Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves

TL;DR

Tite-Seq is a novel high-throughput method for measuring antibody binding affinities across thousands of variants, providing insights into the sequence-affinity landscape and structural basis of antigen binding.

Contribution

The paper introduces Tite-Seq, a new experimental approach that accurately measures titration curves and affinities for many antibody variants simultaneously, overcoming limitations of previous methods.

Findings

Tite-Seq successfully measures binding titration curves for thousands of antibody variants.

Data reveals the structural role of secondary CDR loops in antibody stability.

The method can be applied to other protein systems to study sequence-affinity relationships.

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.