Cited by 21Monoclonal and Polyclonal Antibodies ResearchProtein purification and stabilityChemical Synthesis and Analysis

In Vitro Antibody Affinity Maturation Targeting Germline Hotspots

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Ho, Mitchell, Pastan, Ira·2009·Methods in Molecular Biology™

Abstract

Affinity-matured antibodies can exhibit increased biological efficacy. Regardless of whether an antibody is isolated from a hybridoma or a human Fv phage library, the antibody affinity for its target may need improvement for therapeutic applications. An increased affinity may allow for a reduced dosage of a therapeutic antibody; toxic side effects may also be reduced. In the immune system, affinity maturation is a process involving somatic hypermutations in B cells. Therefore, germline hotspot residues are most likely to have a major impact on antibody affinity. Here, we describe procedures for germline hotspot mutagenesis with an emphasis on strategies for randomizing hotspots with PCR and phage display, using as an example the anti-CD22 monoclonal antibody.

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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).”

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Authors
Ho, M., & Pastan, I.
Affiliations
National Institutes of Health (US)
Corresponding author
Ho, Mitchell
Venue
Methods in Molecular Biology™
Publisher
Humana Press
Published
2008-12-10
Volume
525
Pages
293-308
ISSN
  • 1064-3745 (ISSN-L)
  • 1940-6029
OpenAlex
W10761404
Cited by
21
References
21
Citations by year
2024: 2 · 2023: 2 · 2022: 2 · 2020: 2 · 2019: 2 · 2018: 3 · 2017: 2 · 2014: 2 · 2013: 1
FWCI
4.83
Type
book chapter
Language
en
Open access
Locations
  • Methods in molecular biology (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health (Z99 CA999999)
  • National Cancer Institute (Z01BC010891, Y99 CA999999, ZIABC010891)
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 › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
Keywords
affinity maturation, somatic hypermutation, anti-CD22 monoclonal antibody, phage display, random mutagenesis, therapeutic antibodies, B cells
MeSH terms
Amino Acid Sequence, Antibodies, Monoclonal, Antibody Affinity, Bacteriophages, Base Sequence, Enzyme-Linked Immunosorbent Assay, Escherichia coli, Flow Cytometry, Germ Cells, Models, Molecular, Molecular Biology, Molecular Sequence Data, Sequence Alignment, Sequence Analysis, DNA, Electroporation, Peptide Library

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