Mono and bivalent binding of a scFv and covalent diabody to murine laminin-1 using radioiodinated proteins and SPR measurements: Effects on tissue retention in vivo
Mono and bivalent binding of a scFv and covalent diabody to murine laminin-1 using radioiodinated proteins and SPR measurements: Effects on tissue retention in vivo
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DOI：
10.1016/j.jim.2006.04.006
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发表时间：
2006-06-30
影响因子：
2.2
通讯作者：
Kennel, Stephen J.
中科院分区：
文献类型：
--
作者：
Huang, Bao-Cheng;Davern, Sandra;Kennel, Stephen J.
噬菌体展示技术鉴定出一个scFv，15-9，它与鼠层粘连蛋白-1结合，并选择性地在肿瘤中积聚。在这项研究中，通过将VH44和VL100位的氨基酸残基改变为半胱氨酸残基，构建了一个共价双生体，从而通过二硫键稳定了双生体。共价双功能蛋白在Pichiapastoris中表达，亲和层析纯化。通过表面等离子体共振和固相结合1251-diabody和scFv测定其结合性能。用等离子体共振法计算的K-DS为4.4×10(-10)M，计算的单链抗体的Kos为9.9×10(-8)M，分别为1.7-2.1×10(-10)M和2.1-2.4×10(-8)M。固相层粘连蛋白的解离速率与层粘连蛋白的浓度无关，而125-1二联体的解离既依赖于层粘连蛋白的浓度又依赖于固相层粘连蛋白的浓度。具体地说，高浓度的层粘连蛋白产生非常缓慢的二重体解离速率，这表明已经形成了二价连接。当使用基本上饱和了单价结合的层粘连蛋白位点的较高剂量的Diabody时，解离速率与指示单价结合的ScFv相似。在荷瘤的SCID小鼠中的生物分布研究表明，共价Diabody使肿瘤/肌肉的比例比scFv提高了2倍，尽管两种形式结合到肿瘤部位的蛋白质的绝对量没有显著差异。数据还显示，与scFv相比，Diabody在肿瘤和肾脏中的滞留时间要长得多，而在肿瘤和肾脏中，层粘连蛋白的浓度较高。这些数据符合这样的假设，即在体内，scFv和diabody形式都以相似的结合动力学与可用层粘连蛋白结合，但在高靶标浓度的情况下，diabody可以二价结合，因此在结合部位的结合时间比scFv长得多。(C)2006爱思唯尔B.V.保留所有权利。
Phage display techniques identified a scFv, 15-9, which binds to murine laminin-1 and accumulated selectively in tumors. In this study, a covalent diabody was constructed by changing the amino acid residues at positions VH44 and VL100 to cysteine residues so that the diabody form could be stabilized via a disulfide bond. The covalent diabody was expressed in Pichiapastoris and purified by affinity chromatography. The binding properties were measured by surface plasmon resonance and solid phase binding of 1 25 1 diabody and scFv. Data from the plasmon resonance method yielded calculated K-DS of 4.4 x 10(-10) M for the covalent diabody and 9.9 x 10(-8) M for the scFv Kos calculated from solid phase binding of radioiodinated proteins were 1.7-2.1 x 10(-10) M and 2.1-2.4 x 10(-8) M respectively. The rate of dissociation of 125, scFv from solid phase laminin was independent of laminin concentration; however, the dissociation of the 125 1 diabody was dependent both on the concentration of laminin and on the concentration of the diabody. Specifically, high concentrations of laminin yielded very slow rates of diabody dissociation indicating that bivalent attachments had formed. When higher amounts of diabody were used that essentially saturated the laminin sites with univalent binding, the dissociation rate was similar to that for the scFv indicating univalent binding. Biodistribution studies in tumor-bearing SCID mice showed that the covalent diabody improved the ratio of tumor/muscle 2 fold over that obtained with the scFv, although the absolute amount of protein bound to the tumor site was not significantly different for the two forms. The data also showed that retention of the diabody in the tumor and kidney, sites where laminin is present in high concentration, was much longer compared to that of scFv. These data are consistent with the hypothesis that both scFv and diabody forms bind to available laminin in vivo with similar association kinetics, but that in situations of high target concentration, the diabody can bind bivalently and is thus retained at the binding site much longer than the scFv. (c) 2006 Elsevier B.V. All rights reserved.