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. Here we investigate 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 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 F test. Sensitization ratios were determined based on mean inhibitory dose.
Human KB and NCI-H292 lung cancer cells treated with the folate-conjugated scFv show significant radiosensitization (P<0.001). Sensitization enhancement ratios were 1.92 ± 0.42 for KB cells and 1.63 ± 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of γ-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056.
Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.
Keywords: Radiosensitization, scFv, DNA-dependent protein kinase, folate-mediated delivery, nonhomologous end joining, DNA repair