title: Off-rate screening for selection of high-affinity anti-drug antibodies.
abstract: The rapidly increasing number of therapeutic antibodies in clinical development and on the market requires corresponding detection reagents for monitoring the concentration of these drugs in patient samples and as positive controls for measurement of anti-drug antibodies. Phage display of large recombinant antibody libraries has been shown to enable the rapid development of fully human anti-idiotypic antibodies binding specifically to antibody drugs, since the in vitro panning approach allows for incorporation of suitable blockers to drive selection toward the paratope of the drug. A typical bottleneck in antibody generation projects is ranking of the many candidates obtained after panning on the basis of antibody binding strength. Ideally, such method will work without prior labeling of antigens and with crude bacterial lysates. We developed an off-rate screening method of crude Escherichia coli lysates containing monovalent Fab fragments obtained after phage display of the HuCAL PLATINUM® antibody library. We used the antibody drugs trastuzumab and cetuximab as antigen examples. Using the Octet® RED384 label-free sensor instrument we show that antibody off rates can be reliably determined in crude bacterial lysates with high throughput. We also demonstrate that the method can be applied to screening for high-affinity antibodies typically obtained after affinity maturation.
authors.affiliation: Bio-Rad AbD Serotec GmbH, 82178 Puchheim, Germany. Electronic address: francisco.ylera@abdserotec.com.
authors.forename: Francisco
authors.initials: F
authors.lastname: Ylera
authors.forename: Stefan
authors.initials: S
authors.lastname: Harth
authors.forename: Dirk
authors.initials: D
authors.lastname: Waldherr
authors.forename: Christian
authors.initials: C
authors.lastname: Frisch
authors.forename: Achim
authors.initials: A
authors.lastname: Knappik
mesh_terms: D000888:Antibodies, Anti-Idiotypic / Q000235:genetics / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology*; D000915:Antibody Affinity; D000068818:Cetuximab; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D000068878:Trastuzumab
chemical_list: D000888:Antibodies, Anti-Idiotypic; D061067:Antibodies, Monoclonal, Humanized; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D000068878:Trastuzumab; D000068818:Cetuximab
keywords: Affinity screening; Anti-idiotype; Biolayer interferometry; Cetuximab; HuCAL; Trastuzumab