Efficient production of bispecific antibody by FAST-IgTM and its application to NXT007 for the treatment of hemophilia A.

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.

A protein's conformational stability is an immunologically dominant factor: evidence that free-energy barriers for protein unfolding limit the immunogenicity of foreign proteins.

Foreign protein Ags are incorporated into APCs and then degraded by endosomal proteases. The peptides are then mounted on MHC II molecules on the surfaces of APCs. The T cell-triggering response and, therefore, the immune response, were suggested to be governed by the degree of conformational stability of the foreign protein Ags. However, there is little evidence that a protein's conformational stability is an immunologically dominant factor. In this study, we show that a protein has a threshold of conformational stability to prevent the immunogenicity of foreign proteins. Inverse and linear correlations were found between the amount of IgG production against lysozymes and the free-energy change for the unfolding of lysozymes, based on the correlation between the free-energy changes of the protein unfolding and the amount of IgG production against lysozymes with different stabilities in mice using hen egg white lysozyme derivatives and mutant mouse lysozymes, in which the sequence between 107 and 116 is replaced with that of hen egg white lysozyme, which can produce autoantibodies in mice. Interestingly, the thresholds of free-energy changes for both lysozymes to prevent their immunogenicity were almost identical (21-23 kcal/mol). To confirm the results, we also showed that the cross-linking of Phl p 7, in which intact Phl p 7 has stability greater than ∼20 kcal/mol under physiological conditions, induced minimal IgG production in mice, whereas intact Phl p 7 was antigenic. From the above results, we suggest that protein conformational stability was an immunologically dominant factor.