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Selection and Application of Human Single Chain Fv Antibody Fragments from a Semi-synthetic Phage Antibody Display Library with Designed CDR3 Regions
Abstract
We have constructed a large (3.6 x 10(8) clones) phage display library of human single chain Fv (scFv) antibody fragments by combining 49 germline VH genes with synthetic heavy chain CDR3 (HCDR3) regions and seven light chains. The HCDR3 regions varied in length between 6 and 15 residues and were designed to contain fully randomized stretches of amino acid residues flanked by regions of limited residue variability that were composed of amino acid residues that frequently occur in natural antibodies. We reasoned that this approach would increase the frequency of functional molecules in our library and, in addition, permit us to efficiently utilize available cloning space. By direct selection on solid phase-bound antigens we obtained phage antibodies with binding activities to 13 different antigens, including Von Willebrand factor, the DNA-binding HMG box of transcription factor TCF-1 and the tumor antigen EGP-2. In addition, we applied a competitive selection procedure to target phage antibodies to the desired portion of a recombinant fusion protein and to select phage antibodies capable of discriminating between the two highly homologous homeobox proteins PBX1a and PBX2. The functional capacity of monoclonal phage antibodies was assessed in immuno-histochemical staining of tissue specimens. Western blotting assays and immunofluorescent analysis of cells by flow cytometry. The results demonstrate that this large human phage antibody library contains a broad assortment of binding specificities that can be applied in a variety of biochemical assays.
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“Due to their central role in antigen recognition and binding, CDR loops undergo the most extensive engineering during development of monoclonal antibody (mAb) therapeutics [36, 37].”
From Computational approaches to therapeutic antibody design: established methods and emerging trends (2020) · cites this paper as [37]
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Self-citation
Metadata
- Authors
- de Kruif, J., Boel, E., & Logtenberg, T.
- Affiliations
- Utrecht University (NL)
- Corresponding author
- Logtenberg, Ton
- ORCID
- Boel, Edwin
- Venue
- Journal of Molecular Biology
- Publisher
- Elsevier BV
- Published
- 1995-04
- Volume
- 248
- Issue
- 1
- Pages
- 97-105
- ISSN
- 0022-2836 (ISSN-L)
- OpenAlex
- W2091855294
- Cited by
- 173
- References
- 0
- Citations by year
- 2025: 2 · 2024: 1 · 2023: 1 · 2022: 2 · 2021: 1 · 2019: 5 · 2018: 2 · 2017: 1 · 2015: 3 · 2014: 4 · 2013: 1 · 2012: 7
- FWCI
- 10.91
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
- Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
- Keywords
- phage display library, human antibody fragments, competitive selection, von Willebrand factor, PBX2, homeodomain transcription factor, monoclonal antibodies
- MeSH terms
- Amino Acid Sequence, Animals, Haplorhini, Antibodies, Monoclonal, Bacteriophages, Base Sequence, Cell Line, Transformed, Cloning, Molecular, DNA-Binding Proteins, Herpesvirus 4, Human, Fluorescent Antibody Technique, Humans, Immunoglobulin Fragments, Immunoglobulin Fab Fragments, Molecular Sequence Data, Plasmids, Thyroid Gland, Blotting, Western, Gene Library, Receptor-CD3 Complex, Antigen, T-Cell, Sequence Homology, Amino Acid, DNA Primers, Intercellular Adhesion Molecule-1, Macromolecular Substances
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