Single-domain antibodies as potent inhibitors of clinically relevant β-lactamases in multidrug-resistant bacteria

Abstract Antimicrobial resistance (AMR) represents a critical threat to global health, largely driven by the dissemination of β-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 β-lactams and carbapenems. Current β-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 β-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 (K i ≈ 1.5 µM) 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 K i 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 β-lactamases, with B2 emerging as the most broadly effective candidate. By expanding the utility of existing β-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.