title: VH shuffling can be used to convert an Fv fragment of anti-hen egg lysozyme specificity to one that recognizes a T cell receptor V alpha.
abstract: This study describes the isolation and characterization of Fv fragments that recognize a T cell receptor V alpha (V alpha 1934.4). A VH gene repertoire from an immunized mouse was recombined with the anti-hen egg lysozyme (HEL) V kappa D1.3 gene as single chain (sc)Fvs, and an Fv with reasonable affinity for binding to V alpha 1934.4 isolated. The Fv (VH14/V kappa D1.3) does not bind to HEL, indicating that the heavy chain shuffling has converted an anti-HEL specificity to one that recognizes the unrelated V alpha 1934.4. The association constant for the Fv-V alpha 1934.4 interaction has been determined using surface plasmon resonance (SPR) and is 1.2 x 10(7) M-1. Recombinant antibodies of reasonable affinity can therefore be generated by combining a VH library with a 'fixed' V kappa. To improve the affinity further, light chain shuffling has been used to generate an Fv (VH14/V kappa 9) that has a 30-fold higher affinity for binding to V alpha 1934.4 than the parent (VH14/V kappa D1.3) Fv, and SPR measurements demonstrate that the affinity improvement is due to an increase in on-rate. Unexpectedly, V kappa 9 differs from V kappa D1.3 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.
authors.affiliation: Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas 75235-8576.
authors.forename: E S
authors.initials: ES
authors.lastname: Ward
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D004527:Egg Proteins / Q000276:immunology; D015723:Gene Library; D015322:Gene Rearrangement, B-Lymphocyte / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009113:Muramidase / Q000276:immunology*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013057:Spectrum Analysis / Q000379:methods
chemical_list: D004527:Egg Proteins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins; D009113:Muramidase