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Background Monoclonal antibodies (mAbs) generated in rodents against mammalian proteins typically lack cross-species reactivity, hindering preclinical validation and testing in common mammalian models of disease. Membrane proteins have many pan-mammalian conserved epitopes; however, these epitopes are poorly immunogenic in traditional host species like mice or rabbits due to self-tolerance. Consequently, antibodies generated in these species are often limited to species-specific epitopes. As a result, separate antibody discovery campaigns are often required for each model organism of interest, driving up costs and limiting the use of reagents across species.Methods We present an approach of immunizing chickens, an evolutionarily divergent host, with mRNA and Lipoparticles™ (membrane proteins displayed in virus-like particles) to present antigens in their native-structure. 1 Because of their evolutionary distance from mammals, chickens can successfully elicit antibodies against conserved epitopes shared across mammalian orthologs, including those not accessible with traditional hosts like mice or rabbits.2 Antibodies are isolated under stringent conditions to identify only high-affinity mAbs. Resulting mAbs are recombinantly cloned and tested for cross-reactivity with membrane protein orthologs from multiple mammalian species. Each mAb is validated across major research applications, including flow cytometry (the gold standard for membrane protein antibodies), immunofluorescence, immunocytochemistry, and western blot. Antibody specificity is assessed using the Membrane Proteome Array™ (MPA), a cell-based array displaying 6,000 human membrane proteins.Results This approach consistently generates high-quality, pan-reactive monoclonal antibodies that cross-react with multiple mammalian species. For example, a single mAb isolated against CD56 showed reactivity to over 10 mammalian orthologs tested to date ( figure 1). This antibody was further validated to be specific to CD56 using the Membrane Proteome Array™ (MPA), produces a single band on western blot, and is compatible with immunofluorescence imaging. This example reflects the broader success of dozens of antibodies generated using our high-throughput discovery platform.Conclusions This strategy successfully yielded pan-reactive mAbs against therapeutically relevant proteins, including GPRC5D, CD56, and CCR8. By combining a multi-step immunization approach using native antigens in a divergent host with high-stringency phage panning, this platform overcomes traditional barriers in antibody discovery to generate mAbs against even the most challenging targets. The result is high-quality, reliable antibodies that work across mammalian models—enabling target validation and improved therapeutic translation.References Tucker DF, Sullivan JT, Mattia K-A, Fisher CR, Barnes T, Mabila MN, Wilf R, Sulli C, Pitts M, Payne RJ. Isolation of state-dependent monoclonal antibodies against the 12-transmembrane domain glucose transporter 4 using virus-like particles. Proceedings of the National Academy of Sciences 2018;115:E4990-E4999.Banik SS, Kushnir N, Doranz BJ, Chambers R. Breaking barriers in antibody discovery: harnessing divergent species for accessing difficult and conserved drug targets, mAbs. Taylor & Francis 2023, pp. 2273018.Abstract 8 Figure 1CD56 mAb validation and specificity. A) A single mAb binds nearly every CD56 orthologs (flow cytometry). B) Western blot shows a single band at expected size. C) CD56 mAb is compatible with IF. D) MPA screening demonstrates monospecificity for human CD56