American Association for Cancer Research
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Supplemental Figures S1-S6 from Imaging Active Urokinase Plasminogen Activator in Prostate Cancer

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journal contribution
posted on 2023-03-30, 23:26 authored by Aaron M. LeBeau, Natalia Sevillano, Kate Markham, Michael B. Winter, Stephanie T. Murphy, Daniel R. Hostetter, James West, Henry Lowman, Charles S. Craik, Henry F. VanBrocklin

Supplemental Figures S1-S6. U33 Fab Sequence (S1); Inhibition constant calculation of U33 Fab (S2); U33 does not bind to the uPA-PAI1 complex (S3); Selectivity of U33 IgG for active uPA versus inactive uPA (S4); Dependence of U33 IgG binding on an accessible uPA active site (S5); Selectivity of U33 IgG for human uPA compared to highly related serine proteases (S6).



The increased proteolytic activity of membrane-bound and secreted proteases on the surface of cancer cells and in the transformed stroma is a common characteristic of aggressive metastatic prostate cancer. We describe here the development of an active site-specific probe for detecting a secreted peritumoral protease expressed by cancer cells and the surrounding tumor microenvironment. Using a human fragment antigen-binding phage display library, we identified a human antibody termed U33 that selectively inhibited the active form of the protease urokinase plasminogen activator (uPA, PLAU). In the full-length immunoglobulin form, U33 IgG labeled with near-infrared fluorophores or radionuclides allowed us to noninvasively detect active uPA in prostate cancer xenograft models using optical and single-photon emission computed tomography imaging modalities. U33 IgG labeled with 111In had a remarkable tumor uptake of 43.2% injected dose per gram (%ID/g) 72 hours after tail vein injection of the radiolabeled probe in subcutaneous xenografts. In addition, U33 was able to image active uPA in small soft-tissue and osseous metastatic lesions using a cardiac dissemination prostate cancer model that recapitulated metastatic human cancer. The favorable imaging properties were the direct result of U33 IgG internalization through an uPA receptor–mediated mechanism in which U33 mimicked the function of the endogenous inhibitor of uPA to gain entry into the cancer cell. Overall, our imaging probe targets a prostate cancer–associated protease, through a unique mechanism, allowing for the noninvasive preclinical imaging of prostate cancer lesions. Cancer Res; 75(7); 1225–35. ©2015 AACR.

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