Human exposure to ionizing radiation
Human exposure to ionizing radiation comes from terrestrial sources, such as naturally occurring radioisotopes (222Rn, 40K, 14C, 3H), and from cosmic sources, including solar protons and galactic cosmic radiation. Together, these sources of radiation account for an exposure of 3 milliSievert annually (mSv; 1 mSv=1 mGy for X-rays and gamma rays). For many people, exposure from anthropogenic sources vastly exceeds this natural background. Medical diagnostic procedures, especially nuclear medicine and CT scans, are associated with exposure on the order of 5-50 mSv. Cancer radiotherapy, administered to 750,000 patients annually in the United State alone, is associated with exposures, limited to the tumor region, on the order of 50-75 Sv, or 25,000-fold above the natural background.
Modifying the function of DNA repair nanomachines in radiotherapy patients
An ability to modify the function of DNA repair nanomachines in radiotherapy patients would bring an obvious benefit. As an example, lung cancer is the most common cancer in both men and women in the US, and only 30% of cases are operable for cure at the time of presentation [30
]. Radiotherapy is the primary mode of treatment for the remaining cases, but its ability to cure lung cancer is severely limited by the susceptibility of normal lung tissue to radiation-induced damage. Although there is no doubt that better tumor control can be achieved with higher radiation doses, radiation levels that can be tolerated by normal lung tissue are generally insufficient to eradicate the cancer [31
]. NHEJ appears to be important in determining levels of tumor radioresistance. Particularly striking evidence has been seen in a study of multiple human lung cancer cell lines, where radioresistance was directly proportional to the level of the NHEJ protein, DNA-PKcs [32
How might the ability to affect NHEJ increase the efficacy of radiation therapy? Clearly, if one could deliver a repair inhibitor specifically to tumor cells, and not normal cells, one could achieve better tumor control at a given dose, without increasing the risk of harm to normal tissue. We hypothesize, however, that even without a specific delivery mechanism, modification of the NHEJ nanomachine could lead to therapeutic gain. Tumor cells are intrinsically more sensitive to radiation than normal tissue because they divide rapidly and because they are compromised with respect to DNA damage-dependent cell-cycle checkpoints. Modifying the DNA repair machine to introduce a time lag in the repair process could potentiate this intrinsic sensitivity by increasing the probability that the tumor cell will divide prior to repair, leading to irreparable loss of genetic material and thus to clonogenic death. We hypothesize that normal cells, because of their slower rate of division and intact checkpoints, should be less affected by a delay in repair.
Single chain antibodies as a platform for modifying nanomachine function
We have explored the use of single-chain antibody (abbreviated as scFv, for single-chain fragment, variable) technology as a platform for modifying nanomachine function. Naturally occurring antibodies contain multiple antigen-binding sites, formed at the interface of separate heavy and light polypeptide chains, grafted onto a large, invariant structural framework [33
]. ScFvs are recombinant molecules containing a single antigen binding site and only a minimal portion of the invariant framework. As the name suggests, the heavy and light chains are connected, via a flexible linker, to form a single polypeptide chain that can be readily expressed in recombinant form. Single-chain antibodies are more “drug-like” than the parent antibodies from which they are derived. A scFv has only about 20% of the mass of an immunoglobulin G molecule (the most common type of naturally occurring antibody), facilitating uptake by cells and entry into the nucleus. In addition, because they are recombinant molecules, scFvs can be genetically modified or chemically derivatized to add desired functionalities.
A single chain antibody directed against DNA-PKcs (scFv 18-2)
A single-chain antibody (scFv 18-2) was derived from a parent monoclonal antibody previously shown to bind to DNA-PKcs and to partially inhibit kinase function [34
]. The epitope recognized by scFv 18-2 was mapped by immunoreactivity with successively smaller regions of the protein obtained by proteolytic cleavage, in vitro
transcription-translation, and chemical synthesis. These studies showed that scFv 18-2 recognizes a 25-residue peptide near the center of the 4127-residue DNA-PKcs molecule [35
The significance of this location became apparent only recently. A very extensive modeling effort, drawing on a variety of sources, has allowed the mapping of the primary sequence of DNA-PKcs into the electron density model obtained from single-particle reconstruction [22
]. It appears that the scFv 18-2 epitope lies in an extended arm domain connecting a large DNA binding “palm” region, at the N terminus, with a catalytic head, at the C terminus. The arm undergoes a large conformational change upon DNA binding [36
]. We hypothesize that the ability of scFv 18-2 to block or delay NHEJ may be linked to its ability to block this conformational change ().
Figure 2 Epitope mapping and mechanism of action of scFv 18-2. A. Diagram shows location of fragments and peptides used for epitope mapping relative to kinase catalytic domain. For details and supporting data, see ref . Epitope was mapped to an N-terminal (more ...)
Inhibition of NHEJ by scFv 18-2 in vitro and in vivo
Evidence that scFv 18-2 interferes with NHEJ is based on three findings:
- scFv 18-2 inhibits DNA end joining in a cell-free system containing linearized plasmid substrate, HeLa cell nuclear extract, and recombinant DNA ligase IV/XRCC4 .
- Microinjection of scFv 18-2 into telomerase-immortalized human retinal pigment epithelial (RPE) cells expressing the adenovirus E1A oncoprotein , which are a model for early-stage cancer, greatly increased the toxicity of a single, relatively low dose (200 cGy) of ionizing radiation . This dose is approximately equal to a single clinical radiotherapy fraction.
- Microinjection of scFv 18-2 into human melanoma cells markedly prolonged the lifetime of ionizing radiation-induced repair foci , suggesting that it acts directly to introduce a time lag in the DNA repair process – the desired behavior for a clinically useful radiosensitizer. , taken from ref , illustrates this ability to prolong the lifetime of repair foci.
Figure 3 ScFv 18-2 prolongs the lifetime of γ-H2AX foci. SK-MEL28 melanoma cells were co-injected with a reporter plasmid, pEGFP-N1, and either scFv 18-2 or a control single-chain antibody, scFv 147. Cells were irradiated at 1.5 Gy to induce approximately (more ...)
Together, these data suggest that single-chain antibodies can provide a useful technology platform for modifying the function of the NHEJ nanomachine. A particular advantage of scFv 18-2 is its high target specificity. The sequence in the epitope region is unique to DNA-PKcs, not present in other members of the same protein kinase family (the phosphatidyl inositol 3-kinases). In this respect, it differs from small-molecule inhibitors directed against the phylogenetically conserved kinase active site [38
]. Indeed, scFv 18-2 has no mechanism of action in cells that are outside a radiation field, suggesting that systemic toxicity will be minimal if administered as a radiosensitizer.
The next challenge in development of scFv 18-2 will be to address the problem of delivery to its intracellular target. Although single-chain antibodies are more “drug-like” than their parent monoclonal antibodies, they do not efficiently penetrate the cell membrane. We anticipate that this problem may be overcome in a variety of ways, including the use of liposomes and other nanoparticle carriers. A general review of nanoparticle-based drug discovery is beyond our present scope. We note, however, that elegant recent studies have demonstrated the value of cell-penetrating and nuclear localization peptides in promoting delivery of nanoparticles to intracellular compartments [40
]. Conjugation to biofunctionalized particles offers a promising path for further development of the scFv platform.