Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides 1 1Edited by I. A. Wilson
Abstract
By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.
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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 therapeutics.
“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 [36]
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Self-citationPhage display of disulfide-stabilized Fv fragments
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Flagged first, then by citations
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Binz, H. K., Stumpp, M. T., Forrer, P., Amstutz, P., & Plückthun, A. (2003) · Journal of Molecular Biology · doi:10.1016/s0022-2836(03)00896-9 · Cited by 589 · see citing works
Self-citationActive and passive immunotherapy for neurodegenerative disorders
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“during development of monoclonal antibody (mAb) therapeutics [36, 37].”
BackgroundAdhiron: a stable and versatile peptide display scaffold for molecular recognition applications
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Understanding the human antibody repertoire
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A human SARS-CoV neutralizing antibody against epitope on S2 protein
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Synthetic antibodies designed on natural sequence landscapes
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Jain, T., Boland, T., Lilov, A., Burnina, I., Brown, M., Xu, Y., & Vásquez, M. (2017) · Bioinformatics · doi:10.1093/bioinformatics/btx519 · Cited by 61 · see citing works
Looking for therapeutic antibodies in next generation sequencing repositories
Krawczyk, K., Raybould, M., Kovaltsuk, A., & Deane, C. M. (2019) · doi:10.1101/572958 · Cited by 29 · see citing works
- Authors
- Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wölle, J., Plückthun, A., & Virnekäs, B.
- Affiliations
- MorphoSys (Germany) (DE)
- University of Zurich (CH)
- Corresponding author
- Knappik, Achim
- Venue
- Journal of Molecular Biology
- Publisher
- Elsevier BV
- Published
- 2000-02
- Volume
- 296
- Issue
- 1
- Pages
- 57-86
- ISSN
- 0022-2836 (ISSN-L)
- OpenAlex
- W2144886557
- Cited by
- 728
- References
- 117
- Citations by year
- 2026: 5 · 2025: 7 · 2024: 16 · 2023: 13 · 2022: 20 · 2021: 22 · 2020: 26 · 2019: 18 · 2018: 19 · 2017: 30 · 2016: 20 · 2015: 24 · 2014: 23 · 2013: 30 · 2012: 37
- Type
- book review
- Language
- en
- Open access
- closed
- Locations
- Journal of Molecular Biology (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 › Chemical Synthesis and Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
- Keywords
- human antibody repertoire, hypervariable region, CDR3 diversity, phage display library, periplasmic expression
- MeSH terms
- Amino Acid Sequence, Antibody Affinity, Antibody Diversity, Cloning, Molecular, Escherichia coli, Genes, Immunoglobulin, Humans, Immunoglobulin Variable Region, Kinetics, Models, Molecular, Molecular Sequence Data, Oligodeoxyribonucleotides, Protein Conformation, Random Allocation, Solubility, Thermodynamics, Reproducibility of Results, Mutagenesis, Insertional, Consensus Sequence, Sequence Alignment, Protein Folding, Peptide Library, Combinatorial Chemistry Techniques
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
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