Screening And Identification Of Anti-DNA-PKcs ScFv-antibody And Its Radiosensitization On HeLa Cells

Radiotherapy is one of the most important means for cancer therapy. However, there are also some weak-points, which lead to the limitation for the clinical practice of radiotherapy, including the radiation induced severe side effects resulted from the damage in normal tissues around the radiation-targeted cancer, and radiation-toleration of tumor cells. For a long time, it is an uphill task in the field of radiation biology and oncology how to increase the sensitivity of cancer cells to radiotherapy and decrease the damage to normal tissue.DNA double-strand break (DSB) is a critical damage induced by ionizing radiation(IR), and mostly related to cell death and other genomic events after irradiation. It is well known that the unrepaired or misrepaired DSBs can cause genomic instability, mutations, cancer-prone, cell death, and so on. In the other side, if the cells have the supra-capability to repair the DNA DSBs, the cells will display radiation tolerance. Eukaryotic cells have evolved several DSBs repair mechanistic pathways, including the non-homologous end joining (NHEJ) pathway and homologous recombination (HR) pathway. Although not all components of the NHEJ system have been identified, it is clear that the DNA-dependent protein kinase (DNA-PK) is a crucial component. This enzyme is composed of the regulatory component, Ku70/Ku80 subunits, and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs takes part in NHEJ mainly through its autophosphorylation and phosphorylating various targets, e.g. Artemis and XRCC-4. In addition, DNA-PKcs can also take part in cell cycle G2 regulation, cell apoptosis and autophagic death, telomere length maintenance and V(D)J recombination. It was recently reported that DNA-PKcs is highly expressed in various cancers, and the increased activity of DNA-PKcs was found to be associated with high-metastatic or radio-resistance of tumor cells, and DNA-PKcs deficient leads to greatly increased sensitivity to IR. The DNA-PKcs deficient also slowed down the cells growth and formation of experimental tumor in nude mouse. These facts indicate targetingDNA-PKcs is a good approach for increasing therapeutic gain from radiation therapy.At present there are several methods to inhibit the activity of DNA-PKcs, such as non-specific PI3K inhibitor (Wortmannin, LY294002), specific DNA-PK inhibitor (OK-1035, NU7026), anti-sense oligonuleotides, monoclonal antibodies and C-terminal region of Ku80. But these means either show low specificity or in vivo toxicity or induction of immunological reactions, which limited their application in clinical practical. The development of human-derived antibody engineering, especially the phage antibody library engineering, the screening of single-chain variable antibody fragment (scFv) provides a new approach in tumor biological therapy. Up to now there are no pragmatic DNA-PKcs inhibitor for the purpose of increasing cancer radiation sensitivity in clinical application and no reports about human scFv antibody targeting DNA-PKcs.In this study, we screened large-scale phage antibody library to obtain human anti-DNA-PKcs scFv-antibodies for the potential use of improving the therapeutic gain from radiation therapy. The main results are as follows:1. Human DNA-PKcs protein fragments with high antigenicity and low homologous to other proteins, named as DPK1, DPK2, DPK3 and DPK4, were defined through antigenicity analysis and BLAST searching; the genes of these four segments were amplified through RT-PCR, and constructed into prokaryotic expression vector. After transfecting E.coli BL21(DE3) and optimizing the protein expression condition, the DPK3 and DPK4 peptides were expressed successfully. The purified peptides were gained through inclusion body degeneration and on-column refolding.2. To clone the genes encoding human anti-DNA-PKcs antibodies, we screened the large-scale phage antibody library conducted by Dr LJ Zhou's group in the Medical Experimental Center, Navy General Hospital, PLA. After the peptides DPK3 and DPK4 were coated onto immuno-tube, panning of large-scale phage library against antigens was conducted to select the specific antibodies against DNA-PKcs. 26 clones were identified with the specific binding ability with DPK3, and 31 clones with DPK4. DNA fingerprinting analyses revealed 5 individual positive clones to DPK3 and 21 individual positive clones to DPK4. Soluble scFv was prepared through transfecting HB2151 with the isolated phage antibody genes and the binding activity and specificity were tested by ELISA method. Two scFv antibodies were identified with the specific binding ability with DPK3, and five scFv antibodies with DPK4. Sequencing analysis showed that the VH genes of anti-DPK3-scFvs belong to VH3,VH4 and the VL genes belong to VL2 subgroups respectively; the VH genes of anti-DPK4-scFv belong to VH1,VH3 and the VL genes belong to VL1,VL2 subgroups respectively.3. Western Blotting was used to verify the specificity of anti-DPK3-scFv-2. The protein extract from HeLa cells was detected by using this soluble scFv or commercial rabbit polyclonal anti-DNA-PKcs antibody. The results show that anti-DPK3-scFv-2 binds DPK3 specially and not binds with DPK4, and its specificity is higher than that of commercial polyclonal antibody.4. The HeLa-DPK3-scFv cell line was constructed by transfecting the gene of anti-DPK3-scFv-2. And the HeLa-DPK3-scFv-2 cell line showed the highest radiation sensitivity, which was demonstrated by colony forming ability analysis. The results of cell proliferation and colony forming experiment showed that the proliferation activity of HeLa-DPK3-scFv is repressed and the radiation sensitivity increases evidently. Moreover, the results of neutral single cell gel electrophoresis (SCGE) assay and the immunofluorescence hybridization in situ ofγH2AX foci indicate that anti-DPK3-scFv attenuates the DSBs repair capability of HeLa cells. The activity of caspase-3 in HeLa- DPK3-scFv elevated evidently compared with control cells, which indicates that apoptosis of HeLa-DPK3-scFv induced by radiation and the radiation sensitivity increases.5. The Western Blotting result shows that the expression of DNA-PKcs is not effected by anti- DPK3-scFv. The phosphorylation level of Akt, the substrate of DNA-PKcs, was detected by Western Blotting. The results show that the phosphorylation level of Akt in HeLa-DPK3-scFv do not alter evidently afterγ-ray radiation. This indicates DNA-PKcs in HeLa-DPK3-scFv could not activate downstream protein after DNA damage, and can not perform the DNA repair, so the DSBs repair is blocked.In conclusion: this study defined 4 human DNA-PKcs protein fragments with high antigenicity and low homologous to other proteins through antigenicity analysis in Biosun software and BLAST searching. Purified protein segments DPK3 and DPK4 were obtained from prokaryotic expression extract. By means of panning of large-scale phage library against antigen, 2 scFvs were identified with the specific affinity to DPK3, 5 scFvs to DPK4. The encoding gene of anti-DPK3-scFv was further cloned and transfected into HeLa cells for the studying of its intracellular activity. The results show that HeLa cells transfected with anti-DPK3-scFv displayed decreased proliferation ability, as well as increased radiosensitivity. Furthermore, anti-DPK3-scFv resulted in deficient of DSBs repair capacity in HeLa cells. Presumably, the mechanism of action involves either hindrance of an essential protein-protein interaction surface on DNA-PKcs or blockage of a conformational change required for processing of NHEJ pathway. This study makes a foundation work for further exploration on the measure of improving the therapeutic gain from radiotherapy.