title: Secondary mAb--vcMMAE conjugates are highly sensitive reporters of antibody internalization via the lysosome pathway.
abstract: Monoclonal antibodies (mAb) selectively recognizing tumor surface antigens are an important and evolving approach to targeted cancer therapy. One application of therapeutic mAbs is drug targeting via mAb-drug conjugate (ADC) technology. Identification of mAbs capable of internalizing following antigen binding has been accomplished by tracking decline of surface-bound mAb or by internalization of a secondary mAb linked to a toxin. These methods may not be sufficiently sensitive for screening nor wholly predictive of the mAbs' capacity for a specific drug delivery. We have developed a highly selective and sensitive method to detect mAbs for cell internalization and drug delivery. This system uses secondary anti-human or anti-murine mAbs conjugated to the high-potency drug monomethyl auristatin E (MMAE) via a highly stable, enzymatically cleavable linker. Prior studies of this drug linker technology demonstrated internalization of a primary ADC leads to trafficking to lysosomes, drug release by lysosomal cathepsin B, and ensuing cell death. A secondary antibody--drug conjugate (2 degrees ADC) capable of binding primary mAbs bound to the surface of antigen-positive cells has comparable drug delivery capability. The system is sufficiently sensitive to detect internalizing mAbs in nonclonal hybridoma supernatants and is predictive of the activity of subsequently produced primary ADC. Because of their high extracellular stability, the noninternalized 2 degrees ADC are 100--1000-fold less toxic to cells over extended periods of time, permitting an assay in which components can be added without need for separate wash steps. This homogeneous screening system is amenable to medium-throughput screening applications and enables the early identification of mAbs capable of intracellular trafficking for drug delivery and release.
authors.affiliation: Department of Biochemistry, Seattle Genetics, Inc., 21823 - 30th Drive Southeast, Bothell, Washington 98021, USA.
authors.forename: Kerry
authors.initials: K
authors.lastname: Klussman
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Mixan
authors.forename: Charles G
authors.initials: CG
authors.lastname: Cerveny
authors.forename: Damon L
authors.initials: DL
authors.lastname: Meyer
authors.forename: Peter D
authors.initials: PD
authors.lastname: Senter
authors.forename: Alan F
authors.initials: AF
authors.lastname: Wahl
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D001692:Biological Transport; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D004705:Endocytosis; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D009842:Oligopeptides / Q000032:analysis* / Q000737:chemistry*; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D009842:Oligopeptides; C495575:monomethyl auristatin E