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Biomedical subjects

G Winter

Publications and source records attributed to G Winter.

At least 91 records · Page 5Linked to original sources
PubMed1994

Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen.

We have developed a strategy for guiding the selection of human antibody fragments from phage display repertoires to a single epitope of an antigen, using rodent monoclonal antibodies as a template. Thus the heavy chain of a rodent antibody (MAb32) directed against human tumor necrosis factor alpha (TNF alpha) was cloned and paired as a template chain with a repertoire of human light chains for display as Fab fragments on filamentous phage. The phage were selected by binding to the antigen. The selected human light chains were in turn paired with a repertoire of human heavy chains displayed on phage, and the phage selected again. The isolated phage displaying human antibody fragments binding to TNF alpha also bound to a peptide comprising the N-terminal region of TNF alpha as with MAb32. One of the human Fab fragments was recloned for expression as a glycosylated human antibody in mammalian cells: Binding to TNF alpha could be competed with MAb32 or with anti-serum to the peptide, indicating the same epitope. The human antibody was found to have a binding affinity (Kd = 15 nM) similar to MAb32 (Kd = 26 nM). The process contrasts with existing means of "humanizing" rodent monoclonal antibodies in that the antibodies derived are completely human.

Amino Acid Sequence↗
PubMed1994

A map of the human immunoglobulin VH locus completed by analysis of the telomeric region of chromosome 14q.

Analysis of the telomeric region of chromosome 14q has enabled us to complete a map of the immunoglobulin VH locus which accounts for almost all VH segments known to rearrange in B-lymphocytes. The human germline VH repertoire consists of approximately 50 functional VH segments--the exact number depending on the haplotype--spanning 1,100 kilobases upstream of the JH segments. A yeast artificial chromosome used to map these segments was isolated by its ability to provide telomere activity in yeast, suggesting that the VH locus may be located within a few kilobases of the 14q telomere. The limited structural diversity encoded by the functional VH segments demonstrates the importance of combinatorial diversity produced by VDJ joining and the association of heavy and light chains in producing the human antibody repertoire.

Amino Acid Sequence↗
PubMed1994

Human immunoglobulin VH and D segments on chromosomes 15q11.2 and 16p11.2.

In addition to the major human immunoglobulin heavy-chain locus on chromosome 14q32.3, VH and D segments are known to be present on chromosomes 15 and 16. We have now amplified and sequenced 24 such VH segments from somatic cell hybrids and have assigned them to 15q11.2 and 16p11.2 using cosmid and yeast artificial chromosome clones. In addition, we have located a cluster of D segments on 15q11.2, previously thought to be located on 14q32.3. We propose that the segments on chromosome 16 arose by an interchromosomal duplication and identify the corresponding region on chromosome 14. Taken together with the completion of a map of the human VH locus on 14q32.3, the total number of VH segments now identified is 117. We can now account for most, if not all human germ-line VH segments.

Base Sequence↗
PubMed1994

Making antibodies by phage display technology.

Antibody fragments of predetermined binding specificity have recently been constructed from repertoires of antibody V genes, bypassing hybridoma technology and even immunization. The V gene repertoires are harvested from populations of lymphocytes, or assembled in vitro, and cloned for display of associated heavy and light chain variable domains on the surface of filamentous bacteriophage. Rare phage are selected from the repertoire by binding to antigen; soluble antibody fragments are expressed from infected bacteria; and the affinity of binding of selected antibodies is improved by mutation. The process mimics immune selection, and antibodies with many different binding specificities have been isolated from the same phage repertoire. Thus human antibody fragments have been isolated with specificities against both foreign and self antigens, including haptens, carbohydrates, secreted and cell surface proteins, viral coat proteins, and intracellular antigens from the lumen of the endoplasmic reticulum and the nucleus. Such antibodies have potential as reagents for research and in therapy.

Animals↗
PubMed1994

Surface antigens of Leishmania mexicana amastigotes: characterization of glycoinositol phospholipids and a macrophage-derived glycosphingolipid.

Amastigotes of the protozoan parasite Leishmania proliferate in phagolysosomes of macrophages. They abundantly express glycoinositol phospholipids (GIPLs), which are considered necessary for parasite survival by providing a shield at the surface against lysosomal hydrolases and by serving as receptors for the interaction with host cells. The structures of four GIPLs of L. mexicana amastigotes were characterized by a combination of gas-liquid chromatography-mass spectrometry, methylation linkage analysis and enzymatic treatments. They contain the glycan structures Man alpha 1-3Man alpha 1-4GlcN (iM2), Man alpha 1-6(Man alpha 1-3)Man alpha 1-4GlcN (iM3), Man alpha 1-2Man alpha 1-6(Man alpha 1-3)-Man alpha 1-4GlcN (iM4) and (NH2-CH2CH2-PO4)Man alpha 1-6(Man alpha 1-3)Man alpha 1-4GlcN (EPiM3), which are linked to alkylacyl-phosphatidylinositol. The predominant amastigote GIPL, EPiM3 (approximately 2 x 10(7) molecules/cell), is located at the parasite cell surface, in the flagellar pocket and in lysosomal membranes, but not on host cell structures as shown by immunofluorescence and immunoelectron microscopy. In addition, amastigotes in infected Balb/c mice contain a glycolipid with similar distribution as EPiM3, which has the same characteristics as the Forssman antigen of mammalian cells. In contrast to EPiM3, there is strong evidence that this glycosphingolipid is not synthesized by amastigotes but by macrophages in the lesion. This suggests a mechanism of lipid transfer from the macrophage to the parasite.

Animals↗
PubMed1993

The contribution of contact and non-contact residues of antibody in the affinity of binding to antigen. The interaction of mutant D1.3 antibodies with lysozyme.

We have analysed the contribution of residues of the D1.3 Fv fragment to binding of hen egg lysozyme. We altered residues at the contact interface by site-directed mutagenesis, and determined the affinity of the mutant Fv fragments for lysozyme by fluorescence quench titration. We found that a band of residues at the centre of the contact interface were much more important for binding affinity than those at the periphery. We also subjected the seFv fragment to random mutagenesis to simulate somatic mutation and affinity maturation. By display of the mutants on the surface of filamentous phages, and selection of the phages with biotinylated lysozyme, we were able to select mutants with modest improvements in binding affinity to lysozyme. By combining the mutations we obtained a scFv fragment with a fivefold improved affinity (Kd approximately 0.6 nM compared to wild-type Kd = 3.3 nM). However, none of the altered residues leading to improved affinity was located in the contact interface. This indicates that the interactions of a few residues at the centre of the contact interface are responsible for the binding affinity to antigen, but that these interactions can be modulated by alterations of residues outside the binding site. This may represent a typical mechanism for the affinity maturation of antibodies.

Animals↗
PubMed1993

HAPPY mapping of a YAC reveals alternative haplotypes in the human immunoglobulin VH locus.

We have identified and sequenced 14 human immunoglobulin VH segments cloned in a yeast artificial chromosome, and have used a rapid PCR-based technique (HAPPY mapping, 12) to derive the order and approximate distances between them. The sequences mapped comprise thirteen germline VH segments and one rearranged VH3 gene. Comparison of our map with other data suggests the existence of at least two distinct haplotypes, differing in the presence or absence of the consecutive genes DP-78, DP-46 and DP-64, and in the duplication of segments DP-49 and DP-65. Screening of ten individuals confirms the existence of both haplotypes, and indicates that both are common amongst the population.

Base Sequence↗
PubMed1993

"Diabodies": small bivalent and bispecific antibody fragments.

Bivalent and bispecific antibodies and their fragments have immense potential for practical application. Here we describe the design of small antibody fragments with two antigen-binding sites. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. As indicated by a computer graphic model of the dimers, the two pairs of domains can pack together with the antigen-binding sites pointing in opposite directions. The dimeric antibody fragments, or "diabodies," can be designed for bivalent or bispecific interactions. Starting from the monoclonal antibodies NQ11.7.22 (NQ11) and D1.3 directed against the hapten phenyloxazolone and hen egg lysozyme, respectively, we built bivalent fragments (VHNQ11-VLNQ11)2 and (VHD1.3-VLD1.3)2 and bispecific fragments VHNQ11-VLD1.3 and VHD1.3-VLNQ11. The fragments were expressed by secretion from bacteria and shown to bind specifically to the hapten and/or antigen. Those with 5- and 15-residue linkers had similar binding affinities to the parent antibodies, but a fragment with the VH domain joined directly to the VL domain was found to have slower dissociation kinetics and an improved affinity for hapten. Diabodies offer a ready means of constructing small bivalent and bispecific antibody fragments in bacteria.

Animals↗
PubMed1993

Retroviral vectors displaying functional antibody fragments.

We have made retrovirus particles displaying a functional antibody fragment. We fused the gene encoding an antibody fragment directed against a hapten with that encoding the viral envelope protein (Pr80env) of the ecotropic Moloney murine leukemia virus. The fusion gene was co-expressed in ecotropic retroviral packaging cells with a retroviral plasmid carrying the neomycin phosphotransferase gene (neo), and retroviral particles with specific hapten binding activities were recovered. Furthermore the hapten-binding particles were able to transfer the neo gene and the antibody-envelope fusion gene to mouse fibroblasts. In principle, the display of antibody fragments on the surface of recombinant retroviral particles could be used to target virus to cells for gene delivery, or to retain the virus in target tissues.

3T3 Cells↗
PubMed1993

Cloning and sequencing of human immunoglobulin V lambda gene segments.

To provide the building blocks for making synthetic antibody fragments we have used the polymerase chain reaction (PCR) to clone human variable (V) gene segments of lambda light chains. The PCR primers were based on the sequences of known human V lambda segments, and were used to isolate 14 new V lambda segments (including 4 pseudogenes) from a single individual. We have compiled a sequence directory from this data and other sources to include all known human V lambda segments with open reading frames and we have identified a new V lambda family (V lambda IX). Almost all of the segments (22/24) have different sequences in the complementarity-determining regions, setting a lower limit to the structural diversity of the antigen binding sites encoded by human V lambda genes in the human population.

Base Sequence↗
PubMed1993

Humanized antibodies.

Hybridoma technology enabled rodent monoclonal antibodies to be created against human pathogens and cells, but these had limited clinical utility. Protein engineering, reviewed her by Greg Winter and William Harris, is now generating antibodies for treatment of infectious disease, autoimmune disease and cancer by 'humanizing' rodent antibodies. Humanized antibodies have improved pharmacokinetics, reduced immunogenicity and have been used to clinical advantage.

Animals↗
PubMed1993

Humanized antibodies.

Hybridoma technology enabled rodent monoclonal antibodies to be created against human pathogens and cells, but these had limited clinical utility. Protein engineering is now generating antibodies for treatment of infectious disease, autoimmune disease and cancer by 'humanizing' rodent antibodies. Humanized antibodies have improved pharmacokinetics, reduced immunogenicity and have been used to clinical advantage.

Animals↗
PubMed1993

Engineering bispecific antibodies.

Bispecific antibodies have immense potential as reagents and in medicine. Until recently, they were made by combining monoclonal antibodies of two different specificities in vitro, or by fusion of the corresponding hybridomas. Protein engineering now offers the chance to construct a range of small 'designer' bispecific antibodies using antibody fragments as building blocks.

Animals↗