title: Optimization of experimental conditions for functional in vitro characterization of humanized antibodies specific for staphylococcal enterotoxin B.
abstract: Staphylococcus aureus is a common nosocomial infection and its resistance to penicillin and methicillin antibiotics is a growing clinical problem. We previously described the development of a humanized anti-Staphylococcus enterotoxin B (SEB) antibody derived from the mouse antibody made by the 20B1 hybridoma. This antibody was humanized and characterized kinetically by surface plasmon resonance demonstrating that the humanized clones retained binding to SEB. Clones were then functionally characterized in an in vitro assay demonstrating that the murine 20B1, chimeric and humanized antibodies potently inhibited SEB-induced murine splenocyte proliferation assay. Here, we describe a human cell-based screening assay, optimized by varying multiple experimental parameters that resulted in an assay that was used to demonstrate full and potent neutralization by the parental, chimeric and humanized antibodies. The replacement of fetal bovine serum (FBS) with normal human serum (NHS) was found to be a crucial factor in the performance of the human cell based screening assay that enabled the calculation of mAb efficacy and potency. In addition, we found that anti-SEB antibodies showed similar efficacy and potency with a triple mutant Fc region (designed to be effector function null) or a wild-type Fc region, which is in contrast to previously described studies.
authors.affiliation: Pfizer Centers for Therapeutic Innovation NY, NY, USA. Electronic address: jennifer.macintyre@pfizer.com.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: MacIntyre
authors.affiliation: Albert Einstein College of Medicine, Bronx, NY, USA.
authors.forename: Avanish K
authors.initials: AK
authors.lastname: Varshney
authors.affiliation: Albert Einstein College of Medicine, Bronx, NY, USA.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Wang
authors.affiliation: Pfizer Global Biotherapeutic Technologies, Cambridge, MA, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Gatto
authors.affiliation: Pfizer Global Biotherapeutic Technologies, Cambridge, MA, USA.
authors.forename: Clarence
authors.initials: C
authors.lastname: Friedman
authors.affiliation: Pfizer Global Biotherapeutic Technologies, Cambridge, MA, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Pfizer Global Biotherapeutic Technologies, Cambridge, MA, USA.
authors.forename: Kelvin
authors.initials: K
authors.lastname: Kerns
authors.affiliation: Pfizer Global Biotherapeutic Technologies, Cambridge, MA, USA.
authors.forename: Oleg
authors.initials: O
authors.lastname: Kovalenko
authors.affiliation: Pfizer Drug Safety Research and Development, Groton, CT, USA.
authors.forename: Karissa
authors.initials: K
authors.lastname: Adkins
authors.affiliation: Pfizer Global Biotherapeutic Technologies, Cambridge, MA, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Zollner
authors.affiliation: Depts. of Medicine, Microbiology and Molecular Genetics, Stony Brook University, Stony Brook, NY, USA.
authors.forename: Bettina C
authors.initials: BC
authors.lastname: Fries
authors.affiliation: Pfizer Centers for Therapeutic Innovation NY, NY, USA.
authors.forename: Fergus R
authors.initials: FR
authors.lastname: Byrne
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology*; D001681:Biological Assay; D049109:Cell Proliferation; D002478:Cells, Cultured; D004768:Enterotoxins / Q000276:immunology*; D006801:Humans; D007962:Leukocytes / Q000276:immunology; D008297:Male; D051379:Mice; D008875:Middle Aged; D017452:Receptors, IgG / Q000276:immunology; D044967:Serum
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D004768:Enterotoxins; D017452:Receptors, IgG; C031818:enterotoxin B, staphylococcal
keywords: Humanized antibodies; In vitro; SEB; Staphylococcus aureus