Intranuclear Delivery of a Novel Antibody-Derived Radiosensitizer Targeting the DNA-Dependent Protein Kinase Catalytic Subunit

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• 作者：Hairong Xiong ; M.D. ; Ph.D.^&lowast ; ; ^&dagger ; ; Robert J. Lee ; Ph.D.^&Dagger ; ; Eric B. Haura ; M.D.^§ ; ; John G. Edwards ; S.M.^¶ ; ; William S. Dynan ; Ph.D.^&lowast ; ; Shuyi Li ; M.D. ; Ph.D.^&lowast ; ; ^¶ ; ; ^{sli@georgiahealth.edu}
• 关键词：Radiosensitization ; scFv ; DNA-dependent protein kinase ; Folate-mediated delivery ; Nonhomologous end joining ; DNA repair
• 刊名：International Journal of Radiation Oncology*Biology*Physics
• 出版年：2012
• 期刊代码：136_03603016
• 类别：med
• 出版时间：1 July, 2012
• 卷：83
• 期：3
• 页码：1023-1030
• 文件大小：1167 K

摘要

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Purpose

To inhibit DNA double-strand break repair in tumor cells by delivery of a single-chain antibody variable region fragment (ScFv 18-2) to the cell nucleus. ScFv 18-2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK), an essential enzyme in the nonhomologous end-joining pathway, and inhibits DNA end-joining in a cell-free system and when microinjected into single cells. Development as a radiosensitizer has been limited by the lack of a method for intranuclear delivery to target cells. We investigated a delivery method based on folate receptor-mediated endocytosis.

Methods and Materials

A recombinant ScFv 18-2 derivative was conjugated to folate via a scissile disulfide linker. Folate-ScFv 18-2 was characterized for its ability to be internalized by tumor cells and to influence the behavior of ionizing radiation-induced repair foci. Radiosensitization was measured in a clonogenic survival assay. Survival curves were fitted to a linear-quadratic model, and between-group differences were evaluated by an m>Fm> test. Sensitization ratios were determined based on mean inhibitory dose.

Results

Human KB and NCI-H292 lung cancer cells treated with folate-conjugated ScFv 18-2 showed significant radiosensitization (m>pm> < 0.001). Sensitization enhancement ratios were 1.92 卤 0.42 for KB cells and 1.63 卤 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of 纬-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056.

Conclusions

Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.