**Antibody-Proteases in the Pathogenesis of Autoimmune Demyelination and Monitoring Patients with Multiple Sclerosis**

 Dmitry Kostyushev1, Dmitry Gnatenko1, Mikhail Paltsev3, Aleksandr Gabibov4 and Sergey Suchkov1,2 *1I.M. Sechenov First Moscow Medical State University 2Moscow State University of Medicine and Dentistry 3National Research Center "Kurchatov Institute" 4M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Russia* 

#### **1. Introduction**

476 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

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Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) resulting in axon loss and development of disability. (Gabibov et al., 2011) Autoantibodies (autoAbs) are one of the major features and crucial mechanisms in MS pathogenesis known to illustrate this autoagression. The major component in the pathogenesis of MS is primary myelin damage, which is mediated by autoAbs, which trigger the release of separate and pathogenically valuable myelin-associated epitopes into the bloodstream. These molecules acting as a group of sensitizing factors may provoke the immune system and drive disease progression. Being identified at the pre- or early stages or the demyelination, such autoAbs dominate during the whole course of the disease.

Natural catalytic antibodies (*catAbs*) or natural *abzymes* today are one of the principal effectors of the adaptive immune system. In constructive sense, catAbs are multivalent immunoglobulins (Igs), presumably, of IgG and IgM isotypes, endowed with a capacity to hydrolyze an antigenic substrate.

Traditionally, the basic structure of the Ab molecule is essentially *Y*-shaped, with the two tips (*Fab*-fragments) designed to recognize and bind non-self agents or cells. Moreover, the catalytic capacity is also present in *Fab*-fragments of the molecule. In general, the mechanisms of Ab-mediated catalytic action include nucleophilic catalysis, induction of conformational strain, coordination with ions, and stabilization of transition states (TS). Agspecific or targeted catAbs are preferentially found in the Ig repertoire of patients with a broad scope of diseases to act as pathogenically valuable tools. Since the discovery of catAbs, a wide spectrum of the disease-related abzymes regardless to their natural history or engineering protocols has been described. Moreover, the immune system was shown to express an intrinsic drive to generate natural abzymes in different pathological states in humans. Among them, proteolytic (*Ab-proteases*) and DNA-hydrolyzing (*DNA-abzymes*) autoAbs are of a special practical value.

Antibody-Proteases in the Pathogenesis of Autoimmune

degradation of myelin sheath. (Gabibov et al., 2011)

2007; Nikbin et al., 2007)

et al., 1998)

Demyelination and Monitoring Patients with Multiple Sclerosis 479

antibodies with the ability to mediate direct catalytic and indirect cytotoxic effects on the target. This property is buried in the Fab fragment of the immunoglobulin molecule. Antibody protease were found in most autoimmune conditions, particulary in MS, accomplishing sequence-specific proteolic cleavage of myelin antigens and controlling the

The worldwide median estimated incidence of MS is 2.5 per 100 000 and prevalence is estimated at approximately 1.5 million cases. Usually onset of MS is between age 20 and 40 years. Men are affected approximately twice as rare as women. (Stuve & Oksenberg, 2006 ) Autoantibody mediated tissue destruction is among the main features of organ-specific autoimmunity. Ample data indicate that a significant portion of MS cases is characterized by the presence in the blood of autoantibodies against myelin protein components. Moreover, myelin-specific autoantibodies are detected by highresolution microscopic analysis in the regions of demyelination plaques in human MS and a MS-like disease of marmosets,

suggesting their direct contribution to myelin destruction. (Ponomarenko et al., 2006) Nonetheless, the mechanisms responsible for the induction of autoantibodies and their possible contributions to MS progression are still unknown and are somewhat controversial. Also clonal expansion of B cells and T cells, hallmarks of inflammation in the CNS, are found in MS. The viral mimicry hypothesis was formulated to explain the initiation of this pathology. (Belogurov et al., 2008) But a poor understanding of the etiology of MS has

Despite strong evidence for the contribution of T cell responses to manifestations of autoimmunity in the CNS of patients with MS, recent findings encouraged investigators to search also for B cell-mediated contributions to the MS pathogenesis. (Klawiter & Cross,

Development of new diagnostics and treating tactics can improve patient life quality and decrease MS treatment cost, as in other autoimmune diseases such as type 1 diabetes. (Hahl

Antibodies can cause demyelination by several effector mechanisms. One of these is the opsonization of myelin for subsequent phagocytosis by macrophages, which has been observed in MS and EAE. Serum anti-myelin antibodies raise macrophage phagocytosis, and the uptake by macrophages of CNS myelin increases after opsonization with complement. Another mechanism of demyelination that involves autoantibodies is through activation of the entire complement cascade leading to membrane attack complex (MAC)

Antibodies with specificity against minor myelin components have also been detected in MS patients. MOG is the most interesting candidate B-cell autoantigen in MS. Because of its location it is an ideal target for antibody-mediated demyelination. Anti-MOG antibodies are indeed able to cause myelin destruction in EAE models, while other antibodies against major myelin proteins such as MBP or PLP, which are both not located on the myelin surface, do not cause myelin destruction on their own. Anti-MOG Abs mediate a characteristic vesicular transformation of compact myelin in acutely demyelinated lesions that also has been documented in human MS lesions strongly suggesting a role of anti-MOG Abs in MS. The B-cell response to MOG is enhanced in MS also supporting the pathogenic importance of anti-MOG Abs. Interestingly, the presence of serum anti-MOG antibodies,

complicated the development of effective therapeutics. (Hafler, 2004)

**2. Antibodies-mediated demyelination in the pathogenesis of MS** 

deposition and complement-mediated cytolysis.

And today catAbs can be used as either therapeutic tools or as vehicles for delivering therapeutic agents to damaged cells in the human body. Recent applications of abzymes have included a broad scope of medical and allied areas, i.e.,


The aim of this review is to compare data on the medical applications and implementations of disease-associated and engineered abzymes as new tools for treatment of MS, and to specifically focus on their potential value for clinical research and for clinical utility and public health as well.

The mechanisms of catAb action include nucleophilic catalysis, induction of conformational strain, coordination with metal ions, and stabilization of TS. Only Abs that stabilize the Ag TS more than the GS can be catalytic, and such Abs are thus usually identified among those that bind tightly to an analogue of the TS of the relevant reaction. Abs would thus provide a unique opportunity to combine specific Ag recognition with enzymatic turnover.

For Ab-proteases, for instance, a typical mechanism of nucleophilic catalysis has been established. Since the catalytic efficiency of Ab-proteases derives substantially from the ability to recognize the GS with high affinity, proteolytic Abs while demonstrating an exclusive targeted specificity can be used to selectively address a wide range of metabolically and pathogenically valuable protein targets.

In case of DNA-abzymes, Ab-induced conformational strain is proposed to activate phosphodiester bonds and result in DNA hydrolysis, aided by coordination of bivalent cations. As being similar to proteolytic sites, the nuclease activity of DNA-hydrolyzing Abs is described to be encoded by germline variable region genes (*V*-genes).In general, the specific nature of the Ab catalysis was demonstrated by the adherence of those reactions to the well-known Michaelis-Menten equation, the complete inhibition by a proper hapten analogue, and the failure of the Ab to catalyze the hydrolysis of the substrate antagonists.

During the last 10 years, it has been found that Abs contribute to the degradation of a number of autoantigens. These and related "antibody-enzymes", also termed abzymes, were shown to be able to cleave DNA, RNA, carbohydrates, peptides, and proteins Recently, abzyme-dependent catalytic degradation of an autoantigen, MBP (myelin basic protein), was associated with the course of the neurodegenerative disease MS and its rodent model, experimental autoimmune encephalomyelitis (EAE). Autoantibody-mediated degradation of MBP was shown to be site specic, with cleavage sites localized to the immunodominant epitopes of the protein. These ndings were supported by studies from others. Interestingly, this reaction was inhibited in vitro by glatiramer acetate (Copaxone), an established treatment for MS. (Belogurov et al., 2008)

There are three groups of autoAbs that are specific for MS: anti-myelin autoAbs (e.g., anti-MBP, anti-MOG (MOG - myelin oligodendrocyte glycoprotein) and anti-neurofilament autoAbs); nonmyelin autoAbs (e.g., anti-HSP autoAbs, among others); and autoAbs demonstrating different levels of specificity and functionality (e.g., catalytic autoAbs [i.e., antibody proteases]). The latter group of anti-myelin antibody proteases is of particular interest in terms of disease monitoring, prognosis and preclinical (pre-early) diagnostics of MS. Catalic antbodies are endowed with a capacity to hydroliyze an antigenic substrate. This has moved antibodies to the level of physiological functionality by providing such

And today catAbs can be used as either therapeutic tools or as vehicles for delivering therapeutic agents to damaged cells in the human body. Recent applications of abzymes

The aim of this review is to compare data on the medical applications and implementations of disease-associated and engineered abzymes as new tools for treatment of MS, and to specifically focus on their potential value for clinical research and for clinical utility and

The mechanisms of catAb action include nucleophilic catalysis, induction of conformational strain, coordination with metal ions, and stabilization of TS. Only Abs that stabilize the Ag TS more than the GS can be catalytic, and such Abs are thus usually identified among those that bind tightly to an analogue of the TS of the relevant reaction. Abs would thus provide a

For Ab-proteases, for instance, a typical mechanism of nucleophilic catalysis has been established. Since the catalytic efficiency of Ab-proteases derives substantially from the ability to recognize the GS with high affinity, proteolytic Abs while demonstrating an exclusive targeted specificity can be used to selectively address a wide range of

In case of DNA-abzymes, Ab-induced conformational strain is proposed to activate phosphodiester bonds and result in DNA hydrolysis, aided by coordination of bivalent cations. As being similar to proteolytic sites, the nuclease activity of DNA-hydrolyzing Abs is described to be encoded by germline variable region genes (*V*-genes).In general, the specific nature of the Ab catalysis was demonstrated by the adherence of those reactions to the well-known Michaelis-Menten equation, the complete inhibition by a proper hapten analogue, and the failure of the Ab to catalyze the hydrolysis of the substrate antagonists. During the last 10 years, it has been found that Abs contribute to the degradation of a number of autoantigens. These and related "antibody-enzymes", also termed abzymes, were shown to be able to cleave DNA, RNA, carbohydrates, peptides, and proteins Recently, abzyme-dependent catalytic degradation of an autoantigen, MBP (myelin basic protein), was associated with the course of the neurodegenerative disease MS and its rodent model, experimental autoimmune encephalomyelitis (EAE). Autoantibody-mediated degradation of MBP was shown to be site specic, with cleavage sites localized to the immunodominant epitopes of the protein. These ndings were supported by studies from others. Interestingly, this reaction was inhibited in vitro by glatiramer acetate (Copaxone), an established

There are three groups of autoAbs that are specific for MS: anti-myelin autoAbs (e.g., anti-MBP, anti-MOG (MOG - myelin oligodendrocyte glycoprotein) and anti-neurofilament autoAbs); nonmyelin autoAbs (e.g., anti-HSP autoAbs, among others); and autoAbs demonstrating different levels of specificity and functionality (e.g., catalytic autoAbs [i.e., antibody proteases]). The latter group of anti-myelin antibody proteases is of particular interest in terms of disease monitoring, prognosis and preclinical (pre-early) diagnostics of MS. Catalic antbodies are endowed with a capacity to hydroliyze an antigenic substrate. This has moved antibodies to the level of physiological functionality by providing such

unique opportunity to combine specific Ag recognition with enzymatic turnover.

have included a broad scope of medical and allied areas, i.e., 1. the conversion of drugs to their inactive (non-toxic) forms;

2. the degradation of drugs and harmful substances; 3. the activation of pro-drugs for targeted chemotherapy;

metabolically and pathogenically valuable protein targets.

treatment for MS. (Belogurov et al., 2008)

4. the inhibition of infectivity;

5. others.

public health as well.

antibodies with the ability to mediate direct catalytic and indirect cytotoxic effects on the target. This property is buried in the Fab fragment of the immunoglobulin molecule. Antibody protease were found in most autoimmune conditions, particulary in MS, accomplishing sequence-specific proteolic cleavage of myelin antigens and controlling the degradation of myelin sheath. (Gabibov et al., 2011)

The worldwide median estimated incidence of MS is 2.5 per 100 000 and prevalence is estimated at approximately 1.5 million cases. Usually onset of MS is between age 20 and 40 years. Men are affected approximately twice as rare as women. (Stuve & Oksenberg, 2006 )

Autoantibody mediated tissue destruction is among the main features of organ-specific autoimmunity. Ample data indicate that a significant portion of MS cases is characterized by the presence in the blood of autoantibodies against myelin protein components. Moreover, myelin-specific autoantibodies are detected by highresolution microscopic analysis in the regions of demyelination plaques in human MS and a MS-like disease of marmosets, suggesting their direct contribution to myelin destruction. (Ponomarenko et al., 2006)

Nonetheless, the mechanisms responsible for the induction of autoantibodies and their possible contributions to MS progression are still unknown and are somewhat controversial. Also clonal expansion of B cells and T cells, hallmarks of inflammation in the CNS, are found in MS. The viral mimicry hypothesis was formulated to explain the initiation of this pathology. (Belogurov et al., 2008) But a poor understanding of the etiology of MS has complicated the development of effective therapeutics. (Hafler, 2004)

Despite strong evidence for the contribution of T cell responses to manifestations of autoimmunity in the CNS of patients with MS, recent findings encouraged investigators to search also for B cell-mediated contributions to the MS pathogenesis. (Klawiter & Cross, 2007; Nikbin et al., 2007)

Development of new diagnostics and treating tactics can improve patient life quality and decrease MS treatment cost, as in other autoimmune diseases such as type 1 diabetes. (Hahl et al., 1998)
