Single-Domain Antibodies as Potent Inhibitors of Clinically Relevant β-Lactamases in Multidrug-Resistant Bacteria

Antimicrobial resistance (AMR) represents a critical threat to global health, largely driven by the dissemination of {beta}-lactamases that inactivate frontline antibiotics. Among the most problematic are BlaMab-2 from Mycobacterium abscessus, KPC-2 and OXA-48 from Klebsiella pneumoniae, and VIM-2 from Pseudomonas aeruginosa, which together confer broad resistance to {beta}-lactams and carbapenems. Current {beta}-lactamase inhibitors face declining efficacy as resistance variants continue to emerge, underscoring the need for innovative strategies. Here, we explored single-domain antibodies (sd-Abs) as enzyme-directed inhibitors of {beta}-lactamases. A library of sd-Abs was screened, and two candidates, B2 and B5, were characterized in vitro and in vivo. Both sd-Abs inhibited BlaMab-2 activity in E. coli expression systems, following a competitive inhibition mechanism, with B2 consistently displaying stronger potency (Ki {approx} 1.5 {micro}M) than B5. Remarkably, B2 also demonstrated broad inhibitory activity against KPC-2, VIM-2, and OXA-48, while B5 showed an alternative inhibition profile, including uncompetitive characteristics against VIM-2 and OXA-48. Comparison with clinically deployed inhibitors revealed that the Ki values of B2 and B5 are of the same order of magnitude--or superior in some cases--highlighting their therapeutic promise. Our findings establish sd-Abs as a versatile platform for the inhibition of diverse {beta}-lactamases, with B2 emerging as the most broadly effective candidate. By expanding the utility of existing {beta}-lactams, sd-Abs could help restore antibiotic efficacy against multidrug-resistant pathogens. This study underscores the potential of antibody-based enzyme inhibitors as a new class of anti-resistance therapeutics.