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.

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.

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.

A scalable high-throughput chemical synthesizer.

A machine that employs a novel reagent delivery technique for biomolecular synthesis has been developed. This machine separates the addressing of individual synthesis sites from the actual process of reagent delivery by using masks placed over the sites. Because of this separation, this machine is both cost-effective and scalable, and thus the time required to synthesize 384 or 1536 unique biomolecules is very nearly the same. Importantly, the mask design allows scaling of the number of synthesis sites without the addition of new valving. Physical and biological comparisons between DNA made on a commercially available synthesizer and this unit show that it produces DNA of similar quality.