Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4-dicarboxylate transporter.

Antimicrobial resistance is emerging as the new healthcare crisis necessitating the development of newer classes of drugs using non-traditional approaches. Pseudomonas aeruginosa, one of the most common pathogens involved in nosocomial infections, is extremely difficult to treat even with the last resort frontline drug, the carbapenems. As the pathogen has the ability to acquire resistance to new small-molecule antibiotics, being deployed, a novel biological approach has been tried using antibody fragments in combination with carbapenems and ß-lactams as adjunct therapy for an enduring solution to the problem. In this study, we developed a camelid antibody fragment (VHH) library against P. aeruginosa and isolated a highly potent hit, PsC23. Mass spectrometry identified the target to be a component of the C4-dicarboxylate transporter that feeds metabolites to the glyoxylate shunt particularly under conditions of oxidative stress. PsC23 is bacteriostatic at a concentration of 1.66 µM (25 µg ml-1) and shows a synergistic effect with both the classes of drugs at an effective concentration of 100-200 nM (1.5-3.0 µg ml-1) when co administered with them. In combination with meropenem the VHH completely cleared the infection from a neutropenic mouse with a carbapenem-resistant P. aeruginosa systemic infection. Blocking the glyoxylate shunt by PsC23 resulted in disruption of energy transduction due to a respiratory shift to the oxygen-depleted TCA cycle causing inhibition of efflux and increased free radical generation from carbapenems and ß-lactams exerting a strong bactericidal effect that reversed the resistance to multiple unrelated drugs.

Understanding the Specific Implications of Amino Acids in the Antibody Development.

As the demand for immunotherapy to treat and manage cancers, infectious diseases and other disorders grows, a comprehensive understanding of amino acids and their intricate role in antibody engineering has become a prime requirement. Naturally produced antibodies may not have the most suitable amino acids at the complementarity determining regions (CDR) and framework regions, for therapeutic purposes. Therefore, to enhance the binding affinity and therapeutic properties of an antibody, the specific impact of certain amino acids on the antibody's architecture must be thoroughly studied. In antibody engineering, it is crucial to identify the key amino acid residues that significantly contribute to improving antibody properties. Therapeutic antibodies with higher binding affinity and improved functionality can be achieved through modifications or substitutions with highly suitable amino acid residues. Here, we have indicated the frequency of amino acids and their association with the binding free energy in CDRs. The review also analyzes the experimental outcome of two studies that reveal the frequency of amino acids in CDRs and provides their significant correlation between the outcomes. Additionally, it discusses the various bond interactions within the antibody structure and antigen binding. A detailed understanding of these amino acid properties should assist in the analysis of antibody sequences and structures needed for designing and enhancing the overall performance of therapeutic antibodies.

Significance of antibody numbering systems in the development of antibody engineering.

Immunotherapy has become increasingly popular in recent years for treating a variety of diseases including inflammatory, neurological, oncological, and auto-immune disorders. The significant interest in antibody development is due to the high binding affinity and specificity of an antibody against a specific antigen. Recent advances in antibody engineering have provided a different view on how to engineer antibodies in silico for therapeutic and diagnostic applications. In order to improve the clinical utility of therapeutic antibodies, it is of paramount importance to understand the various molecular properties which impact antigen targeting and its potency. In antibody engineering, antibody numbering (AbN) systems play an important role to identify the complementarity determining regions (CDRs) and the framework regions (FR). Hence, it is crucial to accurately define and understand the CDR, FR and the crucial residues of heavy and light chains that aid in the binding of the antibody to the antigenic site. Detailed understanding of amino acids positions are useful for modifying the binding affinity, specificity, physicochemical features, and half-life of an antibody. In this review, we have summarized the different antibody numbering systems that are widely used in antibody engineering and highlighted their significance. Here, we have systematically explored and mentioned the various tools and servers that harness different AbN systems.