In vitro antibody maturation. Improvement of a high affinity, neutralizing antibody against IL-1 beta.
Abs as therapeutic antagonists should be of relatively high affinity to effectively neutralize their target Ag. Typically, Abs with nanomolar affinities to protein Ags can be obtained in vivo, however, this may not be the upper limit of affinity because the biologic process attempts to optimize Ab function, of which affinity is only one component. SK48, a high affinity neutralizing murine Fab against human IL-1 beta was used to explore the nature of Ab-Ag interactions and the potential for further affinity improvement in vitro using mutagenesis and selection via phage display. The codons of six amino acids in the third complementarity-determining region of the heavy chain (CDR3-H) were both individually and combinatorialy randomized and the resultant libraries were screened for IL-1 binding phenotype. Mutations that reduced affinity suggested that both the backbone conformation of the CDR3 loop and certain side chains are essential for binding, yet alterations to the canonical salt bridge residues at the base of the loop had minimal effect on affinity. Four rounds of selection of the phage Ab libraries on immobilized IL-1 beta gave predominantly the wild-type sequence, indicating efficient affinity maturation of this CDR in vivo. However, a twofold improvement in affinity was observed for both single and double amino acid changes at two positions in the middle of the CDR. Moreover, a 10-fold increase in affinity for IL-1 beta was achieved by combining two of the phage-selected single amino acid substitutions in CDR3-H, thereby demonstrating that a significant improvement in affinity can be achieved through CDR mutagenesis, even in a matured Ab. The increased affinity of this Fab did not, however, enhance its neutralizing activity in vitro.
Jackson
J R
JR
Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Sathe
G
G
Rosenberg
M
M
Sweet
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity / Q000276:immunology; D001483:Base Sequence; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D007375:Interleukin-1 / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests
D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D007375:Interleukin-1