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Biomedical subjects

G Winter

Publications and source records attributed to G Winter.

At least 73 records · Page 4Linked to original sources
PubMed1996

Radioactive labeling of recombinant antibody fragments by phosphorylation using human casein kinase II and [gamma-32P]-ATP.

A wide range of antibody fragments can be expressed in bacteria and detected immunochemically via peptide tags. Using specially designed tags, we have developed a strategy for radiolabeling antibody fragments secreted from bacteria. Tagged antibody fragments were secreted either into the bacterial periplasm or the culture medium. The tag was not subject to proteolysis either in the broth or in human plasma. After affinity purification the antibody fragments were phosphorylated with [gamma-32P]ATP and casein kinase II. The labeled fragments were used in a gel band-shift assay to measure antigen binding affinities. In contrast to non site-specific methods such as radioiodination, antibodies labeled with casein kinase II retain full immunoreactivity. Radioactively phosphorylated antibody fragments may have many other applications, including radioimmunoassays and radioimmunotherapy.

Adenosine Triphosphate↗
PubMed1996

Open sandwich ELISA: a novel immunoassay based on the interchain interaction of antibody variable region.

We describe an immunoassay that is based on the interchain interaction of separated VL and VH chains from a single chain antibody variable region. In the presence of antigen, the chains reassociate. VL fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 were immobilized on microtiter plates. Samples were coincubated with an M13-displayed VH chain, and assayed with peroxidase-labeled anti-M13 antibody. Signal was detected in direct proportion to the amount of HEL in the sample. Wide dynamic range with < 15 ng/ml sensitivity was attained.

Animals↗
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

Calmodulin as a versatile tag for antibody fragments.

Calmodulin is a highly acidic protein (net charge -24 at pH 8.0 in the absence of calcium) that binds to peptide and organic ligands with high affinity (Ka > 10(9) M-1) in a calcium-dependent manner. We have exploited these properties to develop calmodulin as a versatile tag for antibody fragments. Fusions of calmodulin with single chain Fv fragments (scFv) could be expressed by secretion from bacteria in good yield (5-15 mg/l in shaker flasks), and purified from periplasmic lysates or broth to homogeneity in a single step, either by binding to anion-exchange resin (DEAE-Sephadex), or to an organic ligand of calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide-agarose). The antibody fusions could be detected by binding of fluorescently labeled peptide ligands, as illustrated by their use in confocal microscopy, fluorescent activated cell sorting and "band shift" gel electrophoresis. Moreover, the interaction between calmodulin and peptide ligands could provide a means of heterodimerization of proteins, as illustrated by the assembly of an antibody-calmodulin fusion with maltose binding protein tagged with a peptide ligand of calmodulin.

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↗
PubMed1994

In vitro assembly of repertoires of antibody chains on the surface of phage by renaturation.

Antibodies can be made from repertoires of associated heavy and light chains displayed on the surface of bacteriophage, and are readily diversified by random point mutation or by chain shuffling. To make extensive variations around the "core" antigen binding contacts of a crystallographically solved mouse antibody NQ10/12.5 (gamma l, kappa), the NQ10 light chain was assembled in vitro with a repertoire of about 10(7) human heavy chains displayed on the surface of phage, and selected by binding to hapten. An antibody with a much improved affinity was isolated from the repertoire (K(a) = 10(9) M-1 compared with 10(8) M-1 for NQ10). The sequence of the human heavy chain (VH-IL) was highly related to NQ10. It conserved the same folds for the H1, H2 and H3 loops, six of the seven contact residues for hapten, and also a phOx binding motif (Asp-X-Gly-X-X) in the H3 loop. It appears that the new heavy chain partners for the NQ10 light chain often retain many critical antigen binding features found in the NQ10 heavy chain.

Amino Acid Sequence↗
PubMed1994

Selection of beta-lactamase on filamentous bacteriophage by catalytic activity.

Recently the display of repertoires of peptides and proteins on the surface of filamentous phage, and selection of the phage by binding to a ligand, has allowed the isolation of peptides and proteins with rare binding activities. Furthermore, phages displaying enzymes (phage enzymes) have been selected by affinity of binding to inhibitors. Here we show, using a suicide inhibitor, that phage enzymes can also be selected by their catalytic activity. Two phage enzymes were constructed by fusion to the minor coat protein of the phage (g3p), displaying either an active beta-lactamase or a catalytically inactive mutant in which the essential serine of the active site was mutated to alanine. The phages were then incubated with a beta-lactamase suicide inhibitor connected by a spacer to a biotin moiety. The active (but not the inactive) phages were labelled, and the active phages selected from mixtures with inactive phages by binding and elution from streptavidin-coated beads. The selection ratio for active versus inactive phages (about ten on elution of the phages by reduction of an S-S bond in the spacer between the warhead and biotin) could be improved to about 50 on elution by proteolytic cleavage of beta-lactamase from g3p at an intervening factor X site. Selection of phage-enzymes by catalysis may provide a means of creating new enzymes and refining their catalytic properties.

Alanine↗
PubMed1994

Phage libraries for generation of clinically useful antibodies.

Insertion of antibody genes into filamentous bacteriophage makes it possible to generate and screen libraries of 10(7) or more antibodies. Each phage expresses an antibody on its surface and contains the corresponding antibody gene. Genes that encode antibodies with desired characteristics are readily selected and their antibodies expressed as soluble proteins in Escherichia coli. We used this system to produce an antibody to carcinoembryonic antigen with higher affinity and better tumour specificity than antibodies currently in use.

Animals↗
PubMed1994

Antibody fragments from a 'single pot' phage display library as immunochemical reagents.

The display of repertoires of antibody fragments on the surface of filamentous bacteriophage offers a new way of making antibodies with predefined binding specificities. Here we explored the use of this technology to make immunochemical reagents to a range of antigens by selection from a repertoire of > 10(8) clones made in vitro from human V gene segments. From the same 'single pot' repertoire, phage were isolated with binding activities to each of 18 antigens, including the intracellular proteins p53, elongation factor EF-1 alpha, immunoglobulin binding protein, rhombotin-2 oncogene protein and sex determining region Y protein. Both phage and scFv fragments secreted from infected bacteria were used as monoclonal and polyclonal reagents in Western blots. Furthermore the monoclonal reagents were used for epitope mapping (a new epitope of p53 was identified) and for staining of cells. This shows that antibody reagents for research can be readily derived from 'single pot' phage display libraries.

Amino Acid Sequence↗
PubMed1994

A directory of human germ-line V kappa segments reveals a strong bias in their usage.

From the genomic DNA of a single individual, we have amplified, cloned and sequenced 37 human germ-line V kappa segments. Four of these segments were new. We then compiled a comprehensive directory of all germ-line V kappa segments and identified 50 different sequences with open reading frames. Comparison with 236 rearranged sequences revealed that no more than 24 of these germ-line sequences could be assigned rearranged counterparts, that some of these were rarely used, and that only about 11 sequences are used frequently. This suggests that the expressed V kappa repertoire is mainly derived from a limited number of segments. Most surprisingly, the J kappa-distal region of the locus appears to be rarely used: we could unambiguously assign 162 rearranged sequences to V kappa segments of the J kappa-proximal region, but only 5 to segments of the J kappa-distal region.

Amino Acid Sequence↗