Laboratory of Cell Cycle Genetics, The Rockefeller University, New York, New York, USA.
Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York, USA.
J Biol Chem. 2023 Mar;299(3):102954. doi: 10.1016/j.jbc.2023.102954. Epub 2023 Jan 28.
COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single-chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry-based method, using B-cell complementary DNA from the same immunized animals as a source of VH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions of antiviral nanobodies. The results suggested a surprising degree of modularity to complementarity-determining region function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the mass spectrometry-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.
由冠状病毒 SARS-CoV-2 引起的 COVID-19 是一个严重的全球健康问题，不断出现的新变体挑战着当前的治疗方法。一种有前途的替代治疗途径是纳米抗体，它是从小型骆驼科动物中衍生出来的单链抗体，具有许多优势特性，并且有可能中和病毒。为了鉴定和表征广谱抗 SARS-CoV-2 刺突纳米抗体，我们进一步优化了酵母展示方法，利用之前发表的基于质谱的方法，使用来自同一免疫动物的 B 细胞互补 DNA 作为 VH 序列的来源。酵母展示捕获了之前方法所识别的许多序列，以及许多额外的序列，这些序列证明编码了一个具有高亲和力和中和不同 SARS-CoV-2 变体活性的大型新纳米抗体库。我们评估了 DNA 重排在抗病毒纳米抗体的三个互补决定区的应用。结果表明，互补决定区功能具有惊人的模块化程度。重要的是，应用于免疫动物的纳米抗体文库的酵母展示方法允许对大量纳米抗体进行平行检测。例如，我们采用了改良的酵母展示来进行大规模平行表位分组。当前的酵母展示方法在效率和特异性方面与基于质谱的方法相当，而不需要该方法所需的基础设施和专业知识，因此这两种方法高度互补，似乎可以全面探索表位空间。产生的更大的库最大限度地提高了发现广泛特异性试剂的可能性，以及在混合物中协同作用的试剂的可能性。
