Binding Specificity of Hydrolytic Catalytic Antibody: Lessons from the Diverse Immune System

The diverse immune system has the potential to provide numerous antibodies that catalyze a great variety of reactions and exhibit diversity in mechanism and substrate specificity. However, to find such an antibody is not practical, unless we understand the immune response precisely and utilize it. We applied an alternative screening methodology based on the binding specificity of antibodies for the structure of the transition-state analog p-nitrophenyl phosphonate in order to study the built-in diversity of the immune response. This study uncovered the unpredictable binding and substrate specificity of catalytic antibodies derived from the diverse immune response.     

Augmenting the efficacy of anti-cocaine catalytic antibodies through chimeric hapten design and combinatorial vaccination

Given the need for further improvements in anti-cocaine vaccination strategies, a chimeric hapten (GNET) was developed that combines chemically-stable structural features from steady-state haptens with the hydrolytic functionality present in transition-state mimetic haptens. Additionally, as a further investigation into the generation of an improved bifunctional antibody pool, sequential vaccination with steady-state and transition-state mimetic haptens was undertaken. While GNET induced the formation of catalytically-active antibodies, it did not improve overall behavioral efficacy. In contrast, the resulting pool of antibodies from GNE/GNT co-administration demonstrated intermediate efficacy as compared to antibodies developed from either hapten alone. Overall, improved antibody catalytic efficiency appears necessary to achieve the synergistic benefits of combining cocaine hydrolysis with peripheral sequestration.     

Catalytic antibodies: A critical assessment

In the past few years, antibodies that catalyze a variety of reactions with enzyme-like properties have been produced. The present review is of a critical nature, rather than a survey or an introduction to the field of catalytic antibodies. Here, we examine the performance of catalytic antibodies in light of the features that define an enzyme: substrate specificity, rate enhancement, and turnover. We also refer to some limitations of the technologies currently used for their generation. In the future, antibodies may provide a new repertoire of tailor-made, enzyme-like, catalysts with possible applications in biology, medicine, and biotechnology. In the following sections, we emphasize that these applications will require far more efficient catalysts than are presently available, and we point to several trends for future research that may offer more efficient catalytic antibodies.     

A focused antibody library for improved hapten recognition

The topography of the antigen-binding site as well as the number and the positioning of the antigen contact residues are strongly correlated with the size of the antigen with which the antibody interacts. On the basis of these considerations, we have designed a focused scFv repertoire biased for haptens, designated the cavity library. The hapten-specific scFv, FITC8, was used as a scaffold for library construction. FITC8, like other hapten binders, displays a characteristic cavity in its paratope into which the hapten binds. In five of the six complementarity-determining regions, diversity-carrying residues were selected rationally on the basis of a model structure of FITC8 and on known antibody structure-function relationships, resulting in variation of 11 centrally located, cavity-lining residues. L3 was allowed to carry a more complex type of diversity. In addition, length variation was introduced into H2, as longer versions of this loop have been shown to correlate with increased hapten binding. The library was screened, using phage display, against a panel of five different haptens, yielding diverse and highly specific binders to four of the antigens. Parallel selections were performed with a library having diversity spread onto a greater area, including more peripherally located residues. This resulted in the isolation of binders, which, in contrast to the clones selected from the cavity library, were not able to bind to the soluble hapten in the absence of the carrier protein. Thus, we have shown that by focusing diversity to the hotspots of interaction a library with improved hapten-binding ability can be created. The study supports the notion that it is possible to create antibody libraries that are biased for the recognition of antigens of pre-defined size.     

Catalytic antibodies: balancing between Dr. Jekyll and Mr. Hyde

The immunoglobulin molecule is a perfect template for the de novo generation of biocatalytic functions. Catalytic antibodies, or abzymes, obtained by the structural mimicking of enzyme active sites have been shown to catalyze numerous chemical reactions. Natural enzyme analogs for some of these reactions have not yet been found or possibly do not exist at all. Nowadays, the dramatic breakthrough in antibody engineering and expression technologies has promoted a considerable expansion of immunoglobulin's medical applications and is offering abzymes a unique chance to become a promising source of high‐precision “catalytic vaccines.” At the same time, the discovery of natural abzymes on the background of autoimmune disease revealed their beneficial and pathogenic roles in the disease progression. Thus, the conflicting Dr. Jekyll and Mr. Hyde protective and destructive essences of catalytic antibodies should be carefully considered in the development of therapeutic abzyme applications.     

Design of pyridoxal 5'-phosphate dependent catalytic antibodies

Modern approaches for developing antibodies with coenzyme-dependent activities are discussed for pyridoxal 5"-phosphate dependent transformation of amino acid as an example. A new type of antigens analogous to enzyme–substrate compounds is suggested for the production of such antibodies. Approaches for the development of pyridoxal antiidiotypic antibody using analogs of coenzyme–substrate compounds and corresponding apoenzyme complexes are reviewed.     

Prevention and suppression of Helicobacter felis infection in mice using colostral preparation with specific antibodies

Background. Specific antibodies against Helicobacter were enriched from the colostra of hyperimmunized cows. Efficacies of colostral control preparation and immune preparation containing specific antibodies against Helicobacter felis were studied in the prevention and treatment of experimental H. felis infection in mice. Materials and Methods. H. felis‐infected mice were given either immune or control preparation with or without complement or amoxicillin orally in four different trials. H. felis status was assessed on the basis of bacterial stainings, gastric histology and serum antibodies. Results. Immune, but not control preparation, prevented H. felis infection (p > 0.01), the efficacy being dependent on the presence of specific antibodies. In the trial on infected Balb/c mice treatment with immune preparation (p = 0.029) but not control preparation decreased the colonization of gastric antrum by H. felis. In the further trials with infected SJL‐mice, treatments with colostral preparations did not decrease colonization. Amoxicillin treatment decreased the colonization with trend‐setting significance (p = 0.056; infected mice as controls), whereas amoxicillin combined with immune preparation had a significant effect (p < 0.0005). Conclusions. Specific colostral antibodies were useful in the prevention of Helicobacter infection in a mouse model. The results of the treatment trials were controversial but a similar colostral immune preparation against H. pylori could be effective and useful in preventing infections in humans and during antibiotic treatment.     

Generation of humanized anti-DNA hydrolyzing catalytic antibodies by complementarity determining region grafting

Humanization of nonhuman antibodies (Abs) has been carried out mainly for Abs which bind to antigen without catalytic activity. Here we report humanization of mouse-originated 3D8 (m3D8) mAbs (scFv, VH, and VL) with DNA hydrolyzing catalytic activity by grafting the complementarity determining regions (CDRs) into the corresponding regions of a fixed human framework scaffold, generating humanized 3D8 (h3D8) Abs in the respective format of scFv, VH, and VL. h3D8 Abs retained comparable DNA binding and hydrolyzing activities to those of the corresponding m3D8 Abs. Our results suggest that CDRs of anti-DNA hydrolyzing Abs might possess the intrinsic properties of DNA binding and hydrolyzing activities.     

Naturally occurring catalytic antibodies: evidence for preferred development of the catalytic function in IgA class antibodies

IgG class antibodies express catalytic activities rarely and at very low levels. Here, we studied polyclonal IgA and IgG preparations from healthy human sera and saliva for the ability to hydrolyze model peptidyl-aminomethylcoumarin (peptide-AMC) substrates. These substrates permit objective evaluation of the catalytic potential of the antibody classes with minimal effects of noncovalent interactions occurring at sites remote from the reaction center. The IgA preparations hydrolyzed Glu-Ala-Arg-AMC at rates 3-orders of magnitude greater than IgG preparations from the same individuals. The cleavage occurred preferentially on the C terminal side of a basic residue. The activity was confirmed using monoclonal IgAs isolated from patients with multiple myeloma. Active site-directed inhibitors of serine proteases inhibited the catalytic activity and were bound irreversibly by the IgA, suggesting the involvement of a serine protease-like mechanism similar to that utilized by previously described IgM antibodies. These observations suggest that mechanisms underlying B cell clonal selection favor the retention and improvement of catalytic activity in the IgA, but not the IgG compartment of the immune response.     

An Improved Rapid Method for Selecting Monoclonal Antibodies with High Catalytic Activities

Monoclonal antibodies were raised against a -naphthyl phosphonate hapten (1) to elicit antibodies capable of catalyzing the hydrolysis of -naphthyl acetate (3). After cell fusion, potential catalytic antibody-producing hybridomas were selected, by use of a competitive inhibition assay on the basis of the binding activity for a short transition-state analogue (inhibitor 5), followed by use of high-performance liquid chromatography analysis for the hybridoma supernatants to screen the antibodies processing catalytic activities. It was shown that supernatants of 12 wells had high binding activity with inhibitor and of them, 7 had catalytic activities. After cloning by limiting dilution, we got two hybridoma clones producing monoclonal antibodies which catalyzed the hydrolysis of -naphthyl acetate. This combination of competitive inhibition assay with high-performance liquid chromatography analysis represents an improved rapid approach for the screening of potential catalytic antibodies and significantly increases the possibility of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (K _cat/K _uncat 10⁶) and specificity.     

Enhanced immunization techniques to obtain highly specific monoclonal antibodies

Despite fast advances in genomics and proteomics, monoclonal antibodies (mAbs) are still a valuable tool for areas such as the evolution of basic research in stem cells and cancer, for immunophenotyping cell populations, diagnosing and prognosis of diseases, and for immunotherapy. To summarize different subtractive immunization approaches successfully used for the production of highly specific antibodies, we identified scientific articles in NCBI PubMed using the following search terms: subtractive immunization, monoclonal antibody, tolerization, neonatal, high-zone tolerance, masking immunization. Patent records were also consulted. From the list of results, we included all available reports, from 1985 to present, that used any enhanced immunization technique to produce either polyclonal or monoclonal antibodies. Our examination yielded direct evidence that these enhanced immunization techniques are efficient in obtaining specific antibodies to rare epitopes, with different applications, such as to identify food contaminants or tumor cells.     

Functional characterization of two anti-estradiol antibodies as deduced from modelling and site-directed mutagenesis experiments

Monoclonal antibodies are now widely used to measure the concentration of steroid hormones in human serum samples. The great development of molecular engineering techniques over the past 10 years has made possible the improvement of specificity and/or sensitivity of selected antibodies. We have obtained two monoclonal antibodies, 17E12E5 and 10G6D6, using estradiol-6-ethyl methoxy carbonyl (EMC)-bovine serum albumin (BSA) as immunogen. To tentatively improve their affinities for natural estradiol, we have initiated their structural and functional studies. For this purpose, we have cloned and sequenced the genes encoding the variable fragments of each antibody. Single chain variable fragments (scFv) were produced into the periplasmic space of E. coli using the pLIP6 expression vector. Mapping of the functional structures of both antibodies was obtained by combination of modelling and mutational analyses together with cross-reaction studies. The two binding pockets are described and models of estradiol complexed to 17E12E5 and 10G6D6 are proposed.     

Analysis of the binding loops configuration and surface adaptation of different crystallized single‐domain antibodies in response to various antigens

Monoclonal antibodies have revolutionized the biomedical field through their ubiquitous utilization in different diagnostics and therapeutic applications. Despite this widespread use, their large size and structural complexity have limited their versatility in specific applications. The antibody variable region that is responsible for binding antigen is embodied within domains that can be rescued individually as single‐domain antibody (sdAb) fragments. Because of the unique characteristics of sdAbs, such as low molecular weight, high physicochemical stability, and the ability to bind antigens inaccessible to conventional antibodies, they represent a viable alternative to full‐length antibodies. Consequently, 149 crystal structures of sdAbs, originating from human (VH), camelids (VHH), or sharks (VNAR), were retrieved from the Protein Data Bank, and their structures were compared. The 3 types of sdAbs displayed complementarity determining regions (CDRs) with different lengths and configurations. CDR3 of the VHH and VNAR domains were dominated by pleated and extended orientations, respectively. Although VNAR showed the smallest average molecular weight and molecular surface area compared with VHH and VH antibodies. However, the solvent accessible surface area measurements of the 3 tested sdAbs types were very similar. All the antihapten VHH antibodies showed pleated CDR3, which were sufficient to create a binding pocket to accommodate haptens (methotrexate and azo dyes) in terms of shape and electrostatic potential. The sdAbs that recognized lysozyme showed more diversity in their CDR3 orientation to enable them to recognize various topographies of lysozyme. Subsequently, the three sdAb classes were different in size and surface area and have shown distinguishable ability to optimize their CDR length and orientation to recognize different antigen classes.     

Identification and characterization of single chain anti-cocaine catalytic antibodies

Cocaine is a powerful and addictive stimulant whose abuse remains a prevalent health and societal crisis. Unfortunately, no pharmacological therapies exist and therefore alternative protein-based therapies have been examined. One such approach is immunopharmacotherapy, wherein antibodies are utilized to either bind or hydrolyze cocaine thereby blocking it from exerting its euphoric effect. Towards this end, antibodies capable of binding and hydrolyzing cocaine were identified by phage display from a biased single chain antibody library generated from the spleens of mice previously immunized with a cocaine phosphonate transition state analog hapten. Two classes of antibodies emerged based on sequence homology and mode of action. Alanine scanning mutagenesis and kinetic analysis revealed that residues H97, H99, and L96 are crucial for antibodies 3F5 and 3H9 to accelerate the hydrolysis of cocaine. Antibodies 3F1 through 3F4, which are similar to our previously identified 3A6 class of antibodies, catalyze hydrolysis through transition state stabilization by tyrosine or histidine residues H50 and L94. Mutation of either one or both tyrosine residues to histidine conferred hydrolytic activity on previously inactive antibody 3F4. Mutational analysis of residue H50 of antibody 3F3 resulted in a glutamine mutant with a rate enhancement three times greater than wild-type. A double mutant, containing glutamineH50 and lysineH52, showed a tenfold rate enhancement over wild-type. These results indicate the power of initial selection of catalytic antibodies from a biased antibody library in both rapid generation and screening of mutants for improved catalysis.     

An improved and robust DNA immunization method to develop antibodies against extra-cellular loops of multi-transmembrane proteins

Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.     

An improved and robust DNA immunization method to develop antibodies against extra-cellular loops of multi-transmembrane proteins

Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.     

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes

Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.     

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes

Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.     

Studies on propylamine transfer activity in anti-AdoDATO antibodies

Summary Based on a structural similarity to the transition state of a propylamine transfer reaction involved in polyamine biosynthesis, Sadenosyl-(5)-1,8-diamino-3-thiooctane (AdoDATO), the most potent inhibitor of spermidine synthases, was used as a hapten for mice immunization. From immunized mice sera, the IgG fractions were purified by means of affinity (protein A/G) chromatography. Sera and purified polyclonal antibodies from several mice were found to exert spermidine synthase-like activity. Moreover, by means means of hybridoma technology, 19 anti-AdoDATO hybridoma clones have been screened for propylamine transfer activity and at least 6 were found to produce catalytic antibodies. These findings indicate the presence in the sera of active spermidine synthase-like catalytic antibodies. The reported results for the first time evidence the feasibility of preparation of N-alkylating antibodies, widening the biotechnological perspectives of antibodies as biocatalysts.Abbreviations AdoDATO S-adenosyl-(5)-1,8-diaminothiooctane - TSA Transition state analog - decAdoMet S-adenosyl-(5)-3-methylthiopropylamine (decarboxylated adenosylmethionine) - KLH keyhole limpet hemocyanin - NMR nuclear magnetic resonance - BSA bovine serum albumine - WSC 1-ethyl-3-(dimethylaminopropyl)carbodiimide (water-soluble carbodiimide) - PBS phosphate buffer saline - OPD orthodiphenylenediamine - TCA trichloroacetic acid - SDS-PAGE sodium duodecylsulphate-polyacrylamide gel electrophoresis - SN2 bimolecular nucleophilic substitution; abzyme catalytic antibody - IgG immunoglobulin G     

In Vivo Evolution of a β-lactamase-like Activity Throughout the Idiotypic Pathway

Abstract

An abzyme (9G4H9) displaying a β-lactamase activity was obtained through the idiotypic pathway and has been previously described. Analysis of the catalytic mechanism by kinetic measurements with various substrates, chemical modifications, and three dimensional modeling, led us to conclude that IgG 9G4H9 displays an activity at the crossroads of β-lactamases and penicillin-binding proteins (PBPs). Penicillin-binding proteins and βlactamases are two closely related enzymes arising from a common ancestral gene. We herein propose that the idiotypic network has allowed the generation of a catalyst with unique catalytic properties, but that could behave as an intermediate between both enzymes.