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.

Submission Details

ID: pfN5Rjzn

Submitter: Paul Chang

Submission Date: Nov. 1, 2018, 5:25 p.m.

Version: 3

Publication Details
Adams RM;Mora T;Walczak AM;Kinney JB,Elife (2016) Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves. PMID:28035901
Additional Information

Study Summary

Number of data points 22073
Proteins 4-4-20 (IG*G2A=KAPPA=) FAB FRAGMENT ; anti-fluorescein 4-4-20 scFv
Unique complexes 2825
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)

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

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