Nipple fluid for breast cancer diagnosis using the nanopore of Phi29 DNA-packaging motor.

Detection of cancer in its early stage is a challenging task for oncologists. Inflammatory breast cancer has symptoms that are similar to mastitis and can be mistaken for microbial infection. Currently, the differential diagnosis between mastitis and Inflammatory breast cancer via nipple aspirate fluid (NAF) is difficult. Here, we report a label-free and amplification-free detection platform using an engineered nanopore of the phi29 DNA-packaging motor with biomarker Galectin3 (GAL3), Thomsen-Friedenreich (TF) binding peptide as the probe fused at its C-terminus. The binding of the biomarker in NAF samples from breast cancer patients to the probe results in the connector's conformational change with a current blockage of 32 %. Utilization of dwell time, blockage ratio, and peak signature enable us to detect basal levels of biomarkers from patient NAF samples at the single-molecule level. This platform will allow for breast cancers to be resolved at an early stage with accuracy and thoroughness.

Selection of Cancer Stem Cell-Targeting Agents Using Bacteriophage Display.

There is a growing need to develop tumor targeting agents for aggressive cancers. Aggressive cancers frequently relapse and are resistant to various therapies. Cancer stem cells (CSCs) are believed to be the cause of relapse and the aggressive nature of many cancers. Targeting CSCs could lead to novel diagnostic and treatment options. Bacteriophage (phage) display is a powerful tool developed by George Smith in 1985 to aid in the discovery of CSC targeting agents. Phage display selections are typically performed in vitro against an immobilized target. There are inherent disadvantages with this technique that can be circumvented by performing phage display selections in vivo. However, in vivo phage display selections present new challenges. A combination of both in vitro and in vivo selections, however, can take advantage of both selection methods. In this chapter, we discuss in detail how to isolate a CSC like population of cells from an aggressive cancer cell line, perform in vivo and in vitro phage display selections against the CSCs, and then characterize the resulting phage/peptides for further use as a diagnostic and therapeutic tool.

Carbohydrate antigens in nipple aspirate fluid predict the presence of atypia and cancer in women requiring diagnostic breast biopsy.

BACKGROUND: The goal of this prospective study was to determine (a) concentrations of the carbohydrate biomarkers Thomsen Friedenreich (TF) antigen and its precursor, Tn antigen, in nipple discharge (ND) collected from women requiring biopsy because of a suspicious breast lesion; and (b) if concentration levels predicted pathologic diagnosis. METHODS: Adult women requiring biopsy to exclude breast cancer were enrolled and ND obtained. The samples from 124 women were analyzed using an anti-TF and anti-Tn monoclonal antibodies in direct immunoassay. RESULTS: The highest median concentration in ND for TF and Tn was in women with ductal carcinoma in situ (DCIS). TF was higher in women with 1) cancer (DCIS or invasive) vs. either no cancer (atypia or benign pathology, p = .048), or benign pathology (p = .018); and 2) abnormal (atypia or cancer) versus benign pathology (p = .016); and was more predictive of atypia or cancer in post- compared to premenopausal women. Tn was not predictive of disease. High TF concentration and age were independent predictors of disease, correctly classifying either cancer or abnormal vs. benign pathology 83% of the time in postmenopausal women. CONCLUSIONS: TF concentrations in ND were higher in women with precancer and cancer compared to women with benign disease, and TF was an independent predictor of breast atypia and cancer. TF may prove useful in early breast cancer detection.

Tumor targeting and SPECT imaging properties of an (111)In-labeled galectin-3 binding peptide in prostate carcinoma.

INTRODUCTION: Galectin-3 (gal-3) is a carbohydrate binding protein that has been implicated in cell adhesion, tumor invasion and metastasis. The objective of this study was to evaluate the tumor targeting and imaging properties of a gal-3 binding peptide selected by phage display in a mouse model of metastatic human prostate carcinoma expressing gal-3. METHODS: A gal-3 binding peptide, ANTPCGPYTHDCPVKR, was synthesized with a Gly-Ser-Gly (GSG) spacer and 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and then radiolabeled with (111)In. The in vitro cell binding properties of (111)In-DOTA-(GSG)-ANTPCGPYTHDCPVKR were determined in metastatic human PC3-M prostate carcinoma cells. The pharmacokinetics and single-photon emission computed tomographic (SPECT/CT) imaging with the radiolabeled peptide were evaluated in SCID mice bearing human PC3-M prostate carcinoma tumor xenografts. RESULTS: The radiolabeled peptide bound with a 50% inhibitory concentration of 191+/-10.2 nM to cultured PC3-M prostate carcinoma cells. In vivo tumor uptake and retention coupled with fast whole-body clearance of the peptide were demonstrated in PC3-M tumor-bearing SCID mice. The tumor uptake rates of the radiolabeled peptide were 1.27+/-0.10%ID/g at 30 min, 0.82+/-0.15%ID/g at 1 h and 0.57+/-0.09%ID/g at 2 h. MicroSPECT/CT studies revealed good tumor uptake of (111)In-DOTA-(GSG)-ANTPCGPYTHDCPVKR 2 h postinjection, while uptake in normal organs was low, with the exception of the kidneys. CONCLUSIONS: In vitro cell binding along with tumor uptake of (111)In-DOTA-(GSG)-ANTPCGPYTHDCPVKR in PC3-M human prostate carcinoma tumor-bearing SCID mice suggests the potential of this peptide as a radiopharmaceutical for imaging of gal-3-expressing prostate tumors.

In vivo bacteriophage display for the discovery of novel peptide-based tumor-targeting agents.

A powerful strategy for targeted drug discovery is the use of bacteriophage (phage) display technology for identification of peptide-based tumor targeting agents. Peptide pharmaceuticals may possess clinically desirable properties because of their rapid blood clearance, non-immunogenic nature, and ease of synthesis. Phage display has identified hundreds of different peptide sequences that bind a desired target in vitro. Regrettably, few of these peptides offer good targeting efficacy in vivo. One reason for this is the synthesized peptide may not retain its optimal activity outside the microenvironment of the phage. Another possible explanation is that traditionally, phage selections are performed in vitro outside the complicated milieu of a living animal. Given these shortcomings, we have developed methods to select phage peptide display libraries in living mice, to identify, a priori, phage (and corresponding synthesized peptides) with ideal tumor-targeting propensity.

In-labeled KCCYSL peptide as an imaging probe for ErbB-2-expressing ovarian carcinomas.

Aberrant expression of ErbB-2, a member of the epidermal growth factor family of receptors, has been implicated in the formation of various malignancies including ovarian cancer. The objective of this study was to determine if the bacteriophage (phage) display-selected ErbB-2 targeting peptide, KCCYSL, once radiolabeled with (111)In would serve as a tumor targeting and Single Photon Emission Computed Tomography (SPECT/CT) imaging agent in a mouse model of human ovarian carcinoma expressing ErbB-2. The KCCYSL peptide was synthesized with a chelator 1,4,7,10-tetra-azacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), and a Gly-Ser-Gly (GSG) spacer between DOTA and amino terminus of the peptide and radiolabeled with (111)InC1(3). In vitro cell binding studies indicated that (111)In-DOTA-GSG-KCCYSL bound to cultured ovcar-3 carcinoma cells. Biodistribution studies in scid mice bearing human ovcar-3 tumor xenografts revealed a tumor uptake of 0.50 ± 0.05 percent injected dose per gram (%ID/g) at 1 h, and 0.39 ± 0.1 %ID/g at 2 h. Blocking studies with non-radiolabeled counterpart indicated a partial inhibition (41%) (P = 0.04) in tumor uptake of (111)In-DOTA-GSG-KCCYSL. In vivo tumor uptake of (111)In-DOTA-GSG-KCCYSL was clearly evident through SPECT/CT images after 2 h post injection. These studies suggest the potential of this peptide as a radiopharmaceutical for imaging of ErbB-2-expressing ovarian tumors.

111In-labeled galectin-3-targeting peptide as a SPECT agent for imaging breast tumors.

UNLABELLED: Galectin-3 is a member of the galectin family of beta-galactoside-binding animal lectins. Galectin-3 is overexpressed in a wide range of neoplasms and is associated with tumor growth and metastases. Given this fact, radiolabeled galectin-3-targeting molecules may be useful for the noninvasive imaging of tumors expressing galectin-3, as well as for targeted radionuclide therapy. In this study, the tumor cell-targeting and SPECT properties of a galectin-3-avid peptide identified from bacteriophage display were evaluated in human breast carcinoma cells and in human breast tumor-bearing mice. METHODS: The galectin-3-avid peptide G3-C12 (ANTPCGPYTHDCPVKR) was synthesized with a Gly-Ser-Gly (GSG) linker at the amino terminus. After conjugation with 1,4,7,10-tetra-azacyclododecane-N,N',N''N'''-tetraacetic acid (DOTA), the peptide was labeled with (111)In. The radiochemical purity and stability of the compound was assessed by high-performance liquid chromatography. MDA-MB-435 human breast carcinoma cells expressing galectin-3 were used to characterize the in vitro binding properties of the radiolabeled compound. SCID mice bearing MDA-MB-435 xenografts were used as an in vivo model for biodistribution and imaging studies with the (111)In-labeled peptide. RESULTS: (111)In-DOTA(GSG)-G3-C12 bound specifically to galectin-3-expressing MDA-MB-435 cells. The radiolabeled peptide was stable in serum and was found intact in excreted urine for at least 1 h. Competitive binding experiments indicated that the radiolabeled peptide exhibited an inhibitory concentration of 50% of 200.00+/-6.70 nM for cultured breast carcinoma cells. In vivo biodistribution studies revealed that tumor uptake was 1.2+/-0.24, 0.75+/-0.05, and 0.6+/-0.04 (mean +/- SD) percentage injected dose per gram at 30 min, 1.0 h, and 2.0 h after injection of the radiotracer, respectively. SPECT/CT studies with (111)In-DOTA(GSG)-G3-C12 showed excellent tumor uptake and contrast in the tumor-bearing mice. Specificity of peptide binding was demonstrated by successful blocking (52%) of in vivo tumor uptake of (111)In-DOTA(GSG)-G3-C12 in the presence of its nonradiolabeled counterpart at 2 h after injection. CONCLUSION: This study demonstrated the successful use of a new radiolabeled peptide for the noninvasive imaging of galectin-3-positive breast tumors. This peptide may be a promising candidate for future clinical applications.

Evaluation of an 111In-radiolabeled peptide as a targeting and imaging agent for ErbB-2 receptor expressing breast carcinomas.

PURPOSE: The cellular targeting and tumor imaging properties of a novel ErbB-2-avid peptide, discovered from bacteriophage display, were evaluated in human breast carcinoma cells and in breast carcinoma-xenografted mice. EXPERIMENTAL DESIGN: The affinity of the ErbB-2 targeting peptide KCCYSL and its alanine substituted counterparts for the extracellular domain (ECD) of purified recombinant ErbB-2 (ErbB-2-ECD) was assessed by fluorescence titration. Binding of the KCCYSL peptide to breast and prostate carcinoma cells was analyzed by confocal microscopy. A DOTA(GSG)-KCCYSL peptide conjugate was radiolabeled with 111In, and stability, target binding, and internalization were analyzed in vitro. In vivo biodistribution and single-photon emission computed tomography imaging studies were done with the radiolabeled peptide in MDA-MB-435 human breast tumor-bearing severe combined immunodeficient mice. RESULTS: KCCYSL peptide exhibited high affinity (295 +/- 56 nmol/L) to ErbB-2-ECD. Substitution of alanine for lysine, tryptophan, and cysteine reduced the peptide affinity approximately 1- to 2.4-fold, whereas replacing leucine completely abolished binding. Both biotin-KCCYSL and 111In-DOTA(GSG)-KCCYSL were capable of binding ErbB-2-expressing human breast carcinoma cells in vitro. Approximately 11% of the total bound radioactivity was internalized in the carcinoma cells. Competitive binding studies indicated that the radiolabeled peptide exhibited an IC(50) value of 42.5 +/- 2.76 nmol/L for the breast carcinoma cells. 111In-DOTA(GSG)-KCCYSL was stable in serum and exhibited rapid tumor uptake (2.12 +/- 0.32 %ID/g) at 15 min postinjection and extended retention coupled with rapid whole body disappearance, as observed by biodistribution and single-photon emission computed tomography imaging studies, respectively. CONCLUSIONS: The DOTA(GSG)-KCCYSL peptide has the potential to be used as a tumor-imaging agent and a vehicle for specific delivery of radionuclide or cytotoxic agents for tumors overexpressing ErbB-2.

Phage peptide display.

Molecular imaging is at the forefront in the advancement of in-vivo diagnosis and monitoring of cancer. New peptide-based molecular probes to facilitate cancer detection are rapidly evolving. Peptide-based molecular probes that target apoptosis, angiogenesis, cell signaling and cell adhesion events are in place. Bacteriophage (phage) display technology, a molecular genetic approach to ligand discovery, is commonly employed to identify peptides as tumor-targeting molecules. The peptide itself may perhaps have functional properties that diminish tumor growth or metastasis. More often, a selected peptide is chemically synthesized, coupled to a radiotracer or fluorescent probe, and utilized in the development of new noninvasive molecular imaging probes. A myriad of peptides that bind cancer cells and cancer-associated antigens have been reported from phage library selections. Phage selections have also been performed in live animals to obtain peptides with optimal stability and targeting properties in vivo. To this point, few in-vitro, in-situ, or in-vivo selected peptides have shown success in the molecular imaging of cancer, the notable exception being vascular targeting peptides identified via in-vivo selections. The success of vasculature targeting peptides, such as those with an RGD motif that bind alpha(v)beta(3)integrin, may be due to the abundance and expression patterns of integrins in tumors and supporting vasculature. The discovery of molecular probes that bind tumor-specific antigens has lagged considerably. One promising means to expedite discovery is through the implementation of selected phage themselves as tumor-imaging agents in animals.

Melanoma imaging with pretargeted bivalent bacteriophage.

UNLABELLED: Random bacteriophage (phage) display peptide libraries have traditionally been used for the selection of clones that bind specific tissues, tumors, and antigens. However, once the targeting peptide is synthetically produced, it often displays a lower affinity than the original phage because of a lack of avidity effects and removal from the virion surface. We hypothesized that multivalent bifunctional phage displaying peptides that target novel molecular biomarkers would facilitate the in vivo imaging of cancer. This study provides proof of principle for the use of phage displaying multiple melanocortin-1 receptor-homing peptides for the pretargeting and subsequent imaging of murine melanomas in vivo. METHODS: A 2-step melanoma pretargeting-imaging system was developed by first generating and biotinylating phage that displayed up to 5 copies of alpha-melanocyte-stimulating hormone (alpha-MSH) peptide analogs. Second, streptavidin was conjugated to diethylenetriaminepentaacetic acid for the purpose of radiolabeling with (111)In. RESULTS: The specificity of the MSH2.0 phage for the B16-F1 melanoma was demonstrated both in vitro and in vivo. In vitro micropanning assays with phage at inputs of 10(7) and 10(6) transducing units per milliliter resulted in approximately 200- and approximately 1,000-fold-greater recovery of the MSH2.0 phage over the background, respectively. In vivo distribution studies indicated that melanoma uptake values were 2.6 +/- 1.1, 0.6 +/- 0.2, and 1.0 +/- 0.1 (mean +/- SD) percentage injected dose per gram at 0.5, 6, and 24 h after the injection of (111)In-radiolabeled streptavidin ((111)In-SA). The accumulation of radioactivity within the tumor was 1.8 times greater for the biotinylated MSH2.0 phage than for the biotinylated wild-type phage. These data, combined with reduction by 2.4-fold through competition with a nonradiolabeled alpha-MSH peptide analog, indicated the specific targeting of melanoma tumors in vivo. SPECT/CT image analysis of B16-F1 melanoma-bearing mice showed that intravenously injected biotinylated alpha-MSH phage were retained within melanoma tumors at 4 h after injection of (111)In-SA. CONCLUSION: This study demonstrated the use of multivalent bifunctional phage in a 2-step pretargeting-imaging system.

Specificity of phage display selected peptides for modified anticodon stem and loop domains of tRNA.

Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA(LyS3) having 2-thiouridine (s(2)U34) and pseudouridine (psi39), bound the modified human ASL(Lys3)(s(2)U34;psi39) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL(LYS3)(s(2)U34; psi39), followed by the hypermodified ASL(Lys3) (mcm(5)s(2) U34; ms(2)t(6)A37) and the unmodified ASL(Lys3), but bound poorly to singly modified ASL(Lys3) constructs (psi39, ms(2)t(6)A37, s(2)34), ASL(Lys1,2) (t(6)A37) and Escherichia coli ASL(Glu) (s(2)U34). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.

Characterization of In Vivo Selected Bacteriophage for the Development of Novel Tumor-Targeting Agents with Specific Pharmacokinetics and Imaging Applications.

Bacteriophage (phage) display technology is a powerful strategy for the identification of peptide-based tumor targeting agents for drug discovery. Phage selections performed in vitro often result in many phage clones/peptides with similar properties and often similar sequence. However, these phage and corresponding peptides are selected, validated, and characterized outside the complicated milieu of a living animal. Thus, there is no guarantee that peptides from in vitro selections will successfully meet the requirements of an in vivo targeting compound. In comparison, in vivo phage display selections have the distinct advantage of identifying phage clones with robust pharmacokinetics and tumor/tissue targeting ability. This capacity has allowed for the identification of peptides with specific in vivo localization and/or clearance profiles. However, in vivo phage display selections also have the potential to result in an array of phage clones with various and unknown targets and little to no sequence similarity. Given these shortcomings, we have developed methods to select phage peptide display libraries in living mice to identify phage (and corresponding synthesized peptides) with specific clearance and/or tumor-targeting propensity. Additionally, we describe the use of labeled phage clones for the efficient screening of selected phage/peptides to aid in the identification and characterization of a phage clone with an optimal and specific pharmacokinetic profile.

Targeting aggressive prostate cancer-associated CD44v6 using phage display selected peptides.

There is a crucial need to identify new biomarkers associated with aggressive prostate cancer (PCa) including those associated with cancer stem cells (CSCs). CD44v6, generated by alternative splicing of CD44, has been proposed as a CSC biomarker due to its correlation with aggressive PCa disease. We hypothesized that phage display selected peptides that target CD44v6 may serve as theranostic agents for aggressive PCa. Here, a 15 amino acid peptide ("PFT") was identified by affinity selection against a peptide derived from the v6 region of CD44v6. Synthesized PFT exhibited specific binding to CD44v6 with an equilibrium dissociation constant (Kd) of 743.4 nM. PFT also bound CD44v6 highly expressed on human PCa cell lines. Further, an aggressive form of PCa cells (v6A3) was isolated and tagged by a novel CSC reporter vector. The v6A3 cells had a CSC-like phenotype including enriched CD44v6 expression, enhanced clonogenicity, resistance to chemotherapeutics, and generation of heterogeneous offspring. PFT exhibited preferential binding to v6A3 cells compared to parental cells. Immunohistofluorescence studies with human PCa tissue microarrays (TMA) indicated that PFT was highly accurate in detecting CD44v6-positive aggressive PCa cells, and staining positivity was significantly higher in late stage, metastatic and higher-grade samples. Taken together, this study provides for the first time phage display selected peptides that target CD44v6 overexpressed on PCa cells. Peptide PFT may be explored as an aid in the diagnosis and therapy of advanced PCa disease.

Evidence for structural plasticity of heavy chain complementarity-determining region 3 in antibody-ssDNA recognition.

Anti-DNA antibodies play important roles in the pathogenesis of autoimmune diseases. They also represent a unique and relatively unexplored class of DNA-binding protein. Here, we present a study of conformational changes induced by DNA binding to an anti-ssDNA Fab known as DNA-1. Three crystal structures are reported: a complex of DNA-1 bound to dT3, and two structures of the ligand-free Fab. One of the ligand-free structures was determined from crystals exhibiting perfect hemihedral twinning, and the details of structure determination are provided. Unexpectedly, five residues (H97-H100A) in the apex of heavy chain complementarity-determining region 3 (HCDR3) are disordered in both ligand-free structures. Ligand binding also caused a 2-4A shift of the backbone of Tyr L92 and ordering of the L92 side-chain. In contrast, these residues are highly ordered in the Fab/dT3 complex, where Tyr H100 and Tyr H100A form intimate stacking interactions with DNA bases, and L92 forms the 5' end of the binding site. The structures suggest that HCDR3 is very flexible and adopts multiple conformations in the ligand-free state. These results are discussed in terms of induced fit and pre-existing equilibrium theories of ligand binding. Our results allow new interpretations of existing thermodynamic and mutagenesis data in terms of conformational entropy and the volume of conformational space accessible to HCDR3 in the ligand-free state. In the context of autoimmune disease, plasticity of the ligand-free antibody could provide a mechanism by which anti-DNA antibodies bind diverse host ligands, and thereby contribute to pathogenicity.

Thomsen-Friedenreich and Tn antigens in nipple fluid: carbohydrate biomarkers for breast cancer detection.

PURPOSE: Novel biomarkers would facilitate early and accurate diagnosis of breast cancer. The Thomsen-Freidenreich (TF) and Tn antigens are aberrantly glycosylated carbohydrate cancer-associated antigens found in approximately 80% of adenocarcinomas. Both TF and Tn are expressed on cell-surface glycoproteins and glycolipids. Nipple aspirate fluid (NAF) is concentrated in secreted proteins and lipids from cells that give rise to cancer. The objective of this study was to determine if NAF from breasts with cancer contains elevated levels of TF and Tn compared with NAF from normal breasts. A sensitive and specific antigen capture immunoassay for TF and Tn detection in NAF was developed for this purpose. EXPERIMENTAL DESIGN: Fifty NAF samples, 25 from breasts with cancer and 25 from normal breasts, were examined. Antigen capture immunoassays were done on the samples using monoclonal antibodies that specifically recognized either TF or Tn antigen in NAF. These antibodies captured serially diluted NAF samples, and the concentration of TF or Tn was determined by comparing absorbance values against a standard curve generated from standard sources of TF or Tn. RESULTS: TF and Tn were detected in 19 of 25 and 20 of 25 NAF samples from breasts with cancer, respectively, compared with 0 of 25 and 1 of 25 NAF samples from breasts without cancer (P < 0.001 for both TF and Tn). In 92% of the cancerous breast NAF samples tested, either TF or Tn was found. CONCLUSIONS: Simultaneous measurement of TF and Tn in NAF may facilitate the noninvasive detection of breast cancer and warrants further study.

Intravascular metastatic cancer cell homotypic aggregation at the sites of primary attachment to the endothelium.

The two major theories of cancer metastasis, the seed and soil hypothesis and the mechanical trapping theory, view tumor cell adhesion to blood vessel endothelia and cancer cell aggregation as corresponding key components of the metastatic process. Here, we demonstrate in vitro, ex vivo, and in vivo that metastatic breast and prostate carcinoma cells form multicellular homotypic aggregates at the sites of their primary attachment to the endothelium. Our results suggest that metastatic cell heterotypic adhesion to the microvascular endothelium and homotypic aggregation represent two coordinated subsequent steps of the metastatic cascade mediated largely by similar molecular mechanisms, specifically by interactions of tumor-associated Thomsen-Friedenreich glycoantigen with the beta-galactoside-binding protein, galectin-3. In addition to inhibiting neoplastic cell adhesion to the endothelium and homotypic aggregation, disrupting this line of intercellular communication using synthetic Thomsen-Friedenreich antigen masking and Thomsen-Friedenreich antigen mimicking compounds greatly affects cancer cell clonogenic survival and growth as well. Thus, beta-galactoside-mediated intravascular heterotypic and homotypic tumor cell adhesive interactions at the sites of a primary attachment to the microvascular endothelium could play an important role during early stages of hematogenous cancer metastasis.

Cytotoxic Tumor-Targeting Peptides From In Vivo Phage Display.

We previously utilized an in vivo peptide phage display selection technique, which included the use of detergent elution of phage from excised tumor, to obtain tumor-targeting phage with the ability to extravasate the vasculature and bind directly to prostate tumor tissue. It is hypothesized that this same in vivo phage selection technique can be used to functionally select for molecules that not only bind to cancer cells but also kill them. Here we analyzed two different in vivo phage display selected phage clones, G1 and H5, retrieved from PC-3 human prostate carcinoma xenografted tumors. First, cell de-attachment as an endpoint criterion for apoptosis and cell cycle was examined. After 2.5 hours incubation with G1 phage, PC-3 cell attachment was reduced by 23.8% and the percent of cell population in M phase reduced by 32.1%. In comparison, PC-3 cells incubated with H5 phage had a reduction of 25.0% cell attachment and 33.6% of cell population in M phase. These changes in combination with elevated caspase activation within cells in M phase, and no significant changes to G1/G0 or S phase cell populations suggest that the cytotoxic phages are targeting actively dividing PC-3 cells. Microscopic studies were also performed to further analyze the nature of cytotoxicity of these two phage clones. It was found that G1 phage induced and co- localized with tubulin based projections within apoptotic cells, while H5 phage did not. These phage may form the foundation for a new class of targeted prostate cancer therapeutic agents.

Multiple Bacteriophage Selection Strategies for Improved Affinity of a Peptide Targeting ERBB2.

Due to the heterogeneity of ERBB2-expression between tumors and over the course of treatment, a non-invasive molecular imaging agent is needed to accurately detect overall ERBB2 status. Peptides are a highly advantageous platform for molecular imaging, since they have excellent tumor penetration and rapid pharmacokinetics. One limitation of peptides however, is their traditionally low target affinity, and consequently, tumor uptake. The peptide KCCYSL was previously selected from a bacteriophage (phage) display library to bind ERBB2 and did so with moderate affinity of 295 nM. In order to enhance tumor uptake and clinical utility of the peptide, a novel phage microlibrary was created by flanking the parent sequence with random amino acids, followed by reselection using parallel strategies for high affinity and specific ERBB2 binding in an attempt to affinity maturate the peptide. One limitation of traditional phage display selections is difficulty in releasing the highest affinity phages from the target by incubation of acidic buffer. In an attempt to recover high affinity second-generation peptides from the ERBB2 microlibrary, two elution strategies, sonication and target elution, were undertaken. Sonication resulted in an approximately 50-fold enhancement in recovered phage per round of selection in comparison to target elution. Despite the differences in elution efficiency, the affinities of phage-displayed peptides selected from either strategy were relatively similar. Although both selections yielded peptides with significantly improved affinity in comparison to KCCYSL, the improvements were modest, most likely because the parental peptide binding cannot be improved by additional amino acids.

Structure of an anti-DNA fab complexed with a non-DNA ligand provides insights into cross-reactivity and molecular mimicry.

Antibodies that recognize DNA (anti-DNA) are part of the autoimmune response underlying systemic lupus erythematosus. To better understand molecular recognition by anti-DNA antibodies, crystallographic studies have been performed using an anti-ssDNA antigen-binding fragment (Fab) known as DNA-1. The previously determined structure of a DNA-1/dT5 complex revealed that thymine bases insert into a narrow groove, and that ligand recognition primarily involves the bases of DNA. We now report the 1.75-A resolution structure of DNA-1 complexed with the biological buffer HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid). All three light chain complementarity-determining regions (CDRs) and HCDR3 contribute to binding. The HEPES sulfonate hydrogen bonds to His L91, Asn L50, and to the backbone of Tyr H100 and Tyr H100A. The Tyr side-chains of L32, L92, H100, and H100A form nonpolar contacts with the HEPES ethylene and piperazine groups. Comparison to the DNA-1/dT5 structure reveals that the dual recognition of dT5 and HEPES requires a 13-A movement of HCDR3. This dramatic structural change converts the combining site from a narrow groove, appropriate for the edge-on insertion of thymine bases, to one sufficiently wide to accommodate the HEPES sulfonate and piperazine. Isothermal titration calorimetry verified the association of HEPES with DNA-1 under conditions similar those used for crystallization (2 M ammonium sulfate). Interestingly, the presence of 2 M ammonium sulfate increases the affinities of DNA-1 for both HEPES and dT5, suggesting that non-polar Fab-ligand interactions are important for molecular recognition in highly ionic solvent conditions. The structural and thermodynamic data suggest a molecular mimicry mechanism based on structural plasticity and hydrophobic interactions.