Selection and Application of Human Single Chain Fv Antibody Fragments from a Semi-synthetic Phage Antibody Display Library with Designed CDR3 Regions

Kruif, John de; Boel, Edwin; Logtenberg, Ton

doi:10.1006/jmbi.1995.0204

Cited by 162 publications

(90 citation statements)

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“…As antibodies in such libraries are made up of random VH/VL combinations, almost all of which are novel, many new specificities are formed that allow the selection of antibodies against vast arrays of different targets, including self-antigens. Although phage antibody libraries have been widely used to select antibodies against such targets (45)(46)(47)(48)(49)(50)(51)(52)(53), it is surprising that no attempts have been made (or at least published) to select antibodies against post-translational modifications. The results described here suggest a possible explanation: rather than screening 96 -384 clones, which almost always yields a multiplicity of different clones for most targets, we had to screen almost 8000 different clones before finding two identical clones with the desired characteristics.…”

Section: Discussionmentioning

confidence: 99%

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

Kehoe

Velappan

Walbolt

et al. 2006

Molecular & Cellular Proteomics

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Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Much protein activity is regulated by post-translational modifications (PTMs), 1 over 200 of which have been described (1), with changes in enzymatic activity, protein interaction partners, subcellular localization, and targeted degradation being some of the most important regulated activities. A number of different approaches have been used to study PTMs, most of which rely on either specific isolation of the protein of interest and identification of the PTMs that affect that particular protein (2) or isolation of all proteins containing a specific PTM of interest and subsequent identification of the isolated proteins. There are two basic methods to isolate or identify all proteins containing a specific PTM: chemical derivatization or specific affinity reagents. The former rely on the specific chemical reactivity of the PTM to chemical modification, usually by the addition of an easily recognized tag, such as the biotinylation of nitrosylated cysteines (3) or phosphorylated serine/threonine residues (4), allowing them to be either purified or identified by virtue of specific mass shifts. The latter, specific affinity reagents that recognize PTMs, comprise a number of different molecule types, including IMAC using iron or gallium to bind proteins containing phosphotyrosines (5-8) or phosphorylated threonines or serines (9, 10); lectins, which recognize glycosylated proteins; and antibodies recognizing tyrosine modifications. These have all been used in proteomics studies (11)(12)(13)(14)(15)(16)(17). Although antibodies are by far the most common specific affinity reagents, there are very few that recognize PTMs independently of the proteins to which they are attached. Nitrosylated tyrosine and phosphorylated tyrosine are two exceptions for which antibodies have been used in proteomics studies (13-17), and antibodies against phosphoserine/threonine have also been identified (18), indicating that the utility of such reagents...

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Section: Discussionmentioning

confidence: 99%

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

Kehoe

Velappan

Walbolt

et al. 2006

Molecular & Cellular Proteomics

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“…Numerous protein engineering and mutagenesis strategies have been used for the enhancement of antibody affinity (3,5,6,11,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). For small regions (Ͻ8-10 aa), saturation mutagenesis is typically used to produce all combinations of the 20 naturally occurring amino acids.…”

Section: Discussionmentioning

confidence: 99%

A general method for greatly improving the affinity of antibodies by using combinatorial libraries

Rajpal

Beyaz

Haber

et al. 2005

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

142

100

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Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-␣ antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (Kd ‫؍‬ 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and͞or three CDRs for each V H and VL domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-␣. These beneficial mutations in both VH and VL were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500-and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15-to 30-fold improvement in in vitro TNF-␣ neutralization in an L929 bioassay. Thus, this LTM͞combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins.look-through mutagenesis ͉ maturation ͉ mutagenesis ͉ TNF-␣

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“…Abbreviations: A490nm , UV absorption at 490 nm; cfu, colony-forming units; EDTA, ethylenedinitrilotetraacetic acid; ELISA, enzyme-linked immunosorbent assay; IPTG, isopropyl-l-thio-~-D-galactopyranoside; LDH, lactate dehydrogenase; MES, 2-[N-morpholino]ethanesulphonic acid; MOPS, 3-[N-morpholino]propanesulphonic acid; MPBS, skimmed milk powder in PBS; PBS, phosphate-buffered saline; PMSF, phenylmethylsulphonyl fluoride; scFv, single-chain Fv fragment fled from one single library, by using selection and amplification procedures to mimic the immune system [10][11][12][13][14].…”

Section: Introductionmentioning

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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Selection and Application of Human Single Chain Fv Antibody Fragments from a Semi-synthetic Phage Antibody Display Library with Designed CDR3 Regions

Cited by 162 publications

References 0 publications

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

A general method for greatly improving the affinity of antibodies by using combinatorial libraries

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

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