Next Gen MIC - concurrent selection for affinity and developability of evolved candidates

基于抗体的治疗药物在生产过程中的聚集可能是有问题的，危及它们的成功开发。重要的是，虽然我们快速产生有效mAb候选者的能力允许平台制造，但我们对序列和聚集(从最广泛的意义上)之间关系的根本缺乏正在给制造更复杂的合成模式带来重大障碍。为了解决这一未得到满足的需求，利兹大学的团队与阿斯特拉捷利康一起开发了一种体内细菌聚集筛查(EBO等人。自然通讯，2020)，以有效地重新设计有问题的候选序列。我们已经证明，我们的三方β-内酰胺酶试验(TPBLA)能够(I)识别候选单链抗体(ScFv)；(Ii)使用定向进化来改进易于聚集的序列；(Iii)识别导致聚集的精确残基。在这次学习中，我们将增加TPBLA在生物制药开发流水线和基础研究中的适用性。目的1：我们将调整TPBLA，以允许选择抗聚集序列，同时通过使用氨苄西林抗性和荧光分选的正交筛选来保持靶结合亲和力。目的2：能够探测序列对靶亲和力和蛋白质聚集/稳定性的影响，为探索特异性和亲和力之间的关系以及稳定性/亲和力之间的权衡打开了大门。为了做到这一点，我们将通过对TPBLA进行深度突变测序的方法来生成一个大型数据集，从而量化数千个单个变体中每一个的“适合度”。该项目建立在学术导师和他们的行业合作者之间强大的持续合作的基础上。Astra Zeneca所涉及的广泛技术以及一流的设施和机会为学生提供了一流的培训环境。

The aggregation of antibody-based therapeutics during manufacture can be problematic, jeopardising their successful development. Importantly, while our ability to rapidly generate effective mAb candidates allows platform manufacturing, our fundamental lack of understanding of the relationship between sequence and aggregation (in its broadest sense) is presenting a significant hurdle to the manufacture of more complex, synthetic modalities. To address this unmet need, the team at Leeds, together with Astra Zeneca, have developed an in vivo bacterial aggregation screen (Ebo et al. Nature Commun, 2020) to efficiently re-design problematic candidate sequences. We have shown that our tripartite beta-lactamase assay (TPBLA) is able to (i) identify candidate scFvs (ii) improve aggregation prone sequences using directed evolution and (iii) identify the precise residues that lead to aggregation. In this studentship we will increase the applicability of the TPBLA to biopharmaceutical development pipeline and fundamental research. Aim 1: we will adapt the TPBLA to allow selection of aggregation resistant sequences while maintaining target binding affinity by using orthogonal screens of ampicillin resistance and fluorescence sorting. Aim 2: the ability to probe the effects of sequence on target affinity and protein aggregation/stability opens the door to exploration of the relationship between specificity and avidity and stability/affinity trade-offs. To do this we will generate a large dataset by a deep mutational sequencing approach to the TPBLA whereby the "fitness" of each of thousands of individual variants is quantified. The project builds on strong on-going collaborations between the academic supervisors and their industrial collaborators. The breadth of techniques involved and the excellent facilities and opportunities at Astra Zeneca provide a superb training environment for the student.