Antibodies without immunization.
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Antibodies↗
Biomedical subjects
C F Barbas
Publications and source records attributed to C F Barbas.
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↗
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↗
Antibodies from libraries.
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Antibodies, Monoclonal↗
Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.
A panel of 20 recombinant Fab fragments reactive with the surface glycoprotein gp120 of human type 1 immunodeficiency virus (HIV-1) were examined for their ability to neutralize MN and IIIB strains of the virus. Neutralization was determined as the ability of the Fab fragments to inhibit infection as measured in both a p24 ELISA and a syncytium-formation assay. One group of closely sequence-related Fab fragments was found to neutralize virus in both assays with a 50% neutralization titer at approximately 1 micrograms/ml. Another Fab neutralized in the p24 ELISA but not in the syncytium assay. The other Fab fragments showed weak or no neutralizing ability. The results imply that virion aggregation or crosslinking of gp120 molecules on the virion surface is not an absolute requirement for HIV-1 neutralization. Further, all of the Fab fragments were shown to be competitive with soluble CD4 for binding to gp120 and yet few neutralized the virus effectively, implying that the mechanism of neutralization in this case may not involve receptor blocking. The observation of a preponderance of high-affinity Fab fragments with poor or no neutralizing ability could have implications for vaccine strategies.
Amino Acid Sequence↗
Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem.
The properties of naiveté and large diversity are considered to be essential starting features for combinatorial antibody libraries that eschew immunization by evolution in vitro. We have prepared large libraries with such properties by using random oligonucleotide synthesis, which has the potential to create approximately 10(20) complementarity-determining regions for antibody heavy chains. When combined with light chains and expressed on phage surfaces, high-affinity antibodies could be selected from 5.0 x 10(7) Escherichia coli transformants. Remarkably, antibodies selected only for binding displayed both general structural features known to be important in nature's own antibodies and specific consensus sequences thought to be critical for interaction with the hapten against which the library was selected. Semisynthetic and ultimately totally synthetic combinatorial libraries when coupled with mutation and selection procedures should replace immunization for generation of reagent, therapeutic, and catalytic antibodies.
Amino Acid Sequence↗
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↗
In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library.
We have used a combinatorial immunoglobulin library approach to obtain monoclonal antibodies from nonimmune adult mice, thereby establishing the principles of (i) accessing naive combinatorial antibody libraries for predetermined specificities and (ii) increasing the affinity of the selected antibody binding sites by random mutagenesis. A combinatorial Fab library expressing immunoglobulin mu and kappa light-chain fragments on the surface of filamentous phage was prepared from bone marrow of nonimmunized, adult BALB/c mice with the multivalent display vector pComb8. Phage displaying low affinity Fabs (binding constants, 10(4)-10(5) M-1) binding to a progesterone-bovine serum albumin conjugate were isolated from the library. Random mutagenesis of the heavy- and light-chain variable regions expressed in the mono-valent phage display vector pComb3 was performed by error-prone PCR, and subsequently clones with improved affinity for the hapten conjugate were selected. We demonstrate that antibodies with desirable characteristics from a nonimmune source may be selected and affinity maturation may be achieved by using the twin vectors pComb8 and pComb3, thus opening the route to obtaining specific antibodies from a generic library and bypassing immunization.
Amino Acid Sequence↗
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↗
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↗
On the use of combinatorial antibody libraries to clone the "fossil record" of an individual's immune response.
For about the last century the record of immunological events could only be obtained from serum proteins. We suggest that in the future this will change and the far more detailed nucleic acid record will provide new insights into immunological processes as well as selective access to the complete repertoire.
Antibodies↗
Assembly of combinatorial antibody libraries on phage surfaces: the gene III site.
A phagemid system was developed for the monovalent display of combinatorial antibody Fab libraries on the surface of filamentous phage M13. Fab fragments were fused to the carboxyl-terminal domain of the gene III protein. Phage displaying Fab fragments on their surface, or Phabs, were enriched by 10(3)- to 10(5)-fold on antigen-coated surfaces over nonspecific phage. The method may replace current antibody cloning techniques.
Antibodies, Bacterial↗
Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces.
We describe a method based on a phagemid vector with helper phage rescue for the construction and rapid analysis of combinatorial antibody Fab libraries. This approach should allow the generation and selection of many monoclonal antibodies. Antibody genes are expressed in concert with phage morphogenesis, thereby allowing incorporation of functional Fab molecules along the surface of filamentous phage. The power of the method depends upon the linkage of recognition and replication functions and is not limited to antibody molecules.
Antibodies, Monoclonal↗