Peptide-Mediated Targeting to Tumor Blood Vessels of Lung Cancer for Drug Delivery Tong-Young Lee, 1 Chin-Tarng Lin, 1,2 Szu-Yao Kuo, 3 De-Kuan Chang, 1,3 and Han-Chung Wu 1,3 1 Instituteof Pathology, College of Medicine, National Taiwan University; 2 Department of Pathology, National Taiwan University Hospital; and 3 Institute of Cellularand Organismic Biology, Academia Sinica; Taipei, Taiwan Abstract Antiangiogenesis therapies for the treatment of cancers hold the promise of high efficacy and low toxicity. In vivo phage display was used to identify peptides specifically targeting tumor blood vessels. The peptide SP5-52 recognized tumor neovasculature but not normal blood vessels in severe combined immunodeficiency mice bearing human tumors. Synthetic peptide was shown to inhibit the binding of PC5-52 phage particles to the tumor mass in the competitive inhibition assay. Several selected phage clones displayed the consensus motif, proline-serine-proline, and this motif was crucial for peptide binding to the tumor neovasculature. SP5- 52 peptides also bound vascular endothelial growth factor– stimulated human umbilical vein endothelial cells and blood vessels of human lung cancer surgical specimens. Further- more, this targeting phage was shown to home to tumor tissues from eight different types of human tumor xenografts following in vivo phage display experiments. An SP5-52 peptide-linked liposome carrying doxorubicin enhanced the therapeutic efficacy of the drug, markedly decreased tumor blood vessels, and resulted in higher survival rates of human lung and oral cancer–bearing xenograft mice. The current study indicates that ligand-targeted therapy offers improved therapeutic effects over conventional anticancer drug therapy, and that the peptide SP5-52 specifically targets tumor neovasculature and is a good candidate for targeted drug delivery to solid tumors. [Cancer Res 2007;67(22):10958–65] Introduction During tumor progression, tumor cells require increased supplies of oxygen and metabolites, and the efficient removal of waste products. In fact, establishing access to the host vascular system and the generation of a tumor blood supply are the rate- limiting steps of the tumor progression process (1). Tumor blood vessels are prime targets for therapies involving the inhibition of tumorgrowth,andthesevesselsexpressspecificmarkerswhichare absentinthebloodvesselsofnormaltissues.Manyofthesespecific markermolecules,selectivelyexpressedintumorbloodvessels,are proteins associated with tumor-induced angiogenesis, the sprout- ing of new blood vessels (2). These include the cell adhesion receptors,integrins, a v h 3 and a v h 5 ,whichareoverexpressedinthe tumor vasculature (3). Indeed, a v h 3 was recognized by an RGD peptide previously identified by in vivo phage display for tumor homing(4).Certainpeptideligandsspecifictotheseintegrinshave already been tested for the targeted delivery of anticancer and antiangiogenic agents (5). The finding of heterogeneity in the vasculature of normal and cancerous tissues provides promising opportunities for the targeted delivery of therapies. Conventional chemotherapy is limited by the toxicity of the drugs to normal tissues. However, if these drugs could be preferentially directed to tumor sites or vessels instead of normal tissues, the toxic effects would be limited to the intended tumor tissue. Most small-molecule chemotherapeutic agents have a large volumeofdistributiononi.v.administration(6),oftenleadingtoa narrowtherapeuticindexduetothehightoxicitytonormaltissues. However,throughencapsulationofthedrugsinamacromolecular carrier, such as liposomes, the volume of distribution is signi- ficantly reduced and the concentration of drug in the tumor is increased (7). Liposomal doxorubicin represents a new class of chemotherapy delivery system that significantly improves the therapeutic index of doxorubicin in both preclinical (8–10) and clinical pharmacokinetics (11–13). This decreases the amount and typesofnonspecifictoxicityandincreasestheamountofdrugthat can be effectively delivered to the tumor site (8, 14). A tumor-targeting ligand may be a more effective method of directing liposomes containing drugs to the tumor site, increasing thetherapeuticefficacyofliposomaldrugs.Forsolidmalignancies, which comprise >90% of human cancers, antibodies recognizing tumor-specific antigens have thus far provided little use for drug delivery because the immunoconjugates are too large to penetrate the tumor tissue (15, 16). However, phage-displayed peptide libraries have allowed the identification of small peptides a hundredth the mass of IgG antibodies, which might facilitate the development of such intervention strategies. Phage-displayed random peptide libraries provide opportunities to map B-cell epitopes (17–20) and protein-protein contacts (21–23), and to select bioactive peptides bound to receptors (24, 25) or proteins (22, 26–28). They can be used to search for disease-specific antigen mimics (29–31), and to determine cell- specific (32, 33) and organ-specific peptides (5, 28, 34). Screening phage-displayed peptide libraries against specific target tissues is therefore a direct and fast method for identifying peptide sequences which might be used for the targeting of drug or gene delivery vectors. In this study, we used a phage-displayed random peptidelibrarytoidentifytargetingpeptidesthatcouldspecifically bindtothetumorvesselsofseveraltumortypes.Whencoupledto liposomes containing the anticancer drug doxorubicin (Lipo-Dox), one targeting peptide, SP5-52, enhanced the efficacy of the drug against both human lung and oral cancer xenografts in severe combined immunodeficiency (SCID) mice. Our results indicate that this peptide has clinical potential as a drug delivery agent in the treatment of these tumors. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). T-Y. Lee, C-T. Lin, and S-Y Kuo contributed equally to thiswork. Requests for reprints: Han-Chung Wu, Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan. Phone: 886-22789-9558; Fax: 886-22789-8059; E-mail: hcw0928@gate.sinica. edu.tw. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-2233 Cancer Res 2007; 67: (22). November 15, 2007 10958 www.aacrjournals.org Research Article