Development of affinity ligands for antibody glycoform separations based on the Fc-gamma receptor

生物制药作为药品销售额的一小部分继续增长，寻找效率更高的新产品往往意味着产品的详细糖生物学及其与免疫系统的相互作用至关重要。目前的产品包括抗体和促红细胞生成素，它们的糖基化状态是不同的，尽管众所周知，例如，非岩藻糖基化抗体比岩藻糖基化糖形式更有效地提高抗体依赖细胞细胞毒(ADCC)[1]。目前使用CHO细胞系的产品表达高水平的岩藻糖基化。因此，能够均质糖基化的方法引起了相当大的兴趣和投资。这些方法通常集中在上游，例如敲除CHO[2]中的fut8，并创建缺乏岩藻糖基化的宿主细胞或在酵母中设计人的糖基化[3]。在这里，我们建议采取基于下游处理的解决方案，通过设计能够识别糖形的亲和配体用于层析。这个概念是基于抗体和免疫系统之间自然发生的相互作用，这种相互作用是由Fcγ受体介导的。它已被用于亲和层析，使用野生型蛋白作为配基进行制备分离[4]，并表征Fc伽马/抗体相互作用的强度[5]。为了成为制造这种配体的有用基础，它们的分离性能需要稳定和坚固。因此，该项目将为此目的审查Fc伽马配体的合理蛋白质工程，这是一种在改进蛋白质A配体方面一直有效的战略，这是制造单抗的默认技术。野生型Fcγ3a配体在岩藻糖化和非岩藻糖化抗体糖型之间具有选择性，因此具有临床和商业价值。然而，它有五个糖基化位点，所有这些位点在结构上都表现出相当大的多样性。以此为起点，该项目将：-设计克隆，Fc伽马亲和配基的表达和纯化策略。-使用基于酶的策略产生用于模型分离混合物的成分，以重塑抗体上的糖基化以创建均一的制剂，例如非岩藻糖化结构。-将采用与上述类似的策略来创建定义了糖链结构的Fc伽马3a配基。-然后使用特定部位的化学将配基固定在琼脂糖上，并在使用模型抗体混合物测试其亲和层析中的性能。-然后，该层析将被应用于在CHO表达系统和分级血液中的抗体糖体的分离。参考文献[1]由于抗体脱糖作用而增强了效应器功能，取决于Effector Cell FCY受体分布。Brggeman，CW.，Dekkers，G.等人。《免疫学杂志》。2017,199：204-211。[2]利用CRISPR/Cas9对中国仓鼠卵巢细胞中的FUT8进行功能性敲除，以产生脱糖抗体。陶山，超东，L.，雷，H.，等。英语。生活。SCI。2015，15：660-666。[3]GlycoFi控制重组治疗蛋白糖基化的技术。首页--期刊主要分类--期刊细介绍--期刊题录与期刊详细文摘内容2010，5(1)：95-111。[4]使用工程Fc？受体IIIa固定化柱评估治疗性抗体的Fc-葡聚糖的异质性。科学报告。2018，8：3955[5]体外糖基化IgG1及其对Fc受体结合和ADCC活性的影响。书名/作者/作者：Reinessy/T.Dashivets，T.公共科学图书馆一号。2015年10月8日：e0134949。

Biopharmaceuticals continue to grow as a fraction of pharmaceutical sales, the search for new products with increased efficacy often means the detailed glycobiology of the product and its interactions with the immune system are critical. Current products including antibodies and erythropoietin are heterogeneous in their glycosylation state, even though it is known that for example non-fucosylated antibodies are more potent in raising antibody-dependent cell cytotoxicity (ADCC) [1] than fucosylated glycoforms. Current products using CHO cell lines express high levels of fucosylation. Processes capable of homogeneous glycosylation have therefore generated considerable interest and investment. These approaches have generally been upstream focused e.g. to knock out fut8 in CHO [2] and create a fucosylation deficient host cell or engineer human glycosylation in yeast [3]. Here we propose to take a downstream processing based solution by designing affinity ligands capable of glycoform recognition for use in chromatography. This concept is based on the naturally occurring interaction between antibodies and immune system which is mediated by the Fc gamma receptor. It has been used in affinity chromatography using the wild-type protein as the ligand to perform preparative separations [4] and characterise the strength of the Fc gamma/Ab interactions [5]. To be a useful basis for manufacturing such ligands need to be stable and robust in their separation properties. The project will therefore examine rational protein engineering of the Fc gamma ligand for this purpose a strategy that has been effective in improving the protein A ligand that is the default technology for mAb manufacturing. The wild-type Fc gamma 3a ligand has been shown to have selectivity between fucosylated and non-fucosylated antibody glycoforms hence it is of clinical and commercial interest. However it has five glycosylation sites all of which exhibit a considerable diversity in the structure. Using this as a starting point the project will: -Design a cloning, expression and purification strategy for the Fc gamma affinity ligands.-To generate components for model separation mixtures using an enzyme based strategy to remodel glycosylation on antibodies to create homogeneous preparations e.g. non-fucosylated structures.-A similar strategy to the above will be employed to create Fc gamma 3a ligands of defined glycan structure.-The ligands will then be immobilised onto agarose using site specific chemistry and their performance in affinity chromatography tested using the model antibody mixtures.-The chromatography will then be applied to both the separation of antibody glycoforms in CHO expression systems and in fractionated blood.References[1] Enhanced Effector Functions Due to Antibody Defucosylation Depend on the Effector Cell Fcy Receptor Profile. Bruggeman, CW., Dekkers, G., et al. The Journal of Immunology. 2017, 199:204-211.[2] Functional knockout of FUT8 in Chinese hamster ovary cells using CRISPR/Cas9 to produce a defucosylated antibody. Tao, S., Chaodong, L., Lei, H., et al. Eng. Life. Sci. 2015, 15: 660-666.[3] GlycoFi's technology to control the glycosylation of recombinant therapeutic proteins. Beck A, Cochet O, Wurch T. Expert Opin Drug Discov. 2010, 5(1):95-111.[4] Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered Fc?Receptor IIIa-Immobilized Column. Scientific Reports. 2018, 8:3955 [5] In Vitro Glycoengineering of IgG1 and Its Effect on Fc Receptor Binding and ADCC Activity. Thomann, M., Schlothauer, T., Dashivets, T., et al. Plos One. 2015, 10(8):e0134949.