Engineering Protease-Specific Human Antibodies and Discovering Novel Proteases Expressed in Prostate Cancer Using Phage Display

摘要

Early studies revealed the importance of MT-SP1 in prostate cancer progression and indicated the existence of other serine proteases involved in the cascade. Developing selective and potent protease inhibitors is challenging due to frequent coexpression of other similar proteases. The antibody scaffold is an attractive choice for the development of highly potent and selective inhibitors. A phage-displayed antibody library was screened against a cancer- associated protease membrane-type serine protease 1 (NT-SP1). Six inhibitory antibodies were selected and had K(i)'s against human MT-SP1 ranging from 50 pM to 2 nM, Two antibodies had 800-fold and 1500-fold selectivity for human MT-SP1 when tested against mouse MT-SP1 that exhibits 87% sequence identity. These antibodies did not inhibit other closely related serine proteases including factor Xa, thrombin, kallikrein, tPA, and uPA. Binding kinetics of the antibodies revealed association rates as high as 1.2 x 10(exp 7) s(exp-1)M(exp - 1) and dissociation rates as low as 3.8 x 10(exp 4) s(exp -1). One antibody detected denatured NT-SP1 with no cross reactivity to other proteases in PC3 cells. Another antibody recognized the enzyme in human prostate tissue samples in immunohistochemical staining. These antibodies constitute a new class of highly selective protease inhibitors for exploring the biological roles of proteases.