Enzyme mimicry by the antiidiotypic antibody approach

Abstract

The concept of "internal image" of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transition-state analog revealed structural similarity although both antibodies were elicited by two different approaches.

Figure 1

BIAcore analysis of affinity binding between idiotype AE2 and antiidiotype 9A8. Binding of different concentrations of 9A8 to immobilized AE-2 is presented on the sensorgrams. The binding constant was calculated from the experimental curve by nonlinear regression analysis (k_on/k_off = 2.26 10⁹ M⁻¹).

Scheme 1

Compound I: Coumarinyl ethyl p-trifluoroacetamidophenyl methylphosphonate.

Scheme 2

Compound II: Nitrophenyl methyl p-biotinamidophenylmethylphosphonate.

Figure 2

Modifications of cholinesterase enzymes and antiidiotypic mAb by MCA-phosphonate. (A) Kinetics of interaction of MCA-phosphonate (compound I) with BtChoEase (1) and AcChoE (3) enzymes and with 9A8 (2) and IgM 2H11 (4) antibodies. Assays were performed on an Aminco spectrofluorimeter (excitation, 365 nm, and emission, 450 nm). Protein concentration was 55 nM, and substrate concentration was 1 μM in PBS, pH 7.8. (B) Modification of 9A8 mAb H chain by compound II. Protein concentration was 5.5 pM, and substrate concentration was 100 pM in PBS, pH 7.8. Western blot shown was stained by neutravidin-HRP conjugate. Lanes: 1, 10-kDa molecular mass markers; 2, trypsin modified by biotinylated 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF); 3, 9A8 modified by biotinylated AEBSF; 4, monoclonal IgM 2H11 modified by biotinylated AEBSF; 5, 9A8 modified by compound II; 6, trypsin modified by compound II; 7, monoclonal IgM 2H11 modified by compound II.

Figure 3

(A) Primary structure of variable regions of antiidiotypic mAb 9A8. S, active-site Ser (CDR 3 of H chain); H, active-site His (CDR 1 of H chain). (B) Matrix-assisted laser desorption ionization–time-of-flight mass spectrum of a tryptic digest of reduced and S-alkylated antiidiotypic mAb 9A8. About 5 pmol peptide mixture was used in the dried-droplet method of sample preparation, with 2,5-dihydroxybenzoic acid as a matrix. The spectrum was recorded in reflectron positive-ion mode on Vision 2000 (ThermoBioanalysis, Waltham, MA). Tryptic peptides of H chain variable region found are assigned. ●, Peptide with a Ser modified by compound I.

Figure 4

Assembly of recombinant of 9A8 Fab fragment in E. coli periplasm. Western blot stained by anti-c-myc antibody. Lanes: 1, molecular mass markers; 2, without mercaptoethanol; 3, incubation with mercaptoethanol.

Figure 5

Rendered images of 9A8-combining site. (A) The similarity of His H35–Ser H99 dyad of 9A8 esterolytic antibody (green) and His H35–Ser H99 dyad of 17E8 esterolytic antibody (blue) is illustrated. (B) Rendered image of broad combining site cavity. Catalytic HisH35–Ser H99 dyad of 9A8 is colored green.