Kathlynn C. Brown

Peptidic Tumor Targeting Agents: The Road from Phage Display Peptide Selections to Clinical Applications

Cancer has become the number one cause of death amongst Americans, killing approximately 1,600 people per day. Novel methods for early detection and the development of effective treatments are an eminent priority in medicine. For this reason, isolation of tumor-specific ligands is a growing area of research. Tumor-specific binding agents can be used to probe the tumor cell surface phenotype and to customize treatment accordingly by conjugating the appropriate cell-targeting ligand to an anticancer drug. This refines the molecular diagnosis of the tumor and creates guided drugs that can target the tumor while sparing healthy tissues. Additionally, these targeting agents can be used as in vivo imaging agents that allow for earlier detection of tumors and micrometastasis. Phage display is a powerful technique for the isolation of peptides that bind to a particular target with high affinity and specificity. The biopanning of intact cancer cells or tumors in animals can be used as the bait to isolate peptides that bind to cancer-specific cell surface biomarkers. Over the past 10 years, unbiased biopanning of phage-displayed peptide libraries has generated a suite of cancer targeting peptidic ligands. This review discusses the recent advances in the isolation of cancer-targeting peptides by unbiased biopanning methods and highlights the use of the isolated peptides in clinical applications.

MRI-Visible Micellar Nanomedicine for Targeted Drug Delivery to Lung Cancer Cells

Polymeric micelles are emerging as a highly integrated nanoplatform for cancer targeting, drug delivery and tumor imaging applications. In this study, we describe a multifunctional micelle (MFM) system that is encoded with a lung cancer-targeting peptide (LCP), and encapsulated with superparamagnetic iron oxide (SPIO) and doxorubicin (Doxo) for MR imaging and therapeutic delivery, respectively. The LCP-encoded MFM showed significantly increased alpha(v)beta(6)-dependent cell targeting in H2009 lung cancer cells over a scrambled peptide (SP)-encoded MFM control as well as in an alpha(v)beta(6)-negative H460 cell control. (3)H-Labeled MFM nanoparticles were used to quantify the time- and dose-dependent cell uptake of MFM nanoparticles with different peptide encoding (LCP vs SP) and surface densities (20% and 40%) in H2009 cells. LCP functionalization of the micelle surface increased uptake of the MFM by more than 3-fold compared to the SP control. These results were confirmed by confocal laser scanning microscopy, which further demonstrated the successful Doxo release from MFM and accumulation in the nucleus. SPIO clustering inside the micelle core resulted in high T(2) relaxivity (>400 Fe mM(-1) s(-1)) of the resulting MFM nanoparticles. T(2)-weighted MRI images showed clear contrast differences between H2009 cells incubated with LCP-encoded MFM over the SP-encoded MFM control. An ATP activity assay showed increased cytotoxicity of LCP-encoded MFM over SP-encoded MFM in H2009 cells (IC(50) values were 28.3 +/- 6.4 nM and 73.6 +/- 6.3 nM, respectively; p < 0.005). The integrated diagnostic and therapeutic design of MFM nanomedicine potentially allows for image-guided, target-specific treatment of lung cancer.

Biopanning of Phage Displayed Peptide Libraries for the Isolation of Cell-Specific Ligands

One limitation in the development of biosensors for the early detection of disease is the availability of high specificity and affinity ligands for biomarkers that are indicative of a pathogenic process. Within the past 10 years, biopanning of phage displayed peptide libraries on intact cells has proven to be a successful route to the identification of cell-specific ligands. The peptides selected from these combinatorial libraries are often able to distinguish between diseased cells and their normal counterparts as well as cells in different activation states. These ligands are small and chemical methodologies are available for regiospecific derivatization. As such, they can be incorporated into a variety of different diagnostic and therapeutic platforms. Here we describe the methods utilized in the selection of peptides from phage displayed libraries by biopanning. In addition, we provide methods for the synthesis of the selected peptides as both monomers and tetramers. Downstream uses for the peptides are illustrated.

A Peptide Selected by Biopanning Identifies the Integrin αvβ6 as a Prognostic Biomarker for Nonsmall Cell Lung Cancer

The development of new modes of diagnosis and targeted therapy for lung cancer is dependent on the identification of unique cell surface features on cancer cells and isolation of reagents that bind with high affinity and specificity to these biomarkers. We recently isolated a 20-mer peptide which binds to the lung adenocarcinoma cell line, H2009, from a phage-displayed peptide library. We show here that the cellular receptor for this peptide, TP H2009.1, is the uniquely expressed integrin, αvβ6, and the peptide binding to lung cancer cell lines correlates to integrin expression. The peptide is able to mediate cell-specific uptake of a fluorescent nanoparticle via this receptor. Expression of αvβ6 was assessed on 311 human lung cancer samples. The expression of this integrin is widespread in early-stage nonsmall cell lung carcinoma (NSCLC). Log-rank test and Cox regression analyses show that expression of this integrin is significantly associated with poor patient outcome. Preferential expression is observed in the tumors compared with the surrounding normal lung tissue. Our data indicate that αvβ6 is a prognostic biomarker for NSCLC and may serve as a receptor for targeted therapies. Thus, cell-specific peptides isolated from phage biopanning can be used for the discovery of cell surface biomarkers, emphasizing the utility of peptide libraries to probe the surface of a cell. [Cancer Res 2007;67(12):5889–95]

New approaches for cell-specific targeting: identification of cell-selective peptides from combinatorial libraries

Peptides that recognize specific cell types promise to be valuable tools both in research and clinical applications. Cell-specific peptides can be useful as drug delivery vehicles, diagnostic agents, affinity reagents for cell purification, gene therapy delivery agents, and research tools to probe the nature of a cells surface. Recently, cell-specific targeting-peptides have been identified by phage-display selections against purified cell-surface markers, whole cells in tissue culture, and even tissues within live animals. These methods for identifying cell-targeting peptides will certainly increase the tools available to the scientist for cell-specific targeting.

Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides

The surface of a cell represents a collection of macromolecules, which provides the cell with a unique cellular landscape specific to the type and state of the cell. Ligands that discriminate between subtle differences in cell surface phenotypes have utility in a wide variety of research and clinical applications. In particular, cell-binding ligands that can deliver biologically active cargo to a specific cell type or a diseased cell are highly sought. While the concept of the magic bullet drug was introduced by Paul Erlich over a century ago, the scientific community has yet to fully realize this goal.1 This stems primarily from hurdles in obtaining high-affinity cell-binding ligands with the necessary discriminating power. The difficulty of the problem is realized by considering that the human body contains 210 distinct cell types, not including diseased cells, and is composed of ~1014 cells. Furthermore, once isolated, the ligand must be able to be prepared in large quantities, must be amenable to chemical modification for optimal in vivo biodistribution, and must be able to be tailored to suit a variety of clinical applications. As antibodies typically have high affinity and specificity for their targets, they have garnered attention as cell-targeting agents. Monoclonal antibodies (mAbs) can be generated against differentially expressed cell surface features, and the number of FDA-approved mAbs that bind to cell surface antigens continues to grow.2 mAb therapies are used to treat a variety of diseases. However, most of the clinically approved therapeutic mAbs are not conjugated to drugs or toxins and therefore fall into the category of molecularly targeted therapies. Such antibodies function passively by either blocking the activity of receptors or activating the immune system to destroy the antibody target.3 Only a few clinically approved mAbs carry a deliverable. For example, two radiolabeled antibodies, Zevalin (ibritumomab tiuxetan) and Bexxar (iodine-131 tositumomab), are approved in the United States; both are anti-CD20 antibodies used for select patients with non-Hodgkin’s lymphoma. The only clinically approved antibody–drug conjugate in the United States is Adcetris (brentuximab vedotin). Approved in 2011, Adcetris is an anti-CD30 antibody conjugated to the highly toxic microtubule-disrupting agent monomethyl auristatin E and is utilized for the treatment of Hodgkin’s lymphoma (HL) or systemic anaplastic large-cell lymphoma (sALCL). Mylotarg, a calicheamicin anti-CD33 antibody conjugate, was recently removed from the market after 10 years in the clinic for failing to show efficacy. Despite their successes, mAbs have limitations, especially in their ability to serve as delivery vehicles. Significantly, chemically modifying antibodies is challenging, and production costs are substantial. Additionally, nonspecific clearance of antibodies by the reticuloendothelial system can lead to accumulation of conjugated drugs or toxins in unwanted sites such as the liver and bone, damaging these organs.4,5 Recently, concerns have risen over post-translational modifications on mAbs, especially glycosylation, which can trigger severe hypersensitivity reactions. Due to their long in vivo half-lives, intact mAbs are not well suited for molecular imaging techniques, requiring the use of antibody fragments (Fab’s). Of the approved mAb therapies, only 11 different cell surface biomarkers are targeted. This is a minute fraction of the cell surface repertoire. Peptides are an attractive alternative to antibody-targeting therapies. Unlike antibodies, peptides are easy to synthesize in large quantities,6 and their smaller size improves tissue penetration while preventing nonspecific uptake by the reticuloendothelial system. Additionally, peptides can be chemically modified to alter affinity, charge, hydrophobicity, stability, and solubility and can be optimized for in vivo use through reiterative modifications. Importantly, peptides can display antibody-like affinities for their receptors. The biological half-life of peptides is well matched with that of many clinically used radionuclides, making them attractive probes for molecular imaging. Several naturally occurring peptides have been used as delivery agents. For example, reproductive hormone peptides and their derivatives are useful for tumor targeting, due to overexpression of their receptors on many cancer cells.7,8 However, relying on known peptidic ligands limits the types of cells that can be targeted. For this reason, chemists and biologists have turned to diverse peptide libraries to select additional peptides that bind to specific cell types.

Mechanism of the Rhodium Porphyrin-Catalyzed Cyclopropanation of Alkenes

The rhodium porphyrin-catalyzed cyclopropanation of alkenes by ethyl diazoacetate (EDA) is representative of a number of metal-mediated cyclopropanation reactions used widely in organic synthesis. The active intermediate in these reactions is thought to be a metal carbene complex, but evidence for the involvement of metal-olefin π complexes has also been presented. Low-temperature infrared and nuclear magnetic resonance spectroscopies have been used to characterize a rhodium porphyrin-diazoalkyl adduct that results from the stoichiometric condensation of the catalyst and EDA. Optical spectroscopy suggests that this complex is the dominant steady-state species in the catalytic reaction. This compound decomposes thermally to provide cyclopropanes in the presence of styrene, suggesting that the carbene is indeed the active intermediate. Metal-alkene π complexes have also been detected spectroscopically. Kinetic studies suggest that they mediate the rate of carbene formation from the diazoalkyl complex but are not attacked directly by EDA.

From Phage Display to Nanoparticle Delivery: Functionalizing Liposomes with Multivalent Peptides Improves Targeting to a Cancer Biomarker

Phage display is commonly used to isolate peptides that bind to a desired cell type. While chemical synthesis of selected peptides often results in ligands with low affinity, a multivalent tetrameric presentation of the peptides dramatically improves affinity. One of the primary uses of these peptides is conjugation to nanoparticle-based therapeutics for specific delivery to target cell types. We set out to optimize the path from phage display peptide selection to peptide presentation on a nanoparticle surface for targeted delivery. Here, we examine the effects of peptide valency, density, and affinity on nanoparticle delivery and therapeutic efficacy, using the α(v)β(6)-specific H2009.1 peptide as a model phage-selected peptide and liposomal doxorubicin as a model therapeutic nanoparticle. Liposomes displaying the higher affinity multivalent H2009.1 tetrameric peptide demonstrate 5-10-fold higher drug delivery than liposomes displaying the lower affinity monomeric H2009.1 peptide, even when the same number of peptide subunits are displayed on the liposome. Importantly, a 6-fold greater toxicity is observed toward α(v)β(6)-expressing cells for liposomes displaying tetrameric verses monomeric H2009.1 peptides. Additionally, liposomal targeting and toxicity increase with increasing concentrations of H2009.1 tetrameric peptide on the liposome surface. Thus, both the multivalent peptide and the multivalent liposome scaffold work together to increase targeting to α(v)β(6)-expressing cells. This multilayered approach to developing high affinity targeted nanoparticles may improve the utility of moderate affinity peptides. As tetramerization is known to increase affinity for a variety of phage-selected peptides, it is anticipated that the tetrameric scaffold may act as a general method for taking peptides from phage display to nanoparticle display.

Peptide-Targeted Polyglutamic Acid Doxorubicin Conjugates for the Treatment of αvβ6-Positive Cancers

Most chemotherapeutics exert their effects on tumor cells as well as their healthy counterparts, resulting in dose limiting side effects. Cell-specific delivery of therapeutics can increase the therapeutic window for treatment by maintaining the therapeutic efficacy while decreasing the untoward side effects. We have previously identified a peptide, named H2009.1, which binds to the integrin alpha(v)beta(6). Here, we report the synthesis of a peptide targeted polyglutamic acid polymer in which the high affinity alpha(v)beta(6)-specific tetrameric H2009.1 peptide is incorporated via a thioether at the N-terminus of a 15 amino acid polymer of glutamic acid. Doxorubicin is incorporated into the polymer via an acid-labile hydrazone bond. Payloads of four doxorubicin molecules per targeting agent are achieved. The drug is released at pH 4.0 and 5.6 but the conjugate is stable at pH 7.0. The conjugate is selectively internalized into alpha(v)beta(6) positive cells as witnessed by flow cytometric analysis and fluorescent microscopy. Cellular uptake is mediated by the H2009.1 peptide, as no internalization of the doxorubicin-PG polymer is observed when it is conjugated to a scrambled sequence control peptide. Importantly, the conjugate is more cytotoxic toward a targeted cell than a cell line that does not express the integrin.

Synthesis and characterization of a high-affinity αvβ6-specific ligand for in vitro and in vivo applications

The αvβ6 integrin is an attractive therapeutic target for several cancers due to its role in metastasis and its negligible expression in normal tissues. We previously identified a peptide from a phage-displayed peptide library that binds specifically to αvβ6. The tetrameric version of the peptide has higher affinity for its cellular targets than the corresponding monomers. However, the inefficient synthesis limits its clinical potential. We report here a convergent synthesis producing the tetrameric peptide in high yield and purity. The ease of the synthesis allows for rapid optimization of the peptide. We have optimized this αvβ6 integrin–binding peptide, determining the minimal binding domain and valency. Importantly, the half-maximal binding affinity of the optimal peptide for its target cell is in the 40 to 60 pmol/L range, rivaling the affinity of commonly used antibody-targeting reagents. This peptide mediates cell-specific uptake, is functional in diagnostic formats, is stable in sera, and can home to a tumor in an animal. We anticipate that this high-affinity ligand for αvβ6 will find clinical use as a diagnostic and therapeutic reagent. [Mol Cancer Ther 2009;8(5):1239–49]