Human Single-Chain Antibodies to ETA

Human Single-Chain Antibodies to ETA: Potential Therapeutic Agent against Pseudomonal Infecti

Introduction:

Pseudomonas exotoxin A (ETA) is the most potent virulence determinant produced by Pseudomonas aeruginosa which is one of the foremost causes of life-threatening nosocomial infections. The ETA catalyzes ADP-ribosylation of eukaryotic elongation factor-2 halting protein synthesis and leading to mammalian cell death. Engineered single-chain antibodies (HuscFvs) against exotoxin A should raise a hope for treatment of the fatal entity.

 

Methods:

In this study, ETA antigen (includes binding domain, catalytic domain, full-length, and synthesized biotinylated peptide) were produced and used as antigen in the phage bio-panning for selecting phage clones that bound to the respective antigens from a previously constructed HuscFv phage display library. The antigen-bound phages were transfected to E. coli. The transformed E. coli were grown and induced to express soluble HuscFvs. HuscFvs in the E. coli lysates were tested for target binding by indirect ELISA. Genes coding for the target-bound HuscFvs were subcloned for large scale HuscFv production and tested further for neutralizing activities. Moreover, the homology modelling and molecular docking of ETA to single-chain antibodies were also conducted.

 

Results:

The ETA bound-HuscFvs were successfully produced. From phage bio-panning, 241 E. coli clones were selected from the selective media. The 155 clones were carried huscfv which were determined by PCR. HuscFvs of 37 clones bound to the native ETA. Nucleotide sequencing revealed 16 clones presented complete sequences of HuscFv. Based on the deduced amino acid sequences and numbering according to the Kabat and Chothia scheme, the single-chain antibody clones were characterized into 7 different types. The representative HuscFv clones were successfully investigated for their activities (apoptosis assays) and molecular docking studies.

 

Conclusion:

In conclusions, the three candidate of the transformed E. coli clones containing genes coding for the target-bound HuscFvs that gave high binding ability were sub-cloned for large scale HuscFv production and the inhibitory activities of HuscFv-C41 were significantly higher than others; HuscFv-E44 and HuscFv-P32 using apoptosis assays and correlated with the in silico docking results of the HuscFvs that formed their contact interface with several critical residues of ETA.

 

Disclosure of Interest Statement:
The Royal Golden Jubilee Ph.D. Scholarships are funded by Thailand research fund, Thailand which were received in the development of this study.

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Author(s): Sirijan Santajit

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