An improved and robust DNA immunization method to develop antibodies against extra-cellular loops of multi-transmembrane proteins |
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Authors: | Meredith Hazen Sunil Bhakta Rajesh Vij Steven Randle Dara Kallop Vicki Chiang Isidro H?tzel Bijay S Jaiswal Karen E Ervin Bing Li Robby M Weimer Paul Polakis Richard H Scheller Jagath R Junutula Jo-Anne S Hongo |
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Affiliation: | 1.Departments of Antibody Engineering; Genentech, Inc.; South San Francisco, CA USA;2.Discovery Oncology; Genentech, Inc.; South San Francisco, CA USA;3.Biomedical Imaging; Genentech, Inc.; South San Francisco, CA USA;4.Molecular Biology; Genentech, Inc.; South San Francisco, CA USA;5.Genentech Research and Early Development; Genentech, Inc.; South San Francisco, CA USA |
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Abstract: | Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation. |
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Keywords: | monoclonal antibody DNA immunization multi-transmembrane proteins MRP4 |
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