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

Rhys M Adams,Justin B Kinney,Thierry Mora,Aleksandra M Walczak

doi:10.7554/elife.23156

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.

Highlights

During an infection, the immune system must recognize and neutralize invading pathogens
The composition of this library is such that each cell displays a single antibody variant, and each variant is expressed on the surface of multiple cells
Cells are incubated with the antigen of interest, bound antigen is fluroescently labeled, and fluorescence-activated cell sorting (FACS) is used to sort cells one-by-one into multiple “bins” based on this fluorescent readout (Fig. 1B)

Summary

Introduction

The immune system must recognize and neutralize invading pathogens. B-cells contribute to immune defense by producing antibodies, proteins that bind to foreign antigens. The astonishing capability of antibodies to recognize virtually any foreign molecule has been repurposed by scientists in wide variety of experimental techniques (immunofluorescence, western blots, ELISA, ChIP-Seq, etc.). Much is known about the qualitative mechanisms of antibody generation and function [2]. The antigenic specificity of antibodies in humans, mice, and most jawed vertebrates is primarily governed by six complementarity determining regions (CDRs), each roughly 10 amino acids (aa) long. During B-cell differentiation, these six sequences are randomized through V(D)J recombination, selected for functionality as well as against the ability to recognize host antigens. Upon participation in an immune response, CDR regions can further undergo somatic hypermutation and selection, yielding higher-affinity antibodies for specific antigens. Among the CDRs, CDR3H is the most highly variable and typically contributes the most to antigen specificity; less clear are the functional roles of the other CDRs, which often do not interact with the target antigen directly

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