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

• Published in: International journal of radiation oncology, biology, physics Vol. 83; no. 3; pp. 1023 - 1030
• Main Authors: Xiong, Hairong, M.D., Ph.D, Lee, Robert J., Ph.D, Haura, Eric B., M.D, Edwards, John G., S.M, Dynan, William S., Ph.D, Li, Shuyi, M.D., Ph.D
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.07.2012; Elsevier
• Subjects: ANTIBODIES; Biological and medical sciences; Catalytic Domain; Cell Line, Tumor; CELL NUCLEI; Cell Nucleus - metabolism; Cell Survival - radiation effects; DNA; DNA End-Joining Repair - drug effects; DNA End-Joining Repair - radiation effects; DNA REPAIR; DNA-Activated Protein Kinase - metabolism; DNA-dependent protein kinase; Drug Combinations; Drug Delivery Systems - methods; Endocytosis; ENZYMES; Folate-mediated delivery; Folic Acid - administration & dosage; Folic Acid - analogs & derivatives; Folic Acid - chemical synthesis; Folic Acid - pharmacokinetics; Hematology, Oncology and Palliative Medicine; Humans; IONIZING RADIATIONS; Linear Models; LUNGS; Medical sciences; NEOPLASMS; Nonhomologous end joining; RADIATION DOSES; Radiation therapy and radiosensitizing agent; Radiation Tolerance - drug effects; Radiation-Sensitizing Agents - administration & dosage; Radiation-Sensitizing Agents - chemical synthesis; Radiation-Sensitizing Agents - pharmacokinetics; Radiology; RADIOLOGY AND NUCLEAR MEDICINE; Radiosensitization; RECEPTORS; scFv; Single-Chain Antibodies - administration & dosage; Single-Chain Antibodies - pharmacokinetics; STRAND BREAKS; SURVIVAL CURVES; Treatment with physical agents; Treatment. General aspects; TUMOR CELLS; Tumor Stem Cell Assay - methods; Tumors; Nonhomologous end joining; Radiosensitization; DNA-dependent protein kinase; scFv; DNA repair; Folate-mediated delivery; Human; Catalytic subunit; Targeting; Cell nucleus; Folic acid receptor; Joining; Radiotherapy; Folate; Endocytosis; Cancerology; Radiosensitizing agent; Treatment; Inhibitor; Single chain antibody
• ISSN: 0360-3016; 1879-355X
• DOI: 10.1016/j.ijrobp.2011.08.039

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 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.