The isolation and characterisation of human monoclonal HLA‐A2 antibodies from an immune V gene phage display library

Watkins, Nicholas A.; Brown, Colin J.; Hurd, C.; Navarrete, Cristina; Ouwehand, Willem H.

doi:10.1034/j.1399-0039.2000.550305.x

Cited by 24 publications

(43 citation statements)

References 32 publications

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“…Preparation of Monomeric and Dimeric scFvs-scFvs were expressed with c-Myc and hexahistidine affinity tags as previously described (16) and purified using a Ni 2ϩ -charged HiTrap chelating column (Amersham Biosciences) according to the manufacturer's instructions with 0.5 M NaCl, 50 mM sodium phosphate, pH 7.6 -7.9, as the base buffer, eluting with 275 mM imidazole. This eluate contained a mixture of monomeric and dimeric (Յ5%) forms of each scFv.…”

Section: Methodsmentioning

confidence: 99%

Selective Blockade of Glycoprotein VI Clustering on Collagen Helices

O’Connor

Smethurst

Davies

et al. 2006

Journal of Biological Chemistry

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Platelet activation by collagen relies on the interaction of the receptor glycoprotein VI (GPVI) with collagen helices. We have previously generated two recombinant single chain human antibodies (scFvs) to human GPVI. The first, 10B12, binds to the collagen-binding site on the apical surface between the two immunoglobulin-like domains (D1D2) of the receptor and so directly inhibits GPVI function. The second, 1C3, binds D1D2 independently of 10B12 and has been shown to have a more subtle effect on platelet responses to collagen. Here we have shown that 1C3 potentiates the effect of 10B12 on platelet aggregation induced by collagen and cross-linked collagen-related peptide (CRP-XL). We investigated this by measuring the effect of both scFvs on the binding of D1D2 to immobilized collagen and CRP. As expected, 10B12 completely inhibited binding of GPVI to each ligand in a dose-dependent manner. However, 1C3 inhibited only a proportion of GPVI binding to its ligands, implying that it interferes with another aspect of ligand recognition by GPVI. To further understand the mode of inhibition, we used a unique set of CRPs in which the content of critical glycine-proline-hydroxyproline (GPO) triplets was varied in relation to an "inert" scaffold sequence of GPP motifs. We observed that a stepwise increase in D1D2 binding with (GPO) 2 content was blocked by 1C3. Together these results indicate that 1C3 inhibits clustering of the immunoglobulin-like domains of GPVI on collagen/CRPs, a conclusion that is supported by mapping the 1C3 epitope to the region including isoleucine 148 in D2. Recognition of exposed subendothelial collagen by the receptor glycoprotein VI (GPVI)2 on platelets is a critical early step for platelet activation and subsequent thrombus formation. This interaction strengthens platelet adhesion through activation of integrins ␣ 2 ␤ 1 and ␣ IIb ␤ 3 (1) and induces degranulation, aggregation, and procoagulant activity (2). Hence, elucidation of the mechanism of interaction of GPVI with collagen is valuable for developing effective platelet antagonists.We have identified the primary collagen binding surface of human GPVI on the immunoglobulin (Ig)-like domains (D1D2) of the receptor by comparing binding of human and mouse recombinant monomeric D1D2 to a synthetic collagen-related peptide (CRP) that contains ten glycine-proline-hydroxyproline (GPO) repeats in each strand of a triple helix (3, 4). Prolyl hydroxylation is necessary for recognition of collagen and such peptides by platelet GPVI (5). Both mouse and human D1D2 showed specific, dose-dependent, and saturable binding to CRP, relative to peptide GPP10, a peptide of similar structure in which hydroxyproline is replaced by proline. Using such peptides, the minimum recognition motif for GPVI has recently been identified as a tandem GPO triplet or (GPO) 2 . 3Using human recombinant D1D2 as bait, two scFvs were selected that bind to distinct epitopes on GPVI. 10B12 recognizes the primary collagen binding surface of human GPVI, and 1C3 does not (4). The 1C3 e...

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Section: Methodsmentioning

confidence: 99%

Selective Blockade of Glycoprotein VI Clustering on Collagen Helices

O’Connor

Smethurst

Davies

et al. 2006

Journal of Biological Chemistry

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“…Plasmid pCMT2b-scFv was constructed by cloning the single-chain antibody 3PF12 plus a pelB leader sequence and 3Ј myc and his tags (24) under control of the P L promoter in expression vector pCMT2b. 3PF12 is a human scFv specific for HLA-A2/A28, isolated by variable gene phage display.…”

Section: Methodsmentioning

confidence: 99%

“…In cancer therapy, for example, repeated doses of 1 g per patient are commonplace (12). Our scFv of choice, clone 3PF12 (24), is being developed as a diagnostic reagent for HLA typing and as a therapeutic reagent. It has been used to model the interaction of anti-HLA-A2 with the HLA molecule, and this information is being used to produce antibodies that specifically recognize HLA-plus peptide.…”

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confidence: 99%

“…The supernatant and soluble cytoplasmic protein fractions of cultures expressing 3PF12 were tested for biological activity with a platelet immunofluorescence test (24). scFv binding to HLA-A2-positive blood platelet cells was detected with mouse monoclonal antibody 9E10 and fluorescein isothiocyanate-labeled goat-anti-mouse antibody in a three-step process.…”

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confidence: 99%

“…For all samples, 10,000 events were analyzed and median fluorescence intensity was determined. The amount of active protein in the samples was determined by using a standard curve obtained by titrating purified 3PF12 in a platelet immunofluorescence test (24).…”

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confidence: 99%

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Studies of Single-Chain Antibody Expression in Quiescent Escherichia coli

Mukherjee

Rowe

Watkins

et al. 2004

Appl Environ Microbiol

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Quiescent Escherichia coli cells are generated by overexpressing the Rcd transcript in an hns-205 mutant host. The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures. The expression system is based on two plasmids; one carries the product gene expressed from P L under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from P R . Shifting the culture from 30 to 42°C induces Rcd expression and product expression simultaneously. Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant. In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm [OD 600 ] of 3.5) was 37 mg liter ؊1 , compared to a maximum of 13 mg liter ؊1 in the control culture (final OD 600 of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD 600 of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg liter ؊1 . A control culture with a similar feed reached an OD 600 of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg liter ؊1 . Quiescent cells at an OD 600 of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg liter ؊1 was achieved in 8 h. The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture. This study demonstrates that it is possible to establish high-density quiescent E. coli cultures that are capable of efficient synthesis, folding, and export of proteins.Escherichia coli continues to occupy center stage as a host for large-scale recombinant protein production (21). This is especially true for simple proteins, where posttranslational modifications are not essential for biological activity. Recent developments in our understanding of protein folding have allowed the expression of many proteins in their biologically active soluble form rather than as inclusion bodies (2,3,19). Coupled with more efficient secretion mechanisms, it is now possible to obtain a recombinant protein as a periplasmic or even extracellular fraction, which significantly eases the problems of downstream processing (4,14). While sophisticated vectors with strong and tightly regulated promoters have been developed to maximize product expression, comparatively little attention has been paid to the bacterial host. An improved host could potentially offer gains in product quantity and quality and could be used in conjunction with a wide range of existing vectors.We have been exploring the conflict between the biotechnologist's desire for more and better product and the inclination of the bacterium to produce biomass. In an attempt to shift the balance in favor of the biotechnologist, we developed the quiescent-cell (Q-cell) expression system, in which a plasmid-encoded protein is expressed in nongrowing but metabo...

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