Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies

Abstract

Bispecific antibodies (bsAbs) are of significant importance to the development of novel antibody-based therapies, and heavy chain (Hc) heterodimers represent a major class of bispecific drug candidates. Current technologies for the generation of Hc heterodimers are suboptimal and often suffer from contamination by homodimers posing purification challenges. Here, we introduce a new technology based on biomimicry wherein the protein-protein interfaces of two different immunoglobulin (Ig) constant domain pairs are exchanged in part or fully to design new heterodimeric domains. The method can be applied across Igs to design Fc heterodimers and bsAbs. We investigated interfaces from human IgA CH3, IgD CH3, IgG1 CH3, IgM CH4, T-cell receptor (TCR) α/β, and TCR γ/δ constant domain pairs, and we found that they successfully drive human IgG1 CH3 or IgM CH4 heterodimerization to levels similar to or above those of reference methods. A comprehensive interface exchange between the TCR α/β constant domain pair and the IgG1 CH3 homodimer was evidenced by X-ray crystallography and used to engineer examples of bsAbs for cancer therapy. Parental antibody pairs were rapidly reformatted into scalable bsAbs that were free of homodimer traces by combining interface exchange, asymmetric Protein A binding, and the scFv × Fab format. In summary, we successfully built several new CH3- or CH4-based heterodimers that may prove useful for designing new bsAb-based therapeutics, and we anticipate that our approach could be broadly implemented across the Ig constant domain family. To our knowledge, CH4-based heterodimers have not been previously reported.

Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

“…Nevertheless, as observed here, these differences in CH3 thermal stabilities did not translate into meaningful differences in vivo; the same remark can be made for the BEAT heterodimer whose CH3 stability was previously measured at 70 o C. 15 Taken together, all PK profiles overlaid well and half-lives were very similar to one another, suggesting that heterodimer PK properties were mostly influenced by the Fab-scFv-Fc format rather than the nature of the Fc region.…”

“…For the PA method, we set out to compare the two main strategies to abrogate PA binding, and measured affinities of the IgG dA antibody (N82aS substitution in combination with Fc 133), as well as those of an IgG1 antibody carrying the H435R/ Y436F substitutions (denoted IgG1 RF, Eu numbering), thereby comparing usage of an IgG3 Fc portion vs. the known IgG3derived substitutions that abrogate PA binding. 15,16 KD values for both approaches were consistent with a moderate loss in affinity for FcRn. Affinity was reduced by 33% for the IgG dA and by 24% for the IgG1 H435R/Y436F, although the difference between the two methods was not statistically significant (p value was 0.2).…”

“…At the same time that we were improving PA methods, we set out to explore a difference in PG avidity to prepare Hc heterodimers, a method which, to the best of our knowledge, has never been reported. Using our concept of 3D equivalent positions between Ig domains, 15 structure-guided design, and Fc residues known to modulate FcRn binding, we successfully abolished PG binding in the Fc region of IgG1. Importantly, our engineering did not affect PA binding, and most of the FcRn interaction was preserved (95% of the native binding, Table 1).…”

“…Mimicking the inherent difference in PA avidity found in the mouse-rat heterodimer, recent purification strategies have focused on a heterodimer design wherein one Fc chain originates from human immunoglobulin (Ig) G3, a natural IgG subclass that does not bind PA (at least for most allotypes). 14 Hc heterodimers based on a mixed human IgG1/IgG3 subclass have been developed either by using a complete IgG3 CH3 domain 15 or a pair of IgG3 CH3 derived substitutions 16 in one of the two Hcs. Recently, Tustian et al reported that using VH3 domains in these Hc heterodimers poses challenges because the VH3 subclass binds PA and reduces the avidity difference between homo-and heterodimers, overall interfering with the differential purification process and requiring additional purification steps.…”

“…Furthermore, the methods are compatible with CH3 HD technologies such as knobs-into-holes 20 or BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor). 15 Lastly, the PA method was successfully used to manufacture clinical-grade material for a bispecific T-cell engager antibody, currently in a Phase 1 study. 21…”