虽然小型治疗性抗体的开发很重要，但用于其纯化的亲和标签常常导致不均匀生产和免疫原性。在本研究中，我们将金黄色葡萄球菌蛋白A（Staphylococcus aureus protein A，SpA）的结合能力整合到抗体片段中，实现了便利且无需标签的纯化。由于对Fc区域有较高结合亲和力，SpA亲和层析法被用作传统抗体的全球标准纯化方法。SpA还对某些归属于VH3亚家族的可变重链区域（variable heavy domains，VH）具有结合亲和力。基于SpA-VH3共结晶结构的序列比对与结构建模结果，我们通过突变异构法将SpA结合能力整合到抗CD3单链Fv中。此外，我们将此突变异构方法应用于更复杂的小型双特异性抗体，并成功使用SpA亲和层析法纯化了这些抗体。纯化后的抗体仍然保持其生物学功能。将SpA结合能力整合到传统抗体片段中简化了纯化和生产过程的监控，因此是加速小型治疗性抗体开发的理想策略。此外，由于其免疫活性，具有SpA结合能力的抗CD3可变区域是开发无Fc区域的工程化抗癌治疗抗体的有效构建模块。

Although the development of small therapeutic antibodies is important, the affinity tags used for their purification often result in heterogeneous production and immunogenicity. In this study, we integrated Staphylococcus aureus protein A (SpA) binding ability into antibody fragments for convenient and tag-free purification. SpA affinity chromatography is used as a global standard purification method for conventional antibodies owing to its high binding affinity to the Fc region. SpA also has a binding affinity for some variable heavy domains (VH) classified in the VH3 subfamily. Through mutagenesis based on alignment and structural modeling results using the SpA-VH3 cocrystal structure, we integrated the SpA-binding ability into the anti-CD3 single-chain Fv. Furthermore, we applied this mutagenesis approach to more complicated small bispecific antibodies and successfully purified the antibodies using SpA affinity chromatography. The antibodies retained their biological function after purification. Integration of SpA-binding ability into conventional antibody fragments simplifies the purification and monitoring of the production processes and, thus, is an ideal strategy for accelerating the development of small therapeutic antibodies. Furthermore, because of its immunoactivity, the anti-CD3 variable region with SpA-binding ability is an effective building block for developing engineered cancer therapeutic antibodies without the Fc region.