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Multi-pass transmembrane and multi-meric membrane proteins are targets for the development of therapeutic monoclonal antibodies, but are often challenging or impossible to express in a native, bio-active state with appropriate quaternary structure. Immunization with surrogate forms of the target including peptides, soluble protein domains, proteo-liposomes, or intact cells are not always successful in producing antibodies that are capable of binding to native state proteins.
DNA-based immunization strategies have been shown, with mixed results, to generate antibodies against native conformational epitopes of membrane proteins. Generally, target-specific hybridomas are best obtained when DNAimmunization is combined with injections of target protein or target-expressing cells. To this end we evaluated DNA immunization procedures in combination with a cell immunization to generate hybridomas to PD1 as a model antigen. The optimized procedure was then used to generate hybridoma antibodies to human CD3 epsilon/delta.
Summary
We investigated a variety of DNA immunization methods for the generation of Abs to cell surface antigens. We observed that plasmid DNA delivered as a DNA Vaccine (e.g., with Adjuvant) and syngeneic cells stably expressing the target antigen used as priming and final boost immunogen were optimal for the generation of hybridomas. This antigen format was more effective than other methods in generating monoclonal Abs to PD1 and multimeric CD3 epsilon/delta in human Ab transgenic OmniRatsTM.
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Authors
Joshua K. Lowitz, Glen Lin, Leonel Santibanez-Vargas, Rick Chang, Billy Nguyen, Roland Buelow and John S. Kenney, Antibody Solutions, Sunnyvale, CA, USA & Open Monoclonal Technologies, Palo Alto, CA USA
