Atherothrombosis and Vascular Biology, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia.
Circ Res. 2011 Aug 5;109(4):365-73. doi: 10.1161/CIRCRESAHA.111.249375. Epub 2011 Jun 23.
Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component.
To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation.
The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries.
This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.
抗体靶向递呈成像剂可以提高当前成像技术的灵敏度和准确性。同样，效应细胞归巢至疾病部位可以提高再生细胞治疗的疗效，同时减少所需细胞的数量。目前，通过与特定抗体的化学偶联可以实现靶向，这通常会导致抗体功能丧失和严重的细胞损伤。理想的偶联技术应确保保留靶向生物成分的抗原结合活性和功能。
使用金黄色葡萄球菌 sortase A 酶开发一种生化稳定、高度可重复且具有特异性的偶联方法，用于将单链抗体 (scFv) 偶联到纳米颗粒和细胞上，用于心血管疾病的分子成像和细胞归巢。该 scFv 特异性结合激活的血小板，血小板在血栓形成、动脉粥样硬化和炎症中起关键作用。
偶联过程涉及化学和酶介导的偶联步骤。scFv 成功地偶联到氧化铁颗粒（磁共振成像的对比剂）和模型细胞上。偶联效率在 50%到 70%之间，偶联后 scFv 的生物活性得以保留。在体外静态黏附试验、流动室系统、小鼠活体显微镜检查以及小鼠颈总动脉的体内磁共振成像中，均证实了 scFv 偶联细胞和纳米颗粒对激活血小板的靶向性强且具有特异性。
这种独特的生物技术方法为获取靶向再生细胞治疗和靶向分子成像在心血管和炎症性疾病及其他疾病中的应用提供了一种通用且广泛适用的工具。
