Exotoxin A is one of the most potent virulence factors in P. aeruginosa. With increasing prevalence of multi drug resistant strains, it is necessary to develop alternative therapeutic approaches as a robust pipeline to combat resistant pseudomonas infections. Neutralizing virulence factors such Exo A can be an promising strategy to control bacterial infections. Morever, this strategy maight be affect drug resistance through maintaining the host endogenous microbiome and creating less selective pressure to the bacteria (15). Until now, various antibodies have been developed against different antigens of pseudomonas, but none of them have been approved for clinical use (16).
Theoretically, antibody libraries contain various synthetic and semi synthetic antibodies, facilitating the isolation of antibodies against any given antigens. At present, scFv phage libraries have impactful payload in the development of monoclonal antibodies without the need to experimental animals (17).
Pseudomonas exotoxin A domain I has been shown to be responsible for binding of toxin to animal cell receptors and plays a key role in the toxicity caused by pseudomonas exotoxin. The aim of the study was to develop a full human antibody against domain I of exotoxin A for potential use in neutralizing the toxic effects of Pseudomonas exotoxin to overcome the pseudomonas infection.
In this study, a novel screening strategy was used for elimination of nonspecific clones coupled with enrichment of specific clones during biopanning rounds. In this respect, results of output phage titers monitoring during screening rounds indicated a significant enrichment toward increasing specificity (64-fold) to exotoxin A domain I. During six biopanning rounds, two specific clones with VH-VL correct integrity were identified from fifth round. One of the scFv clones (C9) with high affinity was selected for further studies. In order to generate antibody with high efficiency, the expression and purification of C9 scFv was again performed in E. coli. In a 2019 study by Sirijan Santajit et al, they produced human antibodies against subdomain Ia exotoxin A of Pseudomonas using phage display technology. However, in the present study, domain 1 subdomains (domains Ia and Ib) were targeted for antibody production, which had a greater neutralizing power (18). An older study also produced a mouse antibody against exotoxin A, but due to adverse immune responses we decided to produce a completely human antibody (19). S. NATHAN (20) used phage Display technology for isolation of the antibody against Burkholderia pseudomallei by several biopaning rounds. They observed high similarity (93%) among different clones while the main difference in the sequence was related to CDR3 area. In our study, the highest similarity was 93.9% and the different area in the sequence was related to CDRs (21).
The clone 9 anti -exotoxin A scFv was purified with high solubility from periplasmic fraction. Based on ELISA results, the resultant anti- exotoxin A scFv specifically recognized both recombinant exotoxin A domain I and supernatant of pseudomonas with high affinity. Also, western blotting results confirmed that human anti- exotoxin A scFv binds specifically to exotoxin A domain I protein.