Bispecific antibodies (bsAbs) offer the potential for novel therapeutic applications by simultaneously targeting different antigens. The functionality of a bsAb depends on several interconnected parameters: the properties of individual binders (on/off-rates, epitopes), the arrangement of binders relative to the Fc fragment (format geometry), the distance between paratopes, and the valency of each binder. The format itself significantly impacts function, as evidenced by examples where bivalent binding enhances receptor activation (beneficial in some contexts but detrimental in others, such as cMet signaling inhibition). The geometry and arrangement of binders are crucial for activities like linking clotting factors and achieving blood-brain barrier penetration. Even alterations to the Fab fragment configuration of an existing antibody (e.g., Trastuzumab) can switch its activity from antagonistic to agonistic. Creating bsAbs with optimal functionalities remains challenging because these parameters interact in complex ways, making rational design difficult. Existing technologies for generating and assessing binder combinations, such as Genentech's knob-into-hole and Genmab's IgG4 Fab arm exchange, offer valuable tools but may still face limitations in high-throughput screening of diverse binder/format combinations. The combinatorial matrix of binders and formats needed to comprehensively explore the design space is substantial, necessitating robust and easily automatable production processes. This paper introduces Format Chain Exchange (FORCE), a high-throughput technology aimed at addressing these challenges.

The literature extensively documents the importance of antibody format and binder selection in bispecific antibody design and function. Several studies highlight how different formats influence efficacy and other functional characteristics. The impact of monovalent versus bivalent binding on receptor activation and inhibition, the role of geometry in linking clotting factors, and the relationship between affinity, valency, and blood-brain-barrier penetration have all been explored. However, a common theme is the complexity of predicting optimal bsAb function based solely on individual binder characteristics. Existing technologies such as knob-into-hole and IgG4 Fab arm exchange have been instrumental in generating bsAbs, but these techniques have limitations in high-throughput screening of diverse formats. The current work addresses this gap by presenting a new high-throughput technology called FORCE.

The FORCE method is based on a chain exchange reaction driven by engineered Fc-dummy chains. The process begins with monospecific educt molecules that are knob-into-hole heterodimers, each comprising a productive half-antibody-like component and a knob or hole dummy Fc chain. The dummy chain prevents aggregation by masking the CH3 interface. Engineered mutations in the dummy chains create limited repulsions at the CH3 interface without compromising educt expression or biophysical properties. Mild reduction of the hinge disulfides initiates a spontaneous chain exchange reaction, driven by the partially flawed CH3 interfaces, resulting in fully complementary interfaces in the final bsAbs. Affinity tags on the dummy chains allow for one-step purification of the bsAbs. The educts can incorporate various formats, including Fabs, sterically constrained Fab arms, and other binder types. The combination of different educt formats generates a diverse set of bsAb formats (e.g., N-N, N-C, C-C, etc., based on N- or C-terminal Fab attachment). Even a limited number of initial binders and formats produces a vast combinatorial matrix of bsAbs. The design of the dummy chains involves mutations based on structural models of knob-into-hole Fcs (PDB 4NQS, 5HY9), which were subsequently confirmed by crystal structures of the generated dummy-containing Fcs (PDB 6YTB, 6YT7, 6YSC). These mutations include reverting cysteine residues in the CH3 interface to serine or tyrosine and charge-flip mutations to partially destabilize the interface. Educt molecules were produced in HEK cells and purified using standard methods. The chain exchange reaction was initiated by the addition of TCEP. A single-step purification using an affinity tag on the dummy chain removes unwanted by-products, including dummy dimers and aggregates. The process has been automated using liquid handling systems, enabling the high-throughput generation of bsAb matrices. The simultaneous binding capability of generated bsAbs was assessed by bridging ELISA using surface-captured antibodies and labeled antigens.

The FORCE technology enables efficient generation of large bsAb matrices with high throughput. The method involves a chain exchange reaction driven by engineered Fc-dummy chains in heterodimeric antibody derivatives. The expression yields and biophysical properties of the educt molecules were comparable to regular IgGs, despite the presence of partially repulsive CH3 interfaces. This robustness facilitates automation of the entire process, from educt production and purification to chain exchange and final product purification. A single-step purification using an affinity tag eliminates unwanted byproducts. The study demonstrated the effectiveness of FORCE by generating a matrix of bsAbs that simultaneously bind Her1/Her2 and DR5. The resulting data revealed format-dependent functional differences for identical target combinations, emphasizing the importance of comprehensive binder-format screening. Specific rules and patterns emerged from analyzing the data, such as the preferential N-terminal placement of certain DR5 binders for improved dual binding. Additionally, the study identified exceptions to these general rules, highlighting the need for a complete binder-format matrix analysis to capture optimal bsAb candidates. The automated workflow produced high-quality bsAbs, suitable for downstream screening (demonstrated via SEC and bridging ELISA). The homogeneity and purity of the products were confirmed by SEC analyses, showing high monomer peak percentages across a large number of tested bsAbs. Crystal structures confirmed the design and impact of the CH3 interface mutations.

The results demonstrate the successful development and application of FORCE, a high-throughput technology for generating large libraries of bispecific antibodies with diverse formats. The ability to generate and screen a large number of binder-format combinations allows for the identification of optimal molecules for a given application. The results also highlight the importance of considering not only the affinity of individual binders but also their arrangement and format for achieving desired functionalities. The findings emphasize the limitations of solely relying on rules or predicted features for bsAb design and show the benefit of experimental screening of a complete combination space. FORCE overcomes the limitations of previous technologies in scaling up the generation of large libraries of bispecific antibody candidates, paving the way for efficient discovery of novel therapeutics.

The FORCE technology provides a significant advancement in bispecific antibody discovery by enabling high-throughput generation of diverse binder-format matrices. This approach allows for the identification of optimal binder-format combinations that would have been difficult to find using conventional methods. The one-step purification method and process automation make FORCE highly efficient and scalable. Future work can expand the range of binders and formats compatible with FORCE, furthering the discovery of novel bi- or multispecific biologics with improved functionalities.

While the FORCE technology offers significant advantages, there are limitations to consider. The reliance on a chain exchange reaction may not be universally applicable to all antibody formats or binders. The current automation setup is specific to the platform used, and adapting it to other systems may require adjustments. Further optimization of the exchange reaction conditions may be required for specific binder pairs or formats. The current study focused on a specific set of binders and antigens; further testing is needed to confirm the generalizability of the findings.