Girja S. Shukla

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 patients 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.

Combination treatment with Grb7 peptide and Doxorubicin or Trastuzumab (Herceptin) results in cooperative cell growth inhibition in breast cancer cells

Grb7 has potential importance in the progression of cancer. We have previously identified a novel peptide that binds to the SH2 domain of Grb7 and inhibits its association with several different receptor tyrosine kinases. We have synthesised the Grb7 peptide, G7-18NATE, with two different cell penetrating peptides, Penetratin and Tat. In this study, we have shown that both Penetratin- and Tat-conjugated G7-18NATE peptides are able to inhibit the proliferation of SK-BR-3, ZR-75-30, MDA-MB-361 and MDA-MB-231 breast cancer cells. There was no significant effects on breast cancer MCF-7cells, non-malignant MCF 10A or 3T3 cells. In addition, there was no significant inhibition of proliferation by Penetratin or Tat alone or by their conjugates with arbitrary peptide sequence in any of the cell lines tested. We determined the EC50 of G7-18NATE-P peptide for SK-BR-3 cell proliferation to be 7.663 × 10−6 M. Co-treatment of G7-18NATE-P peptide plus Doxorubicin in SK-BR-3 breast cancer cells resulted in an additional inhibition of proliferation, resulting in 56 and 84% decreases in the Doxorubicin EC50 value in the presence of 5 × 10−6 and 1.0 × 10−5 M G7-18NATE-P peptide, respectively. Importantly, the co-treatment with Doxorubicin and the delivery peptide did not change the Doxorubicin EC50. Since Grb7 associates with ErbB2, we assessed whether the peptide inhibitor would have a combined effect with a molecule that targets ErbB2, Herceptin. Co-treatment of Herceptin plus 1.0 × 10−5 M G7-18NATE-P peptide in SK-BR-3 cells resulted in a 46% decrease in the Herceptin EC50 value and no decrease following the co-treatment with Herceptin and penetratin alone. This Grb7 peptide has potential to be developed as a therapeutic agent alone, in combination with traditional chemotherapy, or in combination with other targeting molecules.

Identification of a small peptide that inhibits the phosphorylation of ErbB2 and proliferation of ErbB2 overexpressing breast cancer cells.

ErbB2 is overexpressed in approximately 30% of breast cancer patients with a correlation to poor prognosis. ErbB2 has been identified as a useful receptor for molecular targeting. A cyclic 20 amino acid phage display random peptide library was constructed using the fUSE5 gene III system. The library was panned against 2 different purified forms of the external domain of ErbB2. This resulted in the identification of several ErbB2‐binding phage clones with variable binding to different ErbB2 preparations. One clone (EC‐1) bound all preparations of ErbB2 including live cells and fresh frozen human breast cancer specimens. The synthetic peptide based on the deduced sequence of the EC‐1 clone and its biotin‐conjugated form retained binding affinity for purified ErbB2 and ErbB2 overexpressing cell lysates. EC‐1 peptide was able to effectively inhibit the phosphorylation of ErbB2 on residues Y1248 and Y877 in a dose‐ and time‐dependent manner. Furthermore, EC‐1 peptide selectively inhibits the proliferation of ErbB2 overexpressing breast cancer cells. The linear portion of the cyclic EC‐1 peptide was shown to be essential for binding ErbB2. In addition, 4 biased phage libraries were constructed allowing 4 different regions of the EC‐1 peptide to have random sequence. Screening these EC‐1 biased libraries did not result in higher affinity peptides but did demonstrate the importance of amino acids at position 1–4 on the N‐terminal flanking arm and 11–15 within the cyclic ring. Interestingly, EC‐1 contains homologous motifs with known ErbB receptor family ligands. We have identified a small peptide that binds to the extracellular domain of ErbB2, inhibits ErbB2 autophosphorylation and inhibits the proliferation of ErbB2 overexpressing cells. This supports the notion that small peptides can bind to targets important in cancer therapy even if a target does not have a natural ligand. Continuing research with this peptide includes increasing its affinity to ErbB2, evaluation of pharmacokinetics and evaluation of anti‐proliferative effects with conjugate anti‐cancer agents.

Phage display selection for cell-specific ligands: Development of a screening procedure suitable for small tumor specimens

Phage display technology has been widely used for developing tumor-targeting agents. Most of the efforts were directed towards identifying phage-displayed ligands against cancer-relevant purified targets and cancer cell lines. Whole cell screening procedures typically use a relatively large sample size and are not ideally suited for complex tumor tissues. We describe here a screening protocol that is suitable for non-adherent tumor cells from biopsy specimens. It requires only ∼20,000 cells/round for biopanning and ∼10,000 cells/well for subsequent clone binding assessment by ELISA. We standardized the newly developed protocol using erbB2-overexpressing SKBR3 breast cancer cells and compared the results with conventional protocols employing about 10-times more plate-adhered fixed or live cells. The selection rate of SKBR3-binding clones from biopanning ∼20,000 non-adherent SKBR3 cells by our filter cup protocol was comparable to that obtained from using ∼200,000 plate-adhered cells. Assessment of clones selected from different phage libraries showed that clones from fixed or live cells, adherent or non-adherent cells, either biopanned in filter cup or plate share specific motifs and binding properties. Some of the clones from each biopanning protocol bound to purified erbB2 and shared motifs with erbB family of receptors and their known ligands. These results demonstrated that the protocol developed in this study was capable of selecting cell-specific ligands using relatively small numbers of cells. Screening cells from a fresh human breast cancer specimen using our protocol showed enrichment of tumor binding clones at successive rounds of selection and some of the selected clones were tumor-specific in comparison to normal breast cells. These protocols have direct application to screen for tumor-binding ligands with small tumor tissue specimens.

Profile of reactive oxygen species generation and antioxidative mechanisms in the maturing rat kidney

The antioxidative potential and reactive oxygen species generation were assessed in rat kidney during early critical periods of development and maturation. Superoxide anion generation was found to be low in kidney during early postnatal days of development, whereas hydrogen peroxide levels remained unaltered during development. The levels of thiobarbituric acid reactive substances and protein carbonyls in developing kidney were higher during early postnatal days, up to 26 days after birth, compared to the adult levels. Kidney sulphydryl contents were significantly less during early periods (9 days postnatally) of development compared to adults but attain adult value by postnatal day 26. The levels of ascorbic acid and ceruloplasmin were also higher in developing kidney than in adults. Among enzymic antioxidants, the levels of glutathione peroxidase (GPx) enzyme in developing kidney were high during the early developmental period of the study as compared to adults; however, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were found to be significantly low at early postnatal days up to 16 days of age, which subsequently attained maturational level by the age of 26 days. The levels of antioxidant enzymes and sulphydryl contents in the developing kidney during early periods after birth are low but they increase subsequently with increasing age. Therefore, the present finding suggests that immature kidneys are in a highly dynamic stage of development during the early period and are equipped with antioxidative defence mechanisms that may have a predominant role in protecting against oxidative challenge. Copyright

Species variation in pesticide-induced blood-brain barrier dysfunction

Neurological disorders following acute or chronic exposure to pesticides have been reported in a number of human cases. However, the mechanism(s) by which pesticides produce central nervous system dysfunction is not clear. The objective of the present study was to examine the functional status of blood-brain barrier (BBB) in rats and mice exposed to selected pesticides of different chemical groups. Adult male albino rats and mice were exposed (1/10 of LD50) daily to dichlorvos (organophosphate), lindane (organochlorine) and carbofuran (carbamate) through oral intubation for 3 days. The status of BBB was evaluated by determining brain sodium fluorescein dye uptake and brain uptake index (BUI) in relation to serum dye level. The brain dye uptake and BUI in pesticide-exposed rats did not differ significantly in comparison to that of controls. However, brain dye uptake and BUI were increased significantly in mice exposed to dichlorvos (85%, 40%), lindane (79%, 26%) and carbofuran (129%, 61%). The results of this study show that mouse BBB system is more sensitive to pesticide-induced breach as compared to that of rat. These variations may have a role in determining the outcome of pesticide neurotoxicity in different species.

Phage-display selection on tumor histological specimens with laser capture microdissection.

A method was developed to obtain phage-display ligands that bind to a select population of cells in histological specimens of freshly harvested solid human cancers. It combines phage-display panning with laser capture microdissection (LCM). This method allows selection of phage ligands bound to subpopulations of specific cells contained in tumor tissue on histological sections. Naïve phage scFv library was incubated directly on a histological section of human breast cancer that was snap frozen immediately after surgical resection. Tumor and stromal cells were captured by LCM and bound phages were recovered by bacterial infection. Individual phage clones selected after panning were evaluated for their binding ability by immunofluorescence staining on tumor tissue from the same patient. One phage-display antibody clone selected on tumor stroma showed selective binding on tumor stroma but did not bind to malignant cell population. The expressed scFv of this clone showed no significant binding to normal tissue, or 13 other breast cancers, or 4 colon cancer samples. Using the same method, phage display antibody clones were selected on tumor cells which showed binding to tumor cells and normal tissue. This method is applicable for selection of ligands to virtually any portion of a histological specimen amenable to LCM. This may speed the process of generating ligands to any subset of cells or noncellular feature present on histological specimens.

Structural basis of binding by cyclic nonphosphorylated Peptide antagonists of grb7 implicated in breast cancer progression

Growth-receptor-bound protein (Grb)7 is an adapter protein aberrantly overexpressed, along with the erbB-2 receptor in breast cancer and in other cancers. Normally recruited to focal adhesions with a role in cell migration, it is associated with erbB-2 in cancer cells and is found to exacerbate cancer progression via stimulation of cell migration and proliferation. The G7-18NATE peptide (sequence: WFEGYDNTFPC cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. Cell-permeable versions of G7-18NATE are effective in the reduction of migration and proliferation in Grb7-overexpressing cells. It thus represents a promising starting point for the development of a therapeutic against Grb7. Here, we report the crystal structure of the G7-18NATE peptide in complex with the Grb7-SH2 domain, revealing the structural basis for its interaction. We also report further rounds of phage display that have identified G7-18NATE analogues with micromolar affinity for Grb7-SH2. These peptides retained amino acids F2, G4, and F9, as well as the YDN motif that the structural biology study showed to be the main residues in contact with the Grb7-SH2 domain. Isothermal titration calorimetry measurements reveal similar and better binding affinity of these peptides compared with G7-18NATE. Together, this study facilitates the optimization of second-generation inhibitors of Grb7.

Selective delivery of therapeutic agents for the diagnosis and treatment of cancer

Research activity aimed towards achieving specific and targeted delivery of cancer therapeutics has expanded tremendously in the last decade, resulting in new ways of directing drugs to tumours, as well as new types of drugs. The available strategies exploit differences in the nature of normal and cancer cells and their microenvironment. The discovery and validation of cancer-associated markers, as well as corresponding ligands, is pivotal for developing selective delivery technology for cancer. Although most current clinical trials are either monoclonal antibody- or gene-based, methodological advances in combinatorial libraries of peptides, single chain variable fragments and small organic molecules are expected to change this scenario in the near future. Nanotechnology platforms today allow systematic and modular combinations of therapeutic agents and tumour-binding moieties that may generate novel, personalised agents for selective delivery in cancer. This paper discusses recent developments and future prospects of targeted delivery technologies in the management of cancer.

A Sensitive and Rapid Chemiluminescence ELISA for Filamentous Bacteriophages

Abstract Filamentous bacteriophage (Ff) displayed random peptide and antibody libraries are widely used to identify specific, high affinity, binding ligands. A critical element in the identification of target‐specific phages is to determine phage titers, not only at every round of selection, but also for normalizing phage titers of a set of individual clones for their comparative binding analysis. The conventional ELISA‐based Ff titration methods require a minimum of 4–5 hr assay time and their lowest reported detection limit is ∼50,000 particles/well. In this report, we present a sandwich ELISA that allows detection of ∼1000 Ff particles/well in less than 2.5 hr. The values of correlation of coefficient (r2) for the curves at low phage concentrations (up to 106 TU/well) were about 0.999 in our ELISA. Experiments conducted at different temperatures suggest using 40°C incubations when titering low phage concentration samples. Experiments were also conducted with conventional ELISA for comparison. Our ELISA method derives an advantage from using a chemiluminescence substrate that gives much larger signals and wide linear range of measurement, thus allowing discrimination between background and low Ff phage concentrations. In conclusion, the Ff titration method presented here is highly sensitive, rapid, and amenable to high throughput analysis.