Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

Lung cancer is the leading cause of cancer-related mortality worldwide. The lack of tumor specificity remains a major drawback for effective chemotherapies and results in dose-limiting toxicities. However, a ligand-mediated drug delivery system should be able to render chemotherapy more specific to tumor cells and less toxic to normal tissues. In this study, we isolated a novel peptide ligand from a phage-displayed peptide library that bound to non-small cell lung cancer (NSCLC) cell lines. The targeting phage bound to several NSCLC cell lines but not to normal cells. Both the targeting phage and the synthetic peptide recognized the surgical specimens of NSCLC with a positive rate of 75% (27 of 36 specimens). In severe combined immunodeficiency (SCID) mice bearing NSCLC xenografts, the targeting phage specifically bound to tumor masses. The tumor homing ability of the targeting phage was inhibited by the cognate synthetic peptide, but not by a control or a WTY-mutated peptide. When the targeting peptide was coupled to liposomes carrying doxorubicin or vinorelbine, the therapeutic index of the chemotherapeutic agents and the survival rates of mice with human lung cancer xenografts markedly increased. Furthermore, the targeting liposomes increased drug accumulation in tumor tissues by 5.7-fold compared with free drugs and enhanced cancer cell apoptosis resulting from a higher concentration of bioavailable doxorubicin. The current study suggests that this tumor-specific peptide may be used to create chemotherapies specifically targeting tumor cells in the treatment of NSCLC and to design targeted gene transfer vectors or it may be used one in the diagnosis of this malignancy.

Colorectal cancer is one of the most commonly diagnosed cancers and a leading cause of cancer mortality worldwide. Current treatment for colorectal cancer results in only limited success, and more effective therapeutic approaches are thus urgently needed. The development of new methods for early detection and effective treatments for cancer is contingent on the identification of biomarkers on the surface of cancer cells, as well as isolation of tumor-specific ligands with high binding affinity to such biomarkers. In vitro biopanning of a phage-displayed peptide library was used to identify specific peptides binding to human colorectal carcinoma cells. The targeting peptide pHCT74 showed the greatest potential for drug delivery in both in vitro and in vivo studies. The use of biotinylated peptides combined with an affinity trapping method and liquid chromatography-tandem mass spectrometry identified the target protein for the pHCT74 peptide as α-enolase. In animal model studies, combined pHCT74-conjugated liposomal doxorubicin (pHCT74-LD) and pHCT74-conjugated liposomal vinorelbine (pHCT74-sLV) therapy exhibited an enhanced antitumor effect and markedly extended the survival of mice with human colorectal cancer in subcutaneous and orthotopic models. Our findings indicate that α-enolase-targeted lipid nanoparticles have great potential for application in targeted drug delivery systems for colorectal cancer therapy.

Nanocarriers, such as liposomes, have the potential to increase the payload of chemotherapeutic drugs while decreasing toxicity to non-target tissues; such advantageous properties can be further enhanced through surface conjugation of nanocarriers with targeting moieties. We previously reported that SP94 peptides, identified by phage display, exhibited higher binding affinity to human hepatocellular carcinoma (HCC) than to hepatocytes and other normal cells. Here, we confirm the tumor-targeting properties of SP94 peptide by near-infrared fluorescence imaging. Non-targeted PEGylated liposomal doxorubicin (LD) and SP94-conjugated PEGylated liposomal doxorubicin (SP94-LD) were compared by assessing pharmacokinetics, tissue distribution, and antitumor efficacy in xenograft-bearing mice, in order to investigate the effectiveness of SP94-mediated targeting for cancer therapy. SP94-LD demonstrated a significant increase in drug accumulation in tumors, while its plasma residence time was the same as its non-targeted equivalent. Consistent with this result, conjugation of targeting peptide SP94 enhances the therapeutic efficacy of liposomal doxorubicin in mouse models with hepatocellular carcinoma xenografts. Furthermore, combination targeted therapy exhibited a significant enhancement against orthotopic tumor growth, and markedly extended the survival of mice compared with all other treatments. Our study shows that SP94-mediated targeting enhances antitumor efficacy by improving tumor pharmacokinetics and tissue distribution, allowing large amounts of antitumor drugs to accumulate in tumors.

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