Improving the affinity of an antibody for its antigen via long-range electrostatic interactions.
To address how long-range electrostatic force can affect antibody-antigen binding, we focused on the interactions between human cardiac troponin I and its specific single-chain antibodies (scFvs). We first isolated two scFvs against two linear epitopes with distinct isoelectric points. For the scFv against the acidic epitope (A1scFv), we mutated five residues of framework region 3 of the light chain to Lys or Arg, designated as the K- or R-mutant, respectively. For the scFv against the basic epitope (A2scFv), we mutated four or three residues in framework region 3 of the light or heavy chain to Asp, to generate the VL- and VH-mutant, respectively. Surface plasmon resonance analyses showed that the kon values of all of the mutants were greater than that of wild type, even though framework region 3 does not make direct contact with the epitope. The affinity of the K-mutant was pM range, and that of the R-mutant improved further by more than two orders of magnitude due to a decrease in the dissociation rate constant. For the A2scFv mutants, the affinity of the VL-mutant for its target improved through an increase in the kon value without a decrease in the koff value. The stability slightly decreased in all mutants. These results suggest that introducing electrostatic interaction can improve the affinity of an antibody for its target, even if the mutation reduces stability of the antibody.
Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku 108-8639, Tokyo, Japan.
Atsushi
A
Fukunaga
Kouhei
K
Tsumoto
D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000235:genetics*; D001665:Binding Sites; D019020:Directed Molecular Evolution; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009994:Osmolar Concentration; D015202:Protein Engineering / Q000379:methods*; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D019210:Troponin I / Q000378:metabolism
D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D019210:Troponin I
SPR; electrostatic interaction; refolding; scFv; troponin