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

生物制药作为药品销售的一部分继续增长，寻找具有更高功效的新产品通常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在糖基化状态下是不均匀的，尽管已知例如非聚焦抗体比聚焦糖型更有效地提高抗体依赖性细胞毒性（ADCC）[1]。目前使用CHO细胞系的产品表达高水平的聚焦化。因此，能够均匀糖基化的工艺产生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，并在酵母[3]中产生聚焦化缺陷的宿主细胞或工程人糖基化。在这里，我们建议采取下游处理为基础的解决方案，通过设计亲和配体能够识别糖型用于色谱。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，这是由Fc γ受体介导的。它已被用于亲和层析，使用野生型蛋白作为配体进行制备分离[4]和表征Fc γ /Ab相互作用[5]的强度。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健壮。因此，该项目将为此目的研究Fc γ配体的合理蛋白质工程，这一策略在改善蛋白a配体方面是有效的，这是单抗制造的默认技术。野生型Fc γ 3a配体已被证明在聚焦和非聚焦的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，它们在结构上都表现出相当大的多样性。以此为起点，本项目将：-设计Fc γ亲和配体的克隆、表达和纯化策略。-生成模型分离混合物的组件，使用基于酶的策略来重塑抗体上的糖基化，以创建均匀的制剂，例如非聚焦结构。-与上述类似的策略将用于创建具有确定聚糖结构的Fc γ 3a配体。-然后使用位点特异性化学将配体固定在琼脂糖上，并使用模型抗体混合物在亲和层析中测试其性能。-色谱法将应用于CHO表达系统和分离血液中抗体糖型的分离。由于抗体去聚焦化而增强的效应细胞功能取决于效应细胞Fcy受体谱。布莱格曼,连续波。Dekkers， G，等。中华免疫学杂志，2017,29 (4):444 - 444利用CRISPR/Cas9基因敲除中国仓鼠卵巢细胞中的FUT8，产生去聚焦抗体。陶绍林，赵东，雷洪，等。Eng。的生活。科学通报，2015,15 (5):666 -666控制重组治疗蛋白糖基化的技术。张建军，张建军，张建军，等。中药制剂的研制与应用。中国药理学杂志，2010,31 (1):391 - 391利用工程Fc评估治疗性抗体Fc-聚糖的异质性？受体iiia -固定化柱。科学通报，2018,(8):39 - 55 [j] . IgG1的体外糖工程及其对Fc受体结合和ADCC活性的影响。Thomann， M., Schlothauer， T., Dashivets， T.等。科学通报，2015,10(8):0134949。

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.