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Catalytic antibodies possess a dual function that enables both antigen recognition and degradation. However, their time-consuming preparation is a significant drawback. This study developed a new method for quickly converting mice monoclonal antibodies into catalytic antibodies using site-directed mutagenesis. Three mice type monoclonal antibodies targeting hemagglutinin molecule of influenza A virus could be transformed into the catalytic antibodies by deleting Pro95 in CDR-3 of the light chain. No catalytic activity was observed for monoclonal antibodies and light chains. In contrast, the Pro95-deleted light chains exhibited a catalytic activity to cleave the antigenic peptide including the portion of conserved region of hemagglutinin molecule. The affinity of the Pro95-deleted light chains to the antigen increased approximately 100-fold compared to the wild-type light chains. In the mutants, three residues (Asp1, Ser92, and His93) come closer to the appropriate position to create the catalytic site and contributing to the enhancement of both catalytic function and immunoreactivity. Notably, the Pro95-deleted catalytic light chains could suppress influenza virus infection in vitro assay, whereas the parent antibody and the light chain did not. This strategy offers a rapid and efficient way to create catalytic antibodies from existing antibodies, accelerating the development for various applications in diagnostic and therapeutic applications., (© 2024. The Author(s).)

Exact mechanisms of autoimmune disease development are still yet unknown. However, it is known that the development of autoimmune diseases is associated with defects in the immune system, namely, the violation of the bone marrow hematopoietic stem cells (HSCs) differentiation profiles. Different characteristics of autoimmune reaction development in experimental autoimmune encephalomyelitis (EAE) prone Th mice characterizing T-lymphocytes response were analyzed using standard approaches. Profiles of several HSCs differentiation of bone marrow (BFU-E, CFU-E, CFU-GM, CFU-GEMM, T- and B-lymphocytes) of Th male and female mice during spontaneous development of EAE were noticeably different. Patterns of total lymphocytes, B- and T-cells proliferation in several different organs (bone marrow, blood, spleen, thymus, and lymph nodes) were also remarkably different. In addition, there were in time noticeable differences in their changes for some organs of male and female mice. Characters of changes in the profiles of CD4 and CD8 cells proliferation in some organs not always coincide with those for total T lymphocytes. The changes in the differentiation profiles of HSCs and the level of lymphocytes proliferation in the bone marrow and other organs were associated with the increase in the concentration of antibodies against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, and catalytic antibodies hydrolyzing these substrates. Despite some differences in changes in the analyzed parameters, in general, the spontaneous development of EAE in male and female mice occurs to some extent in a comparable way.

It was previously shown that several monoclonal light chains corresponding to the phagemid library of recombinant peripheral blood lymphocyte immunoglobulin light chains of patients with systemic lupus erythematosus specifically hydrolyze only myelin basic protein (MBP). Canonical enzymes usually have only one active site catalyzing some kind of chemical reaction. It was shown previously that in contrast to classical enzymes, preparations of one of the light chains (NGTA2-Me-pro-Tr) showed two optimal pH values, two optimal concentrations of metal ions, and two K m values for MBP. One protease active site of NGTA2-Me-pro-Tr was trypsin like, whereas second one was metal dependent. In this article, a search for protein sequences of NGTA2-Me-pro-Tr responsible for catalytic functions was carried out. We performed, for the first time, analysis of the homology of the protein sequence of NGTA2-Me-pro-Tr with those of several classical Zn 2+ - and Ca 2+ -dependent, as well as human serine, proteases. The analysis allowed us to identify the protein sequences of NGTA2-Me-pro-Tr responsible for serine-like activity, the binding of MBP, and chelation of metal ions and catalysis directly. The data obtained are summarized using hypothetical models of the structure of the two active centers of a very unusual light chain of antibodies (Abs). The findings obtained may be very important for understanding possible structure of active centers of very unusual light chain of Abs possessing several enzymatic activities., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

Immunoglobulins are known to combine various effector mechanisms of the adaptive and the innate immune system. Classical immunoglobulin functions are associated with antigen recognition and the initiation of innate immune responses. However, in addition to classical functions, antibodies exhibit a variety of non-canonical functions related to the destruction of various pathogens due to catalytic activity and cofactor effects, the action of antibodies as agonists/antagonists of various receptors, the control of bacterial diversity of the intestine, etc. Canonical and non-canonical functions reflect the extreme human antibody repertoire and the variety of antibody types generated in the organism: antigen-specific, natural, polyreactive, broadly neutralizing, homophilic, bispecific and catalytic. The therapeutic effects of intravenous immunoglobulins (IVIg) are associated with both the canonical and non-canonical functions of antibodies. In this review, catalytic antibodies will be considered in more detail, since their formation is associated with inflammatory and autoimmune diseases. We will systematically summarize the diversity of catalytic antibodies in normal and pathological conditions. Translational perspectives of knowledge about natural antibodies for IVIg therapy will be also discussed.

Over thousands of monoclonal antibodies (mAbs) have been produced so far, and it would be valuable if these mAbs could be directly converted into catalytic antibodies. We have designed a system to realize the above concept by deleting Pro 95 , a highly conserved residue in CDR-3 of the antibody light chain. The deletion of Pro 95 is a key contributor to catalytic function of the light chain. The S35 and S38 light chains have identical amino acid sequences except for Pro 95 . The former, with Pro 95 did not show any catalytic activity, whereas the latter, without Pro 95 , exhibited peptidase activity. To verify the generality of this finding, we tested another light chain, T99wt, which had Pro 95 and showed little catalytic activity. In contrast, a Pro 95 -deleted mutant enzymatically degraded the peptide substrate and amyloid-beta molecule. These two cases demonstrate the potential for a new method of creating catalytic antibodies from the corresponding mAbs., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the specificity of interactions with substrates. In the present work, a structural and functional study of the Fab fragment of antibody A5 and a comparative analysis of its properties with antibody A17 have been carried out. These antibodies were previously selected for their ability to interact with organophosphorus compounds via covalent catalysis. It has been established that antibody A5 has exceptional specificity for phosphonate X with bimolecular reaction rate constants of 510 ± 20 and 390 ± 20 min^(-1)М^(-1) for kappa and lambda variants, respectively. 3D-Modeling of antibody A5 structure made it possible to establish that the reaction residue L-Y33 is located on the surface of the active site, in contrast to the A17 antibody, in which the reaction residue L-Y37 is located at the bottom of a deep hydrophobic pocket. To investigate a detailed mechanism of the reaction, A5 antibody mutants with replacements L-R51W and H-F100W were created, which made it possible to perform stopped-flow kinetics. Tryptophan mutants were obtained as Fab fragments in the expression system of the methylotrophic yeast species Pichia pastoris. It has been established that the effectiveness of their interaction with phosphonate X is comparable to the wild-type antibody. Using the data of the stopped-flow kinetics method, significant conformational changes were established in the phosphonate modification process. The reaction was found to proceed using the induced-fit mechanism; the kinetic parameters of the elementary stages of the process have been calculated. The results present the prospects for the further improvement of antibody-based biocatalysts.

The antigen-binding properties of single chain Fv antibodies (scFvs) can vary depending on the position and type of fusion tag used, as well as the host cells used for expression. The issue is even more complicated with a catalytic scFv antibody that binds and hydrolyses a specific antigen. Herein, we investigated the antigen-binding and -hydrolysing activities of the catalytic anti-nucleic acid antibody 3D8 scFv expressed in Escherichia coli or HEK293f cells with or without additional amino acid residues at the N- and C-termini. DNA-binding activity was retained in all recombinant forms. However, the DNA-hydrolysing activity varied drastically between forms. The DNA-hydrolysing activity of E. coli-derived 3D8 scFvs was not affected by the presence of a C-terminal human influenza haemagglutinin (HA) or His tag. By contrast, the activity of HEK293f-derived 3D8 scFvs was completely lost when additional residues were included at the N-terminus and/or when a His tag was incorporated at the C-terminus, whereas a HA tag at the C-terminus did not diminish activity. Thus, we demonstrate that the antigen-binding and catalytic activities of a catalytic antibody can be separately affected by the presence of additional residues at the N- and C-termini, and by the host cell type., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The existence of catalytic antibodies has been known for decades. Natural antibodies capable of cleaving nucleic acid, protein, and polysaccharide substrates have been described. Although the discovery of catalytic antibodies initially aroused great interest because of their promise for the development of new catalysts, their enzymatic performance has been disappointing due to low reaction rates. However, in the areas of infection and immunity, where processes often occur over much longer times and involve high antibody concentrations, even low catalytic rates have the potential to influence biological outcomes. In this regard, the presence of catalytic antibodies recognizing host antigens has been associated with several autoimmune diseases. Furthermore, naturally occurring catalytic antibodies to microbial determinants have been correlated with resistance to infection. Recently, there has been substantial interest in harnessing the power of antibody-mediated catalysis against microbial antigens for host defense. Additional work is needed, however, to better understand the prevalence, function, and structural basis of catalytic activity in antibodies. Here we review the available information and suggest that antibody-mediated catalysis is a fertile area for study with broad applications in infection and immunity., (Copyright © 2017 American Society for Microbiology.)

Taking advantage of antibody molecules to generate tailor-made binding sites, we propose a new class of protein modifications, termed as "site-directed chemical mutation." In this modification, chemically synthesized catalytic components with a variety of steric and electronic properties can be noncovalently and nongenetically incorporated into specific sites in antibody molecules to induce enzymatic activity. Two catalytic antibodies, 25E2 and 27C1, possess antigen-combining sites which bind catalytic components and act as apoproteins in catalytic reactions. By simply exchanging these components, antibodies 25E2 and 27C1 can catalyze a wide range of chemical transformations including acyl-transfer, β-elimination, aldol, and decarboxylation reactions. Although both antibodies were generated with the same hapten, phosphonate diester 1, they showed different catalytic activity. When phenylacetic acid 4 was used as the catalytic component, 25E2 efficiently catalyzed the elimination reaction of β-haloketone 2, whereas 27C1 showed no catalytic activity. In this work, we focused on the β-elimination reaction and examined the site-directed chemical mutation of 27C1 to induce activity and elucidate the catalytic mechanism. Molecular models showed that the cationic guanidyl group of Arg H52 in 27C1 makes a hydrogen bond with the P═O oxygen in the hapten. This suggested that during β-elimination, Arg H52 of 27C1 would form a salt bridge with the carboxylate of 4, thus destroying reactivity. Therefore, we utilized site-directed chemical mutation to change the charge properties of the catalytic components. When amine components 7-10 were used, 27C1 efficiently catalyzed the β-elimination reaction. It is noteworthy that chemical mutation with secondary amine 8 provided extremely high activity, with a rate acceleration [(k cat /K m 2)/k uncat ] of 1 000 000. This catalytic activity likely arises from the proximity effect, plus general-base catalysis associated the electrostatic interactions. In 27C1, the cationic guanidyl group of Arg H52 is spatially close to the nitrogen of the amine components. In this microenvironment, the intrinsic pK a of the amine is perturbed and shifts to a lower pK a , which efficiently abstracts the α-proton during the reaction. This mechanism is consistent with the observed kinetic isotope effect (E2 or E1cB mechanism). Thus, site-directed chemical mutation provides a better understanding of enzyme functions and opens new avenues in biocatalyst research.

We propose a new method of obtaining of stable Fab-fragments of antibodies in Pichia pastoris expression system. Recently, we obtained Fab-fragments of antibodies neutralizing organophosphorus toxins. However, high yield of the target products was not attained because of high level of proteolytic degradation. In the present study, we identified sites of proteolytic degradation in Fab-fragments and endogenous proteases performing degradation, which allowed obtaining optimized genetic constructs for expression of antibody heavy chains (IgGγ1) and kappa and lambda isotypes of light chains. Co-transformation of these vectors allowed obtaining Fab-fragments of antibodies to organophosphorus toxins without proteolytic degradation of the product.

Polyclonal antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. The small pools of phage particles displaying light chains with different affinity for MBP were isolated by affinity chromatography on MBP-Sepharose. The fraction eluted with 0.5M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27kDa). The clones were expressed in Escherichia coli in a soluble form; MLChs were purified by metal-chelating chromatography followed by gel filtration. In mammalians, there are serine proteases and metalloproteases. These and many other enzymes usually have only one active site and catalyze only one chemical reaction. In contrast to canonical proteases, one MLCh (NGTA2-Me-pro-ChTr) efficiently hydrolyzed MBP (but not other proteins) and four different oligopeptides corresponding to four immunodominant sequences containing cleavage sites of MBP. The proteolytic activity of MLCh was efficiently inhibited only by specific inhibitors of serine-like (phenylmethanesulfonylfluoride, PMSF) and metalloproteases (EDTA). It was shown that MLCh possess independent serine-like and metal-dependent activities. The principal existence of monoclonal antibodies with two different proteolytic activities is unexpected but very important for the further understanding of at present unknown biological functions of human antibodies., (© The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Catalytic antibodies have exhibited interesting functions against some infectious viruses such as HIV, rabies virus, and influenza virus in vitro as well as in vivo. In some cases, a catalytic antibody light chain takes on several structures from the standpoint of molecular size (monomer, dimer, etc.) and/or isoelectronic point. In this study, we prepared a monomeric 23D4 light chain by mutating the C-terminal Cys to Ala of the wild-type. The mutated 23D4 molecule took a simple monomeric form, which could hydrolyze synthetic 4-methyl-coumaryl-7-amide substrates and a plasmid DNA. Because the monomeric 23D4 light chain suppressed the infection of influenza virus A/Hiroshima/37/2001 in an in vitro assay, the corresponding experiments were conducted in vivo, after the virus strain (which was taken from a human patient) was successfully adapted into BALB/cN Sea mice. In the experiments, a mixture of the monomeric 23D4 and the virus was nasally administered 1) with preincubation and 2) without preincubation. As a result, the monomeric 23D4 clearly exhibited the ability to suppress the influenza virus infection in both cases, indicating a potential drug for preventing infection of the influenza A virus., (© FASEB.)

Classical immunization methods do not generate catalytic antibodies (catabodies), but recent findings suggest that the innate antibody repertoire is a rich catabody source. We describe the specificity and amyloid β (Aβ)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Aβ C terminus noncovalently and hydrolyzed Aβ rapidly, with no reactivity to the Aβ precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aβ dipeptide unit. The catabody dissolved preformed Aβ aggregates and inhibited Aβ aggregation more potently than an Aβ-binding IgG. Intravenous catabody treatment reduced brain Aβ deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aβ hydrolysis appears to be an innate immune function that could be applied for therapeutic Aβ removal., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Catalytic antibodies are immunoglobulins endowed with enzymatic properties. Discovered in the second part of the 1980s, the enthusiasm they initially aroused was counterbalanced by the difficulty of their production and their low catalytic rates. Nevertheless, improvements in expression systems and engineering technologies, combined with various studies suggesting that catalytic antibodies play a role in the immune system, have opened the way to new applications for these proteins. Herein we review catalytic antibodies from a biotechnological point of view, focusing our study on the different production methods, expression systems and their potential clinical applications dedicated to these proteins.

Human antibody-ribonuclease (RNase) fusion proteins, referred to as immunoRNases, have been proposed as an alternative to heterologous immunotoxins, without their immunogenicity and unspecific toxicity issues. In this study, we investigated if human pancreatic RNase will be suitable as effector component in a therapeutic antibody development platform. We generated several fusion proteins consisting of tumor-specific human immunoglobulins (IgGs) and human pancreatic RNase. Transient mammalian cell production was efficient and IgG-RNases were purified to homogeneity. Antigen binding was comparable to the parental antibodies and RNase catalytic activity was retained even in the presence of 50-fold molar excess of human cytosolic RNase inhibitor (RI). Serum stability, cell binding and internalization of IgG-RNases were comparable to the parental IgGs. Despite these promising properties, none of the IgG-RNases revealed significant inhibition of tumor cell growth in vitro even when targeting different antigens putatively employing different endocytotic pathways. The introduction of different linkers containing endosomal protease cleavage sites into the IgG-RNase did not enhance cytotoxicity. Similarly, RI evasive human pancreatic RNase variants mediated only small inhibiting effects on tumor cell growth at high concentrations, potentially reflecting inefficient cytosolic translocation. Taken together, human pancreatic RNase and variants did not prove to be generally suitable as effector component for a therapeutic antibody drug development platform.

Catalytic antibodies are currently being investigated in order to understand their role under physio-pathological situations. To this end, the knowledge of structure-function relationships is of great interest. Recombinant scFv fragments are smaller and easier to genetically manipulate than whole antibodies, making them well suited for this kind of study. Nevertheless they are often described as proteins being laborious to produce. This paper describes a highly efficient method to produce large quantities of refolded soluble catalytic scFv. For the first time, the functionality of a refolded catalytic scFv displaying a β-lactamase activity has been validated by three approaches: (1) use of circular dichroism to ensure that the refolded had secondary structure consistent with a native scFv fold, (2) development of enzyme-linked immunosorbant assay and surface plasmon resonance (SPR) approaches for testing that the binding characteristics of an inhibitory peptide have been retained, and (3) proof of the subtle catalytic properties conservation through the development of a new sensitive catalytic assay using a fluorogenic substrate.

Enzymes are remarkable catalysts that lie at the heart of biology, accelerating chemical reactions to an astounding extent with extraordinary specificity. Enormous progress in understanding the chemical basis of enzymatic transformations and the basic mechanisms underlying rate enhancements over the past decades is apparent. Nevertheless, it has been difficult to achieve a quantitative understanding of how the underlying mechanisms account for the energetics of catalysis, because of the complexity of enzyme systems and the absence of underlying energetic additivity. We review case studies from our own work that illustrate the power of precisely defined and clearly articulated questions when dealing with such complex and multifaceted systems, and we also use this approach to evaluate our current ability to design enzymes. We close by highlighting a series of questions that help frame some of what remains to be understood, and we encourage the reader to define additional questions and directions that will deepen and broaden our understanding of enzymes and their catalysis.

By combining computational design and site-directed mutagenesis, we have engineered a new catalytic ability into the antibody scFv2F3 by installing a catalytic triad (Trp(29)-Sec(52)-Gln(72)). The resulting abzyme, Se-scFv2F3, exhibits a high glutathione peroxidase (GPx) activity, approaching the native enzyme activity. Activity assays and a systematic computational study were performed to investigate the effect of successive replacement of residues at positions 29, 52, and 72. The results revealed that an active site Ser(52)/Sec substitution is critical for the GPx activity of Se-scFv2F3. In addition, Phe(29)/Trp-Val(72)/Gln mutations enhance the reaction rate via functional cooperation with Sec(52). Molecular dynamics simulations showed that the designed catalytic triad is very stable and the conformational flexibility caused by Tyr(101) occurs mainly in the loop of complementarity determining region 3. The docking studies illustrated the importance of this loop that favors the conformational shift of Tyr(54), Asn(55), and Gly(56) to stabilize substrate binding. Molecular dynamics free energy and molecular mechanics-Poisson Boltzmann surface area calculations estimated the pK(a) shifts of the catalytic residue and the binding free energies of docked complexes, suggesting that dipole-dipole interactions among Trp(29)-Sec(52)-Gln(72) lead to the change of free energy that promotes the residual catalytic activity and the substrate-binding capacity. The calculated results agree well with the experimental data, which should help to clarify why Se-scFv2F3 exhibits high catalytic efficiency.

Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies.

The ultimate goal of catalytic antibody research is to develop new patient therapies that use the advantages offered by human catalytic antibodies. The establishment of a high-throughput method for obtaining valuable candidate catalytic antibodies must be accelerated to achieve this objective. In this study, based on our concept that we can find antibody light chains with a high probability of success if they include a serine protease-like catalytic triad composed of Ser, His, and Asp on a variable region of the antibody structure, we amplified and cloned DNAs encoding human antibody light chains from germline genes of subgroup II by seminested PCR using two primer sets designed for this purpose. Seven DNA fragments encoding light chains in 17 clones were derived from germline gene A18b, 6 DNA fragments from A3/A19, 2 DNA fragments from A17, and a clone DNA fragment from A5 and O11/O1. All light chains expressed in Escherichia coli and highly purified under nondenaturing conditions exhibited amidolytic activity against synthetic peptides. Some of the light chains exhibited unique features that suppressed the infectious activity of the rabies virus. Furthermore, the survival rate of mice in which a lethal level of the rabies virus was coinoculated directly into the brain with light chain 18 was significantly improved. In the case of humans, these results demonstrate that high-throughput selection of light chains possessing catalytic functions and specificity for a target molecule can be attained from a light-chain DNA library amplified from germline genes belonging to subgroup II.

Among the numerous questions remaining opened about catalytic antibodies (abzymes), the understanding of the origin of the genes encoding them is of vital significance. An original statistical analysis of genes encoding abzymes is described in the present report. Results suggested that these genes display a high conservation degree with their germline counterpart and a limited number of amino acid changes. Hence, on the contrary with high-affinity antibodies, maturation process by accumulation of somatic hypermutations is not required for the catalytic function. We demonstrated that despite a weak somatic mutation rate, the physicochemical properties of mutated amino acid (AA) are predominantly dissimilar with that of the germline AA. Further, we developed a novel approach in order to analyze the nature of genes encoding catalytic antibodies. For the first time, an unexpected and significant high level expression of rare gene subgroups was noticed and emphasized. The data described in this paper would lay the foundation for future studies about origin of genes encoding catalytic antibodies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Previously we established a series of catalytic antibodies (catAbs) capable of hydrolyzing DNA prepared by hybridoma technology. A group of these catAbs exhibited high reactivity and substrate specificity. To determine the molecular basis for these catAbs, we cloned, sequenced, and expressed the variable regions of this group of antibodies as functional F(ab) fragments. The nucleotide and deduced amino acid sequences of the expressed light chain (Vκ) germline gene assignments confidently belonged to germline family Vκ1A, gene bb1.1 and GenBank accession number EF672207 while heavy chain variable region V(H) genes belonged to V(H) 1/V(H) J558, gene V130.3 and GenBank accession number EF672221. A well-established expression system based on the pARA7 vector was examined for its ability to produce catalytically active antibodies. Recombinant F(ab) (rF(ab) ) fragments were purified and their hydrolyzing activity was analyzed against supercoiled pUC19 plasmid DNA (scDNA). The study of rF(ab) provides important information about the potential catalytic activities of antibodies whose structure allows us to understand their basis of catalysis. Molecular surface analysis and docking studies were performed on the molecular interactions between the antibodies and poly(dA9), poly(dG9), poly(dT9), and poly(dC9) oligomers. Surface analysis identified the important sequence motifs at the binding sites, and different effects exerted by arginine and tyrosine residues at different positions in the light and heavy chains. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies. These studies may define important features of DNA catAbs., (Copyright © 2011 John Wiley & Sons, Ltd.)

Expression of recombinant antibodies in mammalian cells is one of key problems in immunobiotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively done in yeast cells. We obtained expression strains of the methylotrophic beast Pichia pastoris producing single chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab) and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant Kd and the first order constant (k2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (Phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and single chain antibody A.17 expressed in CHO and E. coli cells respectively.

9G4H9, a catalytic antibody displaying β-lactamase-like activity, has been developed by the anti-idiotypic approach using β-lactamase as the first antigen. Thus 9G4H9 represents the 'internal image' of β-lactamase. We selected a cyclic peptide anchored to a bacteriophage M13 library using 9G4H9 as the target. Pep90 is a cyclic heptapeptide enclosed between two cysteine residues. We showed that Pep90 could inhibit both TEM-1 β-lactamase (K(i)  = 333 μm) and several penicillin-binding proteins (IC₅₀ values ranging from 6-62 μm). We determined that the tryptophan residue of Pep90 is of crucial importance for its inhibitory activity. Using Pep90 as a scaffold, we generated a new class of peptidomimetics that retained inhibitory activity towards TEM-1 β-lactamase., (© 2010 The Authors Journal compilation © 2010 FEBS.)

Glutathione peroxidase (GPX) is a well-known antioxidant selenoenzyme, which can catalyze the reduction of a variety of hydroperoxides and consequently protect cells and other biological tissues against oxidative damage. Many attempts have been made to mimic its function, and a human catalytic antibody Se-scFv-B3 with GPX activity has been prepared in our previous study. This time, a new clone 2D8 that bound specifically to the glutathione analog GSH-S-DNPBu was selected again by using the technology of phage display antibody library, and then scFv-2D8 was successfully expressed in soluble form and purified using Ni(2+)-immobilized metal affinity chromatography. After being converted into selenium-containing scFv by chemically modification, it showed higher GPX activity than previous abzyme Se-scFv-B3. The heavy chain variable fragment of scFv-2D8 was also prepared and converted into selenium-containing protein using the same method. This selenium-containing single-domain antibody showed some GPX activity and, to the best of our knowledge, is the first human single-domain abzyme with GPX activity, which lays a foundation for preparing GPX abzyme with human origin, lower molecular weight and higher activity.

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 V kappa 1A, bb1.1 and V kappa II, bd2, whose protease and DNase catalytic activities have been reported, while the heavy chain genes were expressed in several germline families (eight of V(H)1/J558, three of V(H)5/V(H)7183, and three of V(H)8/V(H)3609). 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., ((c) 2010 Elsevier Ltd. All rights reserved.)

There is great interest in the antibodies-to-DNA transformation, since this change is characteristic of autoimmune diseases and contributes to its pathology. After immunization and fusions, 14 hybridomas bearing DNA-hydrolysis activity against pUC19 plasmid DNA were obtained. Genes coding for V(H) and V(L) regions of the 14 monoclonal antibodies (mAbs) were cloned and sequenced. The sequences were compared with sequences of the Ig-Blast database to determine their germline and to identify potential mutations responsible for DNA binding and DNase activity. V genes of the H chains' genes expressed four genes of the V(H)1/J558 family, three of V(H)5/V(H)7183, and three of V(H)8/V(H)3609. The genetic repertoire of these mAbs was examined by determining the nucleotide sequences of their H chain V regions. This V(H) and V(L) domain was most similar to an anti-ssDNA (DNA-1) antibody as well as to catalytic autoimmune mAb (m3D8). Computer-generated models of the three-dimensional structures of V(H) and V(L) (VHL4) of the IgG4 combinations were used to define the positions occupied by the important sequence motifs at the binding sites. The modeling structure showed that VHL4 binds to oligo (dT3) primarily by sandwiching thymine bases between Tyr L32, Tyr L49 and Tyr H97 side-chains. Superposing VHL4 with anti-nucleic acid m3D8 catAbs revealed a common ssDNA recognition module consisting of His L93, His H35 residues which are critical for DNA-hydrolyzing antibodies. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies' hydrolyzing activities.

Data on catalytic antibodies (abzymes) having critical roles in pathologies, for example in some auto-immune diseases accumulate at overwhelming pace. Nevertheless, the misunderstanding of how antibodies can mimic a catalytic activity may hamper the development of therapeutic tools. We thus investigated the structure function roles of residues of a catalytic antibody endowed with a beta-lactamase activity. The catalytic antibody 9G4H9 was generated using the internal image properties of anti-idiotypic antibodies. The single-chain fragment was cloned and produced in Escherichia coli. Based on the structure function knowledge of beta-lactamases pattern and on the tridimensional model of the scFv, five residues were selected for mutagenic analysis to learn about the contribution of putative residues in catalysis. Light chain mutants R24A, S26A, S28A, and E98A lost catalytic activity significantly. Mutant K27A retained catalytic activity but the estimated K(M) was affected. Kinetic outcomes support the argument that S26 and S28 function as nucleophile and E98 as general acid/base catalyst. We have selected a peptide Pep90 able to inhibit 9G4H9 catalytic activity. We also demonstrate the tryptophan and proline residues of Pep90 (YHFLWGP) are responsible for binding and inhibitory properties of Pep90 on 9G4H9. A three-dimensional model docked with Pep90 is therefore built in which critical residues of Pep90 are buried at the interface of CDR-L1 and CDR-L3 loops whereas other are exposed to the solvent.

Antibodies with nucleophilic or catalytic properties often have these characteristics encoded in their germ line genes. Because hydrolytic activity has been reported to be associated with light chain V regions, we have begun an analysis of germ line light chain proteins that could be the basis for affinity maturation into hydrolytic or other reactive antibodies. We produced the germ line A18b light chain and characterized its hydrolytic, nucleophilic, and tertiary structural activities. This light chain was purified to >99% purity and found to hydrolyze aminomethylcoumarin-peptide and larger protein substrates and bind a fluorophosphonate probe. Mutation of putative catalytic residues only resulted in loss of activity of a tetrameric but not dimeric form of the light chain. These biochemical properties provide a framework for understanding the structure-function relationships of germ line antibodies.

The antigen recognition site of antibodies consists of the heavy and light chain variable domains (V(L) and V(H) domains). V(L) domains catalyze peptide bond hydrolysis independent of V(H) domains (Mei, S., Mody, B., Eklund, S. H., and Paul, S. (1991) J. Biol. Chem. 266, 15571-15574). V(H) domains bind antigens noncovalently independent of V(L) domains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544-546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathione S-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the V(H) domain of an anti-E2 IgG paired with promiscuously catalytic V(L) domains. The hybrid IgG hydrolyzed the E2 fusion proteins more rapidly than the unpaired light chain. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. The hybrid IgG displayed noncovalent E2 binding in enzyme-linked immunosorbent assay tests. Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located approximately 11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid IgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit.

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se-scFv-B3 (selenium-containing single-chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv-B3 was carried out. A three-dimensional (3D) structure of scFv-B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer-aided docking and energy minimization (EM) calculations of the antibody-GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni(2+)-immobilized metal affinity chromatography (IMAC), then transformed to selenium-containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se-scFv-B3-A180S was significantly increased compared to the original Se-scFv-B3., ((c) 2009 John Wiley & Sons, Ltd.)

In enzymes, multiple structural effects cooperatively lead to the high catalytic activity, while individually these effects can be small. The design of artificial enzymes requires the understanding and ability to manipulate such subtle effects. The 34E4 catalytic antibody, catalyzing Kemp elimination of 5-nitrobenzisoxazole, and its Glu50Asp (E50D) variant are the subject of the present investigation. This removal of only a methylene group yields an approximately 30-fold reduction in the rate for the catalyzed Kemp elimination. Here, the aim is to understand this difference in the catalytic performance. The mechanism of Kemp elimination catalyzed by 34E4 and the E50D mutant is elucidated using QM/MM Monte Carlo simulations and free energy perturbation theory. In both proteins, the reaction is shown to follow a single-step, concerted mechanism. In the mutant, the activation barrier rises by 2.4 kcal/mol, which corresponds to a 62-fold rate deceleration, which is in good agreement with the experimental data. The positions and functionality of the residues in the active site are monitored throughout the reaction. It is concluded that the looser contact with the base, shorter base-Asn58 contact, less favorable pi-stacking with Trp91 in the transition state of the reaction, and different solvation pattern all contribute to the reduction of the reaction rate in the E50D variant of 34E4.

Phage-displayed peptides that selectively bind to aldolase catalytic antibody 93F3 when bound to a particular 1,3-diketone hapten derivative have been developed using designed selection strategies with libraries containing 7-12 randomized amino acid residues. These phage-displayed peptides discriminated the particular 93F3-diketone complex from ligand-free 93F3 and from 93F3 bound to other 1,3-diketone hapten derivatives. By altering the selection procedures, phage-displayed peptides that bind to antibody 93F3 in the absence of 1,3-diketone hapten derivatives have also been developed. With using these phage-displayed peptides, ligand-bound states of the antibody were distinguished from each other. A docking model of one of the peptides bound to the antibody 93F3-diketone complex was created using a sequential divide-and-conquer peptide docking strategy; the model suggests that the peptide interacts with both the antibody and the ligand through a delicate hydrogen bonding network.

The competing reaction pathways and the corresponding free energy barriers for cocaine hydrolysis catalyzed by an anti-cocaine catalytic antibody, mAb15A10, were studied by using a novel computational strategy based on the binding free energy calculations on the antibody binding with cocaine and transition states. The calculated binding free energies were used to evaluate the free energy barrier shift from the cocaine hydrolysis in water to the antibody-catalyzed cocaine hydrolysis for each reaction pathway. The free energy barriers for the antibody-catalyzed cocaine hydrolysis were predicted to be the corresponding free energy barriers for the cocaine hydrolysis in water plus the calculated free energy barrier shifts. The calculated free energy barrier shift of -6.87 kcal/mol from the dominant reaction pathway of the cocaine benzoyl ester hydrolysis in water to the dominant reaction pathway of the antibody-catalyzed cocaine hydrolysis is in good agreement with the experimentally derived free energy barrier shift of -5.93 kcal/mol. The calculated mutation-caused shifts of the free energy barrier are also reasonably close to the available experimental activity data. The good agreement suggests that the protocol for calculating the free energy barrier shift from the cocaine hydrolysis in water to the antibody-catalyzed cocaine hydrolysis may be used in future rational design of possible high-activity mutants of the antibody as anti-cocaine therapeutics. The general strategy of the free energy barrier shift calculation may also be valuable in studying a variety of chemical reactions catalyzed by other antibodies or proteins through noncovalent bonding interactions with the substrates.

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.

The Diels-Alder reaction is one of the most important and versatile transformations available to organic chemists for the construction of complex natural products, therapeutics agents, and synthetic materials. Given the lack of efficient enzymes capable of catalyzing this kind of reaction, it is of interest to ask whether a biological catalyst could be designed from an antibody-combining site. In the present work, a theoretical study of the different behavior of a germline catalytic antibody (CA) and its matured form, 39 A-11, that catalyze a Diels-Alder reaction has been carried out. A free-energy perturbation technique based on a hybrid quantum-mechanics/molecular-mechanics scheme, together with internal energy minimizations, has allowed free-energy profiles to be obtained for both CAs. The profiles show a smaller barrier for the matured form, which is in agreement with the experimental observation. Free-energy profiles were obtained with this methodology, thereby avoiding the much more demanding two-dimensional calculations of the energy surfaces that are normally required to study this kind of reaction. Structural analysis and energy evaluations of substrate-protein interactions have been performed from averaged structures, which allows understanding of how the single mutations carried out during the maturation process can be responsible for the observed fourfold enhancement of the catalytic rate constant. The conclusion is that the mutation effect in this studied germline CA produces a complex indirect effect through coupled movements of the backbone of the protein and the substrate.

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.

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.

We performed a series of site-directed mutagenesis experiments of catalytic antibody, 6D9, which hydrolyzes a prodrug of chloramphenicol, based on our previous directed evolution study [Takahashi et al. (2001) Nat. Biotechnol. 19, 563-567]. Since we previously found that the variants with a mutation of Ser(L27e)Tyr afforded a one order of magnitude increase in catalytic rate, we created a site-directed mutant containing this mutation. The resulting mutant, 6D9-Ser(L27e)Tyr, had 6.5-fold higher k(cat)/k(uncat) and 9.8-fold higher k(cat)/K(m) than wild-type 6D9. We also created 6D9-Thr(L27a)Pro, since this mutation occurred frequently in the previous directed evolution, and it had 2.1-fold higher k(cat)/k(uncat) and k(cat)/K(m) than 6D9. Kinetic and computational analyses suggest that Tyr at L27e contributes to transition-state stabilization, while Pro at L27a does not interact with the transition-state structure directly, but obviously contributes to enhanced catalytic activity. Including double mutants that combined favourable substitutions, we created seven site-directed mutants. However, none of them had higher catalytic activities than some of highly improved variants obtained in the previous directed evolution. The present study gives direct evidence that not only a specific amino acid residue which obviously contributes to transition-state stabilization, but also a group of amino acid residues working in concert is important for efficient catalysis of a given transformation.

The single chain variable fragment (scFv) of an anti-idiotypic catalytic monoclonal antibody, 9G4H9, displaying a beta-lactamase-like activity was cloned. The recombinant protein was expressed through the periplasm in Escherichia coli in the presence or in the absence of FkpA, a chaperone-like enzyme and tested for its hydrolytic activity. The results show that the catalytic parameters for hydrolysis of ampicillin by scFv9G4H9 are clearly influenced by the presence of FkpA, indicating that the correct folding of the fragment represents a crucial step for catalysis.

The HpU-2 monoclonal antibody (mAb) raised against Helicobacter pylori urease mainly recognized the alpha-subunit of the urease. On the other hand, the heavy chain of HpU-2 mAb (HpU-2-H) isolated from the parent mAb recognized both the alpha- and beta-subunit, in which the beta-subunit was recognized more strongly than the beta-subunit. HpU-2-H cleaved a peptide, SVELIDIGGNRRIFGFNALVD, which is the epitope sequence recognized by HpU-2 mAb, showing a double-phase reaction profile at 25 degrees C in a phosphate buffer. After an induction time of 24h, the cleavage of the peptide was initiated by HpU-2-H at a high rate and it was completed at 80 h of incubation. By mass spectroscopy, two main fragmented peptides, SVELIDIGGNRR and SVELIDIGGNRRIFG, were identified. In addition, many small peptide fragments were produced by successive cleavage of the fragmented peptides. Cleavage tests for H. pylori urease by HpU-2-H revealed that the beta-subunit of the urease was cleaved first and completely decomposed at 20 h of incubation. Cleavage of the alpha-subunit started after the complete decomposition of the beta-subunit. These cleavage results were in good agreement with the immunological features of HpU-2-H. The irrelevant proteins, BSA and HSA, were hardly cleaved by HpU-2-H.

Aim: To compare clinicopathogenetic value of DNA-hydrolizing autoantibodies or DNA-abzymes in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA)., Material and Methods: We studied sera from 180 patients with SLE, 180 RA patients and 128 healthy donors matched by age and gender; assessed catalytic and cytotoxic activity of DNA-abzymes in patients with different variants of SLE and RA course., Results: The highest catalytic and cytotoxic activities of DNA abzymes were observed in SLE patients. In SLE catalytic and cytotoxic activities of DNA abzymes ranged widely and their mean values depended on SLE activity in patients with systemic lesions. DNA-abzymes in RA patients showed lower catalytic and cytotoxic activities in relation to substrate DNA and target cells than in SLE. DNA-abzymes occurred most frequently in patients with high activity of RA, slow-progressive and lingering course of RA, especially in early development of visceral (extra-articular) pathology. Characteristic for DNA-abzymes in RA and SLE is the phenomenon of wide-range fluctuations due to factors determinating probability of induction of function of Ab-mediated catalysis and, therefore, incidence rates of DNA-abzymes, probably catalytic autoAb of the other specificity in a population of patients with systemic autoimmune diseases., Conclusion: The data indicate the validity of DNA abzymes use in clinical practice for realization of diagnostic and therapeutic programs in SLE and RA.

It has been suggested that complementary proteins are involved in autoimmunity through a network involving idiotype-anti-idiotype reactions termed 'autoantigen complementarity'. We propose that complementary proteins, which occur naturally or result from cellular dysfunction, might be more common than recognized currently. This implies that the role of complementary proteins in autoimmunity merits increasing investigation. The concept of complementary proteins is reviewed here and, also, new ideas are presented that underscore the role of open-reading frames in frame -1 of recognized genes in the production of complementary proteins (frame -1 is the reverse complement sequence of a gene that uses the antisense of the codons of frame +1). Furthermore, a novel role for palindromic sequences in autoimmunity and a new model explaining how abzymes and autoantigen complementarity might be related are proposed.

Antibody 34E4 catalyzes the conversion of benzisoxazoles to salicylonitriles with high rates and multiple turnovers. The crystal structure of its complex with the benzimidazolium hapten at 2.5-angstroms resolution shows that a combination of hydrogen bonding, pi stacking, and van der Waals interactions is exploited to position both the base, Glu(H50), and the substrate for efficient proton transfer. Suboptimal placement of the catalytic carboxylate, as observed in the 2.8-angstroms structure of the Glu(H50)Asp variant, results in substantially reduced catalytic efficiency. In addition to imposing high positional order on the transition state, the antibody pocket provides a highly structured microenvironment for the reaction in which the carboxylate base is activated through partial desolvation, and the highly polarizable transition state is stabilized by dispersion interactions with the aromatic residue Trp(L91) and solvation of the leaving group oxygen by external water. The enzyme-like efficiency of general base catalysis in this system directly reflects the original hapten design, in which a charged guanidinium moiety was strategically used to elicit an accurately positioned functional group in an appropriate reaction environment and suggests that even larger catalytic effects may be achievable by extending this approach to the induction of acid-base pairs capable of bifunctional catalysis.

Antibody 16E7 catalyzes the carbon protonation of enol ether 2 to hemiacetal 3, and the carbon deprotonation of benzisoxazole 7 to phenol 8. This antibody shows an extreme case of hysteresis, requiring several hours to reach full activity. Antibody 16E7 was expressed as recombinant chimeric Fab in Escherichia coli. A model for the three-dimensional structure was produced by homology modeling and used for a docking procedure to obtain models for antibody-ligand complexes. Site-direct mutagenesis of GluL39, identified as a possible catalytic residue by the model, to either glutamine or alanine abolished catalysis, showing that both the protonation reaction of enol ether 2 and the deprotonation of benzisoxazole 7 are promoted by the same residue. The model furthermore suggested that substrate access to the catalytic site might be hindered by a flexible HCDR3 loop held in closed position by a hydrogen bond between SerH99 and GluL39, which could explain the observed hysteresis effect. In agreement with this model, mutagenesis of SerH99 to alanine, or deletion of this residue, was found to reduce hysteresis by approximately 50%.

Catalytic antibody 34E4 accelerates the conversion of benzisoxazoles to salicylonitriles with surprising efficiency, exploiting a carboxylate base with an elevated pKa for proton abstraction. Mutagenesis of this antibody, produced as a chimeric Fab, confirms the prediction of a homology model that GluH50 is the essential catalytic residue. Replacement of this residue by glutamine, alanine, or glycine reduces catalytic activity by more than 2.6 x 10(4)-fold. By comparing the chemical proficiencies of the parent antibody with the chemical proficiencies of acetate and the mutants, the effective concentration of the catalytic side chain was estimated to be >51 000 M. The 2.1 kcal/mol destabilization of the transition state observed when GluH50 is replaced by aspartate suggests that positional ordering imposed by the antibody active site contributes significantly to the efficiency of proton transfer. The observation that the GluH50Ala and GluH50Gly variants could not be chemically rescued by exogenous addition of high concentrations of formate or acetate further underscores the advantage the antibody derives from covalently fixing its base at the active site. Although medium effects also play an important role in 34E4, for example in enhancing the reactivity of the carboxylate side chain through desolvation, comparison of 34E4 with less proficient antibodies shows that positioning a carboxylate in a hydrophobic binding pocket alone is insufficient for efficient general base catalysis. Our results demonstrate that structural complementarity between the antibody and its substrate in the transition state is an important and necessary component of 34E4's high activity. By harnessing an additional catalytic group that could serve as a general acid to stabilize developing negative charge in the leaving group, overall efficiencies rivaling those of highly evolved enzymes should be accessible.

The catalytic antibody, 6D9, was subjected to directed evolution in the phage-display system using two structurally related transition-state analogs (TSAs) for panning. One analog, TSA 3, was originally used for immunization, and the other, TSA 4, a derivative of TSA 3, was designed to optimize the differential affinity for the transition state relative to the ground state so as to provide variants with improved reaction rates. We previously reported that by panning with TSA 4, we could obtain variants with highly improved catalytic rate enhancement (k(cat)/k(uncat)), and Tyr (L27e) seemed to play a key role in stabilizing the transition-state structure [Nat. Biotechnol. 19 (2001) 563]. Here, we examined in detail a large number of the variants selected by these haptens, in order to elucidate the mechanism of the directed evolution driven by them. ELISA with 3- and 4-bovine serum albumin (BSA) showed that variants selected by these TSAs exhibited distinct binding patterns. All the variants whose rate enhancement was greater than five-fold of that of 6D9 had Tyr (L27e) and were obtained from the library panned with TSA 4, but not from the library panned with TSA 3. Kinetic studies showed that TSA 4 could efficiently select variants with increased differential binding affinity for the transition state relative to the ground state, and these variants exhibited improved rate enhancements. This study verified the difference of in vitro evolution driven by the two structurally related TSAs and stresses the importance of designing an appropriate hapten for panning.

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.

Antibody 15A9 is unique in its ability to catalyze the transamination reaction of hydrophobic D-amino acids with pyridoxal-5'-phosphate (PLP). Both previous chemical modification studies and a three dimensional (3-D) homology model indicated the presence of functionally important tyrosine residues in the antigen-binding cavity of antibody 15A9. To gain further insight into the hapten, ligand binding, and catalytic mechanism of 15A9, all tyrosine residues in the complementarity-determining regions (CDRs) and the single arginine residue in CDR3 of the light chain were subject to an alanine scan. Substitution of Tyr(H33), Tyr(L94), or Arg(L91) abolished the catalytic activity and reduced the affinity for PLP and N(a)-(5'-phosphopyridoxyl)-amino acids, which are close analogues of covalent PLP-substrate adducts. The Tyr(H100b)Ala mutant possessed no detectable catalytic activity, while its affinity for each ligand was essentially the same as that of the wild-type antibody. The binding affinity for the hapten was drastically reduced by a Tyr(L32)Ala mutation, suggesting that the hydroxyphenyl group of Tyr(L32) participates in the binding of the extended side chain of the hapten. The other Tyr --> Ala substitutions affected both binding and catalytic activity only to a minor degree. On the basis of the information obtained from the mutagenesis study, we docked N(alpha)-(5'-phosphopyridoxyl)-D-alanine into the antigen-binding site. According to this model, Arg(L91) binds the alpha-carboxylate group of the amino acid substrate and Tyr(H100b) plays an essential role in the catalytic mechanism of antibody 15A9 by facilitating the Calpha/C4' prototropic shift. In addition, the catalytic apparatus of antibody 15A9 revealed several mechanistic features that overlap with those of PLP-dependent enzymes.