PDF(Pubmed)
Abstract：
Crystal structures of camelid heavy-chain antibody variable domains (VHHs) bound to fragments of the combined repetitive oligopeptides domain of Clostridiodes difficile toxin A (TcdA) reveal that the C-terminus of VHH A20 was located 30 Å away from the N-terminus of VHH A26. Based on this observation, we generated a biparatopic fusion protein with A20 at the N-terminus, followed by a (GS)6 linker and A26 at the C-terminus. This A20-A26 fusion protein shows an improvement in binding affinity and a dramatic increase in TcdA neutralization potency (>330-fold [IC 50]; ≥2,700-fold [IC 99]) when compared to the unfused A20 and A26 VHHs. A20-A26 also shows much higher binding affinity and neutralization potency when compared to a series of control antibody constructs that include fusions of two A20 VHHs, fusions of two A26 VHHs, a biparatopic fusion with A26 at the N-terminus and A20 at the C-terminus (A26-A20), and actoxumab. In particular, A20-A26 displays a 310-fold (IC 50) to 29,000-fold (IC 99) higher neutralization potency than A26-A20. Size-exclusion chromatography-multiangle light scattering (SEC-MALS) analyses further reveal that A20-A26 binds to TcdA with 1:1 stoichiometry and simultaneous engagement of both A20 and A26 epitopes as expected based on the biparatopic design inspired by the crystal structures of TcdA bound to A20 and A26. In contrast, the control constructs show varied and heterogeneous binding modes. These results highlight the importance of molecular geometric constraints in generating highly potent antibody-based reagents capable of exploiting the simultaneous binding of more than one paratope to an antigen.
摘要：
骆驼重链抗体可变域（VHHs）的晶体结构与艰难梭菌毒素A（TcdA）的组合重复寡肽结构域的片段结合，表明VHHA20的C端位于距N-30埃处VHHA26的末端。基于这一观察,我们产生了一个在N端有A20的双互补位融合蛋白，随后是(GS)6接头和在C-末端的A26。与未融合的A20和A26VHH相比，该A20-A26融合蛋白显示出结合亲和力的改善和TcdA中和效力的显着增加（>330倍[IC50];≥2,700倍[IC99]）。A20-A26与一系列包括两个A20VHH融合体的对照抗体构建体相比，也显示出高得多的结合亲和力和中和效力。两个A26VHH的融合，A26在N-末端和A20在C-末端（A26-A20），和actoxumab.特别是,A20-A26显示出比A26-A20高310倍(IC50)至29,000倍(IC99)的中和效力。尺寸排阻色谱-多角度光散射(SEC-MALS)分析进一步显示,A20-A26以1:1化学计量结合TcdA,并且同时接合A20和A26表位,如基于由结合A20和A26的TcdA的晶体结构启发的双位型设计所预期的。相比之下,对照构建体显示出不同的和异质的结合模式。这些结果突出了分子几何约束在产生能够利用多于一个互补位与抗原的同时结合的高效基于抗体的试剂中的重要性。
