title: High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe.
abstract: Antibody-drug conjugates (ADCs) represent a rapidly growing class of biotherapeutics that deliver drugs specifically to target cells by binding of the antibody component to surface receptors. The majority of ADCs require receptor internalization depending on intrinsic features of the specific ADC-antigen interaction. The development of potent ADCs would greatly benefit from the identification of efficiently internalizing antibodies at early stages of discovery. We developed a highly sensitive and rapid antibody internalization assay using an indirect Cypher5E label. The pH-activated CypHer5E label becomes fluorescent upon internalization into the acidic environment of endocytic organelles, whereas background fluorescence of noninternalized CypHer5E is minimal. The pH-dependency of the CypHer5E signal enables robust discrimination of antibody internalization from surface binding. The favorable signal-over-background ratio allows a homogeneous assay design with high-throughput fluorescence imaging in 384- and 1536-well formats. The biophysical readout of the primary internalization event substantially shortens incubation times compared to killing assays using toxin internalization. The assay was validated with tumor-relevant targets, including receptor tyrosine kinases (EGFR and HER2) and a class II cytokine receptor (TF) expressed by A431, AU565, and SKOV-3 cells and transient expression systems (CHO-S). Our method enables functional screening of large antibody libraries to identify therapeutic antibody candidates with internalization characteristics favorable for the development of ADCs.
authors.affiliation: Genmab, Utrecht, the Netherlands.
authors.forename: Thilo
authors.initials: T
authors.lastname: Riedl
authors.affiliation: Genmab, Utrecht, the Netherlands.
authors.forename: Egon
authors.initials: E
authors.lastname: van Boxtel
authors.affiliation: Genmab, Utrecht, the Netherlands.
authors.forename: Martijn
authors.initials: M
authors.lastname: Bosch
authors.affiliation: Genmab, Utrecht, the Netherlands Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Paul W H I
authors.initials: PW
authors.lastname: Parren
authors.affiliation: Genmab, Utrecht, the Netherlands A.Gerritsen@genmab.com.
authors.forename: Arnout F
authors.initials: AF
authors.lastname: Gerritsen
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods; D045744:Cell Line, Tumor; D003952:Diagnostic Imaging / Q000379:methods; D066246:ErbB Receptors / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D018121:Receptors, Cytokine / Q000378:metabolism
chemical_list: D000906:Antibodies; D066246:ErbB Receptors; D005456:Fluorescent Dyes; D018719:Erb-b2 Receptor Tyrosine Kinases; D018121:Receptors, Cytokine
keywords: antibody internalization; antibody-drug conjugates; high-throughput screening; homogenous assay; pH-dependent dye