Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
Institute for Infection and Immunity, St. George's University of London, London SW17 0RE, UK.
Int J Mol Sci. 2024 Jun 22;25(13):6856. doi: 10.3390/ijms25136856.
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 -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 . 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.
分泌型免疫球蛋白 A（SIgA）为黏膜免疫治疗提供了一个很有前途的途径，但在表达、纯化和稳定性方面仍面临挑战。IgA 存在两种主要同种型：IgA1 和 IgA2，IgA2 进一步细分为两种常见同种异型：IgA2m(1)和 IgA2m(2)。IgA1 和 IgA2 的主要区别位于铰链区，IgA1 具有 13 个氨基酸的延伸，其中包括多达 6 个糖基化位点。此外，IgA2m(1)同种异型在重链和轻链之间缺乏共价二硫键，而 IgA1 和 IgA2m(2)则存在这种键。虽然 IgA1 表现出优异的表位结合和病原体中和能力，但 IgA2 表现出增强的效应功能和对黏膜细菌降解的稳定性。然而，IgA2m(1)同种异型中非共价键的存在带来了生产和稳定性方面的挑战。引入独特的单点突变旨在促进替代二硫键的形成，以减轻这些挑战。我们比较了四种不同的 IgA2 版本与 IgA1，以进一步开发针对 SARS-CoV-2 的分泌型 IgA 抗体，用于局部递送至黏膜表面。我们的结果表明，在 中，突变体 SIgA2（P221R）的表达水平和组装效率显著提高。此外，工程化的 SIgA2 在生理和酸性条件下表现出更高的热稳定性，并且可以使用网孔雾化器进行雾化。总之，我们的研究阐明了稳定性增强突变在克服 SIgA 表达和稳定性相关障碍方面的益处。
