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G Winter

Publications and source records attributed to G Winter.

At least 19 recordsLinked to original sources
PubMed1992

The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops.

We have used the polymerase chain reaction and VH family-based primers to clone and sequence 74 human germline VH segments from a single individual and built a directory to include all known germline sequences. The directory contains 122 VH segments with different nucleotide sequences, 83 of which have open reading frames. The directory indicates that the structural diversity of the germline repertoire for antigen binding is fixed by about 50 groups of VH segments: each group encodes identical hypervariable loops. The directory should help in mapping the VH locus, in estimating somatic mutation and VH segment usage and in designing and constructing synthetic antibody libraries.

Amino Acid Sequence↗
PubMed1992

Structural repertoire of the human VH segments.

The VH gene segments produce the part of the VH domains of antibodies that contains the first two hypervariable regions. The sequences of 83 human VH segments with open reading frames, from several individuals, are currently known. It has been shown that these sequences are likely to form a high proportion of the total human repertoire and that an individual's gene repertoire produces about 50 VH segments with different protein sequences. In this paper we present a structural analysis of the amino acid sequences produced by the 83 segments. Particular residue patterns in the sequences of V domains imply particular main-chain conformations, canonical structures, for the hypervariable regions. We show that, in almost all cases, the residue patterns in the VH segments imply that the first hypervariable regions have one of three different canonical structures and that the second hypervariable regions have one of five different canonical structures. The different observed combinations of the canonical structures in the first and second regions means that almost all sequences have one of seven main-chain folds. We describe, in outline, structures of the antigen binding site loops produced by nearly all the VH segments. The exact specificity of the loops is produced by (1) sequence differences in their surface residues, particularly at sites near the centre of the combining site, and (2) sequence differences in the hypervariable and framework regions that modulate the relative positions of the loops.

Amino Acid Sequence↗
PubMed1992

By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro.

By display of antibody repertoires on the surface of a filamentous bacteriophage and selection of the phage by binding to antigen, we can mimic immune selection. Recently, by tapping the repertoire of rearranged V-genes from the peripheral blood lymphocytes of unimmunised donors, we succeeded in making human antibody fragments with different specificities, including both haptens and proteins, from the same library of phage. Now we have built a repertoire of human VH genes from 49 human germline VH gene segments rearranged in vitro to create a synthetic third complementarity determining region (CDR) of five or eight residues. The rearranged VH genes were cloned with a human V lambda 3 light chain as single chain Fv fragments for phage display, and the library of phage panned by binding to each of two haptens, 2-phenyl-5-oxazolone (phOx) or 3-iodo-4-hydroxy-5-nitrophenyl-acetate (NIP) coupled to bovine serum albumin (BSA). Many different antibody fragments were isolated which bound specifically to hapten, some with affinities in the micromolar range. The in vitro "immune response" to the hapten NIP was dominated by the 9-1 segment (VH3 family), and that to phOx by the VH26 segment (VH3 family) with an invariant aromatic residue (Tyr, Phe, Trp) at residue 97 of CDR3. However, the isolation of phage against protein antigens proved more elusive, with a single phage binding to human tumour necrosis factor, and none to bovine serum albumin, turkey egg-white lysozyme or human thyroglobulin. Nevertheless, the work shows that human antibody fragments with specific binding activities can be made entirely in vitro.

Amino Acid Sequence↗
PubMed1992

In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells.

We describe a process for the identification of mRNAs within single cells, as demonstrated with the immunoglobulin (Ig) variable region (V) genes of two mouse hybridoma cell lines and the bcr-abl fusion gene of the human K562 myeloid leukaemia line. The cells were fixed and permeabilised, the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction (PCR). After using fluorescent PCR primers, the amplified DNA could be detected within the cells as demonstrated by confocal fluorescence microscopy and flow cytometry. Furthermore the amplified Ig VH and VL DNA could be assembled within the same cell using suitable PCR primers. We detected no cross-contamination of amplified DNA between cells: the DNA isolated from mixtures of two hybridoma cell lines (B1-8 and NQ10/12.5) treated to in-cell PCR and assembly, was shown by cloning to correspond to the combinations of VH and VL genes of the parent hybridomas. We forsee diverse applications of in-cell assembly by PCR, especially for the analysis of the combinations of chains of rearranged Ig or T cell receptor (TCR) V-genes in a population of cells, and the construction of human antibodies from the V-genes of immune B-lymphocytes.

Animals↗
PubMed1992

Selection of phage antibodies by binding affinity. Mimicking affinity maturation.

We describe a process, based on display of antibodies on the surface of filamentous bacteriophage, for selecting antibodies either by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen. For affinity selection, phage are mixed with small amounts of soluble biotinylated antigen (less than 1 microgram) such that the antigen is in excess over phage but with the concentration of antigen lower than the dissociation constant (Kd) of the antibody. Those phage bound to antigen are then selected using streptavidin-coated paramagnetic beads. The process can distinguish between antibodies with closely related affinities. For off-rate selection, antibodies are preloaded with biotinylated antigen and diluted into excess unlabelled antigen for variable times prior to capture on streptavidin-coated paramagnetic beads. To mimic the affinity maturation process of the immune system, we introduced random mutations into the antibody genes in vitro using an error-prone polymerase, and used affinity selection to isolate mutants with improved affinity. Starting with a small library (40,000 clones) of mutants (average 1.7 base changes per VH gene) of the mouse antibody B1.8, and using several rounds of affinity selection, we isolated a mutant with a fourfold improved affinity to the hapten 4-hydroxy-5-iodo-3-nitrophenacetyl-(NIP)-caproic acid (mutant Kd = 9.4(+/- 0.3) nM compared with B1.8 Kd = 41.9(+/- 1.6) nm). The relative increase in affinity of the mutant is comparable to the increase seen in the anti-4-hydroxy-3-nitrophenylacetyl/NIP-caproic acid murine secondary immune response.

Amino Acid Sequence↗
PubMed1992

Comparative stabilities in vitro and in vivo of a recombinant mouse antibody FvCys fragment and a bisFvCys conjugate.

A murine antibody FvCys fragment with a single additional cysteine residue at the C terminus of the VH domain was expressed in Escherichia coli from a modified expression plasmid containing the structural genes for the VH and VL domains derived from the anti-lysozyme hybridoma D1.3. Chemical cross-linking between the introduced sulfhydryl groups of two FvCys fragments by means of bis-maleimidohexane was used to generate a bisFvCys conjugate. The stability of the bisFvCys conjugate and an FvCys analogue that had been reacted with N-ethyl-maleimide to block the free sulfhydryl group, FvCys(BL), were compared after 125I-labeling. The bisFvCys conjugate was completely stable to incubation in solution at 37 degrees C for 24 h whereas only 60% of the FvCys(BL) fragment remained soluble. After i.v. administration to normal Wistar rats, both Fv proteins were rapidly cleared from the circulation with biphasic kinetics that were best fitted to a two-compartment open pharmacokinetic model. The alpha-phase half-life of the bisFvCys conjugate, 0.32 h, was significantly longer than that of the FvCys(BL) fragment, 0.15 h (p less than 0.001) whereas there was no significant difference between the beta-phase half-lives, 1.4 to 1.6h. No chain cleavage or covalent attachment to serum protein was detected by SDS-PAGE analysis of serum samples. However, gel permeation HPLC revealed that both Fv proteins associated with serum proteins in vivo and in vitro.

Amino Acid Sequence↗
PubMed1992

Antibody framework residues affecting the conformation of the hypervariable loops.

Rodent monoclonal antibodies have been "humanized" or "reshaped" for therapy by transplanting the antigen-binding loops from their variable domains onto the beta-sheet framework regions of human antibodies. However, additional substitutions in the human framework regions are sometimes required for high affinity antigen binding. Here we describe antigen binding by a reshaped antibody derived from the mouse anti-lysozyme antibody D1.3, and several variants in which point mutations had been introduced into framework positions to improve its affinity. The affinities were determined from the relaxation kinetics of reactant mixtures using quenching of fluorescence that occurs upon formation of the antibody-antigen complex. The dissociation constant of lysozyme ranged from 3.7 nM (for D1.3) to 260 nM. Measurement of antibody-antigen association kinetics using stopped-flow showed that D1.3 and most of the reshaped antibodies had bimolecular rate constants of 1.4 x 10(6) s-1 M-1, indicating that differences in equilibrium constant were predominantly due to different rates of dissociation of lysozyme from immune complexes. Mutations in a triad of heavy chain residues, 27, 29 and 71, contributed 0.9 kcal/mol in antigen binding free energy, and a Phe to Tyr substitution of light chain residue 71 contributed an additional 0.8 kcal/mol. The combined effect of all these mutations brought the affinity of the reshaped antibody to within a factor of 4 of D1.3. All of these substitutions were in the beta-sheet framework closely underlying the complementarity-determining regions, and do not participate in a direct interaction with antigen. The informed selection of residues in such positions may prove essential for the success of loop transplants in antibodies. Variation of these sites may also have a role in shaping the diversity of structures found in the primary repertoire, and in affinity maturation.

Amino Acid Sequence↗
PubMed1992

Cell selection strategies for making antibodies from variable gene libraries: trapping the memory pool.

The B cells of immunized animals can be used as a source of variable region (V) gene libraries. Such libraries offer a way of making antibodies directly in bacteria: rearranged V genes are amplified using the polymerase chain reaction, cloned and expressed as soluble fragments in bacteria, and then screened for antigen binding. Here we have used a model system to investigate antigen-selected B cells as a source of V gene libraries. Mice were immunized with (4-hydroxyl-3-nitrophenyl)acetyl (NP)-chicken gammaglobulin, and the splenocytes harvested seven days after primary immunization. We prepared a heavy chain variable (VH) gene library from the DNA of cells selected on antigen-coated magnetic beads, and two other libraries from the DNA or mRNA of unselected cells. The VH gene libraries were combined with the V lambda 1 gene (as this light chain dominates the primary response to NP), expressed as Fv fragments in Escherichia coli and screened for binding to (4-hydroxy-3-iodo-5-nitrophenyl)acetyl-bovine serum albumin. The frequency of antigen-binding clones was much greater (greater than 50 fold) in the library from the DNA of antigen-selected cells (17/282) or from the mRNA of unselected cells (29/282) compared to the DNA from unselected cells (0/940). Sequencing of the antigen-binding clones revealed that they almost invariably used the V-186.2 heavy chain, as expected from analysis of primary response hybridomas. The D segments from the mRNA library were entirely DFL16.1 (29/29), as in primary response hybridomas, whereas those from the DNA of selected cells were more diverse, using in addition to DFL16.1, other D segments (5/17) as in later response hybridomas. This suggests that the DNA library from selected cells is derived at least in part from cells destined for the memory compartment. Given the long life of memory cells, they may prove a useful source of antibody libraries in the absence of recent immunization.

Amino Acid Sequence↗
PubMed1992

Blood clearance in the rat of a recombinant mouse monoclonal antibody lacking the N-linked oligosaccharide side chains of the CH2 domains.

The serum half-lives of a wild-type recombinant mouse monoclonal antibody of the IgG2b isotype and a mutant antibody differing from the wild-type antibody by a single amino acid substitution introduced into the CH2 domain, the replacement of Asn 297 by Ala to delete the conserved site of heavy chain glycosylation, were determined in the rat. The biological half-life of the aglycosyl Asn 297-Ala mutant recombinant antibody (4.8 days) was significantly shorter than that of the normally glycosylated wild-type antibody (7.4 days) by enzyme immunoassay. A similar difference between the biological half-lives of 125I-labelled aglycosyl and wild-type antibodies (2.9 and 4.0 days, respectively) was determined by gamma counting. Analysis of serum samples demonstrated that both recombinant antibodies were present in the circulation predominantly as intact monomeric IgG and revealed no differences that could account for the more rapid elimination of the aglycosyl antibody. The results of this investigation indicate that the carbohydrate residues contribute only in part to the survival of IgG in vivo and suggest that the diminished half-life of the aglycosyl antibody is due to increased catabolism in the extravascular tissues.

Animals↗
PubMed1992

Recombinant mouse monoclonal antibodies with single amino acid substitutions affecting Clq and high affinity Fc receptor binding have identical serum half-lives in the BALB/c mouse.

The serum half-lives of three recombinant mouse monoclonal antibodies, differing radically in their ability to bind to Clq or FcRI but only minimally in structure, were determined in the BALB/c mouse following intravenous administration. The wild-type antibody, a chimaeric antibody comprising variable domains binding 3-iodo-4-hydroxy-5-nitrophenylacetate and constant domains of the mouse IgG2b isotype, was eliminated from the bloodstream with biphasic kinetics: alpha-phase, 0.5 days; beta-phase, 7.0 days. The alpha- and beta-phase half-lives of mutant recombinant antibodies with single amino acid substitutions, either Glu 235-Leu allowing binding to the mouse FcRI, or Lys 322-Ala reducing Clq binding 30-fold, were indistinguishable from those of the wild-type antibody demonstrating that the biological half-life of intact mouse IgG is independent of the ability to bind Clq or FcRI. The major implication of the present study is that IgG molecules which have been genetically engineered to eliminate interaction with other components of the immune system should retain the long half-life typical of natural antibodies.

Animals↗
PubMed1992

Multiple binding sites on the CH2 domain of IgG for mouse Fc gamma R11.

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (Fc gamma R). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fc gamma R. This domain was mapped for interaction with mouse Fc gamma R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human Fc gamma R1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237----Ala, Asn 297----Ala, and Glu 318----Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse Fc gamma R11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by Fc gamma R11 of the P388D1 cell line.

Animals↗
PubMed1992

Protein engineering.

Ten years of protein engineering have seen the synthesis of novel therapeutic agents and the analysis of the structure, activity, specificity, stability and folding pathways of proteins. It is hoped that protein engineering will eventually lead to the design of novel catalytic sites on either novel or existing proteins.

Catalysis↗
PubMed1992

By-passing immunization: building high affinity human antibodies by chain shuffling.

Diverse antibody libraries can be displayed on the surface of filamentous bacteriophage, and selected by panning of the phage with antigen. This allows human antibodies to be made directly in vitro without prior immunization, thus mimicking the primary immune response. Here we have improved the affinity of one such "primary" antibody by sequentially replacing the heavy and light chain variable (V) region genes with repertoires of V-genes (chain shuffling) obtained from unimmunized donors. For a human phage antibody for the hapten 2-phenyloxazol-5-one (phOx) (Kd = 3.2 x 10(-7) M), we shuffled the light chains and isolated an antibody with a 20 fold improved affinity. By shuffling the first two hypervariable loops of the heavy chain, we isolated an antibody with a further 15-fold improved affinity. The reshuffled antibody differed in five of the six hypervariable loops from the original antibody and the affinity for phOx (Kd = 1.1 x 10(-9) M) was comparable to that of mouse hybridomas from the tertiary immune response. Reshuffling offers an alternative to random point mutation for affinity maturation of human antibodies in vitro.

Amino Acid Sequence↗
PubMed1992

Germline variable region gene segment derivation of human monoclonal anti-Rh(D) antibodies. Evidence for affinity maturation by somatic hypermutation and repertoire shift.

To date, there has been no systematic study of the process of affinity maturation of human antibodies. We therefore sequenced the variable region genes (V genes) of 14 human monoclonal antibodies specific for the erythrocyte Rh(D) alloantigen and determined the germline gene segments of origin and extent of somatic hypermutation. These data were correlated with determinations of antibody affinity. The four IgM antibodies (low affinity) appear to be derived from two germline heavy chain variable region gene segments and one or two germline light chain variable region gene segments and were not extensively mutated. The 10 IgG antibodies (higher affinity) appear to be derived from somatic hypermutation of these V gene segments and by use of new V gene segments or V gene segment combinations (repertoire shift). Affinity generally increased with increasing somatic hypermutation; on average, there were 8.9 point mutations in the V gene segments of the four IgM antibodies (Ka = 1-4 x 10(7)/M-1) compared with 19 point mutations in the V gene segments of the 10 IgG antibodies. The four highest affinity antibodies (Ka = 0.9-3 x 10(9)/M-1) averaged 25.5 point mutations. The use of repertoire shift and somatic hypermutation in affinity maturation of human alloantibodies is similar to data obtained in inbred mice immunized with haptens.

Antibodies, Monoclonal↗