Esterolytic Antibodies Research Articles

Molecular analysis of multicatalytic monoclonal antibodies

Recently, our first report demonstrated that Cucumber mosaic virus (CMV)-specific monoclonal antibodies (mAbs) possess DNase-like activity against DNA. In the present study, we show for the first time ever how one mAb (mAb-5) has polyreactive (protease, DNase, and RNase) catalytic activities (catAbs). Amino acid sequence analysis of the encoded variable-genes showed that the light chains of the hybridomas expressed the germline family genes Vκ1A, bb1.1 and VκII, bd2, whose protease and DNase catalytic activities have been reported, while the heavy chain genes were expressed in several germline families (eight of V H1/J558, three of V H5/V H7183, and three of V H8/V H3609). Interestingly, these germline genes have been well studied in esterolytic antibodies. Here we present for the first time convincing evidence showing that highly purified mAb-5 catalyze both single- and double-stranded DNA and exhibit RNase and protease activity. The greatest therapeutic potential of catAbs could lie in selective prodrug activation. Furthermore, catAbs offer excellent or unique specificity for individual and defined antigenic targets. Therefore, the phenomenon of autoantibody catalysis can potentially be applied to isolate efficient catalytic domains directed against pathogenetically and clinically relevant autoimmune epitopes.

Mechanistic Analysis of the Phosphonate Transition-state Analogue-derived Catalytic and Non-catalytic Antibody

The esterolytic catalytic antibody (catAb) has the positive charged region interacting with the carbonyl group of the ester substrate. To examine how such a region interacts with the substrate, we compared the catAb with the non-catalytic antibody (non-catAb) for interaction with the non-cleavable amide substrate (a mimic of the ester substrate) and the two end products. Surface plasmon resonance (SPR) analysis revealed that the amide substrate gave the equivalent K(d) values for the two antibodies, whereas both the on-rate and off-rate of the catAb were five-times lower than those of the non-catAb. In agreement with SPR analysis, saturation transfer difference (STD) NMR spectroscopy detected the STD signals only between the catAb and one of the product, suggesting the slower exchange rates of the amide substrate in the catAb as compared with the mixing times, whereas it was not the case with the non-catAb. Transferred nuclear Overhauser effect NMR spectroscopy showed the negative signals for only between the non-catAb and the amide substrate or the product, again suggesting the lower off-rates of the catAb as compared with the mixing times. The decreased interaction rates should be the primary consequence of the positively charged region in the combining site in the catAb.

Structural basis for preferential binding of non-ortho-substituted polychlorinated biphenyls by the monoclonal antibody S2B1

Polychlorinated biphenyls (PCBs) are a family of 209 isomers (congeners) with a wide range of toxic effects. In structural terms, they are of two types: those with and those without chlorines at the ortho positions (2, 2', 6 and 6'). Only 20 congeners have no ortho chlorines. Three of these are bound by the aryl hydrocarbon receptor and are one to four orders of magnitude more toxic than all others. A monoclonal antibody, S2B1, and its recombinant Fab have high selectivity and nanomolar binding affinities for two of the most toxic non-ortho-chlorinated PCBs, 3,4,3',4'-tetrachlorobiphenyl and 3,4,3',4',5'-pentachlorobiphenyl. To investigate the basis for these properties, we built a three-dimensional structure model of the S2B1 variable fragment (Fv) based on the high-resolution crystallographic structures of antibodies 48G7 and N1G9. Two plausible conformations for the complementarity-determining region (CDR) H3 loop led to two putative PCB-binding pockets with very different shapes (models A and B). Docking studies using molecular mechanics and potentials of mean force (PMF) indicated that model B was most consistent with the selectivity observed for S2B1 in competition ELISAs. The binding site in model B had a deep, narrow pocket between V(L) and V(H), with a slight constriction at the top that opened into a wider pocket between CDRs H1 and H3 on the antibody surface. This binding site resembles those of esterolytic antibodies that bind haptens with phenyl rings. One phenyl ring of the PCB fits into the deep pocket, and the other ring is bound in the shallower one. The bound PCB is surrounded by the side chains of TyrL91, TyrL96 and TrpH98, and it has a pi-cation interaction with ArgL46. The tight fit of the binding pocket around the ortho positions of the bound PCBs indicates that steric hindrance of ortho chlorines in the binding site, rather than induced conformational change of the PCBs, is responsible for the selectivity of S2B1.

Changes in structure and dynamics of the Fv fragment of a catalytic antibody upon binding of inhibitor.

Binding of the product inhibitor p-nitrophenol to the monoclonal esterolytic antibody NPN43C9 has been investigated by performing NMR spectroscopy of the heterodimeric variable-domain fragment (Fv) of the antibody in the presence and absence of inhibitor. Structural information from changes in chemical shift upon binding has been related to the changes in local dynamics in the active site of the catalytic antibody using NMR relaxation measurements. Significant changes in the chemical shifts of the backbone resonances upon binding extend beyond the immediate vicinity of the antigen binding site into the interface between the two associated polypeptides that form the Fv heterodimer, a possible indication that the binding of ligand causes a change in the relative orientations of the component light (V(L)) and heavy (V(H)) chain polypeptides. Significant differences in backbone dynamics were observed between the free Fv and the complex with p-nitrophenol. A number of resonances, including almost all of the third hypervariable loop of the light chain (L3), were greatly broadened in the free form of the protein. Other residues in the antigen-binding site showed less broadening of resonances, but still required exchange terms (R(ex)) in the model-free dynamics analysis, consistent with motion on a slow timescale in the active site region of the free Fv. Binding of p-nitrophenol caused these resonances to sharpen, but some R(ex) terms are still required in the analysis of the backbone dynamics. We conclude that the slow timescale motions in the antigen-binding site are very different in the bound and free forms of the Fv, presumably due to the damping of large-amplitude motions by the bound inhibitor.

Binding and catalysis: a thermodynamic study on a catalytic antibody system.

Binding versus catalysis: Attempts to obtain efficient catalysts by using conventional screening methods raise a question concerning the connection between binding and catalysis. To address this issue, we have determined the thermodynamic parameters for the binding of several phosphonates to the esterolytic antibody 17E8 (see scheme). Our results suggest that there may be thermodynamic differences between a binding site selected from a screen for tight transition-state-analogue binding and one that is designed for efficient catalysis.

Intrabody construction and expression. II. A synthetic catalytic Fv fragment

In general, proteins with structural disulfides cannot be expressed in the reducing environment of the cellular cytoplasm. To overcome this folding problem, we have previously engineered stabilizing mutations, predicted from a consensus sequence analysis, into isolated immunoglobulin V L domains. Here we show that such domains can be used as a framework in the construction of a functional heterodimeric Fv fragment, which was expressed solubly, with high yield in the cytoplasm of Escherichia coli. This designed catalytic intrabody, obtained from grafting the combining site of the esterolytic antibody 17E8, is active in the oxidized and the reduced state. Its construction required no special features on the part of the immunoglobulin, no single-chain linker and introduced no non-natural sequence motifs. The potential to design intrabodies with the recognition sequences of arbitrary immunoglobulins opens novel opportunities for gene therapy, cell biology, metabolic engineering and antibody biotechnology.

Structural Basis for Amide Hydrolysis Catalyzed by the 43C9 Antibody

Among catalytic antibodies, the well-characterized antibody 43C9 is unique in its ability to catalyze the difficult, but desirable, reaction of selective amide hydrolysis. The crystallographic structures that we present here for the single-chain variable fragment of the 43C9 antibody, both with and without the bound product p-nitrophenol, strongly support and extend the structural and mechanistic information previously provided by a three-dimensional computational model, together with extensive biochemical, kinetics, and mutagenesis results. The structures reveal an unexpected extended β-sheet conformation of the third complementarity determining region of the heavy chain, which may be coupled to the novel indole ring orientation of the adjacent Trp H103. This unusual conformation creates an antigen-binding site that is significantly deeper than predicted in the computational model, with a hydrophobic pocket that encloses the p-nitrophenol product. Despite these differences, the previously proposed roles for Arg L96 in transition-state stabilization and for His L91 as the nucleophile that forms a covalent acyl-antibody intermediate are fully supported by the crystallographic structures. His L91 is now centered at the bottom of the antigen-binding site with the imidazole ring poised for nucleophilic attack. His L91, Arg L96, and the bound p-nitrophenol are linked into a hydrogen-bonding network by two well-ordered water molecules. These water molecules may mimic the positions of the phosphonamidate oxygen atoms of the antigen, which in turn mimic the transition state of the reaction. This network also contains His H35, suggesting that this residue may also stabilize the transition-states. A possible proton-transfer pathway from His L91 through two tyrosine residues may assist nucleophilic attack. Although transition-state stabilization is commonly observed in esterolytic antibodies, nucleophilic attack appears to be unique to 43C9 and accounts for the unusually high catalytic activity of this antibody.

An esterolytic antibody with vibrational properties of a catalytic H-chain and a non-catalytic L-chain.

An analysis of the temperature factors of an abenzyme has been performed to gain information on the possible role of deterministic chaos in the catalytic mechanism of such artificial proteins. The H-chain displayed a regular attractor of the dimension 3.0 +/- 0.3, whereas the L-chain showed one of <d> = 7.5 +/- 0.5. The abenzyme also displayed a stochastic attractor of the dimension ca. 0.9. The H-chain attractor has one dimension more than those of the native hydrolases chymotrypsin and lysozyme. The additional degree of freedom of the abenzyme offers a likely explanation of the low specific catalytic activities of these artificial enzymes. The dimension of the attractor in the L-chain falls in the range found for other antibodies. Hence, a clear dichotomy seems to rule in this abenzyme; the H-chain displays the vibrational properties of an enzyme and the L-chain those of an antibody. The new data supports the hypothesis of an important role of attractors in biochemical mechanisms by reduction of the number of degrees of freedom (entropy) of reaction partners. A hierarchy of attractors is associated with specific protein functions.

Comparison of phosphonate transition state analogs for inducing catalytic antibodies and evaluation of key structural factors by an ab initio study

The relation between the structure of haptens and the esterolytic activities of antibodies was investigated. We synthesized two phenylalanine analogs, the negatively charged phosphonate derivative 1 and the neutral phosphonamidate derivative 2. Seventeen out of 41 monoclonal antibodies generated against the hapten 1 hydrolyzed the relevant phenylalanine ester R- 12 . On the contrary, none of 27 monoclonal antibodies generated against the hapten 2 had catalytic activity. An ab initio study of the structural and electronic properties of the modeled haptens showed that the value of the negative electrostatic potential around the phosphonyl oxygen was an important factor affecting the induction of esterolytic antibodies.

Probing the importance of second sphere residues in an esterolytic antibody by phage display

We have used phage display to generate a panel of closely related catalytic antibodies. Seeking to improve the catalytic activity of an esterolytic antibody, we displayed libraries derived from the humanized Fab fragment of the antibody 17E8 (h17E8) on filamentous phage and sorted for binding to an immobilized transition-state analog (TSA). Previous work had suggested that residues outside the antibody active site contribute to TSA binding and catalytic efficiency, and we tested this notion by generating libraries containing such “second sphere” residues. Selected variants of h17E8 retained esterolytic activity and showed variations in affinity within 40-fold and kinetic parameters within tenfold of wild-type antibody, indicating that residues remote from the active site do modulate catalytic activity. In order to understand which mutations were responsible for the properties of phage-selected variants, we designed a series of site-directed mutants. From this series, we identified a double mutant in which Tyr97 was changed to Arg in the heavy chain (Y97 HR) and the heavy chain Tyr100a was mutated to Asn (Y100a HN). This variant showed a tenfold improvement in catalytic efficiency ( k cat/ K M) relative to wild-type h17E8. These mutations were additive; Y97 HR increases the catalytic turnover ( k cat) by three- to fourfold, while Y100a HN has been shown to lower the Michaelis constant ( K M) by three- to fivefold. TSA binding was correlated with catalytic turnover for variants that differed by single mutations, but less so for variants that differed by many mutations. Thus, future selections based on TSA binding should focus on mutating a small number of residues at a time.

Crystal structures of the free and liganded form of an esterolytic catalytic antibody

The crystal structure of the esterase catalytic antibody 48G7 has been determined in the presence of hapten at 2.0 Å resolution and in the absence of hapten at 2.7 Å resolution. The root-mean-square difference between the two structures is 0.6 Å for the variable domain and 0.7 Å for the constant domain. Comparison of the active site shows that no significant changes occur upon hapten binding as main-chain and side-chain displacements are negligible. Complex formation occurs as hapten fits into a pre-formed pocket about 10 Å deep. Although 151 water molecules were modeled into the 48G7-hapten structure, none are bound in the active site. Comparison of the 48G7 structures with those of other published ester hydrolysis antibodies illustrates an emerging theme used by esterolytic antibodies in binding their (nitro-)phenyl haptens and in hydrolysing their cognate esters and carbonates: hapten is bound with the aryl end buried deep in the binding pocket, and the phosphonate moiety is responsible for the majority of the binding energy to the antibody-hapten interaction.

The immunological evolution of catalysis.

The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

ChemInform Abstract: Esterolytic Antibodies Induced to Haptens with a 1,2‐Amino Alcohol Functionality.

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Differences in the biochemical properties of esterolytic antibodies correlate with structural diversity

A prerequisite to the design and engineering of catalytic antibodies is the knowledge of their structure and in particular which residues are involved in binding and catalysis. We compared the structure and catalytic properties of a series of six monoclonal antibodies which were all raised against a p-nitrophenyl (PNP) phosphonate and which catalyze the hydrolysis of p-nitrophenyl esters. Three of the antibodies (Group I) have similar light and heavy chain variable regions. The other three antibodies have similar V L regions of which two (Group II) have V H regions from the MOPC21 gene family and the remaining one (Group III) a V H from the MC101 gene family making a total of three different groups based on their V region sequences. The structural division into groups is paralleled by the differences in binding constants to hapten analogs, substrate specificity and the susceptibility of the catalytic activity of the antibodies to chemical modification of tryptophan and arginine residues. The relative binding of a transition state analog to the binding of substrate is much higher for the Group I antibodies than for the other groups. Only the Group I antibodies can catalyze the hydrolysis of a carbonate substrate. However all of the antibodies lose catalytic activity upon specific tyrosine modification which highlights the importance of tyrosine in the active site of the antibodies. Thus, antibodies raised against a single hapten can give antibodies with different structures, and correspondingly different specificities and catalytic properties.

Esterolytic antibodies induced to haptens with a 1,2-amino alcohol functionality

Three structurally related haptens 1-3 were designed and synthesized with the goal of generating antibodies for the hydrolysis of ester 4a and amide 4b. These haptens contain a 1,2-amino alcohol functionality which replaces the ester Jamide moiety of the substrates. A number of catalytic antibodies were generated, and the Michaelis-Menten kinetics constants of three representative catalytic antibodies induced to each of haptens 1-3 were determined. These catalytic antibodies accelerated the hydrolysis of ester 4a with k cat /k un =∼3×10 3 , and their catalytic activities were effectively inhibited by the addition of their respective haptens

A mode of product inhibition of an esterolytic antibody

A catalytic antibody capable of stereoselective ester hydrolysis was obtained. Chemical modification revealed that an Arg residue in the antibody combining site was responsible not only for the catalytic activity. but also for product inhibition by charge interaction with product acid 4. A substrate design allowing for relieving this antibody of product inhibition is described.

In vivo catalysis of a metabolically essential reaction by an antibody.

We have established a growth selection requirement for a catalytic antibody with modest chorismate mutase activity. Conversion of (-)-chorismate into prephenate is the key step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine. Strains of the yeast Saccharomyces cerevisiae containing an insertion mutation in the structural gene for the enzyme chorismate mutase (EC 5.4.99.5) require exogenous supplements of these two amino acids for efficient growth. Intracellular expression of the heterologous antibody catalyst in one such strain, identified by random mutagenesis and genetic selection, provides a substantial growth advantage under auxotrophic conditions; complementation was not observed with an unrelated esterolytic antibody. In addition to demonstrating that tailored immunoglobulin catalysts can carry out vital biochemical reactions in vivo, these experiments provide a powerful selection assay for identifying genetic changes within the antibody molecule itself that augment chemical efficiency.