背景:最近,采用了新的和先进的技术来设计和生产纳米体,用于诊断和免疫治疗。传统上,纳米抗体是从需要动物治疗的骆驼免疫文库制备的。然而,这样的方法需要大的文库大小和复杂的选择程序。当前的研究已经采用CDR移植和定点诱变技术来创建针对白血病治疗中使用的肿瘤标志物CD20(抗CD20纳米抗体)的基因工程纳米抗体。
结果:在这项研究中,我们利用交换方法将VH利妥昔单抗抗体的CDR移植至VHHCDR.我们旨在通过取代VHH-CDR3中的氨基酸(Y101R-Y102R-Y107R)来增强纳米抗体的结合亲和力。为了评估突变纳米抗体的结合能力,我们进行了酶联免疫吸附试验。此外,通过流式细胞术分析,我们比较了Raji细胞中移植的CD20和突变纳米抗体与市售人抗CD20的荧光强度。结果显示,与市售的人抗CD20相比,接枝纳米体和突变纳米体的荧光强度存在显著差异。
结论:我们在本研究中遵循的方法使得可以产生具有不同亲和力的多个抗CD20纳米抗体,而不需要广泛的选择努力。此外,我们的研究表明,计算工具在设计功能性纳米体方面是高度可靠的。
BACKGROUND: Recently, new and advanced techniques have been adopted to design and produce nanobodies, which are used in diagnostic and immunotherapy treatments. Traditionally, nanobodies are prepared from camelid immune libraries that require animal treatments. However, such approaches require large library sizes and complicated selection procedures. The current study has employed CDR grafting and site-directed mutagenesis techniques to create genetically engineered nanobodies against the tumor marker CD20 (anti-CD20 nanobodies) used in leukemia treatment.
RESULTS: In this study, we utilized the swapping method to graft CDRs from the VH Rituximab antibody to VHH CDRs. We aimed to enhance the binding affinity of the nanobodies by substituting the amino acids (Y101R-Y102R-Y107R) in the VHH-CDR3. To assess the binding capacity of the mutated nanobodies, we conducted an ELISA test. Moreover, through flow cytometry analysis, we compared the fluorescence intensity of the grafted CD20 and mutant nanobodies with that of the commercially available human anti-CD20 in Raji cells. The results showed a significant difference in the fluorescence intensity of the grafted nanobodies and mutant nanobodies when compared to the commercially available human anti-CD20.
CONCLUSIONS: The approach we followed in this study makes it possible to create multiple anti-CD20 nanobodies with varying affinities without the need for extensive selection efforts. Additionally, our research has demonstrated that computational tools are highly reliable in designing functional nanobodies.