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Purpose
To inhibit DNA
double-stran
d break repair in tu
mor cells by
delivery of a single-chain antibo
dy variable region frag
ment (ScFv 18-2) to the cell nucleus. ScFv 18-2 bin
ds to a regulatory region of the DNA-
depen
dent protein kinase (DNA-PK), an essential enzy
me in the nonho
mologous en
d-joining pathway, an
d inhibits DNA en
d-joining in a cell-free syste
m an
d when
microinjecte
d into single cells. Develop
ment as a ra
diosensitizer has been li
mite
d by the lack of a
metho
d for intranuclear
delivery to target cells. We investigate
d a
delivery
metho
d base
d on folate receptor-
me
diate
d en
docytosis.
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.