Белогуров Алексей Анатольевич

Кандидат химических наук

Старший научный сотрудник (Лаборатория биокатализа)

Тел.: +7 (495) 7273860

Эл. почта:

Избранные публикации

  1. Terekhov S.S., Smirnov I.V., Stepanova A.V., Bobik T.V., Mokrushina Y.A., Ponomarenko N.A., Belogurov A.A. Jr, Rubtsova M.P., Kartseva O.V., Gomzikova M.O., Moskovtsev A.A., Bukatin A.S., Dubina M.V., Kostryukova E.S., Babenko V.V., Vakhitova M.T., Manolov A.I., Malakhova M.V., Kornienko M.A., Tyakht A.V., Vanyushkina A.A., Ilina E.N., Masson P., Gabibov A.G., Altman S. (2017). Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity. Proc. Natl. Acad. Sci. U.S.A. 114 (10), 2550–2555 [+]

    Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.

  2. Lomakin Y., Arapidi G.P., Chernov A., Ziganshin R., Tcyganov E., Lyadova I., Butenko I.O., Osetrova M., Ponomarenko N., Telegin G., Govorun V.M., Gabibov A., Belogurov A. (2017). Exposure to the Epstein-Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo. Front Immunol 8, 777 [+]

    Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20-50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

  3. Ivanova V.V., Khaiboullina S.F., Gomzikova M.O., Martynova E.V., Ferreira A.M., Garanina E.E., Sakhapov D.I., Lomakin Y.A., Khaibullin T.I., Granatov E.V., Khabirov F.A., Rizvanov A.A., Gabibov A., Belogurov A. (2017). Divergent Immunomodulation Capacity of Individual Myelin Peptides-Components of Liposomal Therapeutic against Multiple Sclerosis. Front Immunol 8, 1335 [+]

    Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination and consequent neuron injury. Although the pathogenesis of MS is largely unknown, a breach in immune self-tolerance to myelin followed by development of autoreactive encephalitogenic T cells is suggested to play the central role. The myelin basic protein (MBP) is believed to be one of the main targets for autoreactive lymphocytes. Recently, immunodominant MBP peptides encapsulated into the mannosylated liposomes, referred as Xemys, were shown to suppress development of experimental autoimmune encephalomyelitis, a rodent model of MS, and furthermore passed the initial stage of clinical trials. Here, we investigated the role of individual polypeptide components [MBP peptides 46-62 (GH17), 124-139 (GK16), and 147-170 (QR24)] of this liposomal peptide therapeutic in cytokine release and activation of immune cells from MS patients and healthy donors. The overall effects were assessed using peripheral blood mononuclear cells (PBMCs), whereas alterations in antigen-presenting capacities were studied utilizing plasmacytoid dendritic cells (pDCs). Among three MBP-immunodominant peptides, QR24 and GK16 activated leukocytes, while GH17 was characterized by an immunosuppressive effect. Peptides QR24 and GK16 upregulated CD4 over CD8 T cells and induced proliferation of CD25(+) cells, whereas GH17 decreased the CD4/CD8 T cell ratio and had limited effects on CD25(+) T cells. Accordingly, components of liposomal peptide therapeutic differed in upregulation of cytokines upon addition to PBMCs and pDCs. Peptide QR24 was evidently more effective in upregulation of pro-inflammatory cytokines, whereas GH17 significantly increased production of IL-10 through treated cells. Altogether, these data suggest a complexity of action of the liposomal peptide therapeutic that does not seem to involve simple helper T cells (Th)-shift but rather the rebalancing of the immune system.

  4. Smirnov I.V., Golovin A.V., Chatziefthimiou S.D., Stepanova A.V., Peng Y., Zolotareva O.I., Belogurov A.A. Jr, Kurkova I.N., Ponomarenko N.A., Wilmanns M., Blackburn G.M., Gabibov A.G., Lerner R.A. (2016). Robotic QM/MM-driven maturation of antibody combining sites. Sci Adv 2 (10), e1501695 [+]

    In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis.

  5. Belogurov A., Zakharov K., Lomakin Y., Surkov K., Avtushenko S., Kruglyakov P., Smirnov I., Makshakov G., Lockshin C., Gregoriadis G., Genkin D., Gabibov A., Evdoshenko E. (2016). CD206-Targeted Liposomal Myelin Basic Protein Peptides in Patients with Multiple Sclerosis Resistant to First-Line Disease-Modifying Therapies: A First-in-Human, Proof-of-Concept Dose-Escalation Study. Neurotherapeutics , [+]

    Previously, we showed that CD206-targeted liposomal delivery of co-encapsulated immunodominant myelin basic protein (MBP) sequences MBP46-62, MBP124-139 and MBP147-170 (Xemys) suppressed experimental autoimmune encephalomyelitis in dark Agouti rats. The objective of this study was to assess the safety of Xemys in the treatment of patients with relapsing-remitting multiple sclerosis (MS) and secondary progressive MS, who failed to achieve a sustained response to first-line disease-modifying therapies. In this phase I, open-label, dose-escalating, proof-of-concept study, 20 patients with relapsing-remitting or secondary progressive MS received weekly subcutaneously injections with ascending doses of Xemys up to a total dose of 2.675 mg. Clinical examinations, including Expanded Disability Status Scale score, magnetic resonance imaging results, and serum cytokine concentrations, were assessed before the first injection and for up to 17 weeks after the final injection. Xemys was safe and well tolerated when administered for 6 weeks to a maximum single dose of 900 μg. Expanded Disability Status Scale scores and numbers of T2-weighted and new gadolinium-enhancing lesions on magnetic resonance imaging were statistically unchanged at study exit compared with baseline; nonetheless, the increase of number of active gadolinium-enhancing lesions on weeks 7 and 10 in comparison with baseline was statistically significant. During treatment, the serum concentrations of the cytokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and interleukin-7 decreased, whereas the level of tumor necrosis factor-α increased. These results provide evidence for the further development of Xemys as an antigen-specific, disease-modifying therapy for patients with MS.

  6. Ziganshin R.H., Ivanova O.M., Lomakin Y.A., Belogurov A.A. Jr, Kovalchuk S.I., Azarkin I.V., Arapidi G.P., Anikanov N.A., Shender V.O., Piradov M.A., Suponeva N.A., Vorobyeva A.A., Gabibov A.G., Ivanov V.T., Govorun V.M. (2016). The pathogenesis of demyelinating form of Guillain-Barre syndrome: proteo-peptidomic and immunological profiling of physiological fluids. Mol. Cell Proteomics , [+]

    Acute inflammatory demyelinating polyneuropathy (AIDP) - the main form of Guillain-Barre syndrome (GBS) - is a rare and severe disorder of the peripheral nervous system (PNS) with an unknown etiology. One of the hallmarks of the AIDP pathogenesis is a significantly elevated cerebrospinal fluid (CSF) protein level. In this paper CSF peptidome and proteome in AIDP were analyzed and compared with multiple sclerosis (MS) and control patients. A total protein concentration increase was shown to be due to even changes in all proteins rather than some specific response, supporting the hypothesis of protein leakage from blood through the blood-nerve barrier. The elevated CSF protein level in AIDP was complemented by activization of protein degradation and much higher peptidome diversity. Due to the studies of the acute motor axonal form, GBS as a whole is thought to be associated with autoimmune response against neurospecific molecules. Thus, in AIDP, autoantibodies against cell adhesion (CAM) proteins localized at Ranvier's nodes were suggested as possible targets in AIDP. Indeed, AIDP CSF peptidome analysis revealed CAM proteins degradation, however no reliable dependence on the corresponding autoantibodies levels was found. Proteome analysis revealed overrepresentation of Gene Ontology groups related to responses to bacteria and virus infections, which were earlier suggested as possible AIDP triggers. Immunoglobulin blood serum analysis against most common neuronal viruses did not reveal any specific pathogen; however AIDP patients were more immunopositive in average and often had polyinfections. Cytokine analysis of both AIDP CSF and blood did not show a systemic adaptive immune response or general inflammation, while innate immunity cytokines were upregulated. To supplement the widely-accepted though still unproven autoimmunity-based AIDP mechanism we propose a hypothesis of the primary PNS damaging initiated as an innate immunity-associated local inflammation following neurotropic viruses egress, while the autoantibody production might be an optional complementary secondary process.

  7. Stepanov A., Belyy A., Kasheverov I., Rybinets A., Dronina M., Dyachenko I., Murashev A., Knorre V., Sakharov D., Ponomarenko N., Tsetlin V., Tonevitsky A., Deyev S., Belogurov A., Gabibov A. (2016). Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs. Biotechnol. Lett. , [+]

    Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment.

  8. Eroshkin F.M., Nesterenko A.M., Borodulin A.V., Martynova N.Y., Ermakova G.V., Gyoeva F.K., Orlov E.E., Belogurov A.A. Jr, Lukyanov K.A., Bayramov A.V., Zaraisky A.G. (2016). Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis. Sci Rep 6, 23049 [+]

    Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. In this study, we demonstrate that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/β -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, we directly estimated the affinity of Noggin4 for Wnt8 in living embryos and determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. Our results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development.

  9. Belogurov A., Kuzina E., Kudriaeva A., Kononikhin A., Kovalchuk S., Surina Y., Smirnov I., Lomakin Y., Bacheva A., Stepanov A., Karpova Y., Lyupina Y., Kharybin O., Melamed D., Ponomarenko N., Sharova N., Nikolaev E., Gabibov A. (2015). Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity. FASEB J. , [+]

    Recent findings indicate that the ubiquitin-proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain-derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin-independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE-SJL mice is caused by a dramatic shift in the balance between constitutive and β1i(high) immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, β1i is increased in resident CNS cells, whereas β5i is imported by infiltrating lymphocytes through the blood-brain barrier. Peptidyl epoxyketone specifically inhibits brain-derived β1i(high) immunoproteasomes in vitro (kobs/[I] = 240 M(-1)s(-1)), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the β1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.-Belogurov Jr., A., Kuzina, E., Kudriaeva, A., Kononikhin, A., Kovalchuk, S., Surina, Y., Smirnov, I., Lomakin, Y., Bacheva, A., Stepanov, A., Karpova, Y., Lyupina, Y., Kharybin, O., Melamed, D., Ponomarenko, N., Sharova, N., Nikolaev, E., Gabibov, A. Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity.

  10. Belogurov A., Kudriaeva A., Kuzina E., Smirnov I., Bobik T., Ponomarenko N., KravtsovaIvantsiv Y., Ciechanover A., Gabibov A. (2014). Multiple Sclerosis Autoantigen Myelin Basic Protein Escapes Control by Ubiquitination During Proteasomal Degradation. J. Biol. Chem. , [+]

    The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility towards proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.

  11. Ponomarenko N., Chatziefthimiou S.D., Kurkova I., Mokrushina Y., Stepanova A., Smirnov I., Avakyan M., Bobik T., Mamedov A., Mitkevich V., Belogurov A., Fedorova O.S., Dubina M., Golovin A., Lamzin V., Friboulet A., Makarov A.A., Wilmanns M., Gabibov A. (2014). Role of κ→λ light-chain constant-domain switch in the structure and functionality of A17 reactibody. Acta Crystallogr. D Biol. Crystallogr. 70 (Pt 3), 708–19 [+]

    The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was found that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the VL and VH domains. These VL/VH domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.

  12. Lomakin Y.A., Zakharova M.Y., Stepanov A.V., Dronina M.A., Smirnov I.V., Bobik T.V., Pyrkov A.Y., Tikunova N.V., Sharanova S.N., Boitsov V.M., Vyazmin S.Y., Kabilov M.R., Tupikin A.E., Krasnov A.N., Bykova N.A., Medvedeva Y.A., Fridman M.V., Favorov A.V., Ponomarenko N.A., Dubina M.V., Boyko A.N., Vlassov V.V., Belogurov A.A. Jr, Gabibov A.G. (2014). Heavy-light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation. Mol. Immunol. , [+]

    The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.

  13. Kuzina E., Kudriaeva A., Smirnov I., Dubina M.V., Gabibov A., Belogurov A. (2014). Glatiramer Acetate and Nanny Proteins Restrict Access of the Multiple Sclerosis Autoantigen Myelin Basic Protein to the 26S Proteasome. Biomed Res Int 2014, 926394 [+]

    We recently showed that myelin basic protein (MBP) is hydrolyzed by 26S proteasome without ubiquitination. The previously suggested concept of charge-mediated interaction between MBP and the proteasome led us to attempt to compensate or mimic its positive charge to inhibit proteasomal degradation. We demonstrated that negatively charged actin and calmodulin (CaM), as well as basic histone H1.3, inhibit MBP hydrolysis by competing with the proteasome and MBP, respectively, for binding their counterpart. Interestingly, glatiramer acetate (GA), which is used to treat multiple sclerosis (MS) and is structurally similar to MBP, inhibits intracellular and in vitro proteasome-mediated MBP degradation. Therefore, the data reported in this study may be important for myelin biogenesis in both the normal state and pathophysiological conditions.

  14. Ilyushin D.G., Smirnov I.V., Belogurov A.A. Jr, Dyachenko I.A., Zharmukhamedova T.I.u., Novozhilova T.I., Bychikhin E.A., Serebryakova M.V., Kharybin O.N., Murashev A.N., Anikienko K.A., Nikolaev E.N., Ponomarenko N.A., Genkin D.D., Blackburn G.M., Masson P., Gabibov A.G. (2013). Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo. Proc. Natl. Acad. Sci. U.S.A. 110 (4), 1243–8 [+]

    The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD(50) of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.

  15. Belogurov A.A. Jr, Stepanov A.V., Smirnov I.V., Melamed D., Bacon A., Mamedov A.E., Boitsov V.M., Sashchenko L.P., Ponomarenko N.A., Sharanova S.N., Boyko A.N., Dubina M.V., Friboulet A., Genkin D.D., Gabibov A.G. (2012). Liposome-encapsulated peptides protect against experimental allergic encephalitis. FASEB J. , [+]

    Multiple sclerosis (MS) is a severe inflammatory and neurodegenerative disease with an autoimmune background. Despite the variety of therapeutics available against MS, the development of novel approaches to its treatment is of high importance in modern pharmaceutics. In this study, experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats has been treated with immunodominant peptides of the myelin basic protein (MBP) encapsulated in mannosylated small unilamellar vesicles. The results show that liposome-encapsulated MBP(46-62) is the most effective in reducing maximal disease score during the first attack, while MBP(124-139) and MBP(147-170) can completely prevent the development of the exacerbation stage. Both mannosylation of liposomes and encapsulation of peptides are critical for the therapeutic effect, since neither naked peptides nor nonmannosylated liposomes, loaded or empty, have proved effective. The liposome-mediated synergistic effect of the mixture of 3 MBP peptides significantly suppresses the progression of protracted EAE, with the median cumulative disease score being reduced from 22 to 14 points, compared to the placebo group; prevents the production of circulating autoantibodies; down-regulates the synthesis of Th1 cytokines; and induces the production of brain-derived neurotrophic factor in the central nervous system. Thus, the proposed formulation ameliorates EAE, providing for a less severe first attack and rapid recovery from exacerbation, and offers a promising therapeutic modality in MS treatment.-Belogurov, A. A., Jr., Stepanov, A. V., Smirnov, I. V., Melamed, D., Bacon, A., Mamedov, A. E., Boitsov, V. M., Sashchenko, L. P., Ponomarenko, N. A., Sharanova, S. N., Boyko, A. N., Dubina, M. V., Friboulet, A., Genkin, D. D., Gabibov, A. G. Liposome-encapsulated peptides protect against experimental allergic encephalitis.

  16. Belogurov A., Smirnov I., Ponomarenko N., Gabibov A. (2012). Antibody-antigen pair probed by combinatorial approach and rational design: bringing together structural insights, directed evolution, and novel functionality. FEBS Lett. 586 (18), 2966–73 [+]

    The unique hypervariability of the immunoglobulin (Ig) superfamily provides a means to create both binding and catalytic antibodies with almost any desired specificity and activity. The diversity of antigens and concept of adaptive response suggest that it is possible to find an antigen pair to any raised Ig. In the current review we discuss combinatorial approaches, which makes it possible to obtain an antibody with predefined properties, followed by 3D structure-based rational design to enhance or dramatically change its characteristics. A similar strategy, but applied to the second partner of the antibody-antigen pair, may result in selection of complementary substrates to the chosen Ig. Finally, 2D screening may be performed solving the "Chicken and Egg" problem when neither antibody nor antigen is known.

  17. Gabibov A.G., Belogurov A.A. Jr, Lomakin Y.A., Zakharova M.Y., Avakyan M.E., Dubrovskaya V.V., Smirnov I.V., Ivanov A.S., Molnar A.A., Gurtsevitch V.E., Diduk S.V., Smirnova K.V., Avalle B., Sharanova S.N., Tramontano A., Friboulet A., Boyko A.N., Ponomarenko N.A., Tikunova N.V. (2011). Combinatorial antibody library from multiple sclerosis patients reveals antibodies that cross-react with myelin basic protein and EBV antigen. FASEB J. 25 (12), 4211–21 [+]

    Multiple sclerosis (MS) is a widespread neurodegenerative autoimmune disease with unknown etiology. It is increasingly evident that, together with pathogenic T cells, autoreactive B cells are among the major players in MS development. The analysis of myelin neuroantigen-specific antibody repertoires and their possible cross-reactivity against environmental antigens, including viral proteins, could shed light on the mechanism of MS induction and progression. A phage display library of single-chain variable fragments (scFvs) was constructed from blood lymphocytes of patients with MS as a potential source of representative MS autoantibodies. Structural alignment of 13 clones selected toward myelin basic protein (MBP), one of the major myelin antigens, showed high homology within variable regions with cerebrospinal fluid MS-associated antibodies as well as with antibodies toward Epstein-Barr latent membrane protein 1 (LMP1). Three scFv clones showed pronounced specificity to MBP fragments 65-92 and 130-156, similar to the serum MS antibodies. One of these clones, designated E2, in both scFv and full-size human antibody constructs, was shown to react with both MBP and LMP1 proteins in vitro, suggesting natural cross-reactivity. Thus, antibodies induced against LMP1 during Epstein-Barr virus infection might act as inflammatory trigger by reacting with MBP, suggesting molecular mimicry in the mechanism of MS pathogenesis.

  18. Stepanov A.V., Belogurov A.A. Jr, Ponomarenko N.A., Stremovskiy O.A., Kozlov L.V., Bichucher A.M., Dmitriev S.E., Smirnov I.V., Shamborant O.G., Balabashin D.S., Sashchenko L.P., Tonevitsky A.G., Friboulet A., Gabibov A.G., Deyev S.M. (2011). Design of targeted B cell killing agents. PLoS ONE 6 (6), e20991 [+]

    B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents.

  19. Belogurov A., Kozyr A., Ponomarenko N., Gabibov A. (2009). Catalytic antibodies: balancing between Dr. Jekyll and Mr. Hyde. Bioessays 31 (11), 1161–71 [+]

    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.

  20. Belogurov A.A. Jr, Zargarova T.A., Turobov V.I., Novikova N.I., Favorova O.O., Ponomarenko N.A., Gabibov A.G. (2009). Suppression of ongoing experimental allergic encephalomyelitis in DA rats by novel peptide drug, structural part of human myelin basic protein 46-62. Autoimmunity 42 (4), 362–4 [+]

    Previously, we demonstrated that autoantibodies (AAb) in multiple sclerosis (MS) reveal site-specific binding and cleavage toward myelin basic protein (MBP) epitope library. We have found several fragments of MBP immunodominant in terms of AAb binding. Here, we applied these peptides to DA rats with induced protracted relapsing experimental allergic encephalomyelitis (EAE) most closely related to MS. DA rats with EAE induced by syngenic spinal cord homogenate in complete Freund's adjuvant were treated by nasal route with human MBP 46-62, 81-102, 124-139, 147-170, and Copaxone. MBP 124-139 and 147-170 displayed only mild therapeutic effects but MBP 46-62 significantly reduced EAE, reflected by lower clinical scores and shorter EAE duration compared to controls.

  21. Belogurov A.A. Jr, Kurkova I.N., Friboulet A., Thomas D., Misikov V.K., Zakharova M.Y., Suchkov S.V., Kotov S.V., Alehin A.I., Avalle B., Souslova E.A., Morse H.C. 3rd, Gabibov A.G., Ponomarenko N.A. (2008). Recognition and degradation of myelin basic protein peptides by serum autoantibodies: novel biomarker for multiple sclerosis. J. Immunol. 180 (2), 1258–67 [+]

    The pathologic role of autoantibodies in autoimmune disease is widely accepted. Recently, we reported that anti-myelin basic protein (MBP) serum Abs from multiple sclerosis (MS) patients exhibit proteolytic activity toward the autoantigen. The aim of this study is to determine MBP epitopes specific for the autoantibodies in MS and compare these data with those from other neuronal disorders (OND), leading to the generation of new diagnostic and prognostic criteria. We constructed a MBP-derived recombinant "epitope library" covering the entire molecule. We used ELISA and PAGE/surface-enhanced laser desorption/ionization mass spectroscopy assays to define the epitope binding/cleaving activities of autoantibodies isolated from the sera of 26 MS patients, 22 OND patients, and 11 healthy individuals. The levels of autoantibodies to MBP fragments 48-70 and 85-170 as well as to whole MBP and myelin oligodendrocyte glycoprotein molecules were significantly higher in the sera of MS patients than in those of healthy donors. In contrast, selective reactivity to the two MBP fragments 43-68 and 146-170 distinguished the OND and MS patients. Patients with MS (77% of progressive and 85% of relapsing-remitting) but only 9% of patients with OND and no healthy donors were positive for catalysis, showing pronounced epitope specificity to the encephalitogenic MBP peptide 81-103. This peptide retained its substrate properties when flanked with two fluorescent proteins, providing a novel fluorescent resonance energy transfer approach for MS studies. Thus, anti-MBP autoantibody-mediated, epitope-specific binding and cleavage may be regarded as a specific characteristic of MS compared with OND and healthy donors and may serve as an additional biomarker of disease progression.

  22. Ponomarenko N.A., Durova O.M., Vorobiev I.I., Belogurov A.A. Jr, Telegin G.B., Suchkov S.V., Misikov V.K., Morse H.C. 3rd, Gabibov A.G. (2006). Catalytic activity of autoantibodies toward myelin basic protein correlates with the scores on the multiple sclerosis expanded disability status scale. Immunol. Lett. 103 (1), 45–50 [+]

    Autoantibodies toward myelin basic protein (MBP) evidently emerge in sera and cerebrospinal fluid of the patients with multiple sclerosis (MS), as well as in a MS rodent model, i.e., experimental autoimmune encephalomyelitis (EAE). The studies of the last two decades have unveiled somewhat controversial data on the diagnostic applicability of anti-MBP autoantibodies as a disease' marker. Here, we present the results of new functional analysis of the anti-MBP autoantibodies isolated from MS (in patients) and EAE (in mice) sera, based on their proteolytic activity against the targeted autoantigen. The activity was shown to be the intrinsic property of the IgG molecule. No activity was found in the sera-derived antibody fraction of healthy donors and control mice. Sera of 24 patients with clinically proven MS at different stages of the disease, and 20 healthy controls were screened for the anti-MBP antibody-mediated proteolytic activity. The activity correlated with the scores on the MS expanded disability status scale (EDSS) (r(2)=0.85, P<0.001). Thus, the anti-MBP autoantibody-mediated proteolysis may be regarded as an additional marker of the disease progression.

  23. Ponomarenko N.A., Durova O.M., Vorobiev I.I., Belogurov A.A. Jr, Kurkova I.N., Petrenko A.G., Telegin G.B., Suchkov S.V., Kiselev S.L., Lagarkova M.A., Govorun V.M., Serebryakova M.V., Avalle B., Tornatore P., Karavanov A., Morse H.C. 3rd, Thomas D., Friboulet A., Gabibov A.G. (2006). Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen. Proc. Natl. Acad. Sci. U.S.A. 103 (2), 281–6 [+]

    Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.