# A High-Affinity Nanobody Selectively Recognizing KPC-2/KPC-3: Biochemical and Structural Insights

## TL;DR

Researchers developed a nanobody that can detect KPC-2 and KPC-3 enzymes in antibiotic-resistant bacteria, offering a potential tool for infection control and treatment.

## Contribution

The study introduces a nanobody that selectively recognizes KPC-2 and KPC-3, providing a new molecular tool for detecting antibiotic-resistant bacteria.

## Key findings

- A nanobody was developed that specifically targets KPC-3 and also recognizes KPC-2 in clinical bacterial strains.
- Structural modeling confirmed favorable interactions between the nanobody and KPC enzymes.
- The nanobody shows promise as a detection tool and a foundation for future therapeutic development.

## Abstract

Carbapenemase-producing bacteria, particularly those expressing the KPC-3 variant, pose a critical global health threat due to their resistance to nearly all β-lactam antibiotics, including carbapenems. Rapid and reliable detection tools are urgently needed to improve infection control and guide patient management. Nanobodies (VHHs) present a promising alternative to conventional antibodies thanks to their high stability, small size, and capacity to access cryptic epitopes. Here, we report the generation and characterization of a nanobody specifically targeting KPC-3. An immune VHH phage display library was constructed, with over 90% of clones containing correctly sized inserts. After three rounds of biopanning, high-specificity binders were identified by ELISA screening. Sequencing identified a nanobody with hallmark VHH features, which was expressed and validated by ELISA and Western blot. Although kinetic assays showed no inhibition of KPC-3 enzymatic activity, interestingly, the nanobody demonstrated high-binding recognition of both KPC-2 and KPC-3 in periplasmic extracts from clinical strains. Structural modeling further supported these results, highlighting favorable interaction surfaces. This study provides the first evidence of a nanobody raised against KPC-3 that recognizes a conserved epitope shared by KPC-3 and KPC-2, underscoring its promising use as a molecular tool for detecting KPC variants and establishing a basis for future affinity maturation toward therapeutic applications.