title: Successful engineering of a highly potent single-chain variable-fragment (scFv) bispecific antibody to target disialoganglioside (GD2) positive tumors.
abstract: Engineering potent bispecific antibodies from single-chain variable fragments (scFv) remains difficult due to the inherent instability and insufficient binding of scFv's compared to their parental immunoglobulin format. Previously, we described a scFv-based bispecific antibody (scBA) against disialoganglioside (GD2) based on the anti-GD2 murine 5F11-scFv and the anti-CD3 huOKT3-scFv (5F11-scBA). In this study, we substituted the 5F11-scFv with the higher affinity (13-fold) hu3F8-scFv to form hu3F8-scBA. With this modification, hu3F8-scBA redirected T cells to kill GD2(+) cancer cell lines with up to 5,000-fold higher potency (femtomolar EC50) compared with 5F11-scBA (picomolar EC50) in cytotoxicity assays, even against target cells with low GD2 densities. Furthermore, hu3F8-scBA induced stronger T-cell activation than 5F11-scBA, as measured by Ca(2+) flux and cytokine release. Additionally, in vivo, hu3F8-scBA suppressed tumor growth and prolonged mice survival much more effectively than 5F11-scBA, in both neuroblastoma and melanoma xenograft models. We conclude that the functional properties of scBA's can be increased substantially by relatively modest increases in antigen affinity.
authors.affiliation: Department of Pediatrics, Memorial Sloan Kettering Cancer Center , New York, NY, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Cheng
authors.affiliation: Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Brian H
authors.initials: BH
authors.lastname: Santich
authors.affiliation: Department of Pediatrics, Memorial Sloan Kettering Cancer Center , New York, NY, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Pediatrics, Memorial Sloan Kettering Cancer Center , New York, NY, USA.
authors.forename: Mahiuddin
authors.initials: M
authors.lastname: Ahmed
authors.affiliation: Immunology Program, Memorial Sloan Kettering Cancer Center , New York, NY, USA.
authors.forename: Morgan
authors.initials: M
authors.lastname: Huse
authors.affiliation: Department of Pediatrics, Memorial Sloan Kettering Cancer Center , New York, NY, USA.
authors.forename: Nai-Kong V
authors.initials: NK
authors.lastname: Cheung
keywords: Affinity; bispecific; disialoganglioside GD2; immunotherapy; scFv; stability