Tumor-specific intracellular delivery: peptide-guided transport of a catalytic toxin.

There continues to be a need for cancer-specific ligands that can deliver a wide variety of therapeutic cargos. Ligands demonstrating both tumor-specificity and the ability to mediate efficient cellular uptake of a therapeutic are critical to expand targeted therapies. We previously reported the selection of a peptide from a peptide library using a non-small cell lung cancer (NSCLC) cell line as the target. Here we optimize our lead peptide by a series of chemical modifications including truncations, N-terminal capping, and changes in valency. The resultant 10 amino acid peptide has an affinity of <40 nM on four different NSCLC cell lines as a monomer and is stable in human serum for >48 h. The peptide rapidly internalizes upon cell binding and traffics to the lysosome. The peptide homes to a tumor in an animal model and is retained up to 72 h. Importantly, we demonstrate that the peptide can deliver the cytotoxic protein saporin specifically to cancer cells in vitro and in vivo, resulting in an effective anticancer agent.

Modular Three-component Delivery System Facilitates HLA Class I Antigen Presentation and CD8(+) T-cell Activation Against Tumors.

Cell-mediated immunotherapies have potential as stand-alone and adjuvant therapies for cancer. However, most current protocols suffer from one or more of three major issues: cost, safety, or efficacy. Here we present a nanoparticle delivery system that facilitates presentation of an immunogenic measles antigen specifically in cancer cells. The delivery system does not contain viral particles, toxins, or biologically derived material. Treatment with this system facilitates activation of a secondary immune response against cancer cells, bypassing the need to identify tumor-associated antigens or educate the immune system through a primary immune response. The delivery system consists of a stealth liposome displaying a cancer-specific targeting peptide, named H1299.3, on its exterior surface and encapsulating H250, an immunogenic human leukocyte antigen class 1 restricted peptide. This targeted-nanoparticle facilitates presentation of the H250 peptide in major histocompatibility complex class I molecules. Activation is dependent on the targeting peptide, previous antigen exposure, and utilizes a novel autophagy-mediated mechanism to facilitate presentation. Treatment with this liposome results in a significant reduction of tumor growth using an aggressive LLC1 model in vaccinated C57BL/6 mice. These data provide proof-of-principle for a novel cell-mediated immunotherapy that is scalable, contains no biologically derived material, and is an efficacious cancer therapy.

Introduction of plasmid encoding for rare tRNAs reduces amplification bias in phage display biopanning.

Amplification bias is a major hurdle in phage display protocols because it imparts additional, unintended selection pressure beyond binding to the desired target. One potential source of amplification bias is the inherent lack of codon optimization that occurs within phage display libraries. Here we present a method that reduces amplification bias by addition of a plasmid that encodes six low abundance tRNAs into K91 Escherichia coli. This new strain, termed K91+, is used to amplify phage during the selection process. We demonstrate the importance of rare codon usage in phage production, and our method produced an overall increase in uniformity of phage production in a random library. Both of these variables are improved in E. coli K91+ compared with the parental K91 strain. This simple solution, requiring only a commercially available plasmid and an additional antibiotic, can reduce amplification bias in phage display protocols.

Identification of a novel lysosomal trafficking peptide using phage display biopanning coupled with endocytic selection pressure.

Methods to select ligands that accumulate specifically in cancer cells and traffic through a defined endocytic pathway may facilitate rapid pairing of ligands with linkers suitable for drug conjugate therapies. We performed phage display biopanning on cancer cells that are treated with selective inhibitors of a given mechanism of endocytosis. Using chlorpromazine to inhibit clathrin-mediated endocytosis in H1299 nonsmall cell lung cancer cells, we identified two clones, ATEPRKQYATPRVFWTDAPG (15.1) and a novel peptide LQWRRDDNVHNFGVWARYRL (H1299.3). The peptides segregate by mechanism of endocytosis and subsequent location of subcellular accumulation. The H1299.3 peptide primarily utilizes clathrin-mediated endocytosis and colocalizes with Lamp1, a lysosomal marker. Conversely, the 15.1 peptide is clathrin-independent and localizes to a perinuclear region. Thus, this novel phage display scheme allows for selection of peptides that selectively internalize into cells via a known mechanism of endocytosis. These types of selections may allow for better matching of linker with targeting ligand by selecting ligands that internalize and traffic to known subcellular locations.

Dimerization of a phage-display selected peptide for imaging of αvß6- integrin: two approaches to the multivalent effect.

The integrin αvß6 is an emerging biomarker for non-small cell lung cancer (NSCLC). An αvß6-binding peptide was previously selected from a phage-displayed peptide library. Here, we utilize a multivalent design to develop a peptidic probe for positron emission tomography (PET) imaging of αvß6+ NSCLC tumors. Multimeric presentation of this peptide, RGDLATLRQL, on a bifunctional copper chelator was achieved using two approaches: dimerization of the peptide followed by conjugation to the chelator (H2-D10) and direct presentation of two copies of the peptide on the chelator scaffold (H2-(M10)2). Binding affinities of the divalent peptide conjugates are four-fold higher than their monovalent counterpart (H2-M10), suggestive of multivalent binding. PET imaging using the bivalent 64Cu-labeled conjugates showed rapid and persistent accumulation in αvß6+ tumors. By contrast, no significant accumulation was observed in αvß6- tumors. Irrespective of the dimerization approach, all divalent probes showed three-fold higher tumor uptake than the monovalent probe, indicating the role of valency in signal enhancement. However, the divalent probes have elevated uptake in non-target organs, especially the kidneys. To abrogate nonspecific uptake, the peptide's N-terminus was acetylated. The resultant bivalent probe, 64Cu- AcD10, showed drastic decrease of kidney accumulation while maintaining tumor uptake. In conclusion, we developed an αvß6-integrin specific probe with optimized biodistribution for noninvasive PET imaging of NSCLC. Further, we have demonstrated that use of multivalent scaffolds is a plausible method to improve library selected peptides, which would be suboptimal or useless otherwise, for imaging probe development.

Identification and characterization of a suite of tumor targeting peptides for non-small cell lung cancer.

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

A liposomal drug platform overrides peptide ligand targeting to a cancer biomarker, irrespective of ligand affinity or density.

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for αvß6-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to αvß6. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.

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)ß(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)ß(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)ß(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.

Synthesis and biological evaluation of a peptide-paclitaxel conjugate which targets the integrin αvß6.

The integrin α(v)ß(6) is an emergent biomarker for non-small cell lung cancer (NSCLC) as well as other carcinomas. We previously developed a tetrameric peptide, referred to as H2009.1, which binds α(v)ß(6) and displays minimal affinity for other RGD-binding integrins. Here we report the use of this peptide to actively deliver paclitaxel to α(v)ß(6)-positive cells. We synthesized a water soluble paclitaxel-H2009.1 peptide conjugate in which the 2'-position of paclitaxel is attached to the tetrameric peptide via an ester linkage. The conjugate maintains its specificity for α(v)ß(6)-expressing NSCLC cells, resulting in selective cytotoxicity. Treatment of α(v)ß(6)-positive cells with the conjugate results in cell cycle arrest followed by induction of apoptosis in the same manner as free paclitaxel. However, initiation of apoptosis and the resultant cell death is delayed compared to free drug. The conjugate demonstrates anti-tumor activity in a H2009 xenograft model of NSCLC with efficacy comparable to treatment with free paclitaxel.

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.

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.

Synthesis and characterization of a high-affinity {alpha}v{beta}6-specific ligand for in vitro and in vivo applications.

The α(v)ß(6) 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 α(v)ß(6). 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 α(v)ß(6) 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 α(v)ß(6) will find clinical use as a diagnostic and therapeutic reagent.

Epithelial-to-mesenchymal transition in the development and progression of adenocarcinoma and squamous cell carcinoma of the lung.

Epithelial-to-mesenchymal transition is a process in which cells undergo a developmental switch from an epithelial to a mesenchymal phenotype. We investigated the role of this phenomenon in the pathogenesis and progression of adenocarcinoma and squamous cell carcinoma of the lung. Archived tissue from primary tumors (n=325), brain metastases (n=48) and adjacent bronchial epithelial specimens (n=192) were analyzed for immunohistochemical expression by image analysis of E-cadherin, N-cadherin, integrin-alpha v beta 6, vimentin, and matrix metalloproteinase-9. The findings were compared with the patients' clinicopathologic features. High expression of the epithelial-to-mesenchymal transition phenotype (low E-cadherin and high N-cadherin, integrin-alpha v beta 6, vimentin, and matrix metalloproteinase-9) was found in most lung tumors examined, and the expression pattern varied according to the tumor histologic type. Low E-cadherin membrane and high N-cadherin cytoplasmic expression were significantly more common in squamous cell carcinoma than in adenocarcinoma (P=0.002 and 0.005, respectively). Dysplastic lesions had significantly lower expression of the epithelial-to-mesenchymal transition phenotype than the squamous cell carcinomas, and integrin-alpha v beta 6 membrane expression increased stepwise according to the histopathologic severity. Brain metastases had decreased epithelial-to-mesenchymal transition expression compared with primary tumors. Brain metastases had significantly lower integrin-alpha v beta 6 membrane (P=0.04), N-cadherin membrane, and cytoplasm (P<0.0002) expression than the primary tumors. The epithelial-to-mesenchymal transition phenotype is commonly expressed in primary squamous cell carcinoma and adenocarcinoma of the lung; this expression occurs early in the pathogenesis of squamous cell carcinoma. Brain metastases showed characteristics of reversed mesenchymal-to-epithelial transition. Our findings suggest that epithelial-to-mesenchymal transition is a potential target for lung cancer chemoprevention and therapy.

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 Novel Strategy for Surface Modification of Superparamagnetic Iron Oxide Nanoparticles for Lung Cancer Imaging.

Superparamagnetic iron oxide (SPIO) nanoparticles are widely used in magnetic resonance imaging (MRI) as versatile ultra-sensitive nanoprobes for cellular and molecular imaging of cancer. In this study, we report a one-step procedure for the surface functionalization of SPIO nanoparticles with a lung cancer-targeting peptide. The hydrophobic surfactants on the as-synthesized SPIO are displaced by the peptide containing a poly(ethylene glycol)-tethered cysteine residue through ligand exchange. The resulting SPIO particles are biocompatible and demonstrate high T(2) relaxivity. The nanoprobes are specific in targeting α(v)ß(6)-expressing lung cancer cells as demonstrated by MR imaging and Prussian blue staining. This facile surface chemistry and the functional design of the proposed SPIO system may provide a powerful nanoplatform for the molecular diagnosis of lung cancer.

Peptide-targeted polyglutamic acid doxorubicin conjugates for the treatment of alpha(v)beta(6)-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.

Advances in molecular imaging of pancreatic beta cells.

The development of non-invasive imaging methods for early diagnosis of beta cell associated metabolic diseases, including type 1 and type 2 diabetes (T1D and T2D), has recently drawn interest from the molecular imaging community and clinical investigators. Due to the challenges imposed by the location of the pancreas, the sparsely dispersed beta cell population within the pancreas, and the poor understanding of the pathogenesis of the diseases, clinical diagnosis of beta cell abnormalities is still limited. Current diagnostic methods are invasive, often inaccurate, and usually performed post-onset of the disease. Advances in imaging techniques for probing beta cell mass and function are needed to address this critical health care problem. A variety of imaging techniques have been tested for the assessment of pancreatic beta cell islets. Here we discuss current advances in magnetic resonance imaging (MRI), bioluminescence imaging (BLI), and nuclear imaging for the study of beta cell diseases. Spurred by early successes in nuclear imaging techniques for beta cells, especially positron emission tomography (PET), the need for beta cell specific ligands has expanded. Progress for obtaining such ligands is presented. We report our preliminary efforts of developing such a peptidic ligand for PET imaging of pancreatic beta cells.

A peptide selected by biopanning identifies the integrin alphavbeta6 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, alphavbeta6, 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 alphavbeta6 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 alphavbeta6 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.

Novel ligands for cancer diagnosis: selection of peptide ligands for identification and isolation of B-cell lymphomas.

OBJECTIVE: Lymphoma and leukemia account for nearly 8% of cancer fatalities each year. Present treatments do not differentiate between normal and malignant cells. New reagents that distinguish malignant cells and enable the isolation of these cells from the normal background will enhance the molecular characterization of disease and specificity of treatment. METHODS: Peptide ligands were selected from a phage-displayed peptide library by biopanning on the B-cell lymphoma line, A20. The isolated peptides were assessed as reagents for identification and isolation of lymphoma cells by flow cytometry and cell capture with magnetic beads. RESULTS: Two novel peptides and one obtained previously on cardiomyocytes were selected. A20 cells bind phage displaying these peptides 250- to 450-fold over control phage. These phage bind to other bone marrow-derived cancel lines including some macrophage and T cells but do not bind to normal splenocytes. Synthetic constructs of these peptides have binding affinities comparable to B-cell-specific antibodies. Similar to antibodies, these peptides can be used in flow cytometry and magnetic bead capture to distinguish lymphoma cells from normal splenocytes. CONCLUSION: Bone marrow-derived malignant cells express cell surface markers that can be used to distinguish them from normal cells. These results demonstrate the ability to use an unbiased screen to rapidly generate high-affinity peptide ligands for identification and isolation of lymphoma cells.