Functional characterization of an anti‐estradiol antibody by site‐directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the V_L domain in estradiol recognition

Abstract

The high-affinity monoclonal anti-estradiol antibody 9D3 presents a specificity defect towards estradiol-3-sulphate and 3-glucuronide conjugates incompatible with use in direct immunoassays. The corresponding single-chain variable fragment (scFv), cloned and produced in E. coli, exhibited a 10-fold lower affinity for estradiol (K(a)=1.2 x 10(9) M (-1)) and a slightly increased specificity defect for the 3-position. Site-directed mutagenesis revealed critical residues involved in estradiol recognition and produced mutants exhibiting up to a 3-fold increase of the binding affinity for estradiol and up to a 2-fold decrease of the cross-reactivity with estradiol-3-sulphate. A comparative model of the antibody 9D3-estradiol complex was built in which the estradiol D-ring is buried into the binding pocket while the 3-, 6- and 7-positions are solvent exposed, agreeing with the lack of specificity for these three positions. Two potential alternative orientations of the A-ring, one close to CDR H3 and L2 loops, and the other one close to CDR H2 and L3 loops, have been considered for the docking of estradiol, none of which could be unambiguously privileged taking into account data from cross-reactivity measurements, photolabelling and mutagenesis studies. For both orientations, estradiol is stabilized by hydrogen bonding of the 17beta-OH group with TyrL36, His89 and GlnH35 in the first case, or TyrL36, only, in the second case and by van der Waals contacts from TyrL91 with alpha- or beta-face of estradiol, respectively, and from ValH95 and GlyH97 with the opposite face. To elucidate the molecular basis of antibody 9D3 specificity, as compared with that of another anti-estradiol antibody 15H11, single variable domains (V(H) and V(L)) and scFv hybrids have been constructed. The binding activity of V(L)9D3 as well as the specificity of the V(L)9D3/V(H)15H11 hybrid, both similar to antibody 9D3, revealed a prominent role of V(L) in estradiol recognition. These findings establish premises for antibody engineering to reduce cross-reactivity, especially with estradiol-3-conjugates.

Quantitative characterization of protein structure: application to a novel α/β fold

“…26 In particular, he showed that the principal determinant of the binding affinity is the change in compactness and folding increment of the two antibody chains. This effect was also observed in more general studies by Getzoff et al, 36 Pellequer et al, 37 and Coulon et al 38 and is likely related to the hydrophobic interactions between the steroids and DB3. 26 While the above studies support our hypothesis, a more extensive analysis of proteins from multiple families is necessary before we can ascertain the generality of this trend.…”

“…This is supported by recent studies by Estrada and others who have noted a strong correlation between the energy of the folded structure and degree of compactness. 26,[36][37][38] Nonetheless, a comprehensive analysis of a large database of protein structures is necessary before one can establish this relationship. We plan to address this question as well as the applicability of the folding profile to characterize different families of folds in a future study.…”

Two-Step in Vitro Antibody Affinity Maturation Enables Estradiol-17β Assays with More than 10-Fold Higher Sensitivity

“…We have confirmed that a V L -domain protein derived from Ab#E4-4 alone had significant affinity to E 2 -7−BSA conjugates (data not shown). It should be noted that, in another mouse monoclonal anti-E 2 antibody (9D3), the V L domain played a prominent role in E 2 recognition …”

“…It should be noted that, in another mouse monoclonal anti-E 2 antibody (9D3), the V L domain played a prominent role in E 2 recognition. 38 This scFv had a remarkably long 15-residue CDR H3 (the highest occurrence of CDR H3 residues in mouse antibodies was reported to be 9). 39 However, only a few residues of this CDR, located on the border between FRs (e.g., Glu H95 , Arg H96 , and Leu H100g ), seemed to be close to E 2 , and the residues on the tip were not likely.…”

Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin

“…In another study, the VL domain of antibody F11 preserved the antigen-binding function in the absence of VH partner and fusion to barnase increased the solubility of the VL domain both in vivo and in vitro [22,23] . Coulon et al showed that the VL domain of anti-estradiol mAb 9D3 played a prominent role in estradiol recognition [24] . The studies of Twan et al further suggested that it was feasible to create specific single VL domain to diverse targets as is the case for single VH domains in their study [25] .…”