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Sci Adv. 2019 Aug 28;5(8):eaaw1822. doi: 10.1126/sciadv.aaw1822. eCollection 2019 Aug.

Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering.

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Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 26, 6525 GA Nijmegen, Netherlands.
Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, Netherlands.
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands.
Sanquin Research, Department of Experimental Immunohematology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, Plesmanlaan 125, Amsterdam 1066 CX, Netherlands.
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, Netherlands.
Laboratory for Translational Immunology, UMC Utrecht, Utrecht, Netherlands.
Department of Medical Oncology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, Netherlands.


Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

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