IN-VITRO AND IN-VIVO CHARACTERIZATION OF A HUMAN ANTI-C-ERBB-2 SINGLE-CHAIN FV ISOLATED FROM A FILAMENTOUS PHAGE ANTIBODY LIBRARY
IN-VITRO AND IN-VIVO CHARACTERIZATION OF A HUMAN ANTI-C-ERBB-2 SINGLE-CHAIN FV ISOLATED FROM A FILAMENTOUS PHAGE ANTIBODY LIBRARY
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DOI：
10.1016/1380-2933(95)00007-0
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发表时间：
1995-05-01
期刊：
影响因子：
--
通讯作者：
ADAMS, GP
中科院分区：
文献类型：
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作者：
SCHIER, R;MARKS, JD;ADAMS, GP
“背景：基于抗体的试剂未能发挥其作为癌症治疗高度特异性靶向剂的预期作用。用人单链Fv（sFv）分子靶向可以克服鼠IgG的一些限制，但难以用常规杂交瘤技术产生。或者，抗体基因库的噬菌体展示可用于产生人sFv。目的：目的分离和鉴定与c-erB-2结合的人单链抗体，c-erB-2是一种在30-50%的乳腺癌和其他腺癌中过表达的癌基因产物。研究设计：分离出特异性结合c-erB-2的非免疫性人单链Fv（C6.5）。C6.5是全人源序列，在大肠杆菌中以天然蛋白质的形式高水平表达。大肠杆菌中，并且易于在两个步骤中以高收率纯化。C6.5是全人源序列，在大肠杆菌中以天然蛋白质的形式高水平表达。大肠杆菌中，并且易于在两个步骤中以高收率纯化。6.5与固定的c-erB-2细胞外结构域结合的Kd为1.6 × 10（-8）M，与SK-0 V-3细胞上的c-erb-2结合的Kd为2.0 × 10（-8）M，亲和力与从杂交瘤产生的针对相同抗原的sFv相似。生物分布研究表明。在将I-125-C6.5注射到携带SK-OV-3肿瘤的scid小鼠中后24小时，1.47%注射剂量/g肿瘤。肿瘤：正常器官的比例范围从肾脏的8.9：1到肌肉的283：1。结论：这些结果是使用从非免疫人类库分离的sFv的首次体内生物分布研究，并证实了以这种方式产生的sFv的特异性。使用噬菌体展示来产生具有更高亲和力和更慢K-off的C6.5突变体将允许严格评估抗体亲和力和结合动力学在肿瘤靶向中的作用，并且可以导致产生用于放射免疫疗法和其他应用的治疗上有用的靶向蛋白。
`Background: Antibody-based reagents have failed to live up to their anticipated role as highly specific targeting agents for cancer therapy. Targeting with human single-chain Fv (sFv) molecules may overcome some of the limitations of murine IgG, but are difficult to produce with conventional hybridoma technology. Alternatively, phage display of antibody gene repertoires can be used to produce human sFv. Objectives: To isolate and characterize human single chain Fvs which bind to c-erB-2, an oncogene product overexpressed by 30-50% of breast carcinomas and other adenocarcinomas. Study design: A non-immune human single-chain Fv (C6.5) was isolated which binds specifically to c-erB-2. C6.5 is entirely human in sequence, expresses at high level as native protein in E. coli, and is easily purified in high yield in two steps. C6.5 is entirely human in sequence, expresses at high level as native protein in E. coli, and is easily purified in high yield in two steps. 6.5 binds to immobilized c-erB-2 extracellular domain with a Kd of 1.6 x 10(-8) M and to c-erb-2 on SK-OV-3 cells with a K-d of 2.0 x 10(-8) M, an affinity that is similar to sFv produced against the same antigen from hybridomas. Biodistribution studies demonstrate. 1.47% injected dose/g tumor 24 h after injection of I-125-C6.5 into scid mice bearing SK-OV-3 tumors. Tumor:normal organ ratios range from 8.9:1 for kidney to 283:1 for muscle. Conclusion: These results are the first in vivo biodistribution studies using an sFv isolated from a non-immune human repertoire and confirm the specificity of sFv produced in this manner. The use of phage display to produce C6.5 mutants with higher affinity and slower K-off would permit rigorous evaluation of the role of antibody affinity and binding kinetics in tumor targeting and could result in the production of a therapeutically useful targeting protein for radioimmunotherapy and other applications.