Optimization of a broad and potent decoy receptor for SARS-associated viruses

摘要 新冠肺炎的病原体SARS冠状病毒2型的刺突蛋白(S)与血管紧张素-2结合。 转换酶2(ACE2)作为进入受体，触发S的构象变化，从而推动 病毒的包膜和宿主细胞膜。感染是通过中和抗体来阻止感染的 血管紧张素转换酶2结合部位在S，但逃逸突变S迅速出现组织中的单抗 文化。此外，单抗一般是菌株特异性的，许多不能识别高水平的 与人类SARS-CoV-1和SARS-CoV-2都有亲和力，但只有外来的蝙蝠贝塔冠状病毒是宿主 为未来的疫情做准备。作为生物药物开发的替代方案，我们使用了深度诱变来 指导设计一种特别广泛的可溶性诱骗受体，它与紧密的微微克分子/低分子结合。 测试了所有蝙蝠和人类SARS相关冠状病毒对S的纳摩尔亲和力。精心设计的诱饵 有效中和正宗SARS-CoV-1和SARS-CoV-2，其效力可与单克隆体相媲美 商业开发，并具有规模化生产的理想特性。工程设计的诱饵也 催化将血管紧张素II转化为血管扩张多肽产品，可能直接治疗 新冠肺炎，为我们提供了一种独特的潜在治疗方法，具有双重作用机制。我们的建议 调查SARS-CoV-2尖峰是否可以突变以逃避工程诱饵的中和 受体，并解决了工程蛋白在进展之前作为IgG1-Fc融合的最终优化 提供给一个支持IND的程序。为了让SARS-CoV-2对工程诱饵产生抗药性，S的突变 必须降低与诱饵的亲和力，同时保持与人ACE2受体的结合。找出这样的S 变异体，我们已经使用了受体结合域的饱和突变结合选择 在竞争的可溶性诱饵存在的情况下与野生型ACE2紧密结合。在深度测序之后，一个 少数突变被发现是富集的，但目前还不清楚这些突变中是否有任何富含 确实优先结合野生型ACE2，如果是这样的话，它们达到了什么程度的特异性。基于 这一初步数据，(目标1)我们将验证是否可以在S身上发现区分 人ACE2和工程诱饵，并鉴定它们的亲和力和表达水平。 到目前为止，我们可以得出结论，可能的耐药性突变似乎非常罕见，通常需要 一个密码子内有多个核苷酸变化，但还需要进一步的定量表征。 同时，(目标2)我们将快速优化工程诱饵与IgG1 Fc区域的融合，以用于 增强了血清稳定性。我们目前的IgG1-融合构建(基于理性的、结构导向的 设计)高表达、稳定，并与SARS-CoV-2 S结合，具有皮摩尔亲和力。我们将最终敲定 通过扫描工程诱饵与IgG1之间合适的融合位点来优化蛋白质， 通过活性、稳定性和表达来评估蛋白质质量。

ABSTRACT The spike protein (S) of SARS coronavirus 2, the pathogen responsible for COVID-19, binds angiotensin- converting enzyme 2 (ACE2) as an entry receptor, triggering conformational changes in S that drive fusion of the viral envelope and host cell membrane. Infection is inhibited by neutralizing antibodies that block the ACE2-binding site on S, yet escape mutations in S rapidly emerge towards monoclonal antibodies in tissue culture. Furthermore, monoclonal antibodies are generally strain specific, and many do not recognize with high affinity both human SARS-CoV-1 and SARS-CoV-2, yet alone exotic bat betacoronaviruses that are a reservoir for future outbreaks. As an alternative for biologic drug development, we have used deep mutagenesis to guide the engineering of an exceptionally broad soluble decoy receptor that binds with tight picomolar/low- nanomolar affinity to S from all bat and human SARS-associated coronaviruses tested. The engineered decoy potently neutralizes authentic SARS-CoV-1 and SARS-CoV-2 with an efficacy that rivals monoclonals under commercial development, and has desirable properties for manufacture at scale. The engineered decoy also catalytically converts angiotensin II to vasodilatory peptide products that might directly address symptoms of COVID-19, providing us with a unique potential therapeutic that has dual mechanisms of action. Our proposal investigates whether the SARS-CoV-2 spike can mutate to escape neutralization by the engineered decoy receptor, and addresses final optimization of the engineered protein as an IgG1-Fc fusion before advancement to an IND-enabling program. For SARS-CoV-2 to become resistant to the engineered decoy, mutations in S must decrease affinity to the decoy while maintaining binding to human ACE2 receptors. To identify such S variants, we have used saturation mutagenesis of the receptor-binding domain coupled with a selection for tight binding to wild type ACE2 in the presence of competing soluble decoy. Following deep sequencing, a small number of mutations were found to be enriched, but it is unclear whether any of these mutations do indeed preferentially bind wild type ACE2 and if so, to what degree they have achieved specificity. Based on this preliminary data, (Aim 1) we will validate whether mutations in S can be found that discriminate between human ACE2 and the engineered decoy, and characterize the variants for their affinities and expression levels. Thus far, we can conclude that possible resistance mutations appear to be very rare and generally require more than one nucleotide change within a codon, but further quantitative characterization is needed. Simultaneously, (Aim 2) we will rapidly optimize fusions of the engineered decoy with the Fc region of IgG1 for enhanced serum stability. Our current IgG1-fusion construct (which was based on rational, structure-guided design) is highly expressed, stable and binds SARS-CoV-2 S with picomolar affinity. We will finalize optimization of the protein by scanning suitable fusion sites between the engineered decoy and IgG1, assessing protein quality by activity, stability and expression.