A binary plasmid system for shuffling combinatorial antibody libraries.

Collet, Thomas A.; Roben, Paul; O’Kennedy, Richard; Barbas, Carlos F.; Burton, Dennis R.; Lerner, Richard A.

doi:10.1073/pnas.89.21.10026

Cited by 70 publications

(32 citation statements)

References 15 publications

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“…Because the dominant VH3-07 used by AMC-M2 was paired with so many unrelated light chains (both and ), its VH probably makes the major contribution to binding specificity, as has frequently been observed with other antibodies (Collet et al, 1992; reviewed in Dorrington and Tanford, 1970;Guo et al, 1999;Weigert and Potter, 1977;Winter et al, 1994). With AMC-M6, by contrast, the recurring usage of highly mutated and clonally related light chains in exclusive combination with the dominant VH3-21 suggests that they also have a major influence on specificity (as reviewed by Weigert and Potter, 1977).…”

Section: Fetal-specific Abs In Arthrogryposismentioning

confidence: 87%

Antibodies to Acetylcholine Receptor in Parous Women with Myasthenia: Evidence for Immunization by Fetal Antigen

Matthews

Sims

Ledwidge

et al. 2002

Laboratory Investigation

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SUMMARY:The weakness in myasthenia gravis (MG) is mediated by autoantibodies against adult muscle acetylcholine receptors (AChR) at the neuromuscular junction; most of these antibodies also bind to fetal AChR, which is present in the thymus. In rare cases, babies of mothers with MG, or even of asymptomatic mothers, develop a severe developmental condition, arthrogryposis multiplex congenita, caused by antibodies that inhibit the ion channel function of the fetal AChR while not affecting the adult AChR. Here we show that these fetal AChR inhibitory antibodies are significantly more common in females sampled after pregnancy than in those who present before pregnancy, suggesting that they may be induced by the fetus. Moreover, we were able to clone high-affinity combinatorial Fab antibodies from thymic cells of two mothers with MG who had babies with arthrogryposis multiplex congenita. These Fabs were highly specific for fetal AChR and did not bind the main immunogenic region that is common to fetal and adult AChR. The Fabs show strong biases to VH3 heavy chains and to a single V1 light chain in one mother. Nevertheless, they each show extensive intraclonal diversification from a highly mutated consensus sequence, consistent with antigen-driven selection in successive steps. Collectively, our results suggest that, in some cases of MG, initial immunization against fetal AChR is followed by diversification and expansion of B cells in the thymus; maternal autoimmunity will result if the immune response spreads to the main immunogenic region and other epitopes common to fetal and adult AChR. (Lab

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Section: Fetal-specific Abs In Arthrogryposismentioning

confidence: 87%

Antibodies to Acetylcholine Receptor in Parous Women with Myasthenia: Evidence for Immunization by Fetal Antigen

Matthews

Sims

Ledwidge

et al. 2002

Laboratory Investigation

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“…The ability to rapidly screen antibody heavy and light chain assembly from separate plasmids should allow V H -V L chain shuffling 14,35 without recloning steps and without in vivo expression bias.…”

Section: Discussionmentioning

confidence: 99%

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

Yin¹,

Garces²,

Yang³

et al. 2012

mAbs

128

157

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“…and S.J.B., unpublished work). These antibody vectors were derived from pBP107 (21) and pTC01 (22), respectively. The pertinent features of these vectors are described in Figs.…”

Section: Methodsmentioning

confidence: 99%

Combinatorial protein engineering by incremental truncation

Ostermeier

Nixon

Shim

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

125

104

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We have developed a combinatorial approach, using incremental truncation libraries of overlapping N-and C-terminal gene fragments, that examines all possible bisection points within a given region of an enzyme that will allow the conversion of a monomeric enzyme into its functional heterodimer. This general method for enzyme bisection will have broad applications in the engineering of new catalytic functions through domain swapping and chemical synthesis of modified peptide fragments and in the study of enzyme evolution and protein folding. We have tested this methodology on Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and, by genetic selection, identified PurN heterodimers capable of glycinamide ribonucleotide transformylation. Two were chosen for physical characterization and were found to be comparable to the wild-type PurN monomer in terms of stability to denaturation, activity, and binding of substrate and cofactor. Sequence analysis of 18 randomly chosen, active PurN heterodimers revealed that the breakpoints primarily clustered in loops near the surface of the enzyme, that the breaks could result in the deletion of highly conserved residues and, most surprisingly, that the active site could be bisected.

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A binary plasmid system for shuffling combinatorial antibody libraries.

Abstract: We have used a binary system of repliconcompatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries.

Cited by 70 publications

References 15 publications

Antibodies to Acetylcholine Receptor in Parous Women with Myasthenia: Evidence for Immunization by Fetal Antigen

Antibodies to Acetylcholine Receptor in Parous Women with Myasthenia: Evidence for Immunization by Fetal Antigen

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

Combinatorial protein engineering by incremental truncation

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