Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli

Ward, E. Sally; Güssow, Detlef; Griffiths, Andrew D.; Jones, Peter; Winter, Greg

doi:10.1038/341544a0

Cited by 944 publications

(419 citation statements)

References 31 publications

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“…In addition, the significantly higher inherent R:S mutation ratios of the CDRs of V H -gene segments as compared with those of the corresponding regions of V L -gene segments might reflect a function-related evolutionary selection, consistent with the accumulating evidence that H-chain V segments provide the major structural correlate for antigen binding [51][52][53][54] . The fact that the inherent structural hypervariability is maximal within a gene sequence, CDR1, that may also be preferentially targeted by the mutational machinery suggests that the concentration of R mutations observed within CDR1 and flanking areas in high-affinity 'mature' antibodies is not solely due to antigen selection.…”

Section: Discussionsupporting

confidence: 63%

The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement

1994

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The variable (V) genes of antigen-selected antibodies are known to exhibit a higher frequency of amino acid replacement mutations in the sequences encoding the antigen-contacting complementarity-determining regions (CDRs) than in those encoding the 'structural' framework regions (FRs). Here, Bernard Chang and Paolo Casali analyse the impact of regional differences in the codon composition of human germline Ig V H and V L genes on regional differences in the frequency of replacement mutations in the gene products (i.e. the antigen-binding sites of antibody molecules). This analysis reveals that CDR and FR sequences can differ significantly in their inherent susceptibility to amino acid replacement given any single nucleotide change. Thus, the CDR sequences of all the Ig V H genes analysed comprise a higher frequency of codons susceptible to replacement mutations than would be expected for a random sequence. Conversely, the FR sequences comprise codons less susceptible to replacement mutations than expected. Random accumulation of nucleotide changes throughout the coding sequence of an Ig V-gene segment containing CDRs inherently more prone to replacement mutations than the respective FRs would inevitably yield a higher rate of amino acid replacements in the CDRs than in the FRs. This would provide a fertile structural substrate of hypervariability for antigen selection while still maintaining the structural integrity of the FRs.The production of antibodies with progressively higher affinity for antigen is an important feature of B-cell clonotypes recruited by repeated antigenic challenge, and is referred to as affinity maturation 1 . The molecular basis of this process was first unveiled by the analysis of the binding affinity and primary structure of monoclonal antibodies (mAbs) generated at successive stages of murine immune responses to different conjugated haptens and antigens [2][3][4][5][6][7][8][9][10] . The first exposure to antigen results in recruitment of B-cell clonotypes that bind antigen by virtue of the combinatorial and junctional specificity of their unmutated surface receptors (Ig V segments). Subsequent exposures to antigen lead to accumulation of somatic point mutations in the antibody V segments and antigen selection of the highaffinity mutated B-cell clonotypes. Sequential rounds of mutation and selection eventually result in restriction of the response to those B cells with the best 'fit' for antigen 11,12 . Somatic point mutations in Ig V-gene segmentsIn the absence of negative or positive selective pressure on the gene product, a random mutational process would entail an even distribution of nucleotide changes yielding amino HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript acid replacements (R mutations) and nucleotide changes not yielding amino acid replacements (S, or silent, mutations) throughout the coding sequence. However, antigenselected antibodies have been shown to include a higher frequency of R mutations in the Ig V-gene complemen...

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Section: Discussionsupporting

confidence: 63%

The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement

1994

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“…A variety of molecular structures that span a wide range of sizes are available. These include 80-kDa (scFv-CH3)2 minibodies (Hu Shizhen et al, 1996), 50-kDa diabodies (Holliger et al, 1993), 27-kDa scFv (Bird et al, 1988;Huston et al, 1988) and individual 12-to 13-kDa VH or VL chains (Ward et al, 1989). While the smallest molecules will be capable of the greatest diffusion into solid tumours, their administration will require careful management to maintain the blood concentrations required to permit diffusion through tumour.…”

Section: Discussionmentioning

confidence: 99%

Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu

Adams

Schier²,

Am³

et al. 1998

Br J Cancer

184

110

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Summary Single-chain Fv (scFv) molecules exhibit highly specific tumour-targeting properties in tumour-bearing mice. However, because of their smaller size and monovalent binding, the quantities of radiolabelled scFv retained in tumours limit their therapeutic applications. Diabodies are dimeric antibody-based molecules composed of two non-covalently associated scFv that bind to antigen in a divalent manner. In vitro, diabodies produced from the anti-HER2/neu (c-erbB-2) scFv C6.5 displayed approximately 40-fold greater affinity for HER2Ineu by surface plasmon resonance biosensor measurements and significantly prolonged association with antigen on the surface of SK-OV-3 cells (tl,2 cell surface retention of > 5 h vs 5 min) compared with C6.5 scFv. In SK-OV-3 tumour-bearing scid mice, radioiodinated C6.5 diabody displayed a highly favourable balance of quantitative tumour retention and specificity. By as early as 4 h after i.v. administration, significantly more diabody was retained in tumour (10 %ID g-1) than in blood (6.7 %ID ml-') or normal tissue (liver, 2.8 %ID g-'; lung, 7.1 %ID g-1; kidney, 5.2 %ID g-1). Over the next 20 h, the quantity present in blood and most tissues dropped approximately tenfold, while the tumour retained 6.5 %ID g-1 or about two-thirds of its 4-h value. In contrast, the 24-h tumour retention of radioiodinated C6.5 scFv monomer was only 1 %ID g-1. When diabody retentions were examined over the course of a 72-h study and cumulative area under the curve (AUC) values were determined, the resulting tumor-organ AUC ratios were found to be superior to those previously reported for other monovalent or divalent scFv molecules. In conclusion, the diabody format provides the C6.5 molecule with a distinct in vitro and in vivo targeting advantage and has promise as a delivery vehicle for therapeutic agents.

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“…The concept of autonomous, antigen binding-competent sdAbs was first described by Ward et al . in 1989, 2 and several years later, naturally-occurring antibodies lacking light chains were discovered in dromedary camels 3 and nurse sharks. 4 The ~12–15 kDa variable domains of these antibodies (V H Hs and V NAR s, respectively; Figure 1) can be produced recombinantly and can recognize antigen in the absence of the remainder of the antibody heavy chain.…”

Section: Introductionmentioning

confidence: 99%

Antigen recognition by single-domain antibodies: structural latitudes and constraints

Henry

MacKenzie

2018

mAbs

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Single-domain antibodies (sdAbs), the autonomous variable domains of heavy chain-only antibodies produced naturally by camelid ungulates and cartilaginous fishes, have evolved to bind antigen using only three complementarity-determining region (CDR) loops rather than the six present in conventional VH:VL antibodies. It has been suggested, based on limited evidence, that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. Here, we comprehensively surveyed the evidence in support of this hypothesis. We found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies: sdAb paratopes have smaller molecular surface areas and diameters, more commonly have non-canonical CDR1 and CDR2 structures, and have elongated CDR3 length distributions, but have similar amino acid compositions and are no more extended (interatomic distance measured from CDR base to tip) than conventional antibody paratopes. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies.

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Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli

Cited by 944 publications

References 31 publications

The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement

The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement

Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu

Antigen recognition by single-domain antibodies: structural latitudes and constraints

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