Antibodies with infinite affinity

Abstract

Here we report an approach to the design and production of antibody/ligand pairs, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. Choosing a structurally characterized antibody/ligand pair as an example, we engineered complementary reactive groups in the antibody binding pocket and the ligand, so that they would be in close proximity in the antibody/ligand complex. Cross-reactions with other molecules in the medium are averted because of the low reactivity of these groups; however, in the antibody/ligand complex the effective local concentrations of the complementary reactive groups are very large, allowing a covalent reaction to link the two together. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This chemical manipulation of affinity is applicable to other biological binding pairs.

Figure 1

Requirements for an antibody with infinite affinity. (a) When the antibody and ligand are apart, their complementary reactive groups do not react significantly with other molecules in the medium. (b) When the ligand binds to the antibody, the effective concentrations of their complementary reactive groups are sharply elevated, and a covalent link is formed. (c) The covalently linked antibody/ligand complex cannot dissociate.

Figure 2

Crystal structure of antibody/ligand complex, adapted from Love et al. (17). Two residues in the wild-type antibody that are not directly involved in ligand binding but are favorably located close to the para-substituent of the ligand (yellow) are light-chain residues S95 and N96 (Kabat positions 93 and 94).

Figure 3

Ligands containing electrophilic substituents, tested with engineered Fab fragments S95C and N96C. BABE, bromoacetamidobenzyl-EDTA; CABE, chloroacetamidobenzyl-EDTA; CpABE, (S)-1-p-chloropropionamidobenzyl-EDTA.

Figure 4

Conjugation of electrophilic ¹¹¹In-benzyl-EDTA derivatives with engineered Fab S95C. Phosphorimage of 10–20% SDS/PAGE gel of samples of complete culture media incubated with ¹¹¹In labeled (A) chloroacetamidobenzyl-EDTA, (B) (S)-1-p-chloropropionamidobenzyl-EDTA, (C) AABE, and (D) ABE.

Figure 5

Demonstration that engineered Fab S95C retains the ligand-binding selectivity of the parent antibody. (a) Competition ELISA curves. Different metal-ABE chelates compete with an immobilized indium chelate for binding Fab S95C. (b) Competitive binding of a series of metal-ABE chelates, measured by blocking the covalent attachment of ¹¹¹In-AABE to Fab S95C. SDS/PAGE gel bands show increased labeling of the light chain with decreasing competition.

Figure 6

(a) Phosphorimage of a representative SDS/PAGE assay of extent of conjugation of ¹¹¹In-AABE to Fab S95C. (b) Kinetics of formation of the covalent bond between bound ¹¹¹In-AABE and Fab S95C, at 22°C, pH 7.4.