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Updated: Sep 27, 2026
Peptide-based Identification of Functional Motifs and their Binding Partners
Published on: June 30, 2013
Potent and selective inhibition of membrane-type serine protease 1 by human single-chain antibodies
Jeonghoon Sun1, Jaume Pons, Charles S Craik
1Department of Pharmaceutical Chemistry, University of California, San Francisco, 513 Parnassus, San Francisco, California 94143, USA.
Abstract:
Specific human antibodies targeting proteases expressed on cancer cells can be valuable reagents for diagnosis, prognosis, and therapy of cancer. To this end, a phage-displayed antibody library was screened against a cancer-associated serine protease, MT-SP1. A protein inhibitor of serine proteases that binds to a defined surface of MT-SP1 was used in an affinity-based washing procedure. Six antibodies were selected on the basis of their ELISA profiles and ability to serve as useful immunological reagents. The apparent K(i), indicative of the potency of the antibodies at inhibiting human MT-SP1 activity, ranged from 50 pM to 129 nM. Two of the antibodies had approximately 800-fold and 1500-fold selectivity when tested against the most homologous serine protease family member, mouse MT-SP1, that exhibits 86.6% sequence identity. Surface plasmon resonance was used as an independent means of determining the binding constants of the six antibodies. Association rates were as high as 1.15 x 10(7) s(-)(1) M(-)(1), and dissociation rates were as low as 3.8 x 10(-)(4) s(-)(1). One antibody was shown to detect denatured MT-SP1 with no cross reactivity to other family members in HeLa or PC3 cells. Another antibody recognized the enzyme in human prostate tissue samples for immunohistochemistry analysis. The mode of binding among the six antibodies and the protease was analyzed by competition ELISA using three distinctly different inhibitors that mapped the enzyme surface. These antibodies constitute a new class of highly selective protease inhibitors that can be used to dissect the biological roles of proteolytic enzymes as well as to develop diagnostic and therapeutic reagents.
Insights
Researchers developed highly selective human antibodies targeting the cancer-associated protease MT-SP1. These antibodies show potential for cancer diagnosis, prognosis, and therapy, offering new tools for studying proteolytic enzymes.
Area of Science:
- Biochemistry and Molecular Biology
- Cancer Research
- Immunology
Background:
- Cancer-associated proteases are crucial targets for diagnosis, prognosis, and therapy.
- Developing specific antibodies against these proteases is essential for effective cancer treatment strategies.
Purpose of the Study:
- To screen a phage-displayed antibody library to identify human antibodies targeting the cancer-associated serine protease, MT-SP1.
- To characterize the selectivity, potency, and binding kinetics of the selected antibodies.
- To evaluate the antibodies' utility as diagnostic and therapeutic reagents in cancer research.
Main Methods:
- Phage display library screening against MT-SP1 using an affinity-based washing procedure.
- Enzyme-linked immunosorbent assay (ELISA) for antibody selection and potency determination (apparent K(i)).
- Surface plasmon resonance (SPR) for precise binding constant analysis (association and dissociation rates).
- Cross-reactivity testing against homologous proteases and detection in cell lines and tissue samples.
Main Results:
- Six human antibodies targeting MT-SP1 were selected with apparent inhibition constants (K(i)) ranging from 50 pM to 129 nM.
- Two antibodies demonstrated high selectivity (800-fold and 1500-fold) against the homologous mouse MT-SP1.
- SPR analysis revealed high association rates (up to 1.15 x 10(7) s(-)(1) M(-)(1)) and low dissociation rates (as low as 3.8 x 10(-)(4) s(-)(1)).
- One antibody detected denatured MT-SP1 in HeLa and PC3 cells without cross-reactivity, while another recognized the enzyme in human prostate tissue for immunohistochemistry.
Conclusions:
- The identified antibodies represent a novel class of highly selective protease inhibitors.
- These antibodies are valuable reagents for dissecting the biological roles of proteolytic enzymes in cancer.
- The developed antibodies hold significant potential for the development of diagnostic and therapeutic cancer reagents.
