Integrating in silico Humanization and DNA Delivery to engineer anti-Influenza Monoclonal Antibodies for improved in vivo expression 2267274

Abstract Introduction Highly pathogenic avian influenzas (HPAI) of the H5 subtype represent an ongoing zoonotic threat to human and animal health. Broadly neutralizing antibodies (bnAbs) offer a powerful tool for pandemic preparedness, yet translation to the clinic often requires humanization to avoid immune reactions against murine antibodies, often impacting its function across divergent viral strains. Methods We developed a computational pipeline integrating BioPhi with in-house refinement to generate humanized variants of 5C2, a murine antibody elicited against a clade 2.3.2.1 H5N1 virus. To assess whether humanized antibodies could retain the breadth and potency of the parental clone, we designed a panel of unique humanized constructs and compared them to a VH/VL chimera and the murine wild-type mAb, which were all genetically optimized for in vivo expression. Results We confirmed in vitro expression of all constructs and subsequently delivered them as DNA-encoded monoclonal antibody (DMAbs) via injection and in vivo electroporation (EP) to wild-type mice. Sera from DMAb treated mice exhibited strong binding and pseudovirus neutralization to multiple H5N1 clades, including the currently circulating panzootic clade 2.3.4.4b virus. In vivo DMAb delivery supported sustained expression across constructs for several months. Conclusion These data demonstrate that optimized in silico humanization can preserve key functional properties, including cross-reactivity against multiple clades. Our results support the use of this computational workflow for rapid generation of humanized antibodies with broad activity, highlighting its potential application against emerging seasonal or pandemic threats. Funding Source NIH/NIAID CIVICs (75N93019C00051) Topic Categories Vaccines and Immunotherapy (VAC)