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Biomedical subjects

J Wilton

Publications and source records attributed to J Wilton.

At least 19 recordsLinked to original sources
PubMed1999

A fully human antibody neutralising biologically active human TGFbeta2 for use in therapy.

Phage display provides a methodology for obtaining fully human antibodies directed against human transforming growth factor-beta (TGFbeta) suitable for the treatment of fibrotic disorders. The strategy employed was to isolate a human single chain Fv (scFv) fragment that neutralises human TGFbeta2 from a phage display repertoire, convert it into a human IgG4 and then determine its TGFbeta binding and neutralisation properties and its physical characteristics. Several scFv fragments binding to TGFbeta2 were isolated by panning of an antibody phage display repertoire, and subsequent chain shuffling of the selected V(H) domains with a library of V(L) domains. The three most potent neutralising antibodies were chosen for conversion to IgG4 format. The IgG4 antibodies were ranked for their ability to neutralise TGFbeta2 and the most potent, 6B1 IgG4, was chosen for further characterisation. 6B1 IgG4 has a high affinity for TGFbeta2 with a dissociation constant of 0.89 nM as determined using the BIAcore biosensor and only 9% cross-reactivity with TGFbeta3 (dissociation constant, 10 nM). There was no detectable binding to TGFbeta1. 6B1 IgG4 strongly neutralises (IC50 = 2 nM) the anti-proliferative effect of TGFbeta2 in bioassays using TF1 human erythroleukaemia cells. Similarly, there was strong inhibition of binding of TGFbeta2 to cell surface receptors in a radioreceptor assay using A549 cells. 6B1 IgG4 shows no detectable cross-reactivity with related or unrelated antigens by immunocytochemistry or ELISA. The 6B1 V(L) domain has entirely germline framework regions and the V(H) domain has only three non-germline framework amino acids. This, together with its fully human nature, should minimise any potential immunogenicity of 6B1 IgG4 when used in therapy of fibrotic diseases mediated by TGFbeta2.

Amino Acid Sequence↗
PubMed1998

IBC's conference on antibody engineering; new technology, application and commercialisation.

This was the first antibody engineering conference held by IBC Conferences in the UK, and was the European equivalent of the annual US meeting held in San Diego. The conference provided a forum for an update on progress in all aspects of antibody engineering. Topics covered ranged from the design of libraries and new selection techniques to news of the first engineered antibodies to enter clinical trials. Library size and diversity were shown to have increased dramatically in the last few years, and new formats have been introduced. In parallel, improvements to existing applications and the development of novel selection technologies were shown to improve accessibility to new targets. Exciting developments included phenotypic selections, ribosome display, improvements to bispecific design and the design of active intracellular antibodies. The data generated for recombinant antibodies in the clinic are very promising, with some antibodies demonstrating improvements over conventional therapies and others targeting diseases where no treatment is currently available. Presented below is a summary of the highlights of the conference, with particular focus on natural antibody phage libraries, and lead candidates derived from these libraries currently in clinical trials.

Journal Article↗
PubMed1997

An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries.

Phage display is now an established method to select antibody fragments specific for a wide range of diverse antigens. In particular, isolation of human monoclonal antibodies has become a reality and for most purposes bacterial expression of the selected recombinant antibody fragments is sufficient. However, there are some cases where the expression of complete human immunoglobulin in mammalian cells is, if not essential, at least desirable. For this reason we have designed and constructed a set of mammalian expression vectors which permit facile and rapid cloning of antibody genes for both transient and stable expression in mammalian cells. Immunoglobulin genes may be cloned into these expression vectors as V regions or as Fabs for expression as either complete antibodies or as Fab fragments, using restriction sites which are rare in human V genes. All the important elements in the vectors--promoter, leader sequence, constant domains and selectable markers--are flanked by unique restriction sites, allowing simple substitution of elements. The vectors have been evaluated using the variable regions from the neutralizing anti-nerve growth factor (NGF) antibody, alphaD11, and the V regions from 2E10, a scFv selected from a scFv phagemid library.

Animals↗
PubMed1997

Oxaprozin and piroxicam, nonsteroidal antiinflammatory drugs with long half-lives: effect of protein-binding differences on steady-state pharmacokinetics.

The nonsteroidal antiinflammatory drugs (NSAIDs) oxaprozin and piroxicam have long elimination half-lives (t 1/2 approximately 55 hours), permitting once-daily dose regimens. The protein-binding characteristics of these drugs, however, vary widely. This study examines the effect of these binding differences on the drugs' disposition kinetics at steady state. A total of 52 participants (26 young healthy volunteers, and 26 elderly osteoarthritic patients, 15 men and 37 women (2 of them poor metabolizers of debrisoquine [CYP2D6]) completed the two-period, two-treatment, randomized, single-dose and 21-day, once-daily multiple-dose, cross-over study. Doses of oxaprozin and piroxicam were 1,200 mg once daily and 20 mg once daily, respectively. Mean single-dose kinetic parameters of oxaprozin versus piroxicam did not differ more than +/-14% (t1/2, 53.0 versus 57.4 hours; apparent oral clearance adjusted for 70-kg body weight [Clpo], 0.139 versus 0.121 L/hr; apparent volume of distribution adjusted for 70-kg body weight [Vd/F]; 10.2 L versus 9.13 L). Protein binding was plasma-concentration dependent with oxaprozin (range, 10-400 mg/L) but not with piroxicam (range, 1-30 mg/ L). Steady-state conditions were established within 3 days with oxaprozin but took almost 12 days with piroxicam. Compared with the single-dose values, steady-state Clpo (Clpo,ss) and Vd/F of total drug increased with oxaprozin by almost 127% but remained within +/-10% with piroxicam. Post-steady-state apparent t 1/2 of the total and unbound drugs of approximately 62 hours were similarly prolonged with piroxicam but differed substantially with oxaprozin (50.6 hours [total drug] versus 23.8 hours [unbound drug]). Single dose Clpo (Clpo,sd) values of both NSAIDs were significantly correlated in the study populations. With both NSAIDs, Clpo in the two poor metabolizers of debrisoquine was within +/-20% of mean values for the population. Clinically important age- and gender-dependent decreases were not observed in the weight-adjusted, Clpo,sd or Vd/F values of the total drug for either NSAID. Clearances of the two NSAIDs were significantly correlated, suggesting that a common P450 isozyme (most likely CYP2C9, in that piroxicam is a known substrate of this isozyme) may be at least partly involved in the oxidative metabolism of these NSAIDs.

Adult↗
PubMed1996

Generation of a panel of related human scFv antibodies with high affinities for human CEA.

BACKGROUND: A human single chain Fv (scFv) specific for human carcinoembryonic antigen (CEA) has been isolated from a 2.0 x 10(9) phage display library from unimmunised human donors. The dissociation constant of the scFv has been measured by surface plasmon resonance (SPR) and found to be 7.7 x 10(-9) M, with an off-rate component of 6.2 x 10(-3) s-1. In order to investigate directly whether increased affinity leads to improved targeting of CEA-positive tumours, this scFv has been affinity matured by both targeted mutagenesis of the CDRs of heavy and light chains, and by light chain shuffling. STUDY DESIGN: A partial randomisation scheme, biased towards amino acids commonly found as somatic mutations of germline antibody sequences, was used for directed diversification of VH and VL CDR3s. Diversification of the entire VL region was also introduced by light chain shuffling of the parental anti-CEA scFv. Selection of the mutagenised repertoires was carried out to enrich for antibodies with a reduced koff. RESULTS: Sequencing the selected clones identified a number of amino acid changes in the VH CDR3, one of which gave a four-fold reduction in koff. Stringent selection of the light chain shuffled library resulted in several clones with a two- to three-fold reduction in koff. It has been possible to combine the selected changes from both mutagenesis approaches by using the mutagenised heavy chain and a light chain derived by shuffling to give a human scFv with a dissociation constant for human CEA of 6.0 x 10(-10) M. CONCLUSION: A panel of human anti-CEA scFvs has been generated with differing dissociation constants for antigen, which will allow the correlation between tumour targeting efficiency in relation to binding affinity to be assessed directly. The scFv panel will be valuable in the optimisation of human antibodies for immunotherapy.

Amino Acid Sequence↗
PubMed1996

Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library.

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.

Amino Acid Sequence↗
PubMed1996

Directing phage selections towards specific epitopes.

It is possible to direct selections from antibody repertoires displayed on filamentous phage towards unique epitopes on protein antigens by competing with related molecules. A phage display repertoire of human single chain Fvs (scFvs) was panned three times against foetal haemoglobin (HbF). The selection was dominated by one clone with a Kd of 10 nM but yielded at least 17 others, all of which bound HbF but crossreacted with adult haemoglobin (HbA). To direct selection towards HbF-specific epitopes, the repertoire was preincubated with HbA in solution before each panning. Crossreactive scFvs can form complexes with the soluble HbA and thereby be prevented from binding the immobilized HbF. Four clones with preferential binding to HbF emerged under these conditions. One of these (Hb-1), with a Kd of 6 microM, had exquisite specificity for HbF and could distinguish cells expressing HbF from those expressing HbA by immunocytochemistry and flow cytometry. This antibody has an affinity that is 600-fold lower than the dominant crossreactive clone, and so only emerged under conditions of 'competitive deselection'. Thus, competitive deselection is a viable means for directing selections towards useful epitopes. It permits a more effective 'search' of phage display repertoires and allows the emergence of lower affinity clones with useful specificities. These clones may be useful in themselves or may serve as leads for in vitro affinity maturation.

Adult↗
PubMed1996

What do deans of medical schools and heads of departments of general practice think of preregistration rotations in general practice?

BACKGROUND: One general practice has offered preregistration rotations in general practice since 1981. Although popular with doctors who participate in the scheme, and considered a success by the associated teaching hospital, it remains the only established general practice rotation in the United Kingdom. AIM: A study was undertaken to assess the degree of interest in preregistration rotations in general practice expressed by medical school deans and heads of departments of general practice. METHOD: A questionnaire was sent to all 26 deans of medical schools and all 25 heads of departments of general practice of teaching hospitals in the UK that undertook clinical training. RESULTS: A total of 24 deans (92%) and 24 heads of departments of general practice (96%) replied to the questionnaire. The scheme was thought to be of value by 58% of deans and 79% of heads of departments of general practice. Half of the teaching hospitals thought that they might consider such a rotation in the next 3-5 years. The most frequently cited problem initiating such a scheme was financial. CONCLUSION: Given the current interest in medical teaching in the community, preregistration rotations in general practice deserve more attention.

Attitude of Health Personnel↗
PubMed1995

Preregistration house officers in general practice.

OBJECTIVES: To obtain from house officers who had rotated through general practice in their pre-registration year their views about their experience; and, separately, to compare the overall hours and type of work performed by hospital based and general practice based house officers. DESIGN: Postal questionnaire; and self recording of working hours and duties during four consecutive weeks. SETTING: Inner London teaching hospital and nearby general practice. PARTICIPANTS: 28 preregistration house officers in general practice, 1981-91; and 12 preregistration house officers, four each in medicine, surgery, and general practice. RESULTS: 26 out of 28 questionnaires were returned (response rate 93%). Twelve respondents were following or thinking of following a career in general practice. Twenty five respondents were satisfied with the clinical and educational aspects of the general practice rotation and would recommend the rotation, and 25 thought four months was about the right length of time in general practice. With regard to hours and type of work performed, hospital based house officers worked on average 55.5 hours a week (excluding on call), with an average of 12.5 hours (22.5%) spent in clinical activities; general practice based house officers worked about 41 hours a week, of which 24 hours (58%) were in clinical activities. House officers in hospital received less than one hour's specific teaching a week; those in general practice received nearly three hours' a week. CONCLUSIONS: A preregistration rotation in general practice is a popular alternative to the hospital based rotation. Although this is a limited study, other medical schools should consider introducing general practice options for preregistration house officers.

Attitude of Health Personnel↗
PubMed1995

Pharmacokinetics of high-dose intravenous ciprofloxacin in young and elderly and in male and female subjects.

The effects of age and gender on the pharmacokinetics of high-dose intravenous ciprofloxacin in a healthy volunteer study were investigated. Plasma ciprofloxacin concentrations were higher in the elderly than in the young, and the pharmacokinetic parameters were not significantly different between the genders. Ciprofloxacin was well tolerated, with the majority of adverse events related to local reactions at the IV site.

Adolescent↗
PubMed1993

Neisseria meningitidis transferrin-binding protein 1 expressed in Escherichia coli is surface exposed and binds human transferrin.

A gene library of Neisseria meningitidis B15 P1.16 DNA was established in lambda Zap II and clones containing DNA encoding transferrin binding protein 1 (TBP-1) identified following hybridisation with a 63-bp DNA probe based on the codon assignment for the first 21 N-terminal amino acids of TBP-1. Sequencing of the cloned DNA demonstrated that all of the intergenic DNA (i.e. upstream of tbp-1 running through to the 3' end of the transferrin-binding protein 2 gene) and approx. 15% of tbp-1 had been cloned. The complete gene was generated using a polymerase chain reaction, with the primer for the 3' end being based on tbp-A of N. gonorrhoeae, and the approx. 2.9-kb DNA product cloned into pGem-3Z. The expressed protein (approx. 100 kDa) reacted with antiserum to an N-terminal peptide of TBP-1. In addition, the native product was surface-expressed by Escherichia coli and bound human transferrin.

Amino Acid Sequence↗
PubMed1993

Cloning and partial sequence of transferrin-binding protein 2 of Neisseria meningitidis using a novel method: twin N-terminal PCR.

The genes encoding transferrin-binding proteins (TBPs) 1 and 2 of Neisseria meningitidis and N. gonorrhoeae were used as model loci in a novel method of cloning (twin N-terminal polymerase chain reaction; TNT-PCR) involving amplification between the 5' ends of two genes. Primers were based on N-terminal amino-acid sequences. A 2.1-kb product amplified from N. meningitidis strain SD (B15 P1.16) was cloned into a plasmid vector and partially sequenced. Translated sequence immediately downstream of the primer at both ends of this product correlated to the additional known N-terminal amino acids of TBP-1 and 2. The protein encoded by the cloned sequence reacted with TBP-2-specific antiserum. The size of products generated in TNT-PCR correlated exactly with the different sized TBP-2 produced by 10 strains of the Neisseria spp. examined, indicating successful cloning of the gene for TBP-2 and showing it to be adjacent to and preceding TBP-1 on the chromosome for both N. meningitidis and N. gonorrhoeae.

Amino Acid Sequence↗
PubMed1992

Comparative evaluation of a new molecular method for typing Staphylococcus epidermidis.

The discriminatory powers of several techniques for typing Staphylococcus epidermidis were evaluated in an epidemiological study of bacteria isolated from intensive care patients and from neonates. Genomic DNA fingerprinting using BclI restriction endonuclease was an effective epidemiological marker. The distinct restriction fragment profiles produced with this enzyme were highlighted with specific probes in a Southern blot technique. Cloned Escherichia coli rRNA gene probes proved to have lower discriminatory power and be less suitable for intraspecies typing. However, a panel of random genomic Staphylococcus epidermidis DNA clones provided almost the same level of discrimination as the DNA fingerprinting technique and also provided a clearer profile. DNA and gene fingerprinting techniques were reproducible and highly discriminatory compared to typing based on antigen and plasmid profiles, antibiotic susceptibility patterns and biotypes.

Blotting, Southern↗