Zhong Xiaotian, Yan Guoying Grace, Chaturvedi Apurva, Li Xiuling, Gao Yijie, Girgenrath Mahasweta, Corcoran Chris J, Diblasio-Smith Liz, LaVallie Edward R, de Rham Teresse, Zhou Jing, Abel Molica, Riegel Logan, Lim Sean K H, Bloom Laird, Lin Laura, D'Antona Aaron M
BioMedicine Design, Discovery and Early Development, Pfizer Research and Development, 610 Main Street, Cambridge, MA 02139, USA.
Rare Disease Research Unit, Pfizer Research and Development, 610 Main Street, Cambridge, MA 02139, USA.
Antibodies (Basel). 2024 Oct 7;13(4):83. doi: 10.3390/antib13040083.
α-dystroglycanopathies are congenital muscular dystrophies in which genetic mutations cause the decrease or absence of a unique and complex O-linked glycan called matriglycan. This hypoglycosylation of O-linked matriglycan on the α-dystroglycan (α-DG) protein subunit abolishes or reduces the protein binding to extracellular ligands such as laminins in skeletal muscles, leading to compromised survival of muscle cells after contraction. Surrogate molecular linkers reconnecting laminin-211 and the dystroglycan β-subunit through bispecific antibodies can be engineered to improve muscle function in the α-dystroglycanopathies. This study reports the metabolic engineering of a novel glycofusion bispecific (GBi) antibody that fuses the mucin-like domain of the α-DG to the light chain of an anti-β-DG subunit antibody. Transient HEK production with the co-transfection of LARGE1, the glycoenzyme responsible for the matriglycan modification, produced the GBi antibody only with a light matriglycan modification and a weak laminin-211 binding activity. However, when a sugar feed mixture of uridine, galactose, and manganese ion (Mn) was added to the culture medium, the GBi antibody produced exhibited a dramatically enhanced matriglycan modification and a much stronger laminin-binding activity. Further investigation has revealed that Mn in the sugar feeds played a critical role in increasing the matriglycan modification of the GBi antibody, key for the function of the resulting bispecific antibody.
α-肌营养不良糖蛋白病是一类先天性肌营养不良症，其中基因突变导致一种名为基质糖链的独特且复杂的O-连接聚糖减少或缺失。α-肌营养不良糖蛋白（α-DG）蛋白亚基上O-连接基质糖链的这种糖基化不足消除或降低了该蛋白与细胞外配体（如骨骼肌中的层粘连蛋白）的结合，导致肌肉细胞在收缩后存活能力受损。可以设计通过双特异性抗体重新连接层粘连蛋白-211和肌营养不良糖蛋白β亚基的替代分子连接体，以改善α-肌营养不良糖蛋白病中的肌肉功能。本研究报告了一种新型糖融合双特异性（GBi）抗体的代谢工程，该抗体将α-DG的粘蛋白样结构域与抗β-DG亚基抗体的轻链融合。在共转染负责基质糖链修饰的糖基酶LARGE1的情况下进行瞬时HEK生产，所产生的GBi抗体仅具有轻度的基质糖链修饰和较弱的层粘连蛋白-211结合活性。然而，当向培养基中添加尿苷、半乳糖和锰离子（Mn）的糖源混合物时，所产生的GBi抗体表现出显著增强的基质糖链修饰和更强的层粘连蛋白结合活性。进一步研究表明，糖源中的Mn在增加GBi抗体的基质糖链修饰方面起关键作用，这是所得双特异性抗体功能的关键。
