Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering
Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering
Abstract 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 chemo-enzymatic modification. We believe this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research. One Sentence SummaryWe demonstrate a universal CRISPR/HDR based platform for rapid genetic engineering of hybridomas to obtain functionally diverse antibody isotype panels in the species and format of choice.
Becker Anouk M.D.、Dalen Duco van、van Bruggen J. Armando C.、Bentlage Arthur E.H.、Heck Albert J.R.、Scheeren Ferenc A.、Nederend Maaike、Hagemans Iris M.、Fransen Marieke F.、Vidarsson Gestur、der Schoot Johan M.S. van、Dolen Yusuf、Valente Michael、Cevirgel Alper、Le Gall Camille M.、Figdor Carl G.、Leusen Jeanette H.W.、Caval Tomislav、Verdoes Martijn、Engelfriet M、Fennemann Felix L.
Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical CenterDepartment of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical CenterDivision of Immunology, The Netherlands Cancer InstituteSanquin Research, Department of Experimental Immunohematology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of AmsterdamBiomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht UniversityDepartment of Medical Oncology, Department of Surgery and Department of Immunohematology and Blood Transfusion, Leiden University Medical Center (LUMC)Laboratory for Translational Immunology, UMC UtrechtDepartment of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical CenterDepartment of Immunohematology and Blood Transfusion, Leiden University Medical Center (LUMC)Sanquin Research, Department of Experimental Immunohematology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of AmsterdamDepartment of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical CenterDepartment of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical CenterDepartment of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical CenterDepartment of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical CenterDepartment of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical CenterDepartment of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical CenterLaboratory for Translational Immunology, UMC UtrechtBiomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht UniversityDepartment of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical CenterDivision of Immunology, The Netherlands Cancer InstituteDepartment of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center
医学研究方法生物科学研究方法、生物科学研究技术生物工程学
Becker Anouk M.D.,Dalen Duco van,van Bruggen J. Armando C.,Bentlage Arthur E.H.,Heck Albert J.R.,Scheeren Ferenc A.,Nederend Maaike,Hagemans Iris M.,Fransen Marieke F.,Vidarsson Gestur,der Schoot Johan M.S. van,Dolen Yusuf,Valente Michael,Cevirgel Alper,Le Gall Camille M.,Figdor Carl G.,Leusen Jeanette H.W.,Caval Tomislav,Verdoes Martijn,Engelfriet M,Fennemann Felix L..Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering[EB/OL].(2025-03-28)[2026-08-26].https://www.biorxiv.org/content/10.1101/551382.点此复制
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