In vitro assembly of repertoires of antibody chains on the surface of phage by renaturation.
Antibodies can be made from repertoires of associated heavy and light chains displayed on the surface of bacteriophage, and are readily diversified by random point mutation or by chain shuffling. To make extensive variations around the "core" antigen binding contacts of a crystallographically solved mouse antibody NQ10/12.5 (gamma l, kappa), the NQ10 light chain was assembled in vitro with a repertoire of about 10(7) human heavy chains displayed on the surface of phage, and selected by binding to hapten. An antibody with a much improved affinity was isolated from the repertoire (K(a) = 10(9) M-1 compared with 10(8) M-1 for NQ10). The sequence of the human heavy chain (VH-IL) was highly related to NQ10. It conserved the same folds for the H1, H2 and H3 loops, six of the seven contact residues for hapten, and also a phOx binding motif (Asp-X-Gly-X-X) in the H3 loop. It appears that the new heavy chain partners for the NQ10 light chain often retain many critical antigen binding features found in the NQ10 heavy chain.
Figini
M
M
MRC Centre for Protein Engineering, Cambridge, U.K.
Marks
J D
JD
Winter
G
G
Griffiths
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000916:Antibody Diversity / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics* / Q000302:isolation & purification; D015723:Gene Library; D005803:Genes, Immunoglobulin / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006241:Haptens; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010081:Oxazolone / Q000031:analogs & derivatives; D017422:Sequence Analysis, DNA
D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; C018361:2-phenyloxazolone; D010081:Oxazolone