By–Passing Immunization: Building High Affinity Human Antibodies by Chain Shuffling

Marks, James D.; Griffiths, Andrew D.; Malmqvist, Magnus; Clackson, Tim; Bye, Jacqueline M.; Winter, Greg

doi:10.1038/nbt0792-779

Cited by 343 publications

(150 citation statements)

References 36 publications

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“…We therefore addressed the V H :V L pairing problem by synthesizing select V H genes and then performing two to three rounds of phage panning of scFv libraries constructed with amplified V L cDNA (i.e., a V L chain shuffled library for each selected V H ). Phage panning of a fixed V H with a library of V L genes is an established method for identifying functional pairs that bind antigen with high affinity (26). The V H genes corresponding to seven of the most abundant proteomically identified iCDRH3s were synthesized by automated DNA synthesis (18).…”

Section: Resultsmentioning

confidence: 99%

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Wine

Boutz

Lavinder

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

129

156

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We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V H CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigenspecific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V H clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.antibody proteomics | antibody repertoire | serum immunoprofiling | B-cell response | humoral response T he first Nobel Prize in Medicine was awarded to Emil von Behring, who in collaboration with Kitasato Shibasaburo and Paul Ehrlich discovered serum antitoxins (1, 2). Remarkably, after more than 100 y of intense research in immunology, little is known about the clonality, relative concentrations, and binding properties of the monoclonal antibodies that constitute the antigen-specific Ig pool in serum.At steady state, circulating antibodies are produced by terminally differentiated B lymphocytes (plasma cells) within the bone marrow, and thus cannot be accessed in living individuals (3). Although recent single B-cell cloning methods (4, 5) have led to the identification of peripheral antigen-specific B memory and/or antibody-secreting cells (plasmablasts), it is generally unknown whether the Igs encoded by peripheral blood B cells correspond to the antibodies present in circulation and especially whether they are present at physiologically relevant levels (i.e., at serum concentrations above K D corresponding to >1 μg/mL for an average affinity of individual antibodies of 5 nM)...

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Section: Resultsmentioning

confidence: 99%

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Wine

Boutz

Lavinder

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

129

156

View full text Add to dashboard Cite

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“…As this requires selection against various antigens simultaneously, we chose to work with a naive library, which is known to be robust and easy to use, and has been used to select antibodies towards several antigens [11]. The affinity of antibodies identified from this library is normally in the micromolar range, but can be improved by affinity maturation [27][28][29][30][31].…”

Section: Resultsmentioning

confidence: 99%

A model phage display subtraction method with potential for analysis of differential gene expression

et al. 1996

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In order to establish a subtractive procedure that makes it possible to enrich selectively phage displayed antibodies directed against proteins constituting a difference between two populations of cells, a competitive selection strategy utilising two solid phases was developed and tested. Antibodies recognising a defined difference between two otherwise identical protein mixtures were isolated and their specificity confirmed. To test further the efficacy of selection inhibition during the competitive selections, selections towards a total cell extract were performed with and without competition from the same extract. An analysis of the resulting phage antibodies confirmed the subtractive nature of the system described.

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“…Phage ELISA (Marks et al 1992) was used to identify lysozyme-binding D1.3 single-chain antibody fragments (scFvs) present in either pDAN5 or pPAO2 with or without ␤-lactamase.…”

Section: Elisa Analysis and Dot Blotmentioning

confidence: 99%

Selecting Open Reading Frames From DNA

Zacchi

Sblattero²,

Florian³

et al. 2003

Genome Res.

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We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding DNA within the context of a specific vector. Phage display has been used as an example, but any system requiring DNA encoding protein fragments, for example, the yeast two-hybrid system, could be used. By cloning DNA fragments upstream of a fusion gene, consisting of the β-lactamase gene flanked by lox recombination sites, which is, in turn, upstream of gene 3 from fd phage, only those clones containing DNA fragments encoding ORFs confer ampicillin resistance and survive. After selection, the β-lactamase gene can be removed by Cre recombinase, leaving a standard phage display vector with ORFs fused to gene 3. This vector has been tested on a plasmid containing tissue transglutaminase. All surviving clones analyzed by sequencing were found to contain ORFs, of which 83% were localized to known genes, and at least 80% produced immunologically detectable polypeptides. Use of a specific anti-tTG monoclonal antibody allowed the identification of clones containing the correct epitope. This approach could be applicable to the efficient selection of random ORFs representing the coding potential of whole organisms, and their subsequent downstream use in a number of different systems

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By–Passing Immunization: Building High Affinity Human Antibodies by Chain Shuffling

Cited by 343 publications

References 36 publications

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

A model phage display subtraction method with potential for analysis of differential gene expression

Selecting Open Reading Frames From DNA

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