In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library.
We have used a combinatorial immunoglobulin library approach to obtain monoclonal antibodies from nonimmune adult mice, thereby establishing the principles of (i) accessing naive combinatorial antibody libraries for predetermined specificities and (ii) increasing the affinity of the selected antibody binding sites by random mutagenesis. A combinatorial Fab library expressing immunoglobulin mu and kappa light-chain fragments on the surface of filamentous phage was prepared from bone marrow of nonimmunized, adult BALB/c mice with the multivalent display vector pComb8. Phage displaying low affinity Fabs (binding constants, 10(4)-10(5) M-1) binding to a progesterone-bovine serum albumin conjugate were isolated from the library. Random mutagenesis of the heavy- and light-chain variable regions expressed in the mono-valent phage display vector pComb3 was performed by error-prone PCR, and subsequently clones with improved affinity for the hapten conjugate were selected. We demonstrate that antibodies with desirable characteristics from a nonimmune source may be selected and affinity maturation may be achieved by using the twin vectors pComb8 and pComb3, thus opening the route to obtaining specific antibodies from a generic library and bypassing immunization.
Gram
H
H
Scripps Research Institute, Department of Molecular Biology, La Jolla, CA 92037.
Marconi
L A
LA
Barbas
C F
CF
Collet
T A
TA
Lerner
R A
RA
Kang
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D001483:Base Sequence; D001853:Bone Marrow / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D005803:Genes, Immunoglobulin; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D007148:Immunoglobulin mu-Chains / Q000235:genetics*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D007148:Immunoglobulin mu-Chains; D009838:Oligodeoxyribonucleotides