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Biomedical subjects

G Winter

Publications and source records attributed to G Winter.

At least 19 recordsLinked to original sources
PubMed1997

Dimerization of Fab fragments enables ready screening of phage antibodies that affect hepatocyte growth factor/scatter factor activity on target cells.

A number of applications of antibodies in diagnosis and therapy require multivalent reagents either because of the polymeric nature of the antigens or because biological activity depends on an effect on the formation of homodimeric species. Here, we report a procedure for mass screening of phage-derived monomeric antibody fragments that depend on valency for activity. As a model system, a set of 13 phage-derived human Fab fragments were first selected against mouse and human recombinant hepatocyte growth factor/scatter factor (HGF/SF), a high molecular weight polypeptide growth factor related to the blood protease plasminogen and involved in development and cancer. These Fab fragments were subsequently screened for an effect on HGF/SF activity either as monomeric fragments or after dimerization with a monoclonal antibody (9E10) directed against a peptide tag on the fragments. Fab were identified that either inhibited or enhanced HGF/SF activity on target cell lines, but dimerization was required for this effect. The approach proposed should facilitate mass screening of phage-derived antibody fragments that depend on multiple valency for activity.

Amino Acid Sequence↗
PubMed1996

Sequence and evolution of the human germline V lambda repertoire.

We recently completed a map of the human immunoglobulin lambda (IGL) locus on chromosome 22q11.2 and showed that the V lambda genes are arranged in three distinct clusters, each containing members of different V lambda families. We have now sequenced each of these V lambda genes and determined which are functional by comparison with the expressed repertoire. Our analysis indicates that there are approximately 30 functional V lambda genes, depending on the haplotype, that belong to ten V lambda families (five V lambda 1, five V lambda 2, eight V lambda 3, three V lambda 4, three V lambda 5, one V lambda 6, two V lambda 7, one V lambda 8, one V lambda 9 and one V lambda 10). V lambda genes related to the major human V lambda families (V lambda 1, V lambda 2 and V lambda 3) predominate in species that express mainly lambda light chains.

Amino Acid Sequence↗
PubMed1996

Phage antibodies with pan-species recognition of the oncofoetal angiogenesis marker fibronectin ED-B domain.

Fibronectin (FN) exists in several polymorphic forms due to alternative splicing. The B-FN isoform (with ED-B domain inserted by splicing) is present in the stroma of foetal and neoplastic tissues and in adult and neoplastic blood vessels during angiogenesis but is undetectable in mature vessels. This isoform, therefore, represents a promising marker for angiogenesis, as already shown using the mouse monoclonal antibody (MAb) BC-1 directed against an epitope on human B-FN. However, this MAb does not directly recognise the human ED-B domain nor does it recognise B-FN of other species; therefore, it cannot be used as a marker of angiogenesis in animal models. In principle, antibodies directed against the human ED-B domain should provide pan-species markers for angiogenesis as the sequence of this domain is highly conserved in different species (and identical in humans and mice). As it has proved difficult to obtain such antibodies by hybridoma technology, we used phage display technology. Here, we describe the isolation of human antibody fragments against the human ED-B domain that bind to human, mouse and chicken B-FN. As shown by immunohistochemistry, the antibody fragments stain human neoplastic tissues and the human, mouse and chicken neovasculature.

Adult↗
PubMed1996

A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage.

It has been suggested that recombination and shuffling between exons has been a key feature in the evolution of proteins. We propose that this strategy could also be used for the artificial evolution of proteins in bacteria. As a first step, we illustrate the use of a self-splicing group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire (> 10(11) members) of peptides from two different exons. Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides. The repertoire was displayed on filamentous bacteriophage by fusion to the pIII phage coat protein and selected by binding to several proteins, including beta-glucuronidase. One of the peptides selected against beta-glucuronidase was chemically synthesized and shown to inhibit the enzymatic activity (inhibition constant: 17 nM); by further exon shuffling, an improved inhibitor was isolated (inhibition constant: 7 nM). Not only does this approach provide the means for making very large peptide repertoires, but we anticipate that by introducing constraints in the sequences of the peptides and of the linker, it may be possible to evolve small folded peptides and proteins.

Amino Acid Sequence↗
PubMed1996

Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain.

Human antibodies can now be isolated from antibody repertoires displayed on the surface of filamentous bacteriophage in a process that mimics the primary immune response. Here we have attempted to mimic the secondary response, the natural process of affinity maturation of antibodies occurring in germinal centres, by multiple cycles of random mutation and selection. Phage displaying a human antibody fragment recognising the hapten 2-phenyl-5-oxazolone were grown in a mutator strain of bacteria (Escherichia coli: mutD5) to generate a large repertoire of antibodies that should include the majority of possible single nucleotide point mutations. The repertoire of phage antibody mutants was then selected by binding to hapten. By multiple rounds of growth in the mutator strain, and increasingly stringent selection, we succeeded in isolating mutants with improved binding affinities; furthermore, the distribution of mutations and nucleotide substitution preferences strongly resembled those of somatic hypermutation. We then constructed a genealogical tree from the sequences of mutants taken at different rounds, and identified four sequentially acquired mutations that together improve the binding affinity of the antibody by a factor of 100-fold (from Kd 320 nM to 3.2 nM).

Amino Acid Sequence↗
PubMed1996

Phage antibodies against an unstable hapten: oxygen sensitive reduced flavin.

It is difficult to raise antibodies against haptens and antigens that are unstable under the physiological conditions of the serum. Here we have used a phage antibody library to isolate antibody fragments against oxygen sensitive reduced flavin, by selection of the phage under anaerobic and reducing conditions at pH 5 and a pre-elution step with the oxidized flavin. The binding of the reduced hapten to one of the antibody fragments was characterised by time-resolved polarised fluorescence, and shown to be highly specific for the reduced flavin.

Amino Acid Sequence↗
PubMed1996

The imprint of somatic hypermutation on the repertoire of human germline V genes.

In the human immune system, antibodies with high affinities for antigen are created in two stages. A diverse primary repertoire of antibody structures is produced by the combinatorial rearrangement of germline V gene segments and antibodies are selected from this repertoire by binding to the antigen. Their affinities are then improved by somatic hypermutation and further rounds of selection. We have dissected the sequence diversity created at each stage in response to a wide range of antigens. In the primary repertoire, diversity is focused at the centre of the binding site. With somatic hypermutation, diversity spreads to regions at the periphery of the binding site that are highly conserved in the primary repertoire. We propose that evolution has favoured this complementarity as an efficient strategy for searching sequence space and that the germline V gene families evolved to exploit the diversity created by somatic hypermutation.

Antibody Diversity↗
PubMed1996

Specific killing of lymphoma cells by cytotoxic T-cells mediated by a bispecific diabody.

Antibody fragments produced by bacterial fermentation lack natural effector functions. Bispecific antibody fragments, however, can be endowed with effector functions, for example cell-mediated killing, by binding to and retargeting of cytotoxic cells. Diabodies are a class of engineered antibody fragments with two antigen binding sites, consisting of two associated chains; each chain consists of heavy and light chain variable domains linked by a short polypeptide linker. In contrast to IgG, or other antibody fragments in which the two binding sites can take up a range of orientations and spacings, the diabody structure is more rigid and compact, with the two binding sites separated by 65 łA (less than half the distance in IgG). To establish whether diabodies could also be used in cell-mediated killing, we have explored the use of a bispecific diabody binding to an idiotypic marker on mouse B-cell lymphoma (BCL-1) and to mouse CD3. The bispecific diabody activated naive T-cells and also mediated the specific killing of the lymphoma cells by cytotoxic T-cells. The diabody was less active in T-cell activation but 10-fold more active (w/v) in killing than an analogous bispecific IgG.

Animals↗
PubMed1996

Multipurpose high sensitivity luminescence analyzer (LUANA): use in gel electrophoresis.

Many applications in molecular biology require the rapid and high-sensitivity detection of biological macromolecules. Here we describe a luminescence analyzer (LUANA), featuring xenon lamp-based illumination and a cooled CCD camera as detector. Luminescent samples (gels, membranes or microplates) are placed in a light-tight chamber, and computer software is used to control the camera and to display and analyze the images. LUANA can be used with a broad spectrum of excitation and emission wavelengths (200-1000 nm), allowing a choice of many fluorescent dyes and facilitating multicolor imaging. We show that LUANA is readily applicable to gel electrophoresis and highly sensitive; on gels we could detect < 100 fg of Cy5TM-labeled protein and 0.1 ng of plasmid DNA labeled with a fluorescent intercalator. We observed a linear response for sample concentrations differing by two orders of magnitude using a single acquisition time. This range could be further extended by using different acquisition times. We have also used LUANA in two-color imaging of DNA and for screening of antigen-antibody and peptide-protein complexes by gel bandshift.

Cloning, Molecular↗
PubMed1995

An antibody fragment from a phage display library competes for ligand binding to the low density lipoprotein receptor family and inhibits rhinovirus infection.

Recently antibodies with a wide range of binding specificities have been isolated from large repertoires of antibody fragments displayed on filamentous phage, including those that are difficult to raise by immunization. We have used this approach to isolate an antibody fragment against chicken very low density lipoprotein (VLDL) receptor. It binds to the receptor with good affinity (Kaff = 2 x 10(8) M-1) as measured by plasmon surface resonance, and competes for binding of natural ligands (vitellogenin, VLDL, and receptor-associated protein). The antibody also binds to other members of the low density lipoprotein (LDL) receptor family including rat LDL receptor and human and rat low density lipoprotein receptor-related protein (LRP/alpha 2MR), and it competes for binding of receptor-associated protein to LRP/alpha 2MR. Moreover, the antibody fragment inhibits infection of human fibroblasts deficient in LDL-R but expressing LRP/alpha 2MR by human rhinovirus. Binding of the antibody is abolished upon reduction of the receptors and is strictly Ca2+ dependent. The phage antibody thus recognizes the ligand binding site(s) of several members of the LDL receptor family, in contrast to antibodies produced by hybridoma technology.

Amino Acid Sequence↗
PubMed1995

High-affinity antigen binding by chelating recombinant antibodies (CRAbs).

We have developed a strategy for making antibody fragments with high binding affinities by harnessing the chelate effect. We create a bispecific antibody fragment (Chelating Recombinant Antibody or CRAb) that recognizes adjacent and non-overlapping epitopes of the target antigen, and is flexible enough to bind to both epitopes simultaneously. Here the strategy is illustrated with two antibodies that form complexes of known three-dimensional structure against different epitopes of lysozyme. Computer graphic modelling indicated that two single-chain antibody fragments (scFv) derived from antibodies D1.3 (Ka = 10(8) M-1) and mutant HyHEL-10 (Ka = 10(6) M-1) could be linked together on the surface of lysozyme by a flexible and hydrophilic polypeptide between the C terminus of one fragment and the N terminus of the other. The CRAb gene was assembled and the CRAb expressed by secretion from bacteria. The purified CRAb was shown to have a much higher affinity than either of the scFv fragments, as shown by competition ELISA (Kd > 10(9) M-1), bandshift on gels (Ka > 2 x 10(9) M-1) and fluorescence quench (Ka > 1.3 x 10(10) M-1).

Amino Acid Sequence↗
PubMed1995

Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2.

The maps of the human immunoglobulin heavy-chain and kappa light-chain loci have recently been completed. We have now completed a map of the human lambda locus (IGL) located on chromosome 22q11.2. We mapped 52 V lambda genes from 10 V lambda families and 7 J lambda and C lambda genes on a 1140 kb contig constructed from eight YACs and 129 cosmid clones. The V lambda genes are arranged within 800 kb. Genes of the different V lambda families are organized in three clusters, V lambda II and III families (cluster A); V lambda I, V, VII and IX families (cluster B); V lambda IV, VI, VIII and X families (cluster C), in contrast to the dispersed organization of the different VH and V kappa families within the human VH and V kappa loci. We note that the most frequently used V lambda families (V lambda II and III) are proximal to the J lambda and C lambda genes. The VpreB gene, encoding part of the surrogate light chain, the GGT2 gene and the BCRL4 pseudogene were also mapped within the lambda locus.

Base Sequence↗
PubMed1994

Crystal structure of a diabody, a bivalent antibody fragment.

BACKGROUND: Diabodies are dimeric antibody fragments. In each polypeptide, a heavy-chain variable domain (VH) is linked to a light-chain variable domain (VL) but unlike single-chain Fv fragments, each antigen-binding site is formed by pairing of one VH and one VL domain from the two different polypeptides. Diabodies thus have two antigen-binding sites, and can be bispecific. Direct structural evidence is lacking for the connections and dimeric interactions between the two polypeptides of the diabody. RESULTS: The 2.6 A resolution structure has been determined for a bivalent diabody with a flexible five-residue polypeptide linker between the (amino-terminal) VH and (carboxy-terminal) VL domains. The asymmetric unit of the crystal consists of four polypeptides comprising two diabodies; for one of these polypeptides the linker can be traced between the VH and VL domains. Within each diabody the two associated VH and VL domains make back-to-back interactions through the VH domains, and there is an extensive VL-VL interface between the two diabodies in the asymmetric unit. CONCLUSIONS: The structure of the diabody is very similar to that which had been predicted by molecular modelling. Diabodies directed against cell-surface antigens should be capable of bringing together two cells, such as in cell-targeted therapy, because the two antigen-binding sites of the diabody are at opposite ends of the molecule and separated by approximately 65 A.

Amino Acid Sequence↗
PubMed1994

Isolation of a peptide antagonist to the thrombin receptor using phage display.

The thrombin receptor on platelets is an integral membrane protein and is cleaved by thrombin to expose a "tethered ligand" that binds to and triggers the receptor. Here we have explored the power of phage selection technology to make a peptide antagonist of this receptor using platelets directly for the selection. To focus the selection to the thrombin receptor, we eluted the phage with a peptide agonist of the thrombin receptor. A repertoire (1 x 10(7) phage clones) displaying peptide sequences based on the sequence of the tethered ligand, was constructed and selected by binding to the platelets. After several rounds of selection, we identified phage clones that were able to immunoprecipitate the thrombin receptor from platelets and the encoded peptides were sequenced. This revealed some features in common with the tethered ligand, in particular an arginine residue followed by a proline. Several of the peptides were synthesized chemically and one of the peptides was shown to antagonise platelet aggregation triggered by the agonist peptide, and to inhibit serotonin release and tyrosine phosphorylation triggered by either thrombin or the agonist peptide. Anti-aggregatory activity was about ten-fold higher than that of previously reported peptide antagonists of the thrombin receptor.

Amino Acid Sequence↗
PubMed1994

Idiotypic vaccination against human B-cell lymphoma. Rescue of variable region gene sequences from biopsy material for assembly as single-chain Fv personal vaccines.

Idiotypic determinants on neoplastic B cells could provide tumor antigens for vaccination of patients with B-cell tumors. Because this approach requires an individual vaccine for each patient, simple methods for obtaining idiotypic antigen are desirable. Using polymerase chain reaction (PCR) with family-based V-gene and J-region primers, the variable region genes of heavy and light chains (VH and VL) of Ig have been obtained from biopsy material from 13 patients with B-cell tumors. In each case, analysis of random clones derived from the PCR product showed repeated, clonally-related sequences, whereas normal lymphoid tissue generated no repeated sequences. In 3/3 cases, the repeated sequences were found to be the same as those in a tumor-derived hybridoma. Mutational patterns in the V-genes differed among the tumors, with follicular lymphoma tending to be more highly mutated. The individual VH and VL sequences have been assembled with a flexible linker sequence to encode single-chain Fv (scFv). The scFv sequences can be cloned into bacterial expression vectors to produce protein, or into vectors suitable for direct vaccination using naked DNA. In a model system, expressed scFv protein retained all idiotypic determinants defined by a panel of five anti-idiotypic monoclonal antibodies (MoAbs). Similarly, expressed scFv proteins from two patients were shown to react with anti-idiotypic antibodies. This approach allows production of potential vaccines from surgical biopsies within 2 to 3 weeks.

Amino Acid Sequence↗