In vivo phage display for screening peptides specifically target to radiation- or drug- treated tumors. 

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... tumor blood vessels. Low oxygen environment will promote tumor angiogenesis, metastasis and render tumors resistant to radiation treatment (Tatum et al., 2006). Therefore, the tumor vasculature structure and oxygen level are valuable biomarkers for prognosis after treatment. 18 F-fluoromisonidazole is the most widely used PET tracer for detecting tumor hypoxia. After uptake in cell, it is reduced and binds selectively to macromolecules under hypoxic conditions (Team, 2005a). One recent study indicates that 18 F-fluoromisonidazole uptake is correlated with radiation treatment outcome in Head and neck cancer (Thorwarth et al., 2005). As to the tumor vasculature, several studies have been proposed using two different techniques - quantified power Doppler sonography or Dynamic contrast-enhanced MRI (DCE-MRI). And both showed promising results (Hormigo et al., 2007; Kim et al., 2006; Mangla et al., 2010). Since its recognition as one of the major forms of cell death after radiation, apoptosis is being increasingly studied as a biomarker of cellular radiosensitivity and a prognosis marker for radiotherapy outcome. During the apoptosis process, phosphatidylserine (PS) flips from the inner leaflet of the cell membrane to the exterior of the cells. Annexin V, a cellular protein of the Annexin family, binds to the exposed PS. To date, Annexin V has been fluorinated for PET and radioiodinated for SPECT. Annexin V labeled with 99mTc has demonstrated significant uptake in patients suffering form myocardial infarction (Narula et al., 2001). Studies assessing quantitative 99m Tc-Annexin V uptake in human tumors and their relationship to radiotherapy outcome are underway. Recently, advances in phage display-related technologies facilitate the use of small peptide derivatives as probe molecules for recognition and targeting tumors. Phage display enables discovery and optimization of affinity probes for the known tumor-specific biomarkers. Furthermore, this technology makes it possible to de novo discover novel imaging probes, and eventually identify novel diagnostic markers or therapeutic targets of cancer. In vivo screening against heterogeneous tumor targets have generated a diverse group of peptides for cancer-targeted delivery of imaging or therapeutic agents. A phage is a type of viruses that infect bacteria. Typically, phages consist of a protein capsid enclosing genetic materials. Due to its simple structure, phages have been developed into a powerful tool in biological studies. Phage display was originally invented by George P. Smith in 1985 when he demonstrated the display of exogenous peptides on the surface of filamentous phage by fusing the DNA of the peptide on to the capsid gene of filamentous phages (Smith, 1985) (Fig. 1). This technology was further developed and improved to display large proteins such as enzymes and antibodies (Fernandez-Gacio et al., 2003; Han et al., 2004). The connection between genotype and phenotype enables large libraries of peptides or proteins to be screened in a relative fast and economic way. The most common phages used in phage display are M13 filamentous phage and T7 phage (Krumpe et al., 2006; Smith and Petrenko, 1997). The functional moiety on the phage surface can be short peptides, recombinant proteins, engineered antibody fragments or scaffold proteins. Screening can be conducted on the purified organic or inorganic materials, cells, or tissues. The application of phage display technology include determination of binding partners of organic (proteins, polysaccharides, or DNAs) (Gommans et al., 2005) or inorganic materials (Hattori et al., 2010; Whaley et al., 2000). The technique is also used to study enzyme evolution in vitro for engineering biocatalysts (Pedersen et al., 1998). Phage display has been widely applied in drug discovery. It can be used for finding new ligands, such as enzyme inhibitors, receptor agonists and antagonists, to target proteins (Hariri et al., 2008; Pasqualini et al., 1995; Perea et al., 2004; Ruoslahti, 1996; Uchino et al., 2005). Invention of antibody phage display revolutionized the drug discovery (Han et al., 2004). Millions of different single chain antibodies on phages are used for isolating highly specific therapeutic antibody leads. One of the most successful examples was adalimumab (Abbott Laboratories), the first fully human antibody targeted to TNF alpha (Spector and Lorenzo, 1975). Because isolating or producing recombinant membrane proteins for use as target molecules in phage library screening is often facing insurmountable obstacles, innovative selection strategies such as panning against whole cells or tissues were devised (Jaboin et al., 2009; Molek et al., 2011; Pasqualini and Ruoslahti, 1996). Due to cells inside the body may express different surface markers and possess different characteristics from cell lines in culture, in vivo phage bio-panning was developed to identify more physiologically relevant biomarkers (Fig. 2) (Pasqualini and Ruoslahti, 1996). Since its invention, in vivo phage display has been used extensively to screen for novel targets for tumor therapy. Majority of those studies focused on analyzing the structure and molecular diversity of tumor vasculature and selecting tumor stage- and type-specific markers on tumor blood vessels (Arap et al., 2002; Rajotte and Ruoslahti, 1999; Sugahara et al., 2010; Valadon et al., 2006). Recently, the use of this technique was expanded to the field of discovering new biomarkers for evaluation of cancer treatment efficacy. (Han et al., 2008; Passarella et al., 2009). Antibodies, especially monoclonal antibodies, have been successfully utilized as cancer- targeting therapeutics and diagnostics due to their high target specificity and affinity. However, due to antibody large size (150 kDa) and limited tissue permeability, non-specific uptake into the reticuloendothelial system, and immunogenicity, most antibody-based therapeutics are of limited efficacy (Lin et al., 2005; Stern and Herrmann, 2005). In contrast to antibodies, peptides are much smaller molecules (1-2 kDa). Peptides have favorable biodistribution profiles compared to antibody, characterized by high uptake in the tumor tissue and rapid clearance from the blood. In addition, peptides have increased capillary permeability, allowing more efficient penetration into tumor tissues. Also peptides are easy to make and safe to use, they will not elicit an immune response (Ladner et al., 2004). With all these advantages, peptides have been increasingly considered as a good tumor targeted imaging probe (Aloj and Morelli, 2004; Okarvi, 2004; Reubi and Maecke, 2008). To date, a large number of peptides derived from natural proteins have already been successfully identified and characterized for tumor targeting and tumor imaging, such as integrin (RGD), somatostatin, gastrin-releasing peptide, cholecystokinin, glucagon-like peptides-1 and neuropeptide-Y (Cai et al., 2008; Hallahan et al., 2003; Korner et al., 2007; Miao and Quinn, 2007; Reubi, 2003; Reubi, 2007). A list of a few tumor homing peptides isolated using phage display technique is shown in Table 1. been used for magnetic resonance imaging (MRI) (Frangioni, 2003; Reubi and Maecke, 2008). Peptides can also be conjugated to other tumor targeted polymers or nanoparticles and dramatically increase their tumor targeted selectivity and efficiency (Hariri et al., 2010; Lowery et al., 2010; Passarella et al., 2009). In our recent studies, we employed in vivo phage display technique and intended to identify peptides that will specifically home to radiation or drug treatment responsive tumors (Han et al., 2008; Passarella et al., 2009). During the studies, we first treated tumors in mice with radiation and tyrosine kinase inhibitors. Then a peptide phage library was injected from the tail vein of tumor bearing mice for tumor binding screening. After several rounds of in vivo screening and enrichment of phages isolated from the treated tumors (Fig. 2), one phage clone, encoding HVGGSSV peptides, was identified preferentially target to treatment responsive tumors. The binding preference of those phages were confirmed by fluorescence labeled phage or peptide imaging (Han et al., 2008; Passarella et al., 2009). To explore HVGGSSV peptide’s clinical application in noninvasive imaging of tumor response to treatment, fluorescent labeled HVGGSSV peptide were used to target human tumors in several mouse models. Optical imaging studies indicated that the signal intensities of peptide binding within tumors correlate to the overall efficacy of treatment regimens on tumor growth control in multiple tumor models that had been treated with a variety of RTK inhibitors with or without combination of radiation (Han et al., 2008). SPECT/CT provides high spatial resolution and sensitivity in functional imaging. We employed this imaging modality to detect tumor response to radiation by using the HVGGSSV peptide. The mice were treated with radiation alone or combination of radiation and one TKI - Sunitinib (40 mg/Kg,). After the treatment, the HVGGSSV peptide complexed with 125 I-labeled streptavidin was selectively targeted to the tumors treated with radiation or radiation combined with Sunitinib. High resolution SPECT/CT images (Fig. 4) also showed that majority of the imaging probes were located in the peripheral area of the tumors that were treated with radiation alone. However, treatment with radiation and Sunitinib extended the imaging probe binding to both the peripheral and central parts of the subcutaneous tumors. This data might reflect the radiosensitization effect of Sunitinib. The tumor targeting potential of HVGGSSV peptide has been further explored in several drug delivery studies. In these studies, HVGGSSV peptide has been conjugated to different nanoparticles, such as liposome, FePt, and nanoparticle albumin bound (nab) (Hariri et al., 2011; Hariri et al., 2010; ...

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... be screened in a relative fast and economic way. The most common phages used in phage display are M13 filamentous phage and T7 phage (Krumpe et al., 2006; Smith and Petrenko, 1997). The functional moiety on the phage surface can be short peptides, recombinant proteins, engineered antibody fragments or scaffold proteins. Screening can be conducted on the purified organic or inorganic materials, cells, or tissues. The application of phage display technology include determination of binding partners of organic (proteins, polysaccharides, or DNAs) (Gommans et al., 2005) or inorganic materials (Hattori et al., 2010; Whaley et al., 2000). The technique is also used to study enzyme evolution in vitro for engineering biocatalysts (Pedersen et al., 1998). Phage display has been widely applied in drug discovery. It can be used for finding new ligands, such as enzyme inhibitors, receptor agonists and antagonists, to target proteins (Hariri et al., 2008; Pasqualini et al., 1995; Perea et al., 2004; Ruoslahti, 1996; Uchino et al., 2005). Invention of antibody phage display revolutionized the drug discovery (Han et al., 2004). Millions of different single chain antibodies on phages are used for isolating highly specific therapeutic antibody leads. One of the most successful examples was adalimumab (Abbott Laboratories), the first fully human antibody targeted to TNF alpha (Spector and Lorenzo, 1975). Because isolating or producing recombinant membrane proteins for use as target molecules in phage library screening is often facing insurmountable obstacles, innovative selection strategies such as panning against whole cells or tissues were devised (Jaboin et al., 2009; Molek et al., 2011; Pasqualini and Ruoslahti, 1996). Due to cells inside the body may express different surface markers and possess different characteristics from cell lines in culture, in vivo phage bio-panning was developed to identify more physiologically relevant biomarkers (Fig. 2) (Pasqualini and Ruoslahti, 1996). Since its invention, in vivo phage display has been used extensively to screen for novel targets for tumor therapy. Majority of those studies focused on analyzing the structure and molecular diversity of tumor vasculature and selecting tumor stage- and type-specific markers on tumor blood vessels (Arap et al., 2002; Rajotte and Ruoslahti, 1999; Sugahara et al., 2010; Valadon et al., 2006). Recently, the use of this technique was expanded to the field of discovering new biomarkers for evaluation of cancer treatment efficacy. (Han et al., 2008; Passarella et al., 2009). Antibodies, especially monoclonal antibodies, have been successfully utilized as cancer- targeting therapeutics and diagnostics due to their high target specificity and affinity. However, due to antibody large size (150 kDa) and limited tissue permeability, non-specific uptake into the reticuloendothelial system, and immunogenicity, most antibody-based therapeutics are of limited efficacy (Lin et al., 2005; Stern and Herrmann, 2005). In contrast to antibodies, peptides are much smaller molecules (1-2 kDa). Peptides have favorable biodistribution profiles compared to antibody, characterized by high uptake in the tumor tissue and rapid clearance from the blood. In addition, peptides have increased capillary permeability, allowing more efficient penetration into tumor tissues. Also peptides are easy to make and safe to use, they will not elicit an immune response (Ladner et al., 2004). With all these advantages, peptides have been increasingly considered as a good tumor targeted imaging probe (Aloj and Morelli, 2004; Okarvi, 2004; Reubi and Maecke, 2008). To date, a large number of peptides derived from natural proteins have already been successfully identified and characterized for tumor targeting and tumor imaging, such as integrin (RGD), somatostatin, gastrin-releasing peptide, cholecystokinin, glucagon-like peptides-1 and neuropeptide-Y (Cai et al., 2008; Hallahan et al., 2003; Korner et al., 2007; Miao and Quinn, 2007; Reubi, 2003; Reubi, 2007). A list of a few tumor homing peptides isolated using phage display technique is shown in Table 1. been used for magnetic resonance imaging (MRI) (Frangioni, 2003; Reubi and Maecke, 2008). Peptides can also be conjugated to other tumor targeted polymers or nanoparticles and dramatically increase their tumor targeted selectivity and efficiency (Hariri et al., 2010; Lowery et al., 2010; Passarella et al., 2009). In our recent studies, we employed in vivo phage display technique and intended to identify peptides that will specifically home to radiation or drug treatment responsive tumors (Han et al., 2008; Passarella et al., 2009). During the studies, we first treated tumors in mice with radiation and tyrosine kinase inhibitors. Then a peptide phage library was injected from the tail vein of tumor bearing mice for tumor binding screening. After several rounds of in vivo screening and enrichment of phages isolated from the treated tumors (Fig. 2), one phage clone, encoding HVGGSSV peptides, was identified preferentially target to treatment responsive tumors. The binding preference of those phages were confirmed by fluorescence labeled phage or peptide imaging (Han et al., 2008; Passarella et al., 2009). To explore HVGGSSV peptide’s clinical application in noninvasive imaging of tumor response to treatment, fluorescent labeled HVGGSSV peptide were used to target human tumors in several mouse models. Optical imaging studies indicated that the signal intensities of peptide binding within tumors correlate to the overall efficacy of treatment regimens on tumor growth control in multiple tumor models that had been treated with a variety of RTK inhibitors with or without combination of radiation (Han et al., 2008). SPECT/CT provides high spatial resolution and sensitivity in functional imaging. We employed this imaging modality to detect tumor response to radiation by using the HVGGSSV peptide. The mice were treated with radiation alone or combination of radiation and one TKI - Sunitinib (40 mg/Kg,). After the treatment, the HVGGSSV peptide complexed with 125 I-labeled streptavidin was selectively targeted to the tumors treated with radiation or radiation combined with Sunitinib. High resolution SPECT/CT images (Fig. 4) also showed that majority of the imaging probes were located in the peripheral area of the tumors that were treated with radiation alone. However, treatment with radiation and Sunitinib extended the imaging probe binding to both the peripheral and central parts of the subcutaneous tumors. This data might reflect the radiosensitization effect of Sunitinib. The tumor targeting potential of HVGGSSV peptide has been further explored in several drug delivery studies. In these studies, HVGGSSV peptide has been conjugated to different nanoparticles, such as liposome, FePt, and nanoparticle albumin bound (nab) (Hariri et al., 2011; Hariri et al., 2010; Lowery et al., 2011), and selectively targeted those nanoparticles to irradiated tumors. One study also showed >5-fold increase in paclitaxel levels within irradiated tumors in HVGGSSV-nab-paclitaxel-treated groups and significantly increase tumor growth delay as compared with controls (Hariri et al., 2010). To understand the physiology underlines peptides binding, we need to identify the molecular targets of peptides. However, peptides are usually unstable. Their surface charges and structures will change dramatically in different environment. And peptides usually interact with their targets with low binding affinity due to their small sizes. Therefore, traditional affinity purification methods are of little use because of high background of non- specific binding. To date, there are very few identified receptors for peptides in contrast to the great number of discovered cancer targeting peptides (Sugahara et al., 2009). New strategies are needed for identifying peptide’s receptors. In our recent studies of one peptide (HVGGSSV), we utilized a phage cDNA library screening to search for peptide’s receptors. Because several rounds of phage display screening can significantly enrich the low-affinity or low-abundance proteins, we successfully identified a PDZ protein - TIP-1 as the target of HVGGSSV peptide (Wang et al., 2010). Through the PDZ domain, TIP-1 binds to the classic C-terminal PDZ motif within the HVGGSSV peptide. One TIP-1-specific antibody that inhibited the in vitro interaction between TIP-1 and the HVGGSSV peptide attenuated the peptide’s accumulation within irradiated tumors. Imaging with TIP-1-specific antibody recapitulated the pattern of peptide imaging in tumor-bearing mice. Mutation in the classic PDZ binding motif of the HVGGSSV peptide destroyed the specific binding within irradiated tumors. These results also demonstrated the potentials of screening phage-displayed cDNA library in discovery of molecular targets of the peptides with a simple structure and low affinity. With a TIP-1 specific antibody, it was further identified that radiation induced translocation of the basically intracellular TIP-1 protein onto the cell surface in a dose-dependent manner. The treatment-induced TIP-1 expression on the cell surface is detectable in the first few hours after the treatment and before the onset of treatment associated apoptosis or cell death. Majority of the cells expressing TIP-1 on the cell surface are the live albeit such cells are less potent in proliferation and more susceptible to subsequent radiation treatment (Wang et al., 2010). The increased susceptibility to the subsequent irradiation might explain why the peptide binding is predictive in assessing tumor overall responsiveness in the early stage of treatment course. The treatment-inducible TIP-1 translocation before the onset of cell apoptosis or death further suggests potentials of the HVGGSSV peptide in non-invasive imaging assessment of tumor response to radiation and tyrosine kinase inhibitors. The ...