PDF(Pubmed)
Abstract：
Secretory IgA (SIgA) presents a promising avenue for mucosal immunotherapy yet faces challenges in expression, purification, and stability. IgA exists in two primary isotypes, IgA1 and IgA2, with IgA2 further subdivided into two common allotypes: IgA2m(1) and IgA2m(2). The major differences between IgA1 and IgA2 are located in the hinge region, with IgA1 featuring a 13-amino acid elongation that includes up to six O-glycosylation sites. Furthermore, the IgA2m(1) allotype lacks a covalent disulfide bond between heavy and light chains, which is present in IgA1 and IgA2m(2). While IgA1 demonstrates superior epitope binding and pathogen neutralization, IgA2 exhibits enhanced effector functions and stability against mucosal bacterial degradation. However, the noncovalent linkage in the IgA2m(1) allotype raises production and stability challenges. The introduction of distinct single mutations aims to facilitate an alternate disulfide bond formation to mitigate these challenges. We compare four different IgA2 versions with IgA1 to further develop secretory IgA antibodies against SARS-CoV-2 for topical delivery to mucosal surfaces. Our results indicate significantly improved expression levels and assembly efficacy of SIgA2 (P221R) in Nicotiana benthamiana. Moreover, engineered SIgA2 displays heightened thermal stability under physiological as well as acidic conditions and can be aerosolized using a mesh nebulizer. In summary, our study elucidates the benefits of stability-enhancing mutations in overcoming hurdles associated with SIgA expression and stability.
摘要：
分泌型IgA（SIgA）为粘膜免疫治疗提供了一个有希望的途径，但在表达方面面临挑战，净化,和稳定性。IgA存在于两种主要同种型中,IgA1和IgA2,其中IgA2进一步细分为两种常见的同种异型:IgA2m(1)和IgA2m(2)。IgA1和IgA2之间的主要差异位于铰链区，IgA1具有13个氨基酸的延伸，包括多达六个O-糖基化位点。此外，IgA2m（1）同种异型在重链和轻链之间缺乏共价二硫键，它存在于IgA1和IgA2m(2)中。虽然IgA1表现出优越的表位结合和病原体中和，IgA2表现出增强的效应子功能和抗粘膜细菌降解的稳定性。然而,IgA2m（1）同种异型中的非共价键增加了生产和稳定性的挑战。引入不同的单突变旨在促进交替的二硫键形成以减轻这些挑战。我们将四种不同的IgA2版本与IgA1进行比较，以进一步开发针对SARS-CoV-2的分泌性IgA抗体，用于局部递送至粘膜表面。我们的结果表明，SIgA2（P221R）在烟草中的表达水平和组装功效显着提高。此外，工程SigA2在生理和酸性条件下显示出更高的热稳定性，并且可以使用网状雾化器进行雾化。总之,我们的研究阐明了稳定性增强突变在克服与SIgA表达和稳定性相关的障碍方面的益处.
