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PLOS ONE  2014 

Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

DOI: 10.1371/journal.pone.0085349

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The urokinase plasminogen activator receptor (uPAR) plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268–275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR) regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM), a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.


[1]  Friedl P, Wolf K (2003) Tumour-cell invasion and migration: Diversity and escape mechanisms. Nature Reviews Cancer 3: 362–374.
[2]  Hood JD, Cheresh DA (2002) Role of integrins in cell invasion and migration. Nature Reviews Cancer 2: 91–100.
[3]  Degryse B (2011) The Urokinase Receptor System as Strategic Therapeutic Target: Challenges for the 21(st) Century. Current Pharmaceutical Design 17: 1872–1873.
[4]  Hildenbrand R, Allgayer H, Marx A, Stroebel P (2010) Modulators of the urokinase-type plasminogen activation system for cancer. Expert Opinion on Investigational Drugs 19: 641–652.
[5]  Kwaan HC, McMahon B (2009) The Role of Plasminogen-Plasmin System in Cancer. In: Green DKHC, editor. Coagulation in Cancer. pp. 43–66.
[6]  Mazar AP (2008) Urokinase plasminogen activator receptor choreographs multiple ligand interactions: Implications for tumor progression and therapy. Clinical Cancer Research 14: 5649–5655.
[7]  Mazzieri R, Masiero L, Zanetta L, Monea S, Onisto M, et al. (1997) Control of type IV collagenase activity by components of the urokinase-plasmin system: A regulatory mechanism with cell-bound reactants. Embo Journal 16: 2319–2332.
[8]  Murphy G, Atkinson S, Ward R, Gavrilovic J, Reynolds JJ (1992) THE ROLE OF PLASMINOGEN ACTIVATORS IN THE REGULATION OF CONNECTIVE-TISSUE METALLOPROTEINASES. In: Brakman PKC, editor. Plasminogen Activation in Fibrinolysis, in Tissue Remodeling, and in Development. pp. 1–12.
[9]  Lyons RM, Gentry LE, Purchio AF, Moses HL (1990) Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin. J Cell Biol 110: 1361–1367.
[10]  Naldini L, Vigna E, Bardelli A, Follenzi A, Galimi F, et al. (1995) Biological activation of pro-HGF (hepatocyte growth factor) by urokinase is controlled by a stoichiometric reaction. J Biol Chem 270: 603–611.
[11]  Park JE, Keller GA, Ferrara N (1993) The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell 4: 1317–1326.
[12]  Rifkin DB, Moscatelli D, Bizik J, Quarto N, Blei F, et al. (1990) Growth factor control of extracellular proteolysis. Cell Differ Dev 32: 313–318.
[13]  Schmitt M, Janicke F, Moniwa N, Chucholowski N, Pache L, et al. (1992) Tumor-Associated Urokinase-Type Plasminogen-Activator - Biological And Clinical-Significance. Biological Chemistry Hoppe-Seyler 373: 611–622.
[14]  Waltz DA, Natkin LR, Fujita RM, Wei Y, Chapman HA (1997) Plasmin and plasminogen activator inhibitor type 1 promote cellular motility by regulating the interaction between the urokinase receptor and vitronectin. Journal of Clinical Investigation 100: 58–67.
[15]  Chaurasia P, Aguirre-Ghiso JA, Liang OD, Gardsvoll H, Ploug M, et al. (2006) A region in urokinase plasminogen receptor domain III controlling a functional association with alpha 5 beta 1 integrin and tumor growth. Journal of Biological Chemistry 281: 14852–14863.
[16]  Ghosh S, Johnson JJ, Sen R, Mukhopadhyay S, Liu YY, et al. (2006) Functional relevance of urinary-type plasminogen activator receptor-alpha 3 beta 1 integrin association in proteinase regulatory pathways. Journal of Biological Chemistry 281: 13021–13029.
[17]  Xue W, Mizukami I, Todd RF, Petty HR (1997) Urokinase-type plasminogen activator receptors associate with beta(1) and beta(3) integrins of fibrosarcoma cells: Dependence on extracellular matrix components. Cancer Research 57: 1682–1689.
[18]  Hildenbrand R, Wolf G, Bohme B, Bleyl U, Steinborn A (1999) Urokinase plasminogen activator receptor (CD87) expression of tumor-associated macrophages in ductal carcinoma in situ, breast cancer, and resident macrophages of normal breast tissue. Journal of Leukocyte Biology 66: 40–49.
[19]  Pyke C, Ralfkiaer E, Ronne E, Hoyerhansen G, Kirkeby L, et al. (1994) IMMUNOHISTOCHEMICAL DETECTION OF THE RECEPTOR FOR UROKINASE PLASMINOGEN-ACTIVATOR IN HUMAN COLON-CANCER. Histopathology 24: 131–138.
[20]  Thomas C, Wiesner C, Melchior SW, Schmidt F, Gillitzer R, et al. (2009) Urokinase-plasminogen-activator receptor expression in disseminated tumour cells in the bone marrow and peripheral blood of patients with clinically localized prostate cancer. Bju International 104: 29–34.
[21]  Cantero D, Friess H, Deflorin J, Zimmermann A, Brundler MA, et al. (1997) Enhanced expression of urokinase plasminogen activator and its receptor in pancreatic carcinoma. British journal of cancer 75: 388–395.
[22]  Kenny HA, Leonhardt P, Ladanyi A, Yamada SD, Montag A, et al. (2011) Targeting the Urokinase Plasminogen Activator Receptor Inhibits Ovarian Cancer Metastasis. Clinical Cancer Research 17: 459–471.
[23]  He C, He P, Liu LP, Zhu YS (2001) Analysis of expressions of components in the plasminogen activator system in high- and low-metastatic human lung cancer cells. Journal of Cancer Research and Clinical Oncology 127: 180–186.
[24]  Yamamoto M, Sawaya R, Mohanam S, Rao VH, Bruner JM, et al. (1994) Expression and localization of urokinase-type plasminogen-activator receptor in human gliomas. Cancer Research 54: 5016–5020.
[25]  Bene MC, Castoldi G, Knapp W, Rigolin GM, Escribano L, et al. (2004) CD87 (urokinase-type plasminogen activator receptor), function and pathology in hematological disorders: a review. Leukemia 18: 394–400.
[26]  Bauer TW, Liu W, Fan F, Camp ER, Yang A, et al. (2005) Targeting of urokinase plasminogen activator receptor in human pancreatic carcinoma cells inhibits c-Met- and insulin-like growth factor-I receptor-mediated migration and invasion and orthotopic tumor growth in mice. Cancer Res 65: 7775–7781.
[27]  Rabbani SA, Ateeq B, Arakelian A, Valentino ML, Shaw DE, et al. (2010) An Anti-Urokinase Plasminogen Activator Receptor Antibody (ATN-658) Blocks Prostate Cancer Invasion, Migration, Growth, and Experimental Skeletal Metastasis In Vitro and In Vivo. Neoplasia 12: 778–788.
[28]  Van Buren GII, Gray MJ, Dallas NA, Xia L, Lim SJ, et al. (2009) Targeting the Urokinase Plasminogen Activator Receptor With a Monoclonal Antibody Impairs the Growth of Human Colorectal Cancer in the Liver. Cancer 115: 3360–3368.
[29]  Wei Y, Czekay RP, Robillard L, Kugler MC, Zhang F, et al. (2005) Regulation of alpha 5 beta 1 integrin conformation and function by urokinase receptor binding. Journal of Cell Biology 168: 501–511.
[30]  Barinka C, Parry G, Callahan J, Shaw DE, Kuo A, et al. (2006) Structural basis of interaction between urokinase-type plasminogen activator and its receptor. J Mol Biol 363: 482–495.
[31]  Zhou A, Huntington JA, Pannu NS, Carrell RW, Read RJ (2003) How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration. Nat Struct Biol 10: 541–544.
[32]  Bdeir K, Kuo A, Sachais BS, Rux AH, Bdeir Y, et al. (2003) The kringle stabilizes urokinase binding to the urokinase receptor. Blood 102: 3600–3608.
[33]  Huai Q, Mazar AP, Kuo A, Parry GC, Shaw DE, et al. (2006) Structure of human urokinase plasminogen activator in complex with its receptor. Science 311: 656–659.
[34]  Li Y, Parry G, Chen L, Callahan JA, Shaw DE, et al. (2007) An anti-urokinase plasminogen activator receptor (uPAR) antibody: crystal structure and binding epitope. J Mol Biol 365: 1117–1129.
[35]  Otwinowski Z, Minor W (1997) Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods Enzymol. New York: Academic Press. pp. 307–326.
[36]  Vagin A, Teplyakov A (1997) MOLREP: an automated program for molecular replacement. J Appl Cryst 30: 1022–1025.
[37]  CCP4 (1994) The CCP4 suite; Programs for protein crystallography. Acta Cryst D50: 760–763.
[38]  Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallographica Section D-Biological Crystallography 60: 2126–2132.
[39]  Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: 240–255.
[40]  Painter J, Merritt EA (2006) TLSMD web server for the generation of multi-group TLS models. Journal of Applied Crystallography 39: 109–111.
[41]  Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, et al. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54: 905–921.
[42]  Jones TA, Zou J-Y, Cowan SW, Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the locations of errors in three models. Acta Crystallogr A47: 110–119.
[43]  Laskowski RA, MacArthur MW, Mass DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26: 283–291.
[44]  DeLano WL (2004) The PyMol Molecular Graphics System. DeLano Scientific, San Carlos, CA.
[45]  Abagyan RA, Totrov MM, Kuznetsov DN (1994) ICM - a new method for protein modeling and design. Applications to docking and structure prediction from the distorted native conformation. JCompChem 15: 488–506.
[46]  Wei Y, Eble JA, Wang Z, Kreidberg JA, Chapman HA (2001) Urokinase receptors promote β1 integrin function through interactions with integrin α3β1. Molecular Biology of the Cell 12: 2975–2986.
[47]  Huai Q, Zhou A, Lin L, Mazar AP, Parry GC, et al. (2008) Crystal structures of two human vitronectin, urokinase and urokinase receptor complexes. Nat Struct Mol Biol 15: 422–423.
[48]  Li S, Wang H, Peng B, Zhang M, Zhang D, et al. (2009) Efalizumab binding to the LFA-1 alphaL I domain blocks ICAM-1 binding via steric hindrance. Proc Natl Acad Sci U S A 106: 4349–4354.
[49]  Zhou T, Xu L, Dey B, Hessell AJ, Van Ryk D, et al. (2007) Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature 445: 732–737.
[50]  Wei Y, Tang CH, Kim Y, Robillard L, Zhang F, et al. (2007) Urokinase receptors are required for alpha 5 beta 1 integrin-mediated signaling in tumor cells. J Biol Chem 282: 3929–3939.
[51]  Simon DI, Wei Y, Zhang L, Rao NK, Xu H, et al. (2000) Identification of a urokinase receptor-integrin interaction site - Promiscuous regulator of integrin function. Journal of Biological Chemistry 275: 10228–10234.
[52]  Todd RF, Petty HR (1997) beta 2(CD11/CD18) integrins can serve as signaling partners for other leukocyte receptors. Journal of Laboratory and Clinical Medicine 129: 492–498.
[53]  Stewart M, Thiel M, Hogg N (1995) LEUKOCYTE INTEGRINS. Current Opinion in Cell Biology 7: 690–696.
[54]  Shaked Y, Voest EE (2009) Bone marrow derived cells in tumor angiogenesis and growth: are they the good, the bad or the evil? Preface. Biochimica Et Biophysica Acta-Reviews on Cancer 1796: 1–4.
[55]  Chioda M, Peranzoni E, Desantis G, Papalini F, Falisi E, et al. (2011) Myeloid cell diversification and complexity: an old concept with new turns in oncology. Cancer and Metastasis Reviews 30: 27–43.
[56]  Peinado H, Lavotshkin S, Lyden D (2011) The secreted factors responsible for pre-metastatic niche formation: Old sayings and new thoughts. Seminars in Cancer Biology 21: 139–146.
[57]  Bass R, Ellis V (2009) Regulation of urokinase receptor function and pericellular proteolysis by the integrin alpha(5)beta(1). Thromb Haemost 101: 954–962.
[58]  D'Alessio S, Blasi F (2009) The urokinase receptor as an entertainer of signal transduction. Frontiers in Bioscience 14: 4575–4587.


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