In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation
Open accessAbstract
Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (KD<10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.
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In vitro affinity maturation methods, such as random mutagenesis with display technologies, improve antibody binding against target proteins but are time consuming and labor intensive.
“In vitro affinity maturation methods, such as random mutagenesis with display technologies, has been shown to improve antibody binding against target proteins, but such approaches are time consuming and labor intensive (27–32).”
From Deep learning guided optimization of human antibody against SARS-CoV-2 variants with broad neutralization (2022) · cites this paper as [31]
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- Authors
- Li, B., Fouts, A. E., Stengel, K., Luan, P., Dillon, M., Liang, W.-C., Feierbach, B., Kelley, R. F., & Hötzel, I.
- Affiliations
- Genentech (US)
- Corresponding author
- Hötzel, Isidro
- ORCID
- Venue
- mAbs
- Publisher
- Informa UK Limited
- Published
- 2014-01-16
- Volume
- 6
- Issue
- 2
- Pages
- 437-445
- ISSN
- 1942-0862 (ISSN-L)
- 1942-0870
- PMC
- PMC3984332
- OpenAlex
- W2028092943
- Cited by
- 28
- References
- 19
- Citations by year
- 2026: 1 · 2024: 3 · 2023: 3 · 2022: 1 · 2021: 2 · 2020: 1 · 2019: 3 · 2018: 1 · 2017: 6 · 2016: 1 · 2015: 5 · 2014: 1
- FWCI
- 3.38
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- mAbs (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Health Sciences › Medicine › Epidemiology › Cytomegalovirus and herpesvirus research
- Keywords
- human cytomegalovirus, affinity maturation, phage display, codon usage, neutralizing antibodies, Fab fragment
- MeSH terms
- Animals, Antibodies, Monoclonal, Antibody Affinity, Cricetulus, Cytomegalovirus, DNA Mutational Analysis, Glycosylation, Humans, Mutation, Mutagenesis, CHO Cells, Transgenes, Antibodies, Blocking, Cell Surface Display Techniques
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