Human Monoclonal Antibodies for Neutralization of Botulinum Neurotoxin
Abstract
The purpose of this work is to generate neutralizing human monoclonal antibodies to Botulinum neurotoxins (BoNT) A, B, and E. To generate a large panel of antibodies, mice transgenic for the human immunoglobulin were immunized with BoNT/A, B, and E binding domain (Hc). RNA was prepared, the human variable regions amplified by PCR and used to construct human single chain Fv (scFv) antibody fragment gene repertoires. The repertories were cloned to create phage antibody libraries. Selection of the libraries on BoNT/A, B, and E Hc resulted in the isolation of a large panel of human monoclonal scFv antibody fragments. To demonstrate in vivo toxin neutralization, it was necessary to express the scFv as fusions with the human IgG1 Fc region from the yeast Pichia pastoris due to the rapid serum clearance of scFv. ScFv-Fc fusions showed increased serum half life compared to scFv, but had a significantly shorter half life than IgG. Previously isolated murine and human scFv showed toxin neutralization in vivo as Fc fusions, with a combination of two neutralizing scFv-Fc fusions able to neutralize 100 toxin LD50s. Since the serum half life of the Fc fusions was significantly shorter than IgG's, the immunglobulin VH and VL genes of neutralizing scFv were sublconed into a mammalian vector for expression as human IgG (in the case of human scFv) or mouse-human chimeric IgG (in the case of murine scFv). To date, three IgG have been constructed from the three neutralizing scFv and stable cell lines are being constructed. Concurrently, human IgG are being constructed from scFv derived from transgenic mice immunized with BoNT/A, B, and E Hc. Our plan is to purify IgG from each clone and evaluate in vivo neutralization potency for each unique antibody and for combinations of antibodies. In this way, we anticipate identifying panels of antibodies capable of neutralizing BoNT/A, B, and E.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 01, 2000
- Accession Number
- ADA382808
Entities
People
- James D. Marks
Organizations
- University of California, San Francisco