PEGylated DX-1000: pharmacokinetics and antineoplastic activity of a specific plasmin inhibitor.

Novel inhibitors of the urokinase-mediated plasminogen (plg) activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pln) (K(i) = 99 pM). When tested in vitro, DX-1000 blocks plasmin-mediated pro-matrix metalloproteinase-9 (proMMP-9) activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor ( approximately 7 kDa) exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP)-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA) and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogen-activated protein kinase (MAPK) in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries.

We introduce a procedure for the rapid generation of fully human antibodies derived from "Fab-on-phage" display libraries. The technology is based on the compatibility of display vectors and IgG expression constructs, and allows reformatting of individual Fab clones to IgG, as well as reformatting of antibody repertoires. Examples of batch reformatting of an uncharacterized Fab repertoire and of a pool of Fabs, previously analyzed at the phage level, are presented. The average transient expression levels of the IgG constructs in HEK293T cells are above 10 microg/ml, allowing the use of conditioned media in functional assays without antibody purification. Furthermore, we describe a high-throughput purification method yielding IgG amounts sufficient for initial antibody characterization. Our technology allows the generation and production of antigen-specific complete human antibodies as fast or even faster than raising monoclonal antibodies by conventional hybridoma techniques.