title: Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein.
abstract: Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins, XOMA 3B and XOMA 3E, each consisting of three mAbs that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (H(N)). Epitope mapping data were used to design LC-H(N) domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or XOMA 3E. Mutant LC-H(N) domains were cloned, expressed, and purified from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of enzyme-linked immunosorbent assays (ELISAs) that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein.
authors.affiliation: Department of Anesthesia, University of California, San Francisco, San Francisco General Hospital, San Francisco, CA 94110, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Meng
authors.forename: C
authors.initials: C
authors.lastname: Garcia-Rodriguez
authors.forename: G
authors.initials: G
authors.lastname: Manzanarez
authors.forename: M A
authors.initials: MA
authors.lastname: Silberg
authors.forename: F
authors.initials: F
authors.lastname: Conrad
authors.forename: J
authors.initials: J
authors.lastname: Bettencourt
authors.forename: X
authors.initials: X
authors.lastname: Pan
authors.forename: T
authors.initials: T
authors.lastname: Breece
authors.forename: R
authors.initials: R
authors.lastname: To
authors.forename: M
authors.initials: M
authors.lastname: Li
authors.forename: D
authors.initials: D
authors.lastname: Lee
authors.forename: L
authors.initials: L
authors.lastname: Thorner
authors.forename: M T
authors.initials: MT
authors.lastname: Tomic
authors.forename: J D
authors.initials: JD
authors.lastname: Marks
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001905:Botulinum Toxins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D019274:Botulinum Toxins, Type A; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E