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.

Ovarian Cancer Targeting Phage for In Vivo Near-Infrared Optical Imaging.

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.

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.

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.

Development of new PTK7-targeting aptamer-fluorescent and -radiolabelled probes for evaluation as molecular imaging agents: Lymphoma and melanoma in vivo proof of concept.

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.

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.

In vitro high throughput phage display selection of ovarian cancer avid phage clones for near-infrared optical imaging.

Ovarian cancer is among the leading causes of cancer deaths in women, and is the most fatal gynecological malignancy. Poor outcomes of the disease are a direct result of inadequate detection and diagnostic methods, which may be overcome by the development of novel efficacious screening modalities. However, the advancement of such technologies is often time-consuming and costly. To overcome this hurdle, our laboratory has established a time and cost effective method of selecting and identifying ovarian carcinoma avid bacteriophage (phage) clones using high throughput phage display technology. These phage clones were selected from a filamentous phage fusion vector (fUSE5) 15-amino acid peptide library against human ovarian carcinoma (SKOV-3) cells, and identified by DNA sequencing. Two phage clones, pM6 and pM9, were shown to exhibit high binding affinity and specificity for SKOV-3 cells using micropanning, cell binding and fluorescent microscopy studies. To validate that the binding was mediated by the phage-displayed peptides, biotinylated peptides (M6 and M9) were synthesized and the specificity for ovarian carcinoma cells was analyzed. These results showed that M6 and M9 bound to SKOV-3 cells in a dose-response manner and exhibited EC50 values of 22.9 ± 2.0 µM and 12.2 ± 2.1µM (mean ± STD), respectively. Based on this, phage clones pM6 and pM9 were labeled with the near-infrared fluorophore AF680, and examined for their pharmacokinetic properties and tumor imaging abilities in vivo. Both phage successfully targeted and imaged SKOV-3 tumors in xenografted nude mice, demonstrating the ability of this method to quickly and cost effectively develop novel ovarian carcinoma avid phage.

In vivo phage display selection of an ovarian cancer targeting peptide for SPECT/CT imaging.

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.

Development of a peptide by phage display for SPECT imaging of resistance-susceptible breast cancer.

Personalized medicine is at the forefront of cancer diagnosis and therapy. Molecularly targeted therapies such as trastuzumab and tamoxifen have enhanced prognosis of patients with cancers expressing ERBB2 and the estrogen receptor, respectively. One obstacle to targeted therapy is the development of resistance. A targeted peptide that could distinguish resistance-susceptible cancer would aid in treatment. BT-474 human breast cancer cells can be resistant to both tamoxifen and trastuzumab, and may serve as a model for malignancies in which targeted therapy may not work. Bacteriophage (phage) display is a combinatorial technology that has been used to isolate peptides that target a specific cancer subtype. It was hypothesized that in vivo phage display could be used to select a peptide for SPECT imaging of BT-474 human breast cancer xenografts. A phage library displaying random 15 amino acid peptides was subjected to four rounds of selection, after which 14 clones were analyzed for BT-474 binding and specificity. One phage clone, 51, demonstrated superior binding and specificity, and the displayed peptide was synthesized for in vitro characterization. Peptide 51 bound specifically to BT-474 cells with an EC50 = 2.33 µM and was synthesized as a DOTA-conjugated peptide and radiolabeled with (111)In for in vitro and in vivo analysis. The radiolabeled peptide exhibited an IC50 = 16.1 nM to BT-474 cells and its biodistribution and SPECT imaging in BT-474 xenografted mice was analyzed. Although tumor uptake was moderate at 0.11% ID/g, SPECT imaging revealed a distinct tumor vasculature binding pattern. It was discovered that peptide 51 had an identical 5 amino acid N-terminal sequence to a peptide, V1, which bound to Nrp1, a tumor vasculature protein. Peptide 51 and V1 were examined for binding to target cells, and 51 bound both target and endothelial cells, while V1 only bound endothelial cells. Truncated versions of 51 did not bind BT-474 cells, demonstrating that the targeting ability of 51 was independent of the homologous V1 sequence. These results demonstrate that in vivo phage display can effectively identify a peptide that specifically targets a breast cancer cell line that is susceptible to targeted therapy resistance.

In vivo bacteriophage peptide display to tailor pharmacokinetics of biological nanoparticles.

PURPOSE: Clinical use of most radiolabeled targeting agents has been limited because of the uptake and retention in kidney and/or liver. We hypothesized that bacteriophage (phage) display could be exploited to select for peptide sequences with fast clearance and low kidney uptake with the added ability to redirect phage clearance away from the reticuloendothelial system towards the kidney possessing rapid kidney clearance. PROCEDURES: In vivo phage display was performed to identify peptides displayed on phage that were excreted rapidly into the urine of mice. A novel in vitro assay using kidney cells, developed to predict in vivo kidney retention, and in vivo pharmacokinetic analyses were performed to characterize selected peptides/phage clones. RESULTS: Forty-three renal clearance clones (RCC) were identified. In vivo mixing experiments and in vitro kidney cell assays identified RCC1-02 as the lead compound. In vivo analysis of fluorescently labeled phage clones demonstrated the ability of RCC1-02 peptide to redirect the biodistribution of the large phage particle towards excretion via the kidney. Pharmacokinetic analysis of [(111)In]-radiolabeled peptides revealed that kidney retention of the control ErBB-2-avid peptide, [(111)In]DOTA-KCCYSL, at 2-h postinjection was 5.7 ± 0.7 %ID/g. In comparison, [(111)In]DOTA-RCC1-02 had kidney retention values of 1.66 ± 0.43 %ID/g, respectively. CONCLUSIONS: In vivo phage display can identify phage and corresponding peptides that rapidly clear the renal system. In the future, these peptides may be used to impart favorable pharmacokinetics onto a wide range of radioimaging or therapeutic macromolecules.

Affinity maturation of an ERBB2-targeted SPECT imaging peptide by in vivo phage display.

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.

Identification of a Peptide from In vivo Bacteriophage Display with Homology to EGFL6: A Candidate Tumor Vasculature Ligand in Breast Cancer.

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.

Computational screening of the human TF-glycome provides a structural definition for the specificity of anti-tumor antibody JAA-F11.

Recombinant antibodies are of profound clinical significance; yet, anti-carbohydrate antibodies are prone to undesirable cross-reactivity with structurally related-glycans. Here we introduce a new technology called Computational Carbohydrate Grafting (CCG), which enables a virtual library of glycans to be assessed for protein binding specificity, and employ it to define the scope and structural origin of the binding specificity of antibody JAA-F11 for glycans containing the Thomsen-Friedenreich (TF) human tumor antigen. A virtual library of the entire human glycome (GLibrary-3D) was constructed, from which 1,182 TF-containing human glycans were identified and assessed for their ability to fit into the antibody combining site. The glycans were categorized into putative binders, or non-binders, on the basis of steric clashes with the antibody surface. The analysis employed a structure of the immune complex, generated by docking the TF-disaccharide (Galß1-3GalNAcα) into a crystal structure of the JAA-F11 antigen binding fragment, which was shown to be consistent with saturation transfer difference (STD) NMR data. The specificities predicted by CCG were fully consistent with data from experimental glycan array screening, and confirmed that the antibody is selective for the TF-antigen and certain extended core-2 type mucins. Additionally, the CCG analysis identified a limited number of related putative binding motifs, and provided a structural basis for interpreting the specificity. CCG can be utilized to facilitate clinical applications through the determination of the three-dimensional interaction of glycans with proteins, thus augmenting drug and vaccine development techniques that seek to optimize the specificity and affinity of neutralizing proteins, which target glycans associated with diseases including cancer and HIV.

Nanoparticles and phage display selected peptides for imaging and therapy of cancer.

Molecular imaging probes are a special class of pharmaceuticals that target specific biochemical signatures associated with disease and allow for noninvasive imaging on the molecular level. Because changes in biochemistry occur before diseases reach an advanced stage, molecular imaging probes make it possible to locate and stage disease, track the effectiveness of drugs, treat disease, monitor response, and select patients to allow for more personalized diagnosis and treatment of disease. Targeting agents radiolabeled with positron emitters are of interest due to their ability to quantitatively measure biodistribution and receptor expression to allow for optimal dose determinations. (68)Ga is a positron emitter, which allows for quantitative imaging through positron emission chromatography (PET). The availability of (68)Ga from a generator and its ability to form stable complexes with a variety of chelates hold promise for expanding PET utilization to facilities unable to afford their own cyclotron. Nanoparticles conjugated with various proteins and peptides derived from phage display that can be selectively targeted are being developed and evaluated for guided imaging and therapy. Herein we highlight some initial efforts in combining the enhanced selectivity of nanoparticles and peptides with (68)Ga for use as molecular imaging probes.

Inhibition of metastatic tumor formation in vivo by a bacteriophage display-derived galectin-3 targeting peptide.

Galectin-3 (gal-3) is involved in the metastatic cascade and interacts with the cancer-associated carbohydrate, Thomsen-Freidenreich (TF) antigen during early stages of metastatic adhesion and tumor formation. Our laboratory previously utilized bacteriophage display to select a peptide, G3-C12, with high specificity and affinity for gal-3 that was able to inhibit cancer cell adhesion. We hypothesized that G3-C12 would inhibit TF/gal-3 and gal-3/gal-3 interactions in vitro and in vivo and would moderate early steps of the metastatic cascade leading to reduced carcinogenesis in vivo. To test this, adhesion of multiple breast carcinoma cell lines to purified gal-3 and a TF-mimic was measured in the presence/absence of G3-C12 resulting in an average reduction of cellular adhesion by 50 and 59 %, respectively. Sensitive optical imaging experiments were utilized to monitor the fate of intravenously injected MDA-MB-231 human breast carcinoma cells expressing luciferase into athymic nude mice in the presence/absence of G3-C12 in vivo. Intravenous administration of G3-C12 reduced lung colonization of MDA-MB-231-luciferase cells within mice by 72 % when compared to saline, whereas, control peptide treatments resulted in no significant reduction of colonization. Histologic examination of excised lung tissue, at day 70, revealed that mice treated with G3-C12 possessed 4.63 ± 3.07 tumors compared to 14.13 ± 3.56 tumors within mice treated with saline. Also, within both saline and control peptide treatment groups, 37 % of mouse lungs contained tumor thrombi, compared to 0 % within the G3-C12 treatment group. This study demonstrated that G3-C12 significantly reduced metastatic cell deposition and consequent outgrowth within vasculature of mice.

A potencial theranostic agent for EGF-R expression tumors: (177)Lu-DOTA-nimotuzumab.

In this work Nimotuzumab (monoclonal antibody, recognizes the EGF-R) was radiolabeled with (177)Lu as a potential cancer therapy radiopharmaceutical. In-vitro cell binding studies and in-vivo biodistribution and imaging studies were performed to determine the radiochemical stability, targeting specificity and pharmacokinetics of the (177)Lu-labeled antibody. Nimotuzumab was derivatized with DOTA-NHS at room temperature for 2 hours. DOTA-Nimotuzumab was radiolabeled with (177)LuCl3 (15 MBq/mg) at 37°C for 1 h. The radiochemical purity was assessed by ITLC, silica gel and by RP-HPLC. Binding specificity studies were performed with EGF-R positive A431 human epithelial carcinoma and EGF-R negative MDA-MB-435 breast carcinoma cells. Biodistribution studies were performed in healthy female CD-1 mice at 1 h, 4 h, 24 h, and A431 xenografted nude mice at 10 min, 1 h, 4 h, 24 h, 48 h, and 96 h. SPECT-CT imaging studies were performed in A431 xenografted mice at 24 h post injection. DOTA-Nimotuzumab was efficiently labeled with (177) LuCl(3) at 37°C. The in vitro stability of labeled product was optimal over 24 h in buffered saline and mouse serum. Specific recognition of EGF-R by (177)Lu-DOTA-Nimotuzumab was observed in A431 cell binding studies. Biodistribution studies demonstrated increasing tumor uptake of (177)Lu-DOTA-Nimotuzumab over time, with tumor to muscle ratios of 6.26, 10.68, and 18.82 at 4 h, 24 h, and 96 h post injection. Imaging of A431 xenografted mice showed high uptake in the tumor. (177)Lu-DOTA-Nimotuzumab has the potential to be a promising therapy agent, which may be useful in the treatment of patients with EGF-R positive cancer.

(64)Cu-labeled peptide for PET of breast carcinomas expressing the Thomsen-Friedenreich carbohydrate antigen.

UNLABELLED: Thomsen-Friedenreich (TF) antigen is a disaccharide, galactose ß1-3 N-acetylgalactosamine (Galß1-3GalNAc), expressed on the cell surfaces of most human carcinomas including breast. In this study, we synthesized and evaluated the in vitro and in vivo properties of a (64)Cu-radiolabeled TF antigen-specific peptide derived from bacteriophage display for the purpose of breast tumor targeting and PET of human breast tumors in xenografted mice. METHODS: The TF antigen-specific peptide IVWHRWYAWSPASRI was synthesized with the chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) at the amino terminus, followed by a Gly-Ser-Gly (GSG) spacer. Amino acids Asp and Arg were introduced at both ends to enhance its solubility. Purified NO2A-GSG-DRD-IVWHRWYAWSPASRI-DRD (NO2A-TFpep) was radiolabeled with (64)Cu and evaluated for binding to human MDA-MB-435 breast cancer cells, 50% inhibitory concentration (IC(50)), and serum stability. In vivo pharmacokinetic and small-animal PET studies were performed using SCID mice bearing MDA-MB-435 tumor xenografts. RESULTS: (64)Cu-NO2A-TFpep bound to human MDA-MB-435 breast carcinoma cells, whereas almost no binding was observed to normal human breast 184A1 cells. The peptide exhibited an apparent IC(50) value of 70 ± 8.0 nM. In vivo biodistribution studies indicated radiolabeled peptide accumulation in tumors of MDA-MB-435 xenografted SCID mice of approximately 1.10 ± 0.20 percentage injected dose per gram (%ID/g) and 0.90 ± 0.12 %ID/g, at 0.5 and 1 h, respectively. Accumulation of radioactivity was low in other organs, with the exception of liver (1.52 ± 0.12 %ID/g) and kidneys (15.4 ± 1.73 %ID/g) at 1 h. Live imaging studies with (64)Cu-NO2A-TFpep (15 MBq) demonstrated good tumor uptake at 1 h after injection, whereas no tumor uptake was observed with a scrambled radiolabeled peptide (64)Cu-NO2A-GSG-DRD-RWSWWAVHRIPYSAI-DRD. CONCLUSION: (64)Cu-NO2A-TFpep may function as a noninvasive in vivo tumor imaging agent of human breast and other carcinomas expressing the TF carbohydrate antigen. This is the first such TF antigen-targeting peptide used in tumor imaging.