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

SARS 相关病毒广泛有效的诱饵受体的优化

• 批准号: 10258005
• 负责人: Kui Kiu Chan
• 金额: $ 9.6万
• 依托单位: ORTHOGONAL BIOLOGICS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10258005
• 关键词: 2019-nCoV; ACE2; Address; Adrenal Cortex Hormones; Adult Respiratory Distress Syndrome; Affinity; Amino Acids; Angiotensin II; Angiotensins; Animal Model; Animals; Binding; Binding Sites; Biological; Biotechnology; Blocking Antibodies; COVID-19; COVID-19 pandemic; COVID-19 treatment; Cell membrane; Cells; Chimeric Proteins; Chiroptera; Clinic; Clinical; Codon Nucleotides; Competitive Binding; Complex; Coronavirus; Coupled; Data; Development; Dexamethasone; Disease; Disease Outbreaks; Drug Targeting; Engineering; Epitopes; Escape Mutant; FDA approved; Flow Cytometry; Freeze Drying; Freezing; Future; Genetic Drift; Genetic Recombination; Half-Life; Heart; Human; IgG1; Illinois; In Vitro; Infection; Inflammation; Investigational Drugs; Libraries; Lung; Lung Inflammation; Measurement; Medical; Molecular Conformation; Monoclonal Antibodies; Mus; Mutagenesis; Mutate; Mutation; Noise; Nucleotides; Patients; Peptides; Pharmaceutical Preparations; Polymerase; Positioning Attribute; Property; Protein Engineering; Proteins; Publishing; Resistance; SARS coronavirus; SARS-CoV-2 infection; Safety; Satellite Viruses; Scanning; Serum; Severe Acute Respiratory Syndrome; Site; Source; Specificity; Structure; Symptoms; Temperature; Testing; Therapeutic; Toxicology; Universities; Variant; Vendor; Viral; Virus; Virus Replication; base; betacoronavirus; commercialization; deep sequencing; design; drug development; human coronavirus; immunogenicity; lead candidate; lead optimization; lung injury; mutant; nanomolar; neutralizing antibody; neutralizing monoclonal antibodies; nucleoside analog; older patient; pandemic disease; pathogen; peptide hormone; physical property; prevent; programs; protective effect; receptor; receptor binding; remdesivir; resistance mutation; stability testing; tissue culture; zoonotic coronavirus

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.

摘要 SARS冠状病毒2（COVID-19的病原体）的刺突蛋白（S）结合血管紧张素- 转化酶2（ACE 2）作为进入受体，触发S的构象变化，驱动融合 病毒包膜和宿主细胞膜。感染被中和抗体所抑制， S上的ACE 2结合位点，但S中的逃逸突变迅速出现在组织中的单克隆抗体中 文化此外，单克隆抗体通常是菌株特异性的，并且许多不识别具有高特异性的菌株。 人SARS-CoV-1和SARS-CoV-2都具有亲和力，但只有外来的蝙蝠β冠状病毒是一个储存库， 以备将来爆发。作为生物药物开发的替代方案，我们使用了深度诱变， 指导一个非常广泛的可溶性诱饵受体，结合紧密的皮摩尔/低- 纳摩尔亲和力的S从所有蝙蝠和人类SARS相关的冠状病毒测试。设计的诱饵 有效中和真正的SARS-CoV-1和SARS-CoV-2，其效力可与 商业开发，并且具有用于大规模生产的所需性质。设计的诱饵还 催化血管紧张素II转化为血管舒张肽产品，可能直接解决的症状， COVID-19，为我们提供了一种具有双重作用机制的独特潜在治疗方法。我们的建议 研究SARS-CoV-2刺突是否可以突变以逃避工程诱饵的中和 受体，并解决了在推进前作为IgG 1-Fc融合体的工程化蛋白的最终优化 一个IND启动计划为了使SARS-CoV-2对工程诱饵产生抗性，S 必须降低对诱饵的亲和力，同时保持与人ACE 2受体的结合。为了识别这样的S 变体中，我们使用了受体结合结构域的饱和诱变，并选择了 在竞争性可溶性诱饵存在下与野生型ACE 2紧密结合。在深度测序之后， 少数突变被发现是富集的，但尚不清楚这些突变中是否有任何突变是富集的。 确实优先结合野生型ACE 2，如果是这样，它们达到何种程度的特异性。基于 根据这些初步数据，（目标1）我们将验证是否可以发现S中的突变， 人ACE 2和工程化诱饵的变体，并表征变体的亲和力和表达水平。 到目前为止，我们可以得出结论，可能的耐药突变似乎是非常罕见的，一般需要 密码子内有一个以上的核苷酸变化，但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IgG 1 Fc区的融合， 提高血清稳定性。我们目前的IgG 1融合构建体（基于合理的，结构指导的 设计）是高度表达的，稳定的，并以皮摩尔亲和力结合SARS-CoV-2S。我们将最终确定 通过扫描工程化诱饵和IgG 1之间的合适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。