Construction of recombinant antibodies against Pseudomonas aeruginosa lipopolysaccharide

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Tout, Nancy Lynn

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University of Guelph

Abstract

Antibody engineering studies were undertaken for a number of P. aeruginosa LPS-specific murine mAbs of therapeutic interest. A molecular and structural analysis of 4 mAbs against LPS revealed a diverse usage of variable region genes of both heavy and light chains. In addition, a number of significant homologies were observed between the V\sbH and V\sbL chain genes from anti-LPS mAbs and other anti-carbohydrate Abs and anti-DNA autoAbs. From the amino acid sequence information, a 3-D structural model was constructed for the Ab-combining site of mAb 5c-101 using atomic coordinates from known structures of J539 (anti-galactan) and Je142 (anti-phosphocarrier protein for sugar transport). A number of similarities were identified between the topography of the Ab-combining site of 5c-101 and another mAb W3129 whose fine specificities are both for a terminal glucose residue on their respective macromolecules, core oligosaccharide and dextran. A potential N-linked glycosylation site was identified in the CDR-H2 region of 5c-101 which may affect binding to core oligosaccharide and most likely explains weak to negligible binding activity when this mAb was expressed as a r-Fab in E. coli. The feasibility of phage display of recombinant Fabs (r-Fabs) from mAbs MF23-1, N1F10, and 5c-101 was investigated. While each mAb was successfully expressed as r-Fab fragments into the periplasm of E. coli, only the r-Fab from mAb MF23-1 showed significant binding activity towards its nominal Ag, P. aeruginosa O6 O-antigen in an ELISA and immunofluorescence microscopy both as a soluble form secreted into the periplasm of E. coli and on the surface of phage as a fusion protein to cpIII. A reduction in avidity of N1F10 from its natural pentameric form as an IgM Ab to a monomeric Fab highlights the challenges associated with the cloning and expression of Ab fragments from low affinity anti-carbohydrate IgM Abs. A dimeric scFv system was therefore examined to increase the valency of recombinant Ab fragments derived from IgM anti-carbohydrate mAbs N1F10 and 177. However, negligible binding activity was detected from these scFv dimers, suggesting that the increase in valency from one to two was not sufficient to detect binding from these IgM Ab-derived fragments.

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recombinant antibodies, Pseudomonas aeruginosa, lipopolysaccharide, antibody engineering

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