Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina , Chapel Hill, North Carolina 27599-7568, United States.
Biochemistry. 2013 Oct 15;52(41):7283-94. doi: 10.1021/bi400716w. Epub 2013 Oct 3.
The C-terminal coiled-coil region of mouse and human cartilage matrix protein (CMP) self-assembles into a parallel trimeric complex. Here, we report a general strategy for the development of highly stable trimeric targeting ligands (tribodies), against epidermal growth factor receptor (EGFR) and prostate-specific membrane antigen (PSMA) as examples, by fusing a specific target-binding moiety with a trimerization domain derived from CMP. The resulting fusion proteins can efficiently self-assemble into a well-defined parallel homotrimer with high stability. Surface plasmon resonance (SPR) analysis of the trimeric targeting ligands demonstrated significantly enhanced target-binding strength compared with the corresponding monomers. Cellular-binding studies confirmed that the trimeric targeting ligands have superior binding strength toward their respective receptors. Significantly, the EGFR-binding tribody was considerably accumulated in the tumor of mice bearing xenografted EGFR-positive tumors, indicating its effective cancer-targeting feature under in vivo conditions. Our results demonstrate that CMP-based self-assembly of tribodies can be a general strategy for the facile and robust generation of trivalent targeting ligands for a wide variety of in vitro and in vivo applications.
鼠和人软骨基质蛋白（CMP）的 C 端卷曲螺旋区自行装配成平行的三聚体复合物。在这里，我们报告了一种通用策略，用于开发高度稳定的三聚体靶向配体（tribody），以表皮生长因子受体（EGFR）和前列腺特异性膜抗原（PSMA）为例，将特定的靶结合部分与源自 CMP 的三聚体化结构域融合。所得融合蛋白可以有效地自我组装成具有高稳定性的定义良好的平行同源三聚体。表面等离子体共振（SPR）分析表明，与相应的单体相比，三聚体靶向配体的靶结合强度显著增强。细胞结合研究证实，三聚体靶向配体对各自的受体具有更高的结合强度。值得注意的是，EGFR 结合的 tribody 在携带异种移植 EGFR 阳性肿瘤的小鼠肿瘤中大量积累，表明其在体内条件下具有有效的癌症靶向特征。我们的结果表明，基于 CMP 的 tribody 自组装可以成为一种通用策略，用于方便、有效地生成各种用于体外和体内应用的三价靶向配体。
