Termini James M, Martinez-Navio José M, Gao Guangping, Fuchs Sebastian P, Desrosiers Ronald C
Department of Pathology, University of Miami Miller School of Medicine, Miami, FL, USA.
Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA.
Mol Ther Methods Clin Dev. 2020 Nov 11;20:204-217. doi: 10.1016/j.omtm.2020.11.001. eCollection 2021 Mar 12.
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 , 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.
已证明，与岩藻糖基化抗体相比，人免疫球蛋白G（IgG）重链天冬酰胺-297位上缺乏岩藻糖可使抗体依赖性细胞毒性（ADCC）活性增强10至100倍。我们实验室正在研究使用腺相关病毒（AAV）作为载体来递送用于治疗目的的HIV特异性抗体。由于抗体是由载体转导的细胞产生的，因此无法利用当前的糖基工程技术。为了使AAV递送的抗体实现类似的ADCC增强效果，设计、测试了靶向岩藻糖基转移酶-8（FUT8）的短发夹RNA（shRNA），并将其克隆到用于递送HIV特异性广泛中和抗体（bNAb）的AAV载体中。对我们的糖基工程化AAV（GE-AAV）载体产生的抗体进行了岩藻糖含量和ADCC分析。GE-AAV构建体能够实现FUT8敲低超过80%。通过针对α1-6岩藻糖的凝集素免疫印迹法证实了结果，该方法显示GE-AAV产生的抗体上几乎完全没有岩藻糖。GE-AAV产生的抗体显示ADCC增强了10倍以上，同时与其岩藻糖基化对应物相比，中和作用和与gp140三聚体的结合相同。当与已知可增强与FcγRIIIA结合的关键Fc突变结合时，ADCC增强了40至60倍。我们的研究结果定义了一种增强AAV递送的抗HIV抗体的有效方法。
