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.
A comprehensive overview of combinatorial peptide libraries that are used to select cell-binding peptides is offered. The pros and cons of peptide libraries are also detailed.
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 αvβ6-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 αvβ6. 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.
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 αvβ6-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 αvβ6-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 αvβ6-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.
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.
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.
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 αvβ6-dependent cell targeting in H2009 lung cancer cells over a scrambled peptide (SP)-encoded MFM control as well as in an αvβ6-negative H460 cell control. 3H-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 (IC50 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.
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 αvβ6. Here, we report the synthesis of a peptide targeted polyglutamic acid polymer in which the high affinity αvβ6-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 αvβ6 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.
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
. 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
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.
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.
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