Isolation of Human Monoclonal Antibodies to Glucosyltransferase by Phage Display

Objective: Active immunization with streptococcal GTF induces a protective immune response in dental caries models. Antibody to GTF is also protective passively. Timely passive exposure of the oral cavity to such antibody should block entry of mutans streptococci into developing biofilms. Methods: Two human phage display libraries totaling 2.7 X 10¹⁰ members constructed from B cells of 57 donors were used for selection of human single-chain variable region fragments (scFv) binding to S. sobrinus GTF. We anticipated that this library contained phage with scFvs binding GTF since adults have significant levels of antibody to GTF. GTF coated on immunotubes was incubated with 10¹² pfu master phage stock. Bound phage were eluted, amplified, and after two rounds of selections, 96 randomly picked single phage-scFv clones were screened for specific binding to GTF (ELISA). Positive clones were sequenced to determine the VH and VL genes of scFv. The VH and VL gene fragments of these GTF-specific scFvs were then cloned to express scFvs and scFv-Fcs (scFv linked in frame with Fc Hinge-CH2-CH3) of human IgG1), respectively. GTF-binding activities of these antibody fragments were confirmed (ELISA). Results: After the 2nd round of selection, 46 out of 96 ELISA-positive clones were detected. Each clone was sequenced and 5 unique anti-GTF scFvs were obtained corresponding to VH1/VL1, VH3/VL3, VH3/VL2, VH1/VL3 and VH3/VL6 gene families. GTF binding clones are confirmed (ELISA) and tested for GTF inhibition. Antibodies with the most potent inhibition of GTF enzymatic activity are cloned into human IgG1 expression plasmids encoding H and L chain genes. Whole human IgG1 monoclonal antibodies produced in transient, stable mammalian expression systems are used for further functional studies. Conclusion: It is possible to isolate human monoclonal antibodies to GTF for passive procedures using phage display and large antibody libraries. Support NIH/NIDCR DE-04733.