Targeting of bivalent anti-ErbB2 diabody antibody fragments to tumor cells is independent of the intrinsic antibody affinity.
Targeting of bivalent anti-ErbB2 diabody antibody fragments to tumor cells is independent of the intrinsic antibody affinity.
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发表时间：
2000-11
期刊：
影响因子：
11.2
通讯作者：
U. Nielsen;G. Adams;L. Weiner;J. Marks
中科院分区：
文献类型：
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作者：
U. Nielsen;G. Adams;L. Weiner;J. Marks
在免疫缺陷小鼠中，抗肿瘤单链Fv（scFv）分子迅速穿透肿瘤并具有快速血清清除，导致优异的肿瘤：正常器官比。然而，由于血清清除迅速和单价单链抗体结合，保留在肿瘤中的单链抗体的绝对量较低。我们先前证明了额外结合位点的存在破坏了基于scFv的分子与表达相关抗原的肿瘤细胞的体外和体内缔合。每个组分scFv的固有亲和力对二聚体scFv与其靶抗原之间的缔合的贡献在很大程度上是未知的。在此，我们通过将重链（VH）和轻链（VL）可变结构域之间的肽接头从15个氨基酸缩短至5个氨基酸，从人抗ErbB 2（HER 2/neu）scFv分子C6.5的三个亲和突变体构建了二价双抗体分子。较短的接头阻止VH和VL的分子内配对，导致分子间配对和产生具有两个抗原结合位点的二聚Mr 50，000分子。用于产生双抗体的scFv跨越133倍范围的对ErbB 2的相同表位的亲和力[133 nM（C6 G98 A）、25 nM（C6.5）和1 nM（C6 ML 3 -9）]，并且仅相差1至3个氨基酸。通过固定化ErbB 2胞外结构域上的表面等离子体共振测定双抗体结合动力学。对于每个双抗体分子获得的缔合速率常数与亲本（组分）scFv的缔合速率常数相似。然而，对于二价双抗体获得的解离速率常数慢多达15倍。二价解离速率常数的降低幅度与单价相互作用成反比，范围从C6 ML 3 -9双抗体的仅3倍到C6 G98 A双抗体的15倍。这导致二价亲和力的差异仅为22倍，而相应scFv的亲和力差异为133倍。通过表面等离子体共振获得的平衡结合常数与ErbB 2过表达细胞体外测定的平衡结合常数相关。在携带已建立的SKOV 3肿瘤的scid小鼠中进行生物分布研究。在24小时，与亲本scFv单体相比，3-37倍更多的双抗体保留在肿瘤中。这可能是由于二价结合导致的较高表观亲和力和较慢的血清清除。令人惊讶的是，双抗体之间亲和力的差异不会导致定量肿瘤保留或肿瘤与血液比率的差异。事实上，从最低亲和力的scFv构建的双抗体表现出最好的肿瘤靶向特性。我们的结论是，高于阈值亲和力，其他因素调节定量肿瘤保留。此外，低亲和力scFv的直接二聚化导致比耗尽性scFv亲和力成熟显著更大的肿瘤定位。
In immunodeficient mice antitumor single-chain Fv (scFv) molecules penetrate tumors rapidly and have rapid serum clearance, leading to excellent tumor:normal organ ratios. However, the absolute quantity of scFv retained in the tumor is low due to rapid serum clearance and monovalent scFv binding. We previously demonstrated that the presence of an additional binding site prolongs in vitro and in vivo association of scFv-based molecules with tumor cells expressing relevant antigen. The contribution of the intrinsic affinity of each component scFv to the association between a dimeric scFv and its target antigen is largely unknown. Here, we have constructed bivalent diabody molecules from three affinity mutants of the human anti-ErbB2 (HER2/neu) scFv molecule C6.5 by shortening the peptide linker between the heavy (VH) and light (VL) chains variable domains from 15 to 5 amino acids. The shorter linker prevents intramolecular pairing of VH and VL, resulting in intermolecular pairing and creation of a dimeric Mr 50,000 molecule with two antigen-binding sites. The scFv used to create the diabodies span a 133-fold range of affinity for the same epitope of ErbB2 [133 nM (C6G98A), 25 nM (C6.5), and 1 nM (C6ML3-9)] and differ by only one to three amino acids. Diabody binding kinetics were determined by surface plasmon resonance on the immobilized ErbB2 extracellular domain. The association rate constants obtained for each diabody molecule were similar to that of the parental (component) scFv. However, the dissociation rate constants obtained for the bivalent diabodies were up to 15-fold slower. The magnitude of the decrease in the bivalent dissociation rate constant was inversely proportional to the monovalent interaction, ranging from only 3-fold for that of the C6ML3-9 diabody to 15-fold for the C6G98A diabody. This resulted in only a 22-fold difference in bivalent affinity, compared with a 133-fold difference in affinity for the respective scFv. Equilibrium-binding constants obtained by surface plasmon resonance correlated well with the equilibrium-binding constants determined in vitro on ErbB2 overexpressing cells. Biodistribution studies were performed in scid mice bearing established SKOV3 tumors. At 24 h, 3-37-fold more diabody was retained in tumor compared with the parental scFv monomers. This likely results from a higher apparent affinity, because of bivalent binding, and a slower serum clearance. Surprisingly, the differences in affinity between diabodies did not result in differences in quantitative tumor retention or tumor to blood ratios. In fact, the diabody constructed from the lowest affinity scFv exhibited the best tumor-targeting properties. We conclude that, above a threshold affinity, other factors regulate quantitative tumor retention. In addition, straightforward dimerization of a low-affinity scFv leads to significantly greater tumor localization than does exhaustive scFv affinity maturation.