用于可定制靶向纳米药物的点击抗体片段--通过非典型氨基酸结合实现特定位点四嗪和叠氮功能化

IF 6.1 Q1 CHEMISTRY, MULTIDISCIPLINARY Chemistry methods : new approaches to solving problems in chemistry Pub Date : 2023-10-23 DOI:10.1002/cmtd.202300036
Dr. Pie Huda, James Humphries, Dr. Nicholas L. Fletcher, Dr. Christopher B. Howard, Prof. Kristofer J. Thurecht, Dr. Craig A. Bell
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引用次数: 0

摘要

用于开发成像剂和抗体药物结合剂的蛋白质功能化通常仍依赖于通过可触及的赖氨酸和半胱氨酸残基对蛋白质进行统计酰胺化,这需要对蛋白质与蛋白质之间的结合进行优化,并有可能影响整体功能。为了解决这个问题,人们把重点转向开发在蛋白质结构中加入非典型氨基酸的蛋白质,从而实现特定位点的标记和功能化。在这里,我们展示了在分别针对前列腺特异性膜抗原和表皮生长因子受体的生物制剂中加入带有四嗪或叠氮正交偶联模式的非典型氨基酸。将这些生物正交残基置入纳米抗体或单链可变片段(scFvs)是通过对蛋白质编码 DNA 进行定点突变来实现的,这样就可以在表达这些蛋白质时对插入进行控制。我们的研究表明,将模型化合物(如荧光团或聚合物材料)与蛋白质进行生物正交偶联不会显著改变结合亲和力,因此这些蛋白质偶联方法是开发简单、可定制的个性化靶向抗体-药物偶联物的有力工具,其亲和力得以保持。
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Click-on Antibody Fragments for Customisable Targeted Nanomedicines – Site-specific Tetrazine and Azide Functionalisation through Non-canonical Amino Acid incorporation

Protein functionalisation for the development of imaging agents and antibody drug conjugates still often relies on statistical amidation of the protein through accessible lysine and cysteine residues, requiring protein to protein conjugation optimisation and can potentially impact the overall function. To combat this, focus has turned to developing proteins that have noncanonical amino acids incorporated into their structure, allowing for site-specific labelling and functionalisation. Herein we showcase the incorporation of non-canonical amino acids bearing a tetrazine or azide orthogonal coupling modality into biologics targeted to the prostate-specific membrane antigen and epidermal growth factor receptor respectively. The placement of these bioorthogonal residues into nanobody or single chain variable fragments (scFvs) is introduced by site-directed mutagenesis of the protein-coding DNA that allows for controlled insertion when these proteins are expressed. We show that bioorthogonal coupling of model compounds such as fluorophore or polymeric materials onto the protein does not significantly change the binding affinity, making these protein conjugation methods a powerful tool for development of simple customisable personalised targeted antibody-drug conjugates where affinity is retained.

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