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.
Submitter: Paul Chang
Submission Date: Nov. 1, 2018, 5:25 p.m.
|Number of data points||22073|
|Proteins||4-4-20 (IG*G2A=KAPPA=) FAB FRAGMENT ; anti-fluorescein 4-4-20 scFv|
|Assays/Quantities/Protocols||Experimental Assay: Expression ; Experimental Assay: Kd (Tite-Seq) ; Experimental Assay: Kd (flow)|
|Libraries||Binding/Expression from Replicate 3 ; Binding/Expression from Replicate 2 ; Binding/Expression from Replicate 1 ; Kd_flow and Kd_TiteSeq for 4-4-10 scFv variants (Table 1)|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|100.0||H||4-4-20 (IG*G2A=KAPPA=) FAB FRAGMENT||P01863||GCAA_MOUSE|
|100.0||H||4-4-20 (IG*G2A=KAPPA=) FAB FRAGMENT||P01865||GCAM_MOUSE|
|94.0||H||4-4-20 (IG*G2A=KAPPA=) FAB FRAGMENT||P01864||GCAB_MOUSE|
|94.7||L||4-4-20 (IG*G2A=KAPPA=) FAB FRAGMENT||P01631||KV2A7_MOUSE|
|100.0||L||4-4-20 (IG*G2A=KAPPA=) FAB FRAGMENT||P01837||IGKC_MOUSE|