title: Surface plasmon resonance-based competition assay to assess the sera reactivity of variants of humanized antibodies.
abstract: While clinical trials are the only way to evaluate the immunogenicity, in patients, of murine or genetically engineered humanized variants of a potentially therapeutic or diagnostic monoclonal antibody (MAb), ethical and logistical considerations of clinical trials do not permit the evaluation of variants of a given MAb that are generated to minimize its immunogenicity. The most promising variant could be identified by comparing the reactivities of the parental antibody (Ab) and its variants to the sera of patients containing anti-variable region (anti-VR) Abs to the administered parental Ab. We have developed a surface plasmon resonance (SPR) biosensor-based assay to monitor the binding of the sera anti-VR Abs to the parental Ab and the inhibition of this binding by the variants. SPR biosensors allow the real-time detection and monitoring of the binding between an immobilized protein and its soluble ligand without the need for prior purification and labeling of the mobile analyte. This new assay requires no radiolabeling, is relatively less time-consuming, and uses only small amounts of serum (5-20 microl of diluted serum) through a new microfluidic sample handling technique. To validate the assay, we have tested the relative reactivities of the CDR-grafted anti-carcinoma Ab, HuCC49, and its two variants, designated V5 and V10, to the sera of patients who were earlier administered radiolabeled murine CC49 in a clinical trial. A comparison of IC(50)s (the concentrations of the competitor Abs required for 50% inhibition of the binding of sera to immobilized HuCC49) showed that V5 and V10 were less reactive than HuCC49 to the three patients' sera tested. We have also demonstrated, for the first time, the specific detection and comparison of relative amounts of anti-VR Abs present in the sera of different patients without prior removal of anti-murine Fc Abs and/or circulating antigen. This may facilitate the rapid screening, for the presence of anti-VR Abs, of the sera of patients undergoing clinical trials.
authors.affiliation: Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Drive, Room 8B09, Bethesda, MD 20892, USA.
authors.forename: Noreen R
authors.initials: NR
authors.lastname: Gonzales
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Schlom
authors.forename: Syed V S
authors.initials: SV
authors.lastname: Kashmiri
mesh_terms: D000327:Adsorption; D000818:Animals; D000906:Antibodies / Q000097:blood*; D000912:Antibodies, Neoplasm / Q000276:immunology*; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D002986:Clinical Trials as Topic; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D051379:Mice; D020349:Surface Plasmon Resonance
chemical_list: D000906:Antibodies; D000912:Antibodies, Neoplasm; C091202:B72.3 antibody; D007135:Immunoglobulin Variable Region