Towards a ligand targeted enzyme prodrug therapy: single round panning of a beta-lactamase scaffold library on human cancer cells.

A novel beta-lactamase scaffold library in which the target-binding moiety is built into the enzyme was generated using phage display technology. The binding element is composed of a fully randomized 8 amino acid loop inserted at position between Y34 and K37 on the outer surface of Enterobacter cloacae P99 cephalosporinase (beta-lactamase, E.C. 3.5.2.6) with all library members retaining catalytic activity. The frequency and diversity of amino acids distributions in peptide inserts from library clones were analyzed. The complexity of the randomized loop appears consistent with standards of other types of phage display library systems. The library was panned against SKBR3 human breast cancer cells in 1 round using rolling circle amplification of phage DNA to recover bound phage. Individual beta-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound beta-lactamase activity. SKBR3 cell-binding beta-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected beta-lactamase clones were further studied for their protein expression, enzyme activity and binding to nontumor cell-lines. Overall, the approach outlined here offers the opportunity of rapidly selecting targeted beta-lactamase ligands that may have a potential for their use in enzyme prodrug therapy with cephalosporin-based prodrugs. It is expected that a similar approach will be useful in developing tumor-targeting molecules of several other enzyme candidates of cancer prodrug therapy.

Selection of tumor-binding ligands in cancer patients with phage display libraries.

Phage display has been used extensively in vitro and in animal models to generate ligands and to identify cancer-relevant targets. We report here the use of phage-display libraries in cancer patients to identify tumor-targeting ligands. Eight patients with stage IV cancer, including breast, melanoma, and pancreas, had phage-displayed random peptide or scFv library (1.6 x 10(8)-1 x 10(11) transducing units/kg) administered i.v.; tumors were excised after 30 minutes; and tumor-homing phage were recovered. In three patients, repeat panning was possible using phage recovered and amplified from that same patient's tumor. No serious side effects, including allergic reactions, were observed with up to three infusions. Patients developed antiphage antibodies that reached a submaximal level within the 10-day protocol window for serial phage administration. Tumor phage were recoverable from all the patients. Using a filter-based ELISA, several clones from a subset of the patients were identified that bound to a tumor from the same patient in which clones were recovered. The clone-binding to tumor was confirmed by immunostaining, bioassay, and real-time PCR-based methods. Binding studies with noncancer and cancer cell lines of the same histology showed specificity of the tumor-binding clones. Analysis of insert sequences of tumor-homing peptide clones showed several motifs, indicating nonrandom accumulation of clones in human tumors. This is the first reported series of cancer patients to receive phage library for serial panning of tumor targeting ligands. The lack of toxicity and the ability to recover clones with favorable characteristics are a first step for further research with this technology in cancer patients.

Single-stranded breaks in DNA but not oxidative DNA base damages block transcriptional elongation by RNA polymerase II in HeLa cell nuclear extracts.

Transcription and repair of many DNA helix-distorting lesions such as cyclobutane pyrimidine dimers have been shown to be coupled in cells across phyla from bacteria to humans. The signal for transcription-coupled repair appears to be a stalled transcription complex at the lesion site. To determine whether oxidative DNA lesions can block correctly initiated human RNA polymerase II, we examined the effect of site-specifically introduced oxidative damages on transcription in HeLa cell nuclear extracts. We found that transcription was blocked by single-stranded breaks, common oxidative DNA lesions, when present in the transcribed strand of the transcription template. Cyclobutane pyrimidine dimers, which have been previously shown to block transcription both in vitro and in vivo, also blocked transcription in the HeLa cell nuclear transcription assay. In contrast, the oxidative DNA base lesions, 8-oxoguanine, 5-hydroxycytosine, and thymine glycol did not inhibit transcription, although pausing was observed with the thymine glycol lesion. Thus, DNA strand breaks but not oxidative DNA base damages blocked transcription by RNA polymerase II.

Decline of nuclear and mitochondrial oxidative base excision repair activity in late passage human diploid fibroblasts.

There are numerous studies documenting the increase of oxidative DNA damage in the nuclei and mitochondria of senescing cells as well as in tissues of aging animals. Here, we show that in IMR 90 human diploid fibroblasts, DNA repair activity is robust in both nuclear and mitochondrial extracts, however, the levels of activity differed against the three substrates tested. In extracts, cleavage of the 8-oxoguanine substrate, and to a lesser extent the dihydrouracil-containing substrate, occurred in a concerted reaction between the DNA glycosylases and the second enzyme in the reaction, hAPE. Cleavage of both the furan and the dihydrouracil-containing substrates was unchanged when nuclear extracts from early and late passage cells were compared. However, cleavage of the 8-oxoguanine substrate was substantially reduced in the nuclear extracts from late passage cells and significantly reduced transcription from the hOGG1 gene was observed. When mitochondrial extracts were examined, activity on all three substrates was significantly reduced, with the reduction in hAPE activity being the most marked. The reduction in cleavage of the furan substrate was not simply due to inactive mitochondrial AP endonuclease but a substantially reduced amount of hAPE protein; transcription from the hAPE gene was also reduced. Confocal microscopic analysis confirmed that hAPE was present in the mitochondria of early passage cells but greatly reduced in the mitochondria of late passage cells. Cytoplasmic extracts from late passage fibroblasts also showed reduced activity with all three substrates suggesting that the residual hAPE, and activities that recognized dihydrouracil, were preferentially targeted to the nuclei. Taken together the data support the concept that the increase in oxidative damage in the mitochondrial DNA of senescing cells and tissues from aging animals is due to reduced base excision repair activity.