Functional selection of protease inhibitory antibodies

Abstract

Critical for diverse biological processes, proteases represent one of the largest families of pharmaceutical targets. To inhibit pathogenic proteases with desired selectivity, monoclonal antibodies (mAbs) hold great promise as research tools and therapeutic agents. However, identification of mAbs with inhibitory functions is challenging because current antibody discovery methods rely on binding rather than inhibition. This study developed a highly efficient selection method for protease inhibitory mAbs by coexpressing 3 recombinant proteins in the periplasmic space of Escherichia coli—an antibody clone, a protease of interest, and a β-lactamase modified by insertion of a protease cleavable peptide sequence. During functional selection, inhibitory antibodies prevent the protease from cleaving the modified β-lactamase, thereby allowing the cell to survive in the presence of ampicillin. Using this method to select from synthetic human antibody libraries, we isolated panels of mAbs inhibiting 5 targets of 4 main protease classes: matrix metalloproteinases (MMP-14, a predominant target in metastasis; MMP-9, in neuropathic pain), β-secretase 1 (BACE-1, an aspartic protease in Alzheimer’s disease), cathepsin B (a cysteine protease in cancer), and Alp2 (a serine protease in aspergillosis). Notably, 37 of 41 identified binders were inhibitory. Isolated mAb inhibitors exhibited nanomolar potency, exclusive selectivity, excellent proteolytic stability, and desired biological functions. Particularly, anti-Alp2 Fab A4A1 had a binding affinity of 11 nM and inhibition potency of 14 nM, anti-BACE1 IgG B2B2 reduced amyloid beta (Aβ40) production by 80% in cellular assays, and IgG L13 inhibited MMP-9 but not MMP-2/-12/-14 and significantly relieved neuropathic pain development in mice.

Generation of highly selective monoclonal antibodies inhibiting a recalcitrant protease using decoy designs

“…After exposure to cdMMP‐12 wt for 4 hr, Fabs LG4, LH6, and LH11 remained 54%, 56%, and 67% intact, respectively. These data agreed with our previous observations (Lopez et al, 2019) that isolated inhibitory mAbs by periplasmic genetic selection exhibited satisfactory proteolytic stability from the target protease.…”

“…In this study, PROSS designs of cdMMP‐12 variants were structure‐validated, and their periplasmic expression was optimized. Applying the functional selection (Lopez et al, 2019), inhibitory mAb clones were then isolated from human Fab synthetic libraries carrying convex paratopes (Nam et al, 2016). Finally, discovered Fabs were produced and characterized toward both the mutant design and wild‐type of cdMMP‐12.…”

Role of Matrix Metalloproteinases in Angiogenesis and Cancer

“…Currently, monoclonal antibodies are possible candidates to inhibit the activity of MMPs (MMP−14,−12,−9, and−2). However, studies have only managed to identify antibodies against MMP-9 activity, which has biological functions and not for the MMP−14,−12, and−2 (197). In prostate cancer, MMP-9 may amplify local angiogenesis by cleaving membranebound VEGF.…”

Inhibitory antibodies identify unique sites of therapeutic vulnerability in rhinovirus and other enteroviruses

“…YDF, identified from intracellular screening, then, appears to be a previously undisclosed allosteric and noncompetitive antibody inhibitor against a cysteine protease. Recently, another method of selecting protease inhibitory antibodies in the reducing periplasmic of Escherichia coli was reported, which may facilitate anticysteine protease antibody screening in the future (40).…”