Isolation of high-affinity monomeric human Anti-c-erbB-2 single chain Fv using affinity-driven selection
Isolation of high-affinity monomeric human Anti-c-erbB-2 single chain Fv using affinity-driven selection
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DOI：
10.1006/jmbi.1996.0004
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发表时间：
1996-01-12
影响因子：
5.6
通讯作者：
Marks, JD
中科院分区：
文献类型：
--
作者：
Schier, R;Bye, J;Marks, JD
使用抗体靶向肿瘤抗原取得的成功有限，部分原因是 IgG 分子尺寸较大、构建较小单链 Fv (scFv) 抗体片段的困难以及鼠抗体的免疫原性。这些限制可以通过直接从非免疫或半合成噬菌体抗体库中选择人scFv来克服；然而，对于治疗应用而言，亲和力通常太低。对于半抗原抗原，可以从噬菌体抗体库中分离出更高亲和力的 scFv，其中结合 scFv 的 V-H 和 V-L 基因被 V 基因库替换（链改组）。这种方法对蛋白质结合 scFv 的适用性尚不清楚。在这项工作中，链改组用于增加非免疫人 scFv 的亲和力，它以 1.6 x 10(-8) M 的亲和力结合糖蛋白肿瘤抗原 c-erbB-2。通过轻链改组，亲本 scFv 的亲和力增加六倍 (K-d = 2.5 x 10(-9) M)，通过轻链改组增加五倍 (K-d = 3.1 x 10(-9)M) 通过 重链改组，其值与杂交瘤产生的针对相同抗原的抗体的值相当。当对固定在聚苯乙烯上的抗原进行选择时，分离出自发二聚化的scFv，其中最好的scFv仅具有比野生型稍低的K-d（K-d = 1.1 x 10(-8) M）。这些scFv在噬菌体上二聚化，并由于亲合力增加而被优先选择。与仅形成单体的scFv相比，二聚化的scFv在V-H-V-L界面处有突变，表明V-H-V-L互补性决定了二聚化的程度。仅通过使用有限浓度的生物素化抗原在溶液中进行选择，然后通过 BIAcore 中的 k(off) 从细菌周质中筛选突变型 scFv，才能获得更高亲和力的单体 scFv。使用适当的选择和筛选条件，无需免疫即可产生具有与鼠杂交瘤相当的亲和力的蛋白质结合人scFv。 (C) 1996 学术出版社有限公司
The use of antibodies to target tumor antigens has had limited success, partially due to the large size of IgG molecules, difficulties in constructing smaller single chain Fv (scFv) antibody fragments, and immunogenicity of murine antibodies. These limitations can be overcome by selecting human scFv directly from non-immune or semi-synthetic phage antibody libraries; however, the affinities are typically too low for therapeutic application. For hapten antigens, higher-affinity scFv can be isolated from phage antibody libraries where the V-H and V-L genes of a binding scFv are replaced with repertoires of V genes (chain shuffling). The applicability of this approach to protein binding scFv is unknown. For this work, chain shuffling was used to increase the affinity of a non-immune human scFv, which binds the glycoprotein tumor antigen c-erbB-2 with an affinity of 1.6 x 10(-8) M. The affinity of the parental scFv was increased sixfold (K-d = 2.5 x 10(-9) M) by light-chain shuffling and fivefold (K-d = 3.1 x 10(-9)M) by heavy-chain shuffling, values comparable to those for antibodies against the same antigen produced by hybridomas. When selections were performed on antigen immobilized on polystyrene, spontaneously dimerizing scFv were isolated, the best of which had only a slightly lower K-d than wild type (K-d = 1.1 x 10(-8) M). These scFv dimerize on phage and are preferentially selected as a result of increased avidity Compared to scFv which formed only monomer, dimerizing scFv had mutations located at the V-H-V-L interface, suggesting that V-H-V-L complementarity determines the extent of dimerization. Higher-affinity monomeric scFv were only obtained by selecting in solution using limiting concentrations of biotinylated antigen, followed by screening mutant scFv from bacterial periplasm by k(off) in a BIAcore. Using the proper selection and screening conditions, protein binding human scFv with affinities comparable to murine hybridomas can be produced without immunization. (C) 1996 Academic Press Limited