Klonisch T, Delves P J, Berger P, Panayotou G, Lapthorn A J, Isaacs N W, Wick G, Lund T, Roitt I M
Department of Immunology, University College London Medical School, GB.
Eur J Immunol. 1996 Aug;26(8):1897-905. doi: 10.1002/eji.1830260834.
We systematically screened a large panel of well-characterized monoclonal antibodies (mAb) directed towards various epitopes on human chorionic gonadotropin (hCG) for synergistic binding of 125I-hCG when they were adsorbed to a solid phase. The epitope locations involved in synergy were then related to the crystal structure of hCG and discussed in accordance with available data on the hCG epitopes. Enhanced binding of hCG was specific for certain pairs of mAb and was reflected in a 3-50-fold increased apparent functional affinity constant for hCG. Surface plasmon resonance revealed that when the mAb were captured by a polyclonal anti-IgG1 coupled to the Biacore chip, the off rates for hCG were significantly slower with synergistic mAb combinations than for the corresponding single mAb or nonsynergistic pairs of mAb, whereas the on rates did not differ appreciably. Each of the two antibodies involved in synergistic binding of hCG (more than 3-fold compared to additive binding of the two mAb) always belonged to a different epitope cluster in a separate antigenic domain on hCG. Synergistic epitope combinations on holo-hCG were located in similar structural planes. Combinations of mAb directed towards the epitope clusters alpha 2/beta 3/5, alpha 2/hCG beta CTP (C-terminal peptide) and beta 3/5/hCG beta CTP showed the strongest enhancement, with binding more than 10-fold greater than the sum of 125I-hCG bound to the individual mAb, followed by pairs of mAb directed towards the epitope groups beta 1/beta 3/5, c 1/2/beta 3/5, beta 1/alpha 2, and alpha 2/alpha 3/5 (3-9-fold). The greater frequency of synergy obtained with the linear epitopes of the hCG beta CTP can be ascribed to their greater molecular flexibility relative to the constrained discontinuous epitopes on hCG alpha and core-hCG beta (residues 1-112). In general, these studies provide a method for rapid screening of synergistic antibody pairs which also helps to identify non-overlapping epitopes that are accessible in similar structural planes. In turn, this facilitates the design of high-affinity bispecific antibodies targetted to a single antigen molecule.
我们系统地筛选了一大组针对人绒毛膜促性腺激素（hCG）上各种表位的特性明确的单克隆抗体（mAb），以研究它们吸附到固相上时对¹²⁵I-hCG的协同结合情况。然后将协同作用中涉及的表位位置与hCG的晶体结构相关联，并根据关于hCG表位的现有数据进行讨论。hCG结合增强对某些mAb对具有特异性，表现为hCG的表观功能亲和常数增加3至50倍。表面等离子体共振显示，当mAb被偶联到Biacore芯片上的多克隆抗IgG1捕获时，与协同mAb组合相比，hCG与相应单mAb或非协同mAb对的解离速率显著减慢，而结合速率没有明显差异。参与hCG协同结合的两种抗体（与两种mAb的加和结合相比增加超过3倍）总是属于hCG上一个单独抗原结构域中不同的表位簇。全hCG上的协同表位组合位于相似的结构平面中。针对表位簇α2/β3/5、α2/hCGβCTP（C末端肽）和β3/5/hCGβCTP的mAb组合显示出最强的增强作用，其结合比与单个mAb结合的¹²⁵I-hCG总和大10倍以上，其次是针对表位组β1/β3/5、c1/2/β3/5、β1/α2和α2/α3/5的mAb对（3至9倍）。与hCGβCTP的线性表位相比，协同作用出现频率更高可归因于它们相对于hCGα和核心hCGβ（第1至112位残基）上受限制的不连续表位具有更大的分子灵活性。总体而言，这些研究提供了一种快速筛选协同抗体对的方法，这也有助于识别在相似结构平面中可及的非重叠表位。反过来，这便于设计靶向单个抗原分子的高亲和力双特异性抗体。
