and clinically.

Rapid Generation of Murine Bispecific Antibodies Using FAST-IgTM for Preclinical Screening of HER2/CD3 T-Cell Engagers

“…As expected, BsAbs with V1+C19 resulted in significantly high BsAb yields for both mIgG1 (≥90.0%) and mIgG2a (≥81.0%), the two main antibody subclasses used in mouse studies ( Table 3 ). Some of the parent BsAbs, such as m4D5/mHIT3a and m2C4/mHIT3a, initially exhibited high BsAb yields exceeding 96%, which could be attributed to the strong preference for cognate HC/LC pairing in the parent antibodies [ 8 , 16 , 17 , 28 , 33 ]. Since the batches of parent antibodies and BsAbs with FAST-Ig differ, it is difficult to strictly compare expression levels in this study.…”

“…To achieve high BsAb yields in a single cell, it is important not only to strongly promote correct inter-chain associations but also to ensure equal expression of both pairs of antibodies per a BsAb [ 13 , 16 , 17 , 28 ]. Hence, adjusting the H1:L1:H2:L2 plasmid ratio during transfection is preferable, particularly for antibodies with low expression levels, such as the m4D5 and mSP34.…”

“…Recently, a novel technology called FAST-Ig (Four-chain Assembly by electrostatic Steering Technology—Immunoglobulin) was developed to promote the correct assembly of HC/HC and HC/LC within a single cell [ 17 ]. To promote correct HC and LC assembly, FAST-Ig uses charge-based orthogonal Fab design for human IgGs.…”

Design and engineering of bispecific antibodies: insights and practical considerations

“…Analogously, bsAbs with common HC can also be discovered to avoid HC mispairing ( Fischer et al, 2015 ). Other approaches have focused on rational design of Fab steering that prevents HC:LC mispairing during co-expression of the different polypeptide chains, including Crossmab ( Schaefer et al, 2011 ), orthogonal Fab interfaces ( Lewis et al, 2014 ; Liu et al, 2015 ; Dillon et al, 2017 ; Froning et al, 2017 ; Koga et al, 2023 ), swapping of CH1/CL domains ( Wu et al, 2015 ; Wozniak-Knopp et al, 2018 ), engineering of native interchain disulfide bonds ( Mazor et al, 2015b ), and grafting of IgE-derived heterodimerization domains ( Kühl et al, 2022 ). The use of two different HC and LC allows flexible pairing of VH and VL domains and thus unrestricted access to antibody diversification when searching for target-specific bsAbs.…”

Exploring nonreplacement therapies’ impact on hemophilia and other rare bleeding disorders

“…A new emerging technology, FAST-Ig (Four-chain assembly by electrostatic steering technology-immunoglobulin), has been applied to develop NXT007, a new anti-FIXa/FX bispecific humanized IgG4 antibody developed by Chugai [ 38 ]. Preliminary data from a phase 1/2 clinical trial in healthy Japanese volunteers (NXTAGE; JapicCTI-194919), presented at ISTH Congress 2023, showed a mean elimination half-life of approximately 10 weeks and single subcutaneous doses were well tolerated without thromboembolic events [ 39 ].…”