Ivan I. Vorobiev

Catalytic antibodies in clinical and experimental pathology: human and mouse models

Most of the data accumulated through studies on natural catalytic autoantibodies indicate that production scales up markedly in pathological abnormalities. We have previously described an increased level of DNA-hydrolyzing autoantibodies in the sera of patients with various autoimmune disorders [systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma], HIV infection and lymphoproliferative diseases accompanied by autoimmune manifestations. In the present study, we show that an increased level of catalytic activity of autoantibodies can be observed in the sera of autoimmune mice, thus providing a fundamental insight into the medical relevance of abzymes. Polyclonal autoantibodies purified from sera of NZB/W, MRL-lpr/lpr and SJL/J mice show proteolytic and DNA-hydrolyzing activities, as opposed to those harvested from non-autoimmune BALB/c mice. The expressiveness of the catalytic activity was strongly dependent on the age of the animal. The highest levels of catalytic activity were found in the sera of mice aged between 8 and 12 months; the lowest level was typical of younger animals whose age ranged from 6 to 8 weeks. Specific inhibition assays of the catalytic activities were performed to throw light on the nature of the abzyme activity. Within a cohort of aging animals, a strong correlation between marked autoimmune abnormalities and levels of catalytic activities has been established. Nonimmunized SJL/J mice revealed specific immune responses to myelin basic protein (MBP), skeletal muscle myosin (skMyo) and cardiac myosin (Myo), and highly purified antibodies from their serum show specific proteolytic attack against the target antigens. This finding prompted us to undertake a more detailed study of specific antibody-mediated proteolysis in diseased humans. A targeted catalytic response was originally demonstrated against MBP and Myo in multiple sclerosis and myocarditis patients, respectively.

Chemical polysialylation: Design of conjugated human oxyntomodulin with a prolonged anorexic effect in vivo

Recombinant gut hormone oxyntomodulin (OXM) is known to act as a satiety signal in human subjects and has therapeutic potential as an appetite controlling agent. The only form of this hormone that has a prospective use is a modified one, because native OXM has a very short half-life in vivo. Conjugation of OXM and the natural hydrophilic polymer polysialic acid (PSA) may significantly improve its half-life. Chemical polysialylation in vitro was used to create a long-acting form of OXM, the polysialic acid-oxyntomodulin (PSA-OXM) conjugate. The conjugation site was identified using mass shift comparative analysis of Asp-N proteolytic digests. The anorexic effect of the conjugate was tested on the lean, fasted mouse model. A two-stage purification technique was developed to obtain a homogeneous PSA-OXM conjugate, suitable for in vivo testing. The N-terminal backbone primary amino group was found to be the only point of conjugation. The conjugate obtained was resistant to the DPP-IV protease. A single injection of PSA-OXM at 15 μmol/kg dose was sufficient to maintain a steady decrease in food consumption for 8 h (P < 0.05). The length of the anorexic effect achieved is comparable to other long-acting derivatives of OXM but it requires a much higher dose for administration. It is expected that site-directed attachment of the PSA chain to the inner residues of OXM, away from the site of interaction with receptors, would produce a compound with a higher specific activity but comparable stability in the bloodstream. The conjugation technique used may be used to create OXM derivatives and other related hormones to obtain long-lasting variants, with improved suitability for clinical use.

Antibody Proteases: Induction of Catalytic Response

Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP89-104) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an “internal image” of protease is described. The “second order” monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.

New Infestin-4 Mutants with Increased Selectivity against Factor XIIa.

Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in ‘global’ assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its K i for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5–20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics.

Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells.

BackgroundEstablishing highly productive clonal cell lines with constant productivity over 2–3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure.ResultsWe have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative.ConclusionsThe p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1. The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

Chemical Polysialylation of Recombinant Human Proteins

Design of drug with prolonged therapeutic action is one of the rapid developing fields of modern medical science and required implementation of different methods of protein chemistry and molecular biology. There are several therapeutic proteins needing increasing of their stability, pharmacokinetic, and pharmacodynamics parameters. To make long-live DNA-encoded drug PEGylation was proposed. Alternatively polysialic (colominic) acid, extracted from the cell wall of E. coli, fractionated to the desired size by anion-exchange chromatography and chemically activated to the amine-reactive aldehyde form, may be chemically attached to the polypeptide chain. Conjugates of proteins and polysialic acid generally resemble properties of protein-PEG conjugates, but possess significant negative net charge and are thought to be fully degradable after endocytosis due to the presence of intracellular enzymes, hydrolyzing the polysialic acid. Complete biodegradation of the polysialic acid moiety makes this kind of conjugates preferable for creation of drugs, intended for chronic use. Here, we describe two different protocols of chemical polysialylation. First protocol was employed for the CHO-derived human butyrylcholinesterase with optimized for recovery of specific enzyme activity. Polysialic acid moieties are attached at various lysine residues. Another protocol was developed for high-yield conjugation of human insulin; major conjugation point is the N-terminal residue of the insulins light chain. These methods may allow to produce polysialylated conjugates of various proteins or polypeptides with reasonable yield and without significant loss of functional activity.

Natural antibody catalytic activities in mice with autoimmune disorders.

Catalytic antibodies (abzymes) are a new type of nonenzymatic biocatalysts. The mechanism of their formation and possible role in immune disorders are now attracting researchers’ attention. A variety of catalytic antibodies with different activities have been revealed to date; however, the purpose-oriented search of and construction of efficient abzymes with certain specificities still remains a problem. There is evidence for spontaneous catalytic-antibody formation in the human body. This has often been observed in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma, etc. [1–3]. Inbred lines of mice with autoimmune diseases serve as a model to study various aspects of autoimmunity in humans. MRL-lpr/lpr mice develop autoimmune disorders with age; these are similar to those observed in patients with SLE and rheumatoid arthritis [4]. The F 1 hybrids of New Zealand NZB/NZW mice spontaneously develop nephritis resembling that in patients with SLE [5]. In contrast, SJL/J mice are characterized by multiple autoimmune abnormalities, including experimental allergic encephalomyelitis, which is a model of human multiple sclerosis [6]. Of interest is a natural spectrum of catalytic antibodies in these animals. In this work, the catalytic activities of natural antibodies in MRL-lpr/lpr, SJL/J, and NZB/NZW F 1 mice were compared for the first time with those in BALB/c mice. The animals were kept under sterile conditions to minimize the interaction of their immune systems with foreign antigens (the Specific Pathogen-Free status). Polyclonal IgG antibodies were isolated from mouse blood serum using a previously developed chromatographic technique, precluding the contamination of the final preparation with serum enzymes [7]. The antibodies’ capacity to hydrolyze DNA, proteins, and esters was assessed because abzymes were previously shown to catalyze the hydrolysis of these substances [1, 8, 9]. To determine the DNA-hydrolyzing activity of antibodies, their effect on supercoiled plasmid DNA was estimated as described previously [7]. Serum antibodies of MRL-lpr/lpr mice exhibited the highest DNAhydrolyzing activity; they transformed supercoiled DNA not only into the relaxed circular form, but also into the linear form (Fig. 1). Both SJL/J and NZB/NZW F 1 mouse antibodies incubated with supercoiled DNA induced only single-strand nicks in some DNA molecules, converting supercoiled molecules into circular ones. Antibodies isolated from BALB/c mice exhibiting no autoimmunity were almost unable to hydrolyze DNA. The kinetic parameters of plasmid DNA hydrolysis by antibodies isolated from MRL-lpr/lpr mice were studied using the linear dichroism method as described in [10]; the efficiency of catalysis ( k cat / k M ) was 0.12 nM –1 min –1 , which is comparable with data previously obtained on the polyclonal antibodies isolated from the blood of an SLE patient ( k cat / k M = 0.32 nM –1 min –1 ) [10]. Note that, although the autoimmune processes are similar in the MRL-lpr/lpr and NZB/NZW F 1 mice, which have high titers of DNA-binding antibodies in the total pool of immunoglobulins [11], only the antibodies of MRL-lpr/lpr mice exhibit a high DNA-hydrolyzing activity. Proteolytic activity in antibody preparations was measured using both fluorescence [12] and enzymatic methods. The fluorescence method is based on the previously described phenomenon of quenching the fluorescence of a protein overlabeled with fluorophore, which enhanced the fluorescence after the initiation of nicks in a polypeptide chain. Bovine serum albumin overlabeled with fluoresceine isothiocyanate (BSA-FITC) served as a substrate for proteolysis in this test. The activity of antibodies was assayed by a time-dependent increase in fluorescence compared to antibody-free Natural Antibody Catalytic Activities in Mice with Autoimmune Disorders

A modular assembly cloning technique (aided by the BIOF software tool) for seamless and error-free assembly of long DNA fragments

BackgroundMolecular cloning of DNA fragments >5 kbp is still a complex task. When no genomic DNA library is available for the species of interest, and direct PCR amplification of the desired DNA fragment is unsuccessful or results in an incorrect sequence, molecular cloning of a PCR-amplified region of the target sequence and assembly of the cloned parts by restriction and ligation is an option. Assembled components of such DNA fragments can be connected together by ligating the compatible overhangs produced by different restriction endonucleases. However, designing the corresponding cloning scheme can be a complex task that requires a software tool to generate a list of potential connection sites.FindingsThe BIOF program presented here analyzes DNA fragments for all available restriction enzymes and provides a list of potential sites for ligation of DNA fragments with compatible overhangs. The cloning scheme, which is called modular assembly cloning (MAC), is aided by the BIOF program. MAC was tested on a practical dataset, namely, two non-coding fragments of the translation elongation factor 1 alpha gene from Chinese hamster ovary cells. The individual fragment lengths exceeded 5 kbp, and direct PCR amplification produced no amplicons. However, separation of the target fragments into smaller regions, with downstream assembly of the cloned modules, resulted in both target DNA fragments being obtained with few subsequent steps.ConclusionsImplementation of the MAC software tool and the experimental approach adopted here has great potential for simplifying the molecular cloning of long DNA fragments. This approach may be used to generate long artificial DNA fragments such as in vitro spliced cDNAs.

Peculiarities of the Mechanism of Interactions of Catalytic Antibodies with Organophosphorus Substrates

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–1M–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 antiidiotypic approach to obtaining a proteolytic antibody

105 Biocatalysis is one of the most important functions of polymers. Understanding of the mechanisms and structural-functional interactions of biopolymers that ensure their catalytic properties is a nationwide direction of modern biochemistry, molecular biology, and molecular medicine. Such knowledge can help to closely come to the creation of biocatalysts de novo with preset features, which is so essential for the fundamental aspects of the science about life and for the solution of specific problems in biotechnology. Earlier, we were the first to propose a method to create biocatalysts using the hypervariable features of immunoglobulins as antiidiotypic antibodies to the active sites of enzymes [1, 2]. Being an alternative of the known method of obtaining abzymes (catalytic antibodies) to chemical structures, analogues of transient states of enzymatic reactions [3], the suggested approach often ensures a higher specificity and efficiency of catalysis [4]. Despite the acknowledged advances in obtaining specific esterase antibodies [5–7], a real breakthrough in the application of the proposed approach would be the creation of proteases with preset features. Construction of catalytic vaccines that are based on such proteases and are capable of target disintegration of proteins responsible for development of certain pathologies could become one of the ways of obtaining drugs of the future. Recently, we have managed to obtain a proteolytic antibody using subtilisin Carlsberg as a primary antigen [8, 9].