Heydarchi Behnaz, D'Silva Damian B, Wong Huon, Goddard-Borger Ethan D, Wicks Ian P
Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.
Department of Medical Biology, The University of Melbourne, Parkville, Australia.
Blood Adv. 2025 Apr 8;9(7):1522-1533. doi: 10.1182/bloodadvances.2024015082.
Hemolytic disease of the fetus and newborn (HDFN) due to Rhesus D (RhD) antigen mismatch between the mother and fetus has been a significant cause of neonatal jaundice, recurrent miscarriage, and stillbirth throughout history. Polyclonal anti-RhD immunoglobulin G (RhD-pIgG), derived from the plasma of RhD-negative donors immunized with RhD-positive red blood cells (RBCs), has reduced the incidence of HDFN, but this approach is currently restricted to developed countries. Monoclonal antibodies (mAbs) offer a promising alternative to address this pressing need, but prior attempts to develop effective anti-RhD mAbs have failed, in some cases, due to differences in fucosylation patterns between mAbs produced in cell lines and RhD-pIgG. Chinese hamster ovary (CHO) cell lines, commonly used for pharmaceutical protein production, induce high levels of fucosylation, reducing the antibody-dependent cellular cytotoxicity (ADCC) activity crucial for clearing RhD-positive RBCs. In contrast, RhD-pIgG has lower fucosylation levels, which enhances ADCC activity. Regulating the glycan levels of mAbs during production requires specialized cell lines and culture conditions. In this study, we took an alternative approach through antibody engineering. The Fragment crystallizable (Fc) regions of 2 existing anti-RhD mAbs (Brad3 and Fog1) were subjected to mutagenesis to introduce ADCC-enhancing mutations and then expressed in CHO cells under standard conditions. We demonstrate that targeted Fc mutagenesis significantly enhanced ADCC compared with the wild-type mAbs, while preserving RhD binding and efficient production in CHO cells. Furthermore, these Fc variants achieved comparable efficacy with RhD-pIgG, suggesting a new strategy for producing anti-RhD mAbs with improved functionality, without the need for glycoengineering.
由于母亲与胎儿之间存在恒河猴D（RhD）抗原不匹配而导致的胎儿及新生儿溶血病（HDFN），在历史上一直是新生儿黄疸、反复流产和死产的重要原因。多克隆抗RhD免疫球蛋白G（RhD-pIgG）源自用RhD阳性红细胞（RBC）免疫的RhD阴性供体的血浆，它降低了HDFN的发病率，但这种方法目前仅限于发达国家。单克隆抗体（mAb）为满足这一迫切需求提供了一种有前景的替代方案，但此前开发有效的抗RhD mAb的尝试均以失败告终，在某些情况下，原因是细胞系产生的mAb与RhD-pIgG之间岩藻糖基化模式存在差异。常用于生产药用蛋白质的中国仓鼠卵巢（CHO）细胞系会诱导高水平的岩藻糖基化，降低清除RhD阳性RBC至关重要的抗体依赖性细胞毒性（ADCC）活性。相比之下，RhD-pIgG的岩藻糖基化水平较低，这增强了ADCC活性。在生产过程中调节mAb的聚糖水平需要专门的细胞系和培养条件。在本研究中，我们通过抗体工程采用了另一种方法。对2种现有的抗RhD mAb（Brad3和Fog1）的可结晶片段（Fc）区域进行诱变，以引入增强ADCC的突变，然后在标准条件下在CHO细胞中表达。我们证明，与野生型mAb相比，靶向Fc诱变显著增强了ADCC，同时保留了RhD结合能力以及在CHO细胞中的高效生产能力。此外，这些Fc变体与RhD-pIgG具有相当的疗效，这表明了一种无需糖基工程即可生产功能改进的抗RhD mAb的新策略。
