VH shuffling can be used to convert an Fv fragment of anti-hen egg lysozyme specificity to one that recognizes a T cell receptor Vα
VH shuffling can be used to convert an Fv fragment of anti-hen egg lysozyme specificity to one that recognizes a T cell receptor Vα
This study describes the isolation and characterization of Fv fragments that recognize a T cell receptor Vα (Vα 1934.4). A VH gene repertoire from an immunized mouse was recombined with the anti-hen egg lysozyme (HEL) VκD1.3 gene as single chain (sc)Fvs, and an Fv with reasonable affinity for binding to Vα1934.4 isolated. The Fv (VH14/VκD1.3) does not bind to HEL, indicating that the heavy chain shuffling has converted an anti-HEL specificity to one that recognizes the unrelated Vα1934.4. The association constant for the Fv-Vα1934.4 interaction has been determined using surface plasmon resonance (SPR) and is 1.2 × 107 M-1. Recombinant antibodies of reasonable affinity can therefore be generated by combining a VH library with a 'fixed' Vκ. To improve the affinity further, light chain shuffling has been used to generate an Fv (VH14/Vκ9) that has a 30-fold higher affinity for binding to Vα1934.4 than the parent (VH14/VκD1.3) Fv, and SPR measurements demonstrate that the affinity improvement is due to an increase in on-rate. Unexpectedly, Vκ9 differs from VκD1.3 by only two amino acids at positions 30 and 91 and, consistent with the change in binding affinity, both of these residues are located in CDRs.
affinity, bacteriophage display, recombinant Fv, T cell receptor Vα, VH
147-156
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
February 1995
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ward, E. Sally
(1995)
VH shuffling can be used to convert an Fv fragment of anti-hen egg lysozyme specificity to one that recognizes a T cell receptor Vα.
Molecular Immunology, 32 (2), .
(doi:10.1016/0161-5890(94)00119-L).
Abstract
This study describes the isolation and characterization of Fv fragments that recognize a T cell receptor Vα (Vα 1934.4). A VH gene repertoire from an immunized mouse was recombined with the anti-hen egg lysozyme (HEL) VκD1.3 gene as single chain (sc)Fvs, and an Fv with reasonable affinity for binding to Vα1934.4 isolated. The Fv (VH14/VκD1.3) does not bind to HEL, indicating that the heavy chain shuffling has converted an anti-HEL specificity to one that recognizes the unrelated Vα1934.4. The association constant for the Fv-Vα1934.4 interaction has been determined using surface plasmon resonance (SPR) and is 1.2 × 107 M-1. Recombinant antibodies of reasonable affinity can therefore be generated by combining a VH library with a 'fixed' Vκ. To improve the affinity further, light chain shuffling has been used to generate an Fv (VH14/Vκ9) that has a 30-fold higher affinity for binding to Vα1934.4 than the parent (VH14/VκD1.3) Fv, and SPR measurements demonstrate that the affinity improvement is due to an increase in on-rate. Unexpectedly, Vκ9 differs from VκD1.3 by only two amino acids at positions 30 and 91 and, consistent with the change in binding affinity, both of these residues are located in CDRs.
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e-pub ahead of print date: 21 July 1994
Published date: February 1995
Keywords:
affinity, bacteriophage display, recombinant Fv, T cell receptor Vα, VH
Identifiers
Local EPrints ID: 425112
URI: http://eprints.soton.ac.uk/id/eprint/425112
ISSN: 0161-5890
PURE UUID: f4fe56fb-d195-4387-93c3-b7d6dec4fd89
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Date deposited: 11 Oct 2018 16:30
Last modified: 16 Mar 2024 04:37
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