Multitarget selection of catalytic antibodies with β‐lactamase activity using phage display

Abstract

β-lactamase enzymes responsible for bacterial resistance to antibiotics are among the most important health threats to the human population today. Understanding the increasingly vast structural motifs responsible for the catalytic mechanism of β-lactamases will help improve the future design of new generation antibiotics and mechanism-based inhibitors of these enzymes. Here we report the construction of a large murine single chain fragment variable (scFv) phage display library of size 2.7 × 10 with extended diversity by combining different mouse models. We have used two molecularly different inhibitors of the R-TEM β-lactamase as targets for selection of catalytic antibodies with β-lactamase activity. This novel methodology has led to the isolation of five antibody fragments, which are all capable of hydrolyzing the β-lactam ring. Structural modeling of the selected scFv has revealed the presence of different motifs in each of the antibody fragments potentially responsible for their catalytic activity. Our results confirm (a) the validity of using our two target inhibitors for the in vitro selection of catalytic antibodies endowed with β-lactamase activity, and (b) the plasticity of the β-lactamase active site responsible for the wide resistance of these enzymes to clinically available inhibitors and antibiotics.

Genetic background and immunological status influence B cell repertoire diversity in mice

“…The NGS data generated 351 286 sequences specific to immunoglobulins analyzed using IMGT/HighV-QUEST. Within this population, we observed 2 915 pseudogenes (ie 0.83%), which is consistent with our previous estimations 11 , and with findings of other teams 13 . The presence of pseudogenes in our library can be explained by the potential homology of some designed primers with pseudogene sequences, despite the option of taking into account only “functional” genes in the IMGT/LIGM-DB database manipulation during the conception of our library.…”

“…Our library of size 2.6 × 10 9 in size gathers 4 distinct immune repertoires (NB, IB, NS and IS), each reflecting a unique mouse 11 . It is noteworthy that a specific murine strain is generated from a rigorous selection applied over many generations, thus producing mice with identical genes.…”

“…The phagemides pAK100, pAK101, pAK102 and pAK103 were independently prepared as previously described 11 . 100 µL of each phage preparation was added to 100 mL of E. coli bacteria, cultured overnight at 37 °C in LB medium with chloramphenicol 25 µg.mL −1 .…”

“…This strategy is based on a technological optimization relying on Rolling Circle Amplification (RCA), combined with a newly designed set of oligonucleotide primers based on a thorough analysis of the IMGT/LIGM-DB database 10 . In the present study, we have used this strategy to generate libraries form two murine inbred strains were used, namely Balb/C (healthy) and SJL/J (susceptible to autoimmune disease), together representing 4 different IgG immune repertoires: i) healthy and naïve (NB for naïve Balb/C), ii) healthy and immunized (IB for immunized Balb/C), iii) autoimmune prone and naïve (NS for naïve SJL/J), and iv) autoimmune prone and immunized (IS for immunized SJL/J) 11 . We have decidedly chosen to focus on the IgG repertoires, excluding the IgM immunoglobulins present in natural naïve immune repertoires 12 , with the aim of focusing our comparative study on the same isotype originating from the 4 populations.…”

Structural and Functional Mise en Abyme

“…The three scFv have different primary structure. Nevertheless, a structural modeling suggests that all contains potential catalytic residues that could form a catalytic triad (Shahsavarian et al, 2017). So, the use of a small constraining peptide, with only 7 amino acids, as a template for the selection of phage-display antibody fragments, has resulted in the isolation of three scFv fragments with a ß-lactamase activity.…”