title: Epitope characterization of the ADA response directed against a targeted immunocytokine.
abstract: Targeted immunocytokines (TICs) display potent activity in selective tumor suppression. This class of multi domain biotherapeutics (MDBs) is composed of the three major domains Fab, Fc, and a cytokine which may induce a complex polyclonal anti-drug antibody (ADA) response. However, classical ADA assays usually are not suitable to specify ADAs and to identify the immunogenic domains of a TIC. The purpose of the present study was to establish epitope characterization of ADA responses in order to specify immunogenic responses against a TIC and their direct impact on the pharmacokinetic profile, safety, and efficacy. Based on standard ADA screening and confirmation assays, respectively, domain detection assays (DDAs) and domain competition assays (DCAs) were established and compared by the use of 12 ADA-positive samples obtained from a cynomolgus monkey study in early development. Both domain-specific assays were sensitive enough to preserve the positive screening assay result and revealed an overall accordance for the evaluation of domain-specific ADA responses. About half of the samples displayed one ADA specificity, either for the Fab or for the cytokine (Cy) domain, and the remaining samples showed a combination of Fab-specific and Cy-specific ADA fractions. Fc-specific ADAs occurred in only one sample. In-depth comparison of DCAs and DDAs showed that both assays appeared to be appropriate to assess multi-specific ADA responses as well as minor ADA fractions. An advantage of DCAs is typically a fast and easy assay establishment, whereas, DDAs in some cases may be superior to assess low abundant ADAs in multi-specific responses. Our results reveal that both approaches benefit from thorough reagent development as an essential precondition for reliable epitope characterization of ADA responses.
authors.affiliation: Roche Innovation Center Penzberg, Pharma Research & Early Development (pRED), Pharmaceutical Sciences, Large Molecule Bioanalytical Research & Assay Development, Nonnenwald 2, 82377 Penzberg, Germany. Electronic address: kay-gunnar.stubenrauch@roche.com.
authors.forename: Kay
authors.initials: K
authors.lastname: Stubenrauch
authors.affiliation: Roche Innovation Center Penzberg, Pharma Research & Early Development (pRED), Pharmaceutical Sciences, Large Molecule Bioanalytical Research & Assay Development, Nonnenwald 2, 82377 Penzberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Künzel
authors.affiliation: Roche Diagnostics GmbH, Roche Professional Diagnostics R&D, Early Development & Rare Reagents, D-82377 Penzberg, Nonnenwald 2, 82377 Penzberg, Germany.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Vogel
authors.affiliation: Roche Innovation Center Basel, Pharma Research & Early Development (pRED), Pharmaceutical Sciences, DMPK project leaders, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, 4070 Basel, Switzerland.
authors.forename: Dietrich
authors.initials: D
authors.lastname: Tuerck
authors.affiliation: Roche Innovation Center Basel, Pharma Research & Early Development (pRED), Pharmaceutical Sciences, Large Molecule Regulated Bioanalysis, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, 4070 Basel, Switzerland.
authors.forename: Eginhard
authors.initials: E
authors.lastname: Schick
authors.affiliation: Roche Innovation Center Penzberg, Pharma Research & Early Development (pRED), Pharmaceutical Sciences, Large Molecule Bioanalytical Research & Assay Development, Nonnenwald 2, 82377 Penzberg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Heinrich
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000917:Antibody Formation / Q000276:immunology; D000918:Antibody Specificity; D001667:Binding, Competitive; D016207:Cytokines / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D008252:Macaca fascicularis; D008297:Male; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D016207:Cytokines; D000939:Epitopes; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins
keywords: Anti-drug antibody epitope characterization; Domain competition assay; Domain detection assay; Immunogenicity testing; Multi domain biotherapeutic; Targeted immunocytokine