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.

Ovarian cancer is among the leading causes of cancer deaths in women. The low survival rate is in part due to inadequate means of detection, which emphasizes the need to develop effective screening techniques. Peptide J18 was previously discovered using phage display technology and was found to specifically target and image human ovarian tumors in a SKOV-3 xenografted mouse model. Here, affinity maturation of J18 was carried out to improve its binding affinity by conducting an alanine scan of the peptide. A cell-based modified ELISA using SKOV-3 cells was carried out and dose-response curves were modelled using non-linear regression. The results showed that substitution of serine-5 (S5) and aspartic acid-6 (D6) with alanine decreased the EC value of the original peptide J18 from 45.02 µM (39.15-52.94 95% confidence interval; CI) to 19.54 (9.70-38-51 95% CI) and 6.92 (5.2-9.80 95% CI), respectively. Next, a double alanine substitution of S5 and D6 (J18-S5A-D6A) was investigated to further increase the binding affinity. The EC value was determined to be 4.22 (2.16-16.14 95% CI), thus improving the binding affinity approximately 10-fold. To ensure that J18-S5A-D6A maintained specificity for ovarian cancer cells, the peptide was evaluated against ovarian cancer SKOV-3, pancreatic cancer Mia-Paca-2, prostate cancer LNCaP, and embryonic kidney HEK 293 cells using a modified ELISA and fluorescent microscopy. Both assays showed significantly increased binding of J18-S5A-D6A to SKOV-3 cells compared to the other cell lines. Based on these results, peptide J18-S5A-D6A may be used as an improved imaging agent of ovarian cancer.

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.

Ovarian cancer is often diagnosed at late stages due to current inadequate detection. Therefore, the development of new detection methods of ovarian cancer is needed. This may be achieved by phage nanoparticles that display targeting peptides for optical imaging. Here, two such phage clones are reported. Ovarian cancer binding and specificity of phage clones (pJ18, pJ24) and peptides (J18, J24) were investigated using fluorescent microscopy and modified ELISA. Further, AF680-labeled phage particles were subjected to biodistribution and optical imaging studies in SKOV-3 xenografted mice. Fluorescent microscopy and ELISA of phage and peptides showed significantly increased binding to SKOV-3 cells compared to controls. Additionally, these studies revealed that J18 exhibits specificity for ovarian cancer SKOV-3 and OVCAR-3 cell lines. Further, peptides displayed increased SKOV-3 binding compared to N35 (non-relevant peptide) with EC50 values of 22.2 ± 10.6 μM and 29.0 ± 6.9 (mean ± SE), respectively. Biodistribution studies of AF680-labeled phage particles showed tumor uptake after 4 h and excretion through the reticuloendothelial system. Importantly, SKOV-3 tumors were easily localized by optical imaging after 2 h and 4 h and displayed good tumor-to-background contrast. The fluorescent tumor signal intensity was significantly higher for pJ18 compared to wild type (WT) after 2 h.

Phage display permits very large numbers of peptides to be tested simultaneously, and so can be used in a broad range of applications, including the identification of proteins eliciting autoimmunity.

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.

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.

Aptamers are single-stranded oligonucleotides that recognize molecular targets with high affinity and specificity. Aptamer that selectively bind to the protein tyrosine kinase-7 (PTK7) receptor, overexpressed on many cancers, has been labelled as probes for molecular imaging of cancer. Two new PTK7-targeting aptamer probes were developed by coupling frameworks from the fluorescent dye AlexaFluor647 or the 6-hydrazinonicotinamide (HYNIC) chelator-labelled to 99mTc. The derivatizations via a 5′-aminohexyl terminal linker were done at room temperature and under mild buffer conditions. Physicochemical and biological controls for both imaging agents were performed verifying the integrity of the aptamer-conjugates by HPLC. Recognition of melanoma (B16F1) and lymphoma (A20) mouse cell lines by the aptamer was studied using cell binding, flow cytometry and confocal microscopy. Finally, in vivo imaging studies in tumour-bearing mice were performed. The new probes were able to bind to melanoma and lymphoma cell lines in vitro, the in vivo imaging in tumour-bearing mice showed different uptake behaviours showing for the fluorescent conjugate good uptake by B cell lymphoma while the radiolabelled conjugate did not display tumour uptake due to its high extravascular distribution, and both showed rapid clearance properties in tumour-bearing mice.

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.

1190 Objectives Her2 expression in breast cancer and other tumors has prognostic and therapeutic implications.67Gallium chloride is a radionuclide with SPECT imaging capabilities and it is available to most Nuclear Medicine Centers. The aim of this work was to develop 67GaCl3 radiolabeled DOTA-GSG-KCCYSL and perform its chemical and biological evaluation as a potential imaging agent for in vivo HER2 protein expression. Methods The Her2-targeting peptide KCCYSL, previously isolated using bacteriophage display, was synthesized and conjugated with DOTA via a GSG linker sequence using solid phase peptide chemistry. The peptide was radiolabeled with 67GaCl3 at 900C for 20 minutes. Radiochemical purity was evaluated by C-18 reverse phase HPLC. Protein stability was analyzed in serum and buffer solution. Binding studies were performed with MCF-7 breast carcinoma cells and blocking assays were carried out with unlabeled peptide. Biodistribution studies were evaluated in normal CD-1 female mice at several times post-injection (n=4 per time). Results DOTA-GSG-KCCYSL was efficiently labeled with 67GaCl3 yielding a radiochemical purity over 90%. The in vitro radiochemical stability of the radiolabeled peptide showed that the complex was stable. Transchelation was less than 5% at 24 hours. Specific uptake was confirmed by competitive receptor blocking studies (Figure 1) were binding was reduced over 50% at 2 hours. Biodistribution studies revealed high blood purification with renal elimination in more than 95% at 2 h post injection. Conclusions DOTA-GSG-KCCYSL was efficiently radiolabeled, and showed good stability and radiochemical purity. Biological studies showed good affinity for HER2 receptor. The biodistribution profile makes this peptide an interesting agent for in vivo HER 2 expression evaluation. Research Support ANII, Pro.In.Bio, PEDECIBA Quimica, CSIC, Espacio Interdisciplinario-Oncologia Nuclear

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.

Purpose The goal of this study was to improve the pharmacokinetic properties and specificity of an ERBB2-targeted peptide for SPECT imaging. Procedures Bacteriophages (phages) displaying the ERBB2 targeting sequence, KCCYSL, flanked by additional random amino acids were used for in vivo selections in mice-bearing ERBB2-expressing MDA-MB-435 human breast xenografts. Phage-displayed peptides were evaluated for ERBB2 and cancer cell binding affinity and specificity in vitro , and one peptide was radiolabeled with 111 In-DOTA and biodistribution and SPECT imaging properties were compared to the first generation peptide, 111 In-DOTA-KCCYSL. Results In vivo phage display selected two peptides, 1-D03 (MEGPSKCCYSLALSH) and 3-G03 (SGTKSKCCYSLRRSS), with higher breast carcinoma cell specificity and similar ErbB2 affinity (236 and 289 nM, respectively) to the first generation peptide. The corresponding radiolabeled probes bound with higher affinity to target cancer cells than 111 In-DOTA-KCCYSL; however, only 111 In-DOTA-1-D03 demonstrated higher specificity for MDA-MB-435 cells. Biodistribution analysis demonstrated that although 111 In-DOTA-1-D03 had slightly reduced tumor uptake (0.661 % ID/g) in comparison to 111 In-DOTA-KCCYSL (0.78 %/ID/g), its dramatic improvement in blood clearance led to a significantly higher tumor/blood ratio (6.02:1). Non-specific uptake was also reduced in most organs including heart, lung, muscle, bone, and kidneys. SPECT imaging revealed tumor-specific uptake of 111 In-DOTA-1-D03, which was confirmed by blocking with unlabeled 1-D03 peptide. Conclusions This is the first evidence that SPECT imaging peptides with improved tumor specificity and pharmacokinetics can be obtained by in vivo phage display affinity maturation. The combination of ERBB2-specific binding, rapid clearance, and tumor specificity may make 1-D03 a viable candidate for clinical imaging studies.

The often fatal outcome of ovarian cancer (OC) is related to inadequate detection methods, which may be overcome by development of nuclear imaging agents. Cancer targeting peptides have been identified using in vivo bacteriophage (phage) display technology; however, the majority of these ligands target tumor vasculature. To overcome this problem, a two-tier phage display method was employed to select an ovarian cancer targeting peptide with good pharmacokinetic and imaging properties. A fUSE5 15-amino acid peptide library was screened against xenografted human OC SKOV-3 tumors in mice, which was followed by selection against enriched SKOV-3 cells. The selected peptide RSLWSDFYASASRGP (J18) was synthesized with a GSG-spacer and a 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) chelator and radiolabeled with (111)In. SKOV-3 xenografted mice were used to evaluate the biodistribution and single photon emission computed tomography (SPECT) imaging capabilities of the radiolabeled peptide. Competitive binding experiments using (111)In-DOTA-GSG-J18 indicated that the peptide displayed a half maximal inhibitory concentration (IC50) value of 10.5 ± 1.1 μM. Biodistribution studies revealed that tumor uptake was 1.63 ± 0.68, 0.60 ± 0.32, 0.31 ± 0.12 and 0.10 ± 0.02% injected dose/g at 30 min, 1 h, 2 h and 4 h post-injection of (111)In-DOTA-GSG-J18, respectively. SPECT/CT imaging demonstrated good tumor uptake and minimal background binding. This study demonstrated successful utilization of a two-tier phage display selection process to identify an ovarian cancer avid peptide with excellent SPECT/CT imaging capabilities.

Background A crucial step in tumorigenesis is the recruitment of novel vasculature to the site of neoplasia. Currently, a number of high throughput techniques are employed to identify genes, mRNA and proteins that are aberrantly expressed in tumor vasculature. One drawback of such techniques is the lack of functional in vivo data that they provide. Bacteriophage (phage) display has been demonstrated in vivo to select peptides that home to tumors and tumor vasculature. The peptides can be compared to sequences of putative cancer-related proteins, in order to identify novel proteins essential for tumorigenesis. Objectives It was hypothesized that an in vivo selection for phage which targeted human breast cancer xenografts could identify peptides with homology to cancer-related proteins for in vivo imaging of breast cancer. Methods Following four rounds of in vivo selection in human MDA-MB-435 breast cancer xenografted mice, peptide 3-G03 was discovered with significant homology to a putative secreted protein termed EGFL6. Egfl6 mRNA is upregulated in several transcriptomic analyses of human cancer biopsies, and the protein may play a role in tumor vascularization. Results Egfl6 mRNA expression was demonstrated in MDA-MB-435 cells and EGFL6 protein was secreted from these cells. Based on homology of 3-G03 to EGFL6, an EGFL6 peptide was synthesized and shown to target MDA-MB-435 cells. EGFL6 peptide was radiolabeled with 111In and analyzed for biodistribution and tumor imaging capabilities. Single photon emission computed tomography imaging revealed uptake of the peptide in a manner consistent with other tumor vasculature targeting agents.