Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.

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Isolation of high affinity human antibodies directly from large synthetic repertoires.

The discovery of the first neurotransmitter — acetylcholine — was soon followed by the discovery of its hydrolysing enzyme, acetylcholinesterase. The role of acetylcholinesterase in terminating acetylcholine-mediated neurotransmission made it the focus of intense research for much of the past century. But the complexity of acetylcholinesterase gene regulation and recent evidence for some of the long-suspected 'non-classical' actions of this enzyme have more recently driven a profound revolution in acetylcholinesterase research. Although our understanding of the additional roles of acetylcholinesterase is incomplete, the time is ripe to summarize the evidence on a remarkable diversity of acetylcholinesterase functions.

Acetylcholinesterase New roles for an old actor

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination. Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes. Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering.

Methods and compositions for cellular and metabolic engineering

The display of repertoires of antibody fragments on the surface of filamentous bacteriophage offers a new way of making antibodies with predefined binding specificities. Here we explored the use of this technology to make immunochemical reagents to a range of antigens by selection from a repertoire of > 10(8) clones made in vitro from human V gene segments. From the same 'single pot' repertoire, phage were isolated with binding activities to each of 18 antigens, including the intracellular proteins p53, elongation factor EF-1 alpha, immunoglobulin binding protein, rhombotin-2 oncogene protein and sex determining region Y protein. Both phage and scFv fragments secreted from infected bacteria were used as monoclonal and polyclonal reagents in Western blots. Furthermore the monoclonal reagents were used for epitope mapping (a new epitope of p53 was identified) and for staining of cells. This shows that antibody reagents for research can be readily derived from 'single pot' phage display libraries.

G. Flynn

Papers

Antibody fragments from a 'single pot' phage display library as immunochemical reagents.