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 Â 10 8 -1 Â 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. (Cancer Res 2006; 66(15): 7724-33)

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.Transcription-coupled repair (TCR) 1 is a specialized form of DNA repair where damages are repaired preferentially in the transcribed strand of actively transcribed genes (for reviews, see Refs. 1 and 2). TCR was originally believed to be a subpathway of nucleotide excision repair; however, ionizing radiation damage (3), 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol; Tg) (4, 5), and 7,8-dihydro-8-oxoguanine (8-oxoG) (5) are removed in a TCR-dependent manner from human cells that lack nucleotide excision repair. Since Tg and 8-oxoG are small nonbulky lesions that are repaired primarily by the base excision repair (BER) pathway (for reviews, see Refs. 6 -8), TCR of Tg and 8-oxoG in cells that lack nucleotide excision repair (5) links TCR to BER. TCR of 8-oxoG has also been shown to occur in nonreplicating Escherichia coli cells (9). TCR of oxidative damage does not appear to be universal, since in Chinese hamster ovary cells, TCR of oxidative damage produced by photosensitization and oxidizing agents is not observed in the Dhfr and cFos genes (10 -12). Furthermore, DNA strand breaks, oxidative lesions also repaired by BER, do not appear to be repaired by TCR in the Dhfr gene from Chinese hamster ovary cells (13) or human colon cancer cells (14). Interestingly, recent measurements of TCR of cyclobutane pyrimidine dimers in the Hprt gene, integrated at different sites in Chinese hamster ovary cell chromosomes, have suggested that preferential repair of actively transcribed genes, as well as preferential repair of damages in the transcribed strand, is significantly affected by genomic context (15).The proposed signal for TCR is an RNA polymerase transcription complex stalled at a lesion, which recruits the repair proteins to the damage site (16 -18); the ability of a lesion on the transcribed strand to block the RNA polymerase transcription complex has been assumed to be crucial for T...

A novel b-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 (b-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 b-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound b-lactamase activity. SKBR3 cell-binding b-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected b-lactamase clones were further studied for their protein expression, enzyme activity and binding to nontumor celllines. Overall, the approach outlined here offers the opportunity of rapidly selecting targeted b-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. ' 2007 Wiley-Liss, Inc.Key words: phage display; b-lactamase; screening; cancer; targeted enzyme; prodrug therapyThe major problems associated with cytotoxic cancer therapeutic drugs are lack of selectivity for tumor cells over normal cells, insufficient drug concentrations in tumors, systemic toxicity and development of drug resistant cancer cells.1,2 Developing a successful strategy for a targeted delivery of drugs to cancer cells without harming the rest of the body is one of the most important challenges for cancer researchers today. The ability to selectively concentrate or deliver chemotherapeutic drugs to cancer cells may make chemotherapy both more effective and less likely to cause side effects. A variety of strategies are being evaluated to achieve this goal. [3][4][5][6] The majority of cancer-targeting agents developed over the last 2 decades are based on the use of monoclonal antibodies (mAb) to selectively deliver toxic agents such as conventional cytotoxic drugs, radioisotopes, or plantand bacteria-derived toxins for a direct tumor-killing.7-9 These approaches can reduce systemic toxicity; however, the therapeutic results have generally been less than ideal at least in part because of inadequate conjugate uptake into the tumor. 10Another approach that shows promise is targeted prodrug therapy. This is based on the concept that a systemically administered nontoxic prodrug can be converted locally to high concentrations of a cytotoxic drug by an enzyme p...

The A-pathway of development in the basidiomycete fungus Schizophyllum commune may be activated by either of two mating-type loci, A alpha and A beta. A alpha consists of two multiallelic genes, Y and Z. Y contains a putative homeodomain; Z contains a homeodomain-related region. Non-self combinations of Y and Z form heteromultimers which are thought to be transcription factors of developmental genes. To more completely understand A-regulated development it is necessary to address the issue of functional redundancy, i.e., how do two different mating loci, A alpha and A beta, both manage to regulate the same pathway. Here we report the structure of a gene with A beta 6 activity. This gene, denoted A beta V6, encodes a deduced polypeptide of 640 amino-acids with a homeodomain motif. V6 also contains a 20-amino acid sequence that is conserved in A alpha Y1, Y3 and Y4. Except for the homeodomain and the conserved sequence, the deduced V6 polypeptide shows no significant identity to A alpha Y, A alpha Z, or other known proteins. The presence of a homeodomain suggests that V, like Y and Z, may be a regulatory protein for genes in the A-pathway. Thus while A alpha and A beta encode different proteins, the general mechanism by which A alpha and A beta components signal A-regulated development may be similar.

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