UC Riverside

Overwhelming evidence has implicated that protease upregulation plays a critical role in cancer growth and tumorigenesis. Extracellular metalloproteinases (e.g. MMPs, MT-SPs) have been considered as therapeutic targets. However, prior attempts focusing on small compounds against metalloproteainses failed in clinical trials, indicating selectivity is the key for a successful protease inhibition. With a large contact surface by multiple complementarity determining regions (CDRs), antibodies are exquisitely specific thus considered as promising drugs. Unfortunately, at least two obstacles make the routine discovery of protease inhibiting antibodies considerably difficult: (1) low antigenicity of the proteolytic active sites, and (2) lack of a function-based selection method.

To develop therapeutic antibodies that specifically inhibit metalloproteinases, this thesis aims to overcome aforementioned technical hurdles and establish general methodologies for the discovery of inhibitory antibodies. These research objectives were successfully achieved by:

(1) Developing a function-based high-throughput inhibitory antibody screening method using a simple periplasmic preparation and a sensitive FRET assay without antibody purification. Using this method, inhibitory antibodies were rapidly distinguished from non-inhibitory clones with satisfactory Z-factors.

(2) Developing a novel expression strategy for direct production of catalytic domain of MMP-14 in the E. coli periplasm without refolding or activation. Achieving co-expression of both MMP-14 and antibody fragment in the periplasm further facilitated screening of inhibitory antibodies without purification of MMP-14 or Fabs.

(3) Constructing large synthetic human antibody libraries (>109 variants) carrying extended CDR-H3 (23 to 27 aa) and isolating a panel of highly potent and selective inhibitory antibodies from the constructed libraries. Particularly, 3A2 Fab was a competitive inhibitor exhibiting a binding affinity of 4.85 nM by SPR and an inhibition potency of 25 nM.

(4) Converting a low potency peptide inhibitor (IC50=150 µM) into an antibody with an >103-fold improved inhibitory potency by grafting the inhibitory peptide into CDR-H3 of antibody scaffold and optimizing the sequences flanking peptide motif. The resulting 1F8 Fab exhibited an inhibition potency of IC50=120 nM against MMP-14.

In conclusion, we believe these inhibitory antibody discovery approaches have great potentials to apply for proteases and important targets with biological significance.