title: Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves.
abstract: Despite the central role that antibodies play in the adaptive immune system and in biotechnology, much remains unknown about the quantitative relationship between an antibody's amino acid sequence and its antigen binding affinity. Here we describe a new experimental approach, called Tite-Seq, that is capable of measuring binding titration curves and corresponding affinities for thousands of variant antibodies in parallel. The measurement of titration curves eliminates the confounding effects of antibody expression and stability that arise in standard deep mutational scanning assays. We demonstrate Tite-Seq on the CDR1H and CDR3H regions of a well-studied scFv antibody. Our data shed light on the structural basis for antigen binding affinity and suggests a role for secondary CDR loops in establishing antibody stability. Tite-Seq fills a large gap in the ability to measure critical aspects of the adaptive immune system, and can be readily used for studying sequence-affinity landscapes in other protein systems.
authors.affiliation: Laboratoire de Physique Théorique, UMR8549, CNRS, École Normale Supérieure, Paris, France.|Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, United States.
authors.forename: Rhys M
authors.initials: RM
authors.lastname: Adams
authors.affiliation: Laboratoire de Physique Statistique, UMR8550, CNRS, École Normale Supérieure, Paris, France.
authors.forename: Thierry
authors.identifier: 0000-0002-5456-9361
authors.initials: T
authors.lastname: Mora
authors.affiliation: Laboratoire de Physique Théorique, UMR8549, CNRS, École Normale Supérieure, Paris, France.
authors.forename: Aleksandra M
authors.identifier: 0000-0002-2686-5702
authors.initials: AM
authors.lastname: Walczak
authors.affiliation: Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, United States.
authors.forename: Justin B
authors.identifier: 0000-0003-1897-3778
authors.initials: JB
authors.lastname: Kinney
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000032:analysis*; D001666:Binding Sites, Antibody; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D013329:Structure-Activity Relationship
chemical_list: D000936:Antigen-Antibody Complex; D009841:Oligonucleotides; D019151:Peptide Library; D057127:Single-Chain Antibodies
keywords: S. cerevisiae; affinity; antibody; biophysics; deep mutational scan; dissociation constant; structural biology; titration curve