Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves
Open accessPublished version of a preprint
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.
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Yeast surface display has been used to screen thousands of protein variants for binding affinity and function.
“Yeast surface display, in particular, has been used to screen thousands of protein variants for binding affinity and function (65, 66).”
From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [65]
The Results (Proof-of-principle Tite-Seq experiments; no page available) describe yeast-displayed libraries totaling thousands of antibody variants, assayed for antigen binding and surface expression. This supports the scale and binding-affinity portion of the claim, but the paper does not report screening those variants for a separate measure of protein function. (LLM, conf=0.83)
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Starr, T. N., Greaney, A. J., Hilton, S. K., Ellis, D., Crawford, K. H. D., Dingens, A. S., Navarro, M. J., Bowen, J. E., Tortorici, M. A., Walls, A. C., et al. (2020) · Cell · doi:10.1016/j.cell.2020.08.012 · Cited by 1,956 · see citing works
Gauge fixing for sequence-function relationships
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Self-citationMassively parallel functional genomic assays in endocrinology: from promise to delivery
Xu, J., Aslanzadeh, V., Kudla, G., & Semple, R. K. (2026) · Journal of Molecular Endocrinology · doi:10.1530/jme-25-0134
- Authors
- Adams, R. M., Mora, T., Walczak, A. M., & Kinney, J. B.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- Cold Spring Harbor Laboratory (US)
- Venue
- eLife
- Publisher
- eLife Sciences Publications, Ltd
- Published
- 2016-12-30
- Volume
- 5
- ISSN
- 2050-084X (ISSN-L)
- PMC
- PMC5268739
- OpenAlex
- W2238330501
- Cited by
- 137
- References
- 2
- Citations by year
- 2026: 26 · 2025: 11 · 2024: 13 · 2023: 15 · 2022: 19 · 2021: 11 · 2020: 13 · 2019: 11 · 2018: 10 · 2017: 5 · 2016: 3
- FWCI
- 7.30
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- eLife (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Cold Spring Harbor Laboratory Institutional Repository (Cold Spring Harbor Laboratory) (acceptedVersion)
- arXiv (Cornell University) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- arXiv (Cornell University)
- Funders
- European Research Council (StG n. 306312)
- Simons Center for Quantitative Biology
- Topics
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
- Keywords
- antibody affinity, deep mutational scanning, scFv, antibody stability, adaptive immunity, next-generation sequencing
- MeSH terms
- Amino Acid Sequence, Antibody Affinity, Antibody Specificity, Antigen-Antibody Complex, Binding Sites, Antibody, Humans, Kinetics, Models, Molecular, Oligonucleotides, Protein Binding, Saccharomyces cerevisiae, Structure-Activity Relationship, Peptide Library, Single-Chain Antibodies, High-Throughput Screening Assays
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