In vitro antibody maturation. Improvement of a high affinity, neutralizing antibody against IL-1 beta .
Abstract
Abs as therapeutic antagonists should be of relatively high affinity to effectively neutralize their target Ag. Typically, Abs with nanomolar affinities to protein Ags can be obtained in vivo, however, this may not be the upper limit of affinity because the biologic process attempts to optimize Ab function, of which affinity is only one component. SK48, a high affinity neutralizing murine Fab against human IL-1 beta was used to explore the nature of Ab-Ag interactions and the potential for further affinity improvement in vitro using mutagenesis and selection via phage display. The codons of six amino acids in the third complementarity-determining region of the heavy chain (CDR3-H) were both individually and combinatorialy randomized and the resultant libraries were screened for IL-1 binding phenotype. Mutations that reduced affinity suggested that both the backbone conformation of the CDR3 loop and certain side chains are essential for binding, yet alterations to the canonical salt bridge residues at the base of the loop had minimal effect on affinity. Four rounds of selection of the phage Ab libraries on immobilized IL-1 beta gave predominantly the wild-type sequence, indicating efficient affinity maturation of this CDR in vivo. However, a twofold improvement in affinity was observed for both single and double amino acid changes at two positions in the middle of the CDR. Moreover, a 10-fold increase in affinity for IL-1 beta was achieved by combining two of the phage-selected single amino acid substitutions in CDR3-H, thereby demonstrating that a significant improvement in affinity can be achieved through CDR mutagenesis, even in a matured Ab. The increased affinity of this Fab did not, however, enhance its neutralizing activity in vitro.
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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 [29]
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- Authors
- Jackson, J. R., Sathe, G., Rosenberg, M., & Sweet, R.
- Affiliations
- Departments of Molecular Immunology and Molecular Genetics, SmithKline Beecham Pharmaceuticals , King of Prussia, PA 19406
- ORCID
- Rosenberg, M
- Venue
- The Journal of Immunology
- Publisher
- Oxford University Press (OUP)
- Published
- 1995-04-01
- Volume
- 154
- Issue
- 7
- Pages
- 3310-3319
- ISSN
- 0022-1767 (ISSN-L)
- 1550-6606
- OpenAlex
- W2143623518
- Cited by
- 50
- References
- 0
- Citations by year
- 2024: 1 · 2023: 1 · 2022: 3 · 2021: 2 · 2019: 2 · 2018: 1
- FWCI
- 6.37
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- The Journal of Immunology (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
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Keywords
- affinity maturation, IL-1β, neutralizing antibodies, Fab fragment, antigen-antibody reaction
- MeSH terms
- Amino Acid Sequence, Animals, Antibody Affinity, Base Sequence, Binding, Competitive, Enzyme-Linked Immunosorbent Assay, Humans, Immunoglobulin Variable Region, Immunoglobulin Fab Fragments, Interleukin-1, Molecular Sequence Data, Neutralization Tests, Mutagenesis, Site-Directed, Epitope Mapping, Mice
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