ABSTRACT
Human antibodies able to bind with high affinity and specificity to numerous targets have been successfully identified from Fab phage display libraries. A key step in the library selection screening process is the early characterization of library isolates in order to determine which of these isolates to pursue further. Here we describe a Biacore assay that allows isolated clones expressed as soluble Fab fragments in E. coli to be screened and ranked based on their affinity against the target. The assay takes advantage of our ability to measure Fab concentrations in crude bacterial extracts in Biacore using very high density Protein A chips. The procedure allows up to 100 clones per week to be screened and permits the identification of a small number of high-affinity Fabs from a large batch obtained following library selection or affinity maturation.
Subject(s)
Immunoglobulin Fab Fragments/immunology , Receptor, TIE-1/immunology , Antibody Affinity/immunology , Chromatography, Affinity , Escherichia coli/genetics , Humans , Immunoglobulin Fab Fragments/analysis , Immunoglobulin Fab Fragments/genetics , Kinetics , Peptide Library , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Staphylococcal Protein A , Surface Plasmon Resonance/methodsABSTRACT
Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.
Subject(s)
Antibodies, Monoclonal , Antineoplastic Agents/therapeutic use , Cell Division/drug effects , Enzyme Inhibitors/therapeutic use , Matrix Metalloproteinase Inhibitors , Neovascularization, Pathologic/prevention & control , Animals , Antibodies, Monoclonal, Humanized , Breast Neoplasms/pathology , Cell Line, Tumor , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Female , Genes, Reporter , Humans , Immunohistochemistry , Mice , Neoplasm Invasiveness/pathology , Transfection , Transplantation, Heterologous , Umbilical Veins/cytology , Umbilical Veins/drug effectsABSTRACT
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.