title: Intranuclear delivery of a novel antibody-derived radiosensitizer targeting the DNA-dependent protein kinase catalytic subunit.
abstract: OBJECTIVE
  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
  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 F 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 (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.
  
  
  CONCLUSIONS
  Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.
authors.affiliation: Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA 30912, USA.
authors.forename: Hairong
authors.initials: H
authors.lastname: Xiong
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lee
authors.forename: Eric B
authors.initials: EB
authors.lastname: Haura
authors.forename: John G
authors.initials: JG
authors.lastname: Edwards
authors.forename: William S
authors.initials: WS
authors.lastname: Dynan
authors.forename: Shuyi
authors.initials: S
authors.lastname: Li
mesh_terms: D020134:Catalytic Domain; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival / Q000528:radiation effects; D059766:DNA End-Joining Repair / Q000187:drug effects / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004338:Drug Combinations; D016503:Drug Delivery Systems / Q000379:methods; D004705:Endocytosis; D005492:Folic Acid / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000493:pharmacokinetics; D006801:Humans; D016014:Linear Models; D011836:Radiation Tolerance / Q000187:drug effects; D011838:Radiation-Sensitizing Agents / Q000008:administration & dosage / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D057127:Single-Chain Antibodies / Q000008:administration & dosage / Q000493:pharmacokinetics*; D014410:Tumor Stem Cell Assay / Q000379:methods
chemical_list: D004338:Drug Combinations; D011838:Radiation-Sensitizing Agents; D057127:Single-Chain Antibodies; C573937:folate-ScFv 18-2; D005492:Folic Acid; D051747:DNA-Activated Protein Kinase