TY - JOUR
T1 - Glycoengineering of AAV-delivered monoclonal antibodies yields increased ADCC activity
AU - Termini, James M.
AU - Martinez-Navio, José M.
AU - Gao, Guangping
AU - Fuchs, Sebastian P.
AU - Desrosiers, Ronald C.
N1 - Funding Information:
We would like to acknowledge the assistance of the Flow Cytometry Core Facility of the Sylvester Comprehensive Cancer Center at the University of Miami, Miller School of Medicine, for the provision of fluorescence analysis and cell sorting service. We want to thank Michael Alpert for assistance with statistical analysis of our ADCC assays. We want to thank Leydi Machado for administrative assistance. We also want to thank Anabetsy Rivero and William Lauer for proofreading the manuscript. This work was made possible through funding support from the Florida Department of Health Pilot Award program (contract CODMR ), as well as grants R01AI098446 and U19AI149646 from the NIH .
Funding Information:
We would like to acknowledge the assistance of the Flow Cytometry Core Facility of the Sylvester Comprehensive Cancer Center at the University of Miami, Miller School of Medicine, for the provision of fluorescence analysis and cell sorting service. We want to thank Michael Alpert for assistance with statistical analysis of our ADCC assays. We want to thank Leydi Machado for administrative assistance. We also want to thank Anabetsy Rivero and William Lauer for proofreading the manuscript. This work was made possible through funding support from the Florida Department of Health Pilot Award program (contract CODMR), as well as grants R01AI098446 and U19AI149646 from the NIH. Conceptualization, J.M.T.; Methodology, J.M.T. J.M.M.-N. S.P.F. and R.C.D.; Investigation, J.M.T. and G.G.; Validation, J.M.T. and R.C.D.; Formal Analysis, J.M.T.; Writing-Original Draft, J.M.T. and R.C.D.; Writing-Review and Editing J.M.T. R.C.D. S.P.F. and J.M.M.-N.; Funding Acquisition, J.M.T. and R.C.D.; Supervision, R.C.D.; Project Administration, J.M.T. and R.C.D. G.G. is a scientific co-founder of Voyager Therapeutics and holds equity in the company. G.G. is an inventor on patents with potential royalties licensed to Voyager Therapeutics and other biopharmaceutical companies. G.G. is a scientific co-founder of Aspa Therapeutics and holds equity in the company. G.G. is an inventor on patents with potential royalties licensed to Aspa Therapeutics and other bio-pharmaceutical companies. The remaining authors declare no competing interests.
Publisher Copyright:
© 2020 The Authors
PY - 2021/3/12
Y1 - 2021/3/12
N2 - The absence of fucose on asparagine-297 of the human immunoglobulin G (IgG) heavy chain has been shown to enhance antibody-dependent cellular cytotoxicity (ADCC) activity by 10- to 100-fold compared to fucosylated antibody. Our lab is studying the use of adeno-associated virus (AAV) as a vector for the delivery of HIV-specific antibodies for therapeutic purposes. Since the antibody is produced by vector-transduced cells in vivo, current techniques of glycoengineering cannot be utilized. In order to achieve similar enhancement of ADCC with AAV-delivered antibodies, short hairpin RNAs (shRNAs) that target fucosyltransferase-8 (FUT8), were designed, tested, and cloned into AAV vectors used to deliver HIV-specific broadly neutralizing antibodies (bNAbs). Antibodies produced by our glycoengineered-AAV (GE-AAV) vectors were analyzed for fucose content and ADCC. GE-AAV constructs were able to achieve over 80% knockdown of FUT8. Results were confirmed by lectin western blot for α1-6 fucose, which revealed almost a complete absence of fucose on GE-AAV-produced antibodies. GE-AAV-produced antibodies revealed >10-fold enhancement of ADCC, while showing identical neutralization and gp140 trimer binding compared to their fucosylated counterparts. ADCC was enhanced 40- to 60-fold when combined with key Fc mutations known to enhance binding to FcγRIIIA. Our findings define a powerful approach for supercharging AAV-delivered anti-HIV antibodies.
AB - The absence of fucose on asparagine-297 of the human immunoglobulin G (IgG) heavy chain has been shown to enhance antibody-dependent cellular cytotoxicity (ADCC) activity by 10- to 100-fold compared to fucosylated antibody. Our lab is studying the use of adeno-associated virus (AAV) as a vector for the delivery of HIV-specific antibodies for therapeutic purposes. Since the antibody is produced by vector-transduced cells in vivo, current techniques of glycoengineering cannot be utilized. In order to achieve similar enhancement of ADCC with AAV-delivered antibodies, short hairpin RNAs (shRNAs) that target fucosyltransferase-8 (FUT8), were designed, tested, and cloned into AAV vectors used to deliver HIV-specific broadly neutralizing antibodies (bNAbs). Antibodies produced by our glycoengineered-AAV (GE-AAV) vectors were analyzed for fucose content and ADCC. GE-AAV constructs were able to achieve over 80% knockdown of FUT8. Results were confirmed by lectin western blot for α1-6 fucose, which revealed almost a complete absence of fucose on GE-AAV-produced antibodies. GE-AAV-produced antibodies revealed >10-fold enhancement of ADCC, while showing identical neutralization and gp140 trimer binding compared to their fucosylated counterparts. ADCC was enhanced 40- to 60-fold when combined with key Fc mutations known to enhance binding to FcγRIIIA. Our findings define a powerful approach for supercharging AAV-delivered anti-HIV antibodies.
KW - adeno-associated virus
KW - antibody-dependent cellular cytotoxicity
KW - broadly neutralizing antibodies
KW - fucosyltransferase 8
KW - gene therapy
KW - glycoengineering
KW - immunotherapy
KW - viral reservoir
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U2 - 10.1016/j.omtm.2020.11.001
DO - 10.1016/j.omtm.2020.11.001
M3 - Article
AN - SCOPUS:85098648309
VL - 20
SP - 204
EP - 217
JO - Molecular Therapy - Methods and Clinical Development
JF - Molecular Therapy - Methods and Clinical Development
SN - 2329-0501
ER -