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Biomedical subjects

G Winter

Publications and source records attributed to G Winter.

At least 55 records · Page 3Linked to original sources
PubMed1997

Sequence of the human immunoglobulin diversity (D) segment locus: a systematic analysis provides no evidence for the use of DIR segments, inverted D segments, "minor" D segments or D-D recombination.

We have determined the complete nucleotide sequence of the human immunoglobulin D segment locus on chromosome 14q32.3 and identified a total of 27 D segments, of which nine are new. Comparison with a database of rearranged heavy chain sequences indicates that the human antibody repertoire is created by VDJ recombination involving 25 of these 27 D segments, extensive processing at the V-D and D-J junctions and use of multiple reading frames. We could find no evidence for the proposed use of DIR segments, inverted D segments, "minor" D segments or D-D recombination. Conventional VDJ recombination, which obeys the 12/23 rule, is therefore sufficient to explain the wealth of lengths and sequences for the third hypervariable loop of human heavy chains.

Base Sequence↗
PubMed1997

The creation of diversity in the human immunoglobulin V(lambda) repertoire.

Sequence diversity in the human antibody repertoire is generated in two steps: by the combinatorial assembly of V gene segments and by somatic hypermutation. Here, we have characterised these processes for the lambda (lambda) light chain using a library of 7600 lambda cDNA clones from peripheral blood lymphocytes. By hybridisation and sequencing we found that most lambda chains are derived from the cluster of V(lambda) segments closest to the J(lambda)-C(lambda) pairs and that there is considerable variation in the use of individual V(lambda) segments (ranging from 0.02% to 27%): three of the 30 functional V(lambda) segments encode half the expressed V(lambda) repertoire. As a result of these biases, sequence diversity in the primary repertoire is focused at the centre of the antigen binding site. By contrast, somatic hypermutation spreads diversity to the periphery. Comparison with the human kappa (kappa) light chain indicates that both kappa and lambda use the same strategy for searching sequence space and have almost identical patterns of diversity in the mature antibody repertoire.

Gene Frequency↗
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↗
PubMed1997

Immunoglobulin lambda light chain orphons on human chromosome 8q11.2.

We have identified two V lambda genes outside the major lambda locus on chromosome 22q11.2, and shown that they reside on chromosome 8q11.2. One gene (Orphée1), hybridizing strongly to the V lambda probes, was sequenced and found to belong to the V lambda 8 family; the other gene (Orphée2) only hybridized weakly. Orphée1 was present in all individuals tested (140) from three different populations, and was also found in gorillas. We envisage that these genes were generated by duplication and translocation of the V lambda 8a gene (and a V lambda pseudogene) from the major locus, and that this event occurred before the evolutionary divergence of humans and gorillas. As there is no other evidence for V lambda genes outside the major locus, it appears that the human lambda locus has undergone considerably less evolutionary shuffling than either the human light chain kappa locus or the heavy chain locus.

Amino Acid Sequence↗
PubMed1997

Enzyme immunoassays using bispecific diabodies.

BACKGROUND: Bispecific antibodies with a first binding specificity to a target antigen and a second to an enzyme have great potential in enzyme immunoassays. As bispecific antibodies are difficult to make, the use of recombinant bispecific antibody fragments may provide a breakthrough. OBJECTIVES: To make bispecific antibody fragments directed against an enzyme and to demonstrate their application in enzyme immunoassays. STUDY DESIGN: Bispecific antibody fragments were assembled as diabodies (Holliger P., Prospero T., Winter G. Proc. Natl. Acad. Sci. USA 90, 1993, 6444-6448) directed to an enzyme, E. coli beta-galactosidase, and to each of three target antigens, hen-egg lysozyme (HEL), carcinoembryonic antigen (CEA), and HIV gpl20 (HIV). The diabodies were then evaluated in immunoassays. RESULTS: The HEL diabody was shown to recruit beta-galactosidase in a microtiter plate immunoassay in which diabody and enzyme were co-incubated with antigen, washed and enzyme substrate added. The CEA diabody was shown to detect CEA by immunocytochemical staining of transfected, CEA-expressing HeLa cells and of adenocarcinoma colon tissue sections, and the HIV diabody to detect gpl20 in immunoblots of total cell extracts. CONCLUSION: The results illustrate the diagnostic potential of diabodies in enzyme immunoassays.

Amino Acid Sequence↗
PubMed1997

Complement recruitment using bispecific diabodies.

We describe the engineering of antibody fragments produced in bacteria for recruitment of complement effector functions. From a phage display repertoire we isolated human antibody fragments directed against complement C1q, and linked these to lysozyme-specific antibody fragments, creating bispecific antibodies (diabodies). One diabody was able to recruit C1q, resulting in efficient lysis of lysozyme-coated sheep erythrocytes, and also induced rosette-formation of erythrocytes with human monocytes and phagocytosis after phorbol ester stimulation. These diabodies may have therapeutic applications requiring the activation of complement.

Animals↗
PubMed1997

Retargeting serum immunoglobulin with bispecific diabodies.

Monospecific antibody fragments produced in bacteria lack the Fc portion of antibodies, and are therefore unable to recruit natural effector functions. We describe the use of a bispecific antibody fragment (diabody) to recruit the whole spectrum of antibody effector functions by retargeting serum immunoglobulin (Ig). One arm of the diabody was directed against the target antigen, and the other against the serum Ig. The bispecific diabodies were able to recruit complement, induce mononuclear phagocyte respiratory burst and phagocytosis, and promote synergistic cytotoxicity towards colon carcinoma cells in conjunction with CD8+ T-cells. Further, by virtue of binding to serum Ig their half-life (beta-phase) was increased fivefold compared to a control diabody of the same molecular weight. Such bispecific diabodies may provide an attractive alternative to monoclonal antibodies for serotherapy.

Animals↗
PubMed1997

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.

Amino Acid Sequence↗
PubMed1997

Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris.

Diabodies are dimeric antibody fragments held together by associated heavy and light chain variable domains present on different polypeptide chains. To improve their stability we have introduced cysteine residues into the V-domains to promote the disulphide crosslinking of the dimer. A crosslinked bivalent diabody against carcinoembryonic antigen (CEA) and a crosslinked bispecific diabody against CEA and the T-cell co-receptor CD3 were expressed from Pichia pastoris and Escherichia coli by secretion. From Pichia (but not E.coli) the chains were almost quantitatively crosslinked. Compared with the parent diabodies both crosslinked diabodies were more stable to heat (by >7 degrees C) and the crosslinked bivalent diabody showed improved localization to CEA+ human tumour xenografts in nude mice.

Animals↗
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↗