Selection of Monoclonal Antibodies that Bind and Inhibit Tissue-Type Plasminogen Activator

Abstract

Three monoclonal antibodies raised against tissue-type plasminogen activator (t-PA) were selected for their ability to inhibit solid-phase bound t-PA. Each monoclonal antibody blocked the release of p-nitroaniline from H-D-Ile-Pro-Arg-pNA (S-2288). The first antibody 1D2 was a γ2b,κ with K_D = 8 x 10^-9 M, the second antibody 2B9 was a γ1,κ with K_D = 2 x 10^-9 M, and the third antibody 5A9 was a γ1,κ with K_D = 4 x 10^-10 M. In solution-phase format each antibody blocked the conversion of plasminogen to plasmin as judged by a plasmin assay and also inhibited t-PA-mediated lysis of plasma fibrin clot in plasma. The binding of each ¹²⁵I-radiolabeled antibody to t-PA was inhibited by any one of the three antibodies, suggesting that they recognized a common epitope on t-PA which was absent on unfolded t-PA. We concluded these antibodies bind near t-PA active site since PPACK treatment lowered binding of two antibodies. We believe solid-phase chromogenic substrate assay may be a useful way to screen for antibodies directed against the active site of proteases.