Isolation of high affinity human antibodies directly from large synthetic repertoires.
15 Jul 1994-The EMBO Journal (European Molecular Biology Organization)-Vol. 13, Iss: 14, pp 3245-3260
TL;DR: This work created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and generated a large synthetic repertoire of Fab fragments displayed on filamentous phage to help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.
Abstract: Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.
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Patent•
29 Apr 1996
TL;DR: In this paper, a transgenic animal has been modified to produce antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, and various subsequent manipulations can be performed to obtain either antibodies per se or analogs thereof.
Abstract: Fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Various subsequent manipulations can be performed to obtain either antibodies per se or analogs thereof.
2,667 citations
TL;DR: C-terminal fusion to the Aga2p mating adhesion receptor of Saccharomyces cerevisiae has been used for the selection of scFv antibody fragments with threefold decreased antigen dissociation rate from a randomly mutated library.
Abstract: Display on the yeast cell wall is well suited for engineering mammalian cell-surface and secreted proteins (e.g., antibodies, receptors, cytokines) that require endoplasmic reticulum-specific post-translational processing for efficient folding and activity. C-terminal fusion to the Aga2p mating adhesion receptor of Saccharomyces cerevisiae has been used for the selection of scFv antibody fragments with threefold decreased antigen dissociation rate from a randomly mutated library. A eukaryotic host should alleviate expression biases present in bacterially propagated combinatorial libraries. Quantitative flow cytometric analysis enables fine discrimination of kinetic parameters for protein binding to soluble ligands.
1,880 citations
TL;DR: This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.
Abstract: To generate a stable resource from which high affinity human antibodies to any given antigen can be rapidly isolated, functional V-gene segments from 43 non-immunized human donors were used to construct a repertoire of 1.4 x 10(10) single-chain Fv (scFv) fragments displayed on the surface of phage. Fragments were cloned in a phagemid vector, enabling both phage displayed and soluble scFv to be produced without subcloning. A hexahistidine tag has been incorporated to allow rapid purification of scFv by nickel chelate chromatography. This library format reduces the time needed to isolate monoclonal antibody fragments to under two weeks. All of the measured binding affinities show a Kd < 10 nM and off-rates of 10(-3) to 10(-4) s-1, properties usually associated with antibodies from a secondary immune response. The best of these scFvs, an anti-fluorescein antibody (0.3 nM) and an antibody directed against the hapten DTPA (0.8 nM), are the first antibodies with subnanomolar binding affinities to be isolated from a naive library. Antibodies to doxorubicin, which is both immunosuppressive and toxic, as well as a high affinity and high specificity antibody to the steroid hormone oestradiol have been isolated. This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.
1,409 citations
TL;DR: 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.
1,122 citations
Cites background or methods from "Isolation of high affinity human an..."
...This observed expression yield is signi®cantly higher than that reported for antibody fragments from other libraries (Grif®ths et al., 1994; Vaughan et al., 1996)....
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...…of the VH3 germline subfamily, is also the most frequently used VH3 germline gene (see Table 1) and it is very frequently found in antibody phage-display libraries based on human genes (Grif®ths et al., 1994; Vaughan et al., 1996; Dorsam et al., 1997; Boel et al., 1998; Sheets et al., 1998)....
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...The predominance of VH3 occurs also in nature (see Table 1) and is even higher in other libraries (Grif®ths et al., 1994; Vaughan et al., 1996)....
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...Previous phage-display libraries of human antibodies have been generated from immunized donors (Barbas & Burton, 1996), germline sequences (Grif®ths et al., 1994) or, most recently, naive B-cell Ig repertoires (Vaughan et al., 1996; Sheets et al., 1998; De Haard et al., 1999)....
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...The strategy of the synthetic library approach was therefore to represent each family by one representative member, subject to veri®cation of the structural consequence of the distribution of CDR conformations (see the next section)....
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TL;DR: Domain Antibels (dAbs) as discussed by the authors are the smallest known antigen-binding fragments of antibodies, ranging from 11 kDa to 15 kDa, and they are the robust variable regions of the heavy and light chains of immunoglobulins (VH and VL respectively).
1,078 citations
References
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TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
232,912 citations
Book•
01 Jan 1972
TL;DR: Molecular Genetics (Biology): An Overview | Sciencing Experimental in Molecular Genetics Experiments in molecular genetics (1972 edition) | Open ...
Abstract: Molecular Genetics (Biology): An Overview | Sciencing Experiments in Molecular Genetics Experiments in molecular genetics (1972 edition) | Open ... Experimental Molecular Genetics | Biology | MIT OpenCourseWare DNA experiments you can perform at home | SBS Science Experiments in molecular genetics Jeffrey H. Miller ... DNA and Molecular Genetics Experiments in Molecular Biology: Biochemical Applications ... Molecular Genetics Biology Experiment Please help ... Molecular genetics | biology | Britannica Molecular Genetic Experiment : Biology Lab 1793 Words ... Miller, J.H. (1972) Experiments in Molecular Genetics ... Griffith's experiment Wikipedia DNA as genetic material: Revisiting classic experiments ... Experiments in molecular genetics (Book, 1972) [WorldCat.org] Measuring βGalactosidase Activity in Bacteria: Cell ... Classic Experiments in
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TL;DR: New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors and mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences.
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TL;DR: Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase.
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