Modular generation of multispecific antibodies using protein trans-splicing.

Multispecific antibodies are exhibiting rapid growth in drug development due to their ability to engage multiple therapeutic targets and deliver unique functional properties inaccessible to conventional monoclonal antibodies. The development of these complex modalities, however, is limited by the large design space created by the numerous possible combinations associated with multiple binding moieties and molecular formats. Here, we present a high-throughput platform for the efficient production and screening of complex combinatorial multispecific antibody libraries using intein-mediated protein splicing. To validate the fidelity of this screening approach, we demonstrate that spliced bispecific molecules exhibit indistinguishable biochemical and functional properties compared to bispecific antibodies generated through conventional production processes. We further demonstrate that intein splicing can be combined with the DuetMab platform to create trispecific antibodies and perform dual-splicing reactions to generate bivalent bispecific and trispecific formats. This work establishes intein-based protein splicing as a powerful strategy for the efficient generation and screening of multispecific antibodies libraries, enabling the rapid identification of optimal binding configurations, combinations, and molecular formats that elicit the desired mechanism of action.