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Biomedical subjects

D R Burton

Publications and source records attributed to D R Burton.

At least 19 recordsLinked to original sources
PubMed1992

A binary plasmid system for shuffling combinatorial antibody libraries.

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis.

Amino Acid Sequence↗
PubMed1992

Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity.

Respiratory syncytial virus (RSV) is the most important cause, throughout the world, of severe viral lower respiratory tract illness in young children. Antibodies are known to mediate resistance to RSV infection and illness. We have isolated a number of human monoclonal Fab fragments to RSV F glycoprotein from a combinatorial antibody library expressed on the surface of phage. One of these neutralized a wide range of virus isolates, 10 subgroup A and 9 subgroup B isolates, with a titer (60% neutralization) of approximately 0.1-1.0 micrograms/ml. Another Fab neutralized diverse isolates at a concentration somewhat higher. These human Fab fragments show great promise for use in the prophylaxis or therapy of serious RSV lower respiratory tract disease. For intramuscular or intravenous administration, whole antibodies will be required, whereas for aerosol application, F(ab')2 or Fab fragments may suffice.

Amino Acid Sequence↗
PubMed1992

Human monoclonal antibodies: achievement and potential.

The concept of monoclonal antibodies evoked great excitement when first described, but usefulness has been limited by their murine origin and consequent immunogenicity. Human monoclonal antibodies which would obviate xenotypic responses-can now be produced. Clinical implications-including use against HIV-are discussed.

Animals↗
PubMed1992

Human combinatorial antibody libraries to hepatitis B surface antigen.

Human antibody Fab fragments that bind to hepatitis B surface antigen (HBsAg) were generated by using a recombinant phage surface-display expression system. Characterization of HBsAg-specific Fab fragments isolated from two vaccinated individuals reveals diversity in specificity of antigen binding and in the sequences of the complementarity-determining region. The sequence results show examples of human light-chain promiscuity that result in fine specificity changes and a strong relationship to a human germ-line gene. This application illustrates further that this technique is a powerful tool to isolate distinct human antibodies against immunogenic viral targets.

Amino Acid Sequence↗
PubMed1992

Immunization of hu-PBL-SCID mice and the rescue of human monoclonal Fab fragments through combinatorial libraries.

Antibodies are usually prepared from recently boosted animals and reflect ongoing immune responses. In humans, this is restrictive as ethical constraints generally prevent antigen-boosting. Therefore the rich memory compartment of human antibody responses remains largely untapped. Severe combined immune deficiency (SCID) mice populated with human cells allow the stimulation of human antibody memory without the usual constraints. Here we show how peripheral blood lymphocytes can be stimulated by antigen to produce large secondary responses after transfer to SCID mice. Specific monoclonal human Fab fragments can then be isolated from the mice by repertoire cloning even when the human donor's last contact with antigen was more than 17 years ago.

Amino Acid Sequence↗
PubMed1992

Cloning of a human autoimmune response: preparation and sequencing of a human anti-thyroglobulin autoantibody using a combinatorial approach.

Thyroid lymphocyte RNA from a Hashimoto patient exhibiting high titre serum IgG autoantibodies against thyroglobulin (Tg) has been used to construct a Fab library in phage lambda. Screening of this library with radioiodinated Tg has permitted the cloning of an anti-Tg antibody (MH52) with an affinity of 4.5 x 10(9) molar-1 as determined by inhibition ELISA. Sequence analysis showed MH52 to be an authentic antibody of the IgG1/K isotype with variable region genes from the VHI and VKIII families in combination with the JH3, DK4 and JK2 gene segments. The MH52 light chain gene showed high sequence homology (93%) with the germline gene used by several rheumatoid factors and some DNA autoantibodies. Greater divergence from the germline was observed in the case of the MH52 heavy chain gene which showed 86% homology with a germline heavy chain gene isolated from human liver. Overall the similarity between the genes coding for MH52 and the genes coding for some other autoantibodies of non-related specificity might suggest that similar regulatory processes control the formation of these different autoantibodies.

Amino Acid Sequence↗
PubMed1991

Antibody redesign by chain shuffling from random combinatorial immunoglobulin libraries.

A number of experiments on the shuffling of heavy and light chains from antibodies of defined specificity for the transition-state analogue hapten nitrophenyl phosphonamidate are described. The experiments report on the promiscuity of heavy and light chains in binding antigen and the feasibility of antibody redesign by this shuffling process. The concepts of incestuous and extraclonal promiscuous association are described. Shuffling opens the possibility of generating panels of antibodies with related specificity but of distinct idiotypic composition that may have significance in the use of human monoclonal antibodies in therapy.

Animals↗
PubMed1991

A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals.

A panel of human monoclonal antibody Fab fragments has been generated against the surface glycoprotein gp120 of type 1 human immunodeficiency virus (HIV) by antigen selection from a random combinatorial library expressed on the surface of filamentous phage. The library was prepared from 5 ml of bone marrow from an asymptomatic individual who has been HIV-positive for 6 years. The antibodies have high affinity for antigen (mostly with affinity constants of greater than 10(8) M-1) and notable sequence diversity. Given appropriate donor selection, the methods described should allow the generation of antibodies for the evaluation of passive immunization as a therapy for AIDS.

Amino Acid Sequence↗
PubMed1991

Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG.

Cellular receptors for IgG (Fc gamma R) mediate important protective functions. By using site-specific mutants of a chimeric antibody (mouse V H domain and L chain; human IgG3 C H domains), we have demonstrated that human Fc gamma RI interacts with a site in the lower hinge of human IgG (residues 234 to 237) and that this interaction dictates Fc gamma RI-mediated superoxide generation. Mutations at position 235 resulted in the most profound reductions in Fc gamma RI recognition. We have also mapped an interaction site for Fc gamma RII to the same region; however, mutations at position 234 and 237 resulted in the greatest reductions in Fc gamma RII recognition. The two receptors appear to recognize overlapping but nonidentical sites on the lower hinge of IgG. Deviations from the optimal motif 234-Leu-Leu-Gly-Gly-237 may then explain the human IgG subclass specificity profile for human Fc gamma RI and Fc gamma RII.

Antigens, Differentiation↗
PubMed1991

Expression of a human monoclonal anti-(rhesus D) Fab fragment in Escherichia coli with the use of bacteriophage lambda vectors.

A human anti-(rhesus D) antibody (IgG1 lambda) Fab fragment was cloned from an Epstein-Barr-virus-transformed cell line and expressed in Escherichia coli with the use of bacteriophage lambda vectors. The cloned protein is active in binding to human erythrocytes and permits the development of a recombinant reagent for the prevention of haemolytic disease of the newborn. The method offers a rapid and effective means of rescuing human Fabs from potentially unstable cell lines secreting human antibodies.

Antibodies, Monoclonal↗
PubMed1991

The regulation of actin polymerization in differentiating U937 cells correlates with increased membrane levels of the pertussis-toxin-sensitive G-protein Gi2.

Undifferentiated U937 cells appear to lack a capacity of increase cellular F-actin. However, electropermeabilized cells gain the ability to respond in this way to a guanine nucleotide analogue, guanosine 5'-[gamma-thio]trisphosphate (GTP[S]) after 1 h of treatment with dibutyryl cyclic AMP (db-cAMP). The results reported here show that the levels of membrane association of the G-protein Gi2 alpha increase with a time course identical with that of the GTP[S]-sensitivity of electropermeabilized cells. These results suggest that Gi2 alpha may be involved in the signal-transduction pathway leading to actin polymerization in db-cAMP-differentiated U937 cells.

Actins↗
PubMed1991

Generation of diverse high-affinity human monoclonal antibodies by repertoire cloning.

Combinatorial libraries of antibody heavy and light chains derived from the peripheral blood lymphocytes of an individual immunized with tetanus toxoid have been expressed in Escherichia coli by using phage lambda vectors. Screening of the libraries allowed identification of a large number of human monoclonal Fab fragments specific for tetanus toxoid. Initial studies suggested considerable sequence diversity in these antibodies. The method should allow the generation of many human monoclonal antibodies of interest and the dissection of human humoral immune responses.

Amino Acid Sequence↗
PubMed1991

Expanded transition state analogues.

Stable analogues of transition states are used as haptens to elicit antibodies that will catalyse the reaction under investigation. The present failure of such antibodies to equal the catalytic efficiency of enzymes has prompted a more detailed analysis of the structure of transition states. Calculations suggest that for many types of reaction, including the Diels-Alder reaction and addition-elimination reactions, interatomic distances in the transition state are longer than those in analogues hitherto used to elicit catalytic antibodies. Three possible solutions are proposed for the design and synthesis of stable analogues that match the longer interatomic distances of transition states: atom substitution, atom insertion and double atom insertion. Some applications of these ideas to specific reactions are described. The concept of expanded transition states is also valuable for associative processes as a way of avoiding product inhibition of the catalytic antibody; this is being explored for a phenylalanine transcarbamylase antibody.

Antibodies↗