Bélanger Kasandra, Wu Cunle, Sulea Traian, van Faassen Henk, Callaghan Deborah, Aubry Annie, Sasseville Marc, Hussack Greg, Tanha Jamshid
Human Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Ottawa, Ontario, Canada.
Medical Devices Research Centre, Life Sciences Division, National Research Council Canada, Montréal, Quebec, Canada.
Protein Sci. 2025 May;34(5):e70114. doi: 10.1002/pro.70114.
An attractive feature of human Vs over camelid VHs as immunotherapeutics is their perceived lower risk of immunogenicity. While human Vs can readily be obtained from synthetic phage display libraries, they often suffer from low affinity and poor solubility compared to VHs derived from immune libraries. Using SARS-CoV-2 spike protein as a model antigen, we screened a synthetic human V phage display library and identified a diverse set of antigen-specific Vs. However, the Vs exhibited low affinity, and many had low solubility; that is, they were prone to aggregation. To explore the feasibility of improving the affinity, we subjected a representative V to in vitro affinity maturation. We created a yeast surface display library of V variants employing a site-saturated mutagenesis approach targeting complementarity-determining regions and selected against the target antigen. Next-generation sequencing of the selected variants, combined with structural modeling, identified a set of Vs as potentially improved candidates. Characterization of these candidates revealed several Vs with improved affinities of up to 100-fold (Ks as low as 3 nM) and potent neutralization capabilities; however, they still showed significant aggregation. By introducing as few as two camelid residues into the framework region 2 of a high-affinity V (a process referred to as camelization), we were able to completely solubilize the V without compromising its affinity and other important attributes, including thermostability and protein A binding. This study demonstrates the feasibility of generating high-affinity, -solubility, and -stability human Vs from synthetic libraries through a combination of in vitro affinity maturation and minimal camelization.
人源V区作为免疫治疗药物相对于骆驼科动物V区的一个吸引人的特点是其被认为具有较低的免疫原性风险。虽然人源V区可以很容易地从合成噬菌体展示文库中获得，但与从免疫文库中获得的V区相比，它们常常具有低亲和力和低溶解性。以严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白作为模型抗原，我们筛选了一个合成人源V噬菌体展示文库，并鉴定出了一系列不同的抗原特异性V区。然而，这些V区表现出低亲和力，并且许多具有低溶解性；也就是说，它们易于聚集。为了探索提高亲和力的可行性，我们对一个代表性的V区进行了体外亲和力成熟。我们采用靶向互补决定区的位点饱和诱变方法创建了一个V区变体的酵母表面展示文库，并针对目标抗原进行筛选。对所选变体的二代测序，结合结构建模，鉴定出一组可能得到改善的候选V区。对这些候选物的表征揭示了几个亲和力提高高达100倍（解离常数低至3 nM）且具有有效中和能力的V区；然而，它们仍然表现出明显的聚集。通过在高亲和力V区的框架区2中引入少至两个骆驼科动物残基（这一过程称为骆驼化），我们能够使V区完全溶解，同时不损害其亲和力和其他重要特性，包括热稳定性和与蛋白A的结合能力。这项研究证明了通过体外亲和力成熟和最小化骆驼化相结合，从合成文库中产生高亲和力、高溶解性和高稳定性人源V区的可行性。
