**5. Prediction of MS**

The use of assays for Abs to MBP and MOG for diagnostic and prognostic purposes in patients with a clinically isolated syndrome (CIS), a frequent precursor to clinically definite MS (CDMS), has yielded conflicting results. One study showed that the presence of myelin Abs in the sera of CIS patients was predictive of a shorter time course to the development of CDMS. (Tomassini et al., 2007) Others, however, indicated that positive tests had no prognostic value for progression to CDMS. (Kuhle et al., 2007)

Symbols indicate OD450 values for Abs from individual subjects within each EDSS category for specific MBP peptides or intact MBP and MOG proteins. Lines indicate the relation between EDSS value and the binding activity of specific peptides/proteins: MBP 81-103 (R2 0.332; p 0.005), MBP 130-156 (R2 0.381; p 0.011), MBP 146-170 (R2 0.310; p 0.033), intact MBP

Antibody-Proteases in the Pathogenesis of Autoimmune

4. verification of the diagnosis and prediction for the future.

**5.2 Sequence specificity and clinical implementation** 

antibody proteases). (Ponomarenko et al., 2006)

molecule as well;

action at the *preclinical* stages.

Demyelination and Monitoring Patients with Multiple Sclerosis 485

3. determination of sequence-specificity of the individual Ab-proteases towards MBP as the whole molecule and separate peptides (epitope-bearing) fragmented from the

A phenomenon of the *pre-clinical* formation of the abzyme-based armamentarium and Abproteases, in particular, would be interpreted as a portion of the proper immune response of the body to the environmental and other shifts. So, a selection of the mode of action for proteolytic antibodies would depend on the metabolic motivation occurring at a particular stage of the disease. Of great interest would be driving motivation to control the mode of

The occurrence of antibody proteases among healthy individuals is due to the development of the pre-early immune imbalances and formation of the preclinical conditions. So, when bursts of the antibody-associated proteolytic activity or the tendency for the latter were evident, the pre-early stages preceding formation of CIS and the exacerbation of the disease could be predicted, even with no observable clinical manifestations. There was definite correlation between the interval before CIS and autoAb (including antibody proteases) status. The occurrence of antibody proteases and the degree of their activity were significantly associated with symptomatology/severity of CIS and clinical course of the disease.

Sequence specificity of Ab-proteases is the capacity to distinguish or not distinguish between particular epitopes in the MBP molecule. Serum anti-MBP auto-Abs obtained from MS patients exhibited sequence-specific proteolytic cleavage of the MBP molecule. Such antibody proteases revealed significant correlations with different manifestations of MS (i.e., progressive and/or remittance phases of the disease), with Expanded Disability Status Scale scores and thus with scales of demyelination and, moreover, with the degree of disability of MS patients. For instance, anti-MBP antibody proteases that are able to recognize 43-68 and 146-170 amino acid sequences within the MBP molecule are predominantly more typical of MS patients, but not of patients with neurodegenerative diseases other than MS. In MS patients with low disease activity (in remission), a family of antibody proteases has been detected with low proteolytic activity that predominantly targets 43-68 and 146-170 amino acid sequences within MBP. Progression of disability in MS patients is accompanied by the bursts of anti-MBP antibody-associated sequence-specific proteolytic activity. Moreover, signs of a trend in the specificity of the activity were found (i.e., specificity of the sequence recognition from 43-68 and 146-170 amino acid sites with low affinity to 81-103 and 82-98 amino acid sites, demonstrating that high-affinity indices are recognized by anti-MBP

**5.3 Perspectives in the application of catalytic antibodies for clinical medicine** 

More than a hundred of abzyme-catalyzed reactions have been described. Catalytic efficacy of some abzymes is comparable with those of enzymes, but the specificity of abzymes is even higher. The practical relevance of abzymes is due to the unique feature drastically distinguishing them from other biocatalysts, i.e., abzymes could be produced for the catalysis of not only all the reactions occuring in the living systems but also be designed for the development of principally new catalysts with no natural counterparts. (Gabibov et al., 2002; Zhou et al., 2002) In this sense, we can anticipate that manmade catalytic antibodies

(R2 0.458; p 0.005), intact MOG (R2 0.052; p 0.001), and control Trx carrier protein (R2 0.055; p 0.376)

Fig. 2. Correlation of the EDSS of MS patients with the levels of autoantibodies to MBP fragments.

The latter results are in keeping with our demonstration that the binding activities of Abs to intact MBP and MOG proteins were indistinguishable for patients with MS and other neurological diseases (OND). In addition, a screen of MBP peptides showed that the reactivity of Abs from patients with MS and OND could be distinguished only by their differential binding to MBP 43–68 and MBP 146–170. Follow-up studies of patients with MS using assays based on the use of these fragments will be needed to determine whether it provides an improved prognostic tool for patients with CIS.

Interestingly, the parameters of Ab binding associated with disease progression were different from those with potential prognostic value. Disease severity, as determined by Expanded disability status scale (EDSS), correlated with level of autoantibodies to intact MBP and MOG proteins as well as MBP fragments 81–103, 130–156 and 146–170. These findings are in keeping with the demonstrated correlations between levels of anti-MOG and anti-MBP Abs and inflammatory signs revealed by MRI and cerebrospinal fluid analyses. (Kuhle et al., 2007)

#### **5.1 Diagnostic protocol**

The appearance of Ab-proteases at the pre-early (*preclinical*) stages of demyelination to illustrate myelin degradation has been documented.

The results we had obtained would allow for approaching to a novel generation of diagnostic technologies based on the protocols of clinical and preclinical diagnostics to exploit Ab-proteases and their sequence-specificity.

The protocols would include:


(R2 0.458; p 0.005), intact MOG (R2 0.052; p 0.001), and control Trx carrier protein (R2 0.055;

Fig. 2. Correlation of the EDSS of MS patients with the levels of autoantibodies to MBP

provides an improved prognostic tool for patients with CIS.

illustrate myelin degradation has been documented.

exploit Ab-proteases and their sequence-specificity.

2. quantification of the proteolytic activity of Ab-proteases;

for suspicious MS (e.g., the relatives);

The latter results are in keeping with our demonstration that the binding activities of Abs to intact MBP and MOG proteins were indistinguishable for patients with MS and other neurological diseases (OND). In addition, a screen of MBP peptides showed that the reactivity of Abs from patients with MS and OND could be distinguished only by their differential binding to MBP 43–68 and MBP 146–170. Follow-up studies of patients with MS using assays based on the use of these fragments will be needed to determine whether it

Interestingly, the parameters of Ab binding associated with disease progression were different from those with potential prognostic value. Disease severity, as determined by Expanded disability status scale (EDSS), correlated with level of autoantibodies to intact MBP and MOG proteins as well as MBP fragments 81–103, 130–156 and 146–170. These findings are in keeping with the demonstrated correlations between levels of anti-MOG and anti-MBP Abs and inflammatory signs revealed by MRI and cerebrospinal fluid analyses.

The appearance of Ab-proteases at the pre-early (*preclinical*) stages of demyelination to

The results we had obtained would allow for approaching to a novel generation of diagnostic technologies based on the protocols of clinical and preclinical diagnostics to

1. isolation of individual Ab-proteases from sera of MS patients or persons to be at risks

p 0.376)

fragments.

(Kuhle et al., 2007)

**5.1 Diagnostic protocol** 

The protocols would include:


A phenomenon of the *pre-clinical* formation of the abzyme-based armamentarium and Abproteases, in particular, would be interpreted as a portion of the proper immune response of the body to the environmental and other shifts. So, a selection of the mode of action for proteolytic antibodies would depend on the metabolic motivation occurring at a particular stage of the disease. Of great interest would be driving motivation to control the mode of action at the *preclinical* stages.

The occurrence of antibody proteases among healthy individuals is due to the development of the pre-early immune imbalances and formation of the preclinical conditions. So, when bursts of the antibody-associated proteolytic activity or the tendency for the latter were evident, the pre-early stages preceding formation of CIS and the exacerbation of the disease could be predicted, even with no observable clinical manifestations. There was definite correlation between the interval before CIS and autoAb (including antibody proteases) status. The occurrence of antibody proteases and the degree of their activity were significantly associated with symptomatology/severity of CIS and clinical course of the disease.

#### **5.2 Sequence specificity and clinical implementation**

Sequence specificity of Ab-proteases is the capacity to distinguish or not distinguish between particular epitopes in the MBP molecule. Serum anti-MBP auto-Abs obtained from MS patients exhibited sequence-specific proteolytic cleavage of the MBP molecule. Such antibody proteases revealed significant correlations with different manifestations of MS (i.e., progressive and/or remittance phases of the disease), with Expanded Disability Status Scale scores and thus with scales of demyelination and, moreover, with the degree of disability of MS patients. For instance, anti-MBP antibody proteases that are able to recognize 43-68 and 146-170 amino acid sequences within the MBP molecule are predominantly more typical of MS patients, but not of patients with neurodegenerative diseases other than MS. In MS patients with low disease activity (in remission), a family of antibody proteases has been detected with low proteolytic activity that predominantly targets 43-68 and 146-170 amino acid sequences within MBP. Progression of disability in MS patients is accompanied by the bursts of anti-MBP antibody-associated sequence-specific proteolytic activity. Moreover, signs of a trend in the specificity of the activity were found (i.e., specificity of the sequence recognition from 43-68 and 146-170 amino acid sites with low affinity to 81-103 and 82-98 amino acid sites, demonstrating that high-affinity indices are recognized by anti-MBP antibody proteases). (Ponomarenko et al., 2006)

#### **5.3 Perspectives in the application of catalytic antibodies for clinical medicine**

More than a hundred of abzyme-catalyzed reactions have been described. Catalytic efficacy of some abzymes is comparable with those of enzymes, but the specificity of abzymes is even higher. The practical relevance of abzymes is due to the unique feature drastically distinguishing them from other biocatalysts, i.e., abzymes could be produced for the catalysis of not only all the reactions occuring in the living systems but also be designed for the development of principally new catalysts with no natural counterparts. (Gabibov et al., 2002; Zhou et al., 2002) In this sense, we can anticipate that manmade catalytic antibodies

Antibody-Proteases in the Pathogenesis of Autoimmune

EAE - experimental autoimmune encephalomyelitis MOG - myelin oligodendrocyte glycoprotein

List of abbreviations: MS - Multiple sclerosis CNS - central nervous system autoAbs – Autoantibodies catAbs *-* catalytic antibodies Igs – immunoglobulins TS - transition states MBP - myelin basic protein

HSP – heat shock protein

PLP - Proteolipid protein

**7. References** 

67]

MAC - membrane attack complex

SLE - system lupus erythematosis CIS - clinically isolated syndrome

104(13):5584-5589

CDMS - clinically definite multiple sclerosis EDSS - Expanded disability status scale MRI – magnetic resonance imaging

Demyelination and Monitoring Patients with Multiple Sclerosis 487

circulating drugs before initiating toxic effects on nervous system and other tissues. Catalytic Abs directly affecting the physiologic reconstruction of tissues and organ systems with complex architectonics including neuroglia are of special value. Neurology is estimated as an interesting area for abzyme application since Abs can be used for the induction of

Abraham S., Guo F., Li L.S., Rader C., Liu C., Barbas C.F. 3rd., Lerner R.A., Sinha S.C.

Altmann D. (2005) Evaluating the evidence for multiple sclerosis as an autoimmune disease.

Angclucci F, MirabeIIa M, Frisullo G, Caggiula M, Tonali PA & Batocchi AP, (2005) Scrum

Belogurov AA, Kurkova IN, Friboulct A et al.' (2008). Recognition and degradation of

Berger, T., Rubner, P., Schautzer, F., Egg, R., Ulmer, H., Mayringer, I., Dilitz, E.,

England journal of medicine Vol.349, No.2 (July 2003) ISSN 139-45

Archives of neurology. Vol.62, No.4 (April 2005) ISSN 688-9

*markers.* Vol.21, No.2 (2005) ISSN 49-55.]

Synthesis of the next-generation therapeutic antibodies that combine cell targeting and antibody-catalyzed prodrug activation. Proc. Natl. Acad. Sci. USA 2007,

levels of anti-myelin antibodies in relapsing— remitting multiple sclerosis patients during different phases of disease activity and immunomodulatory therapy. *Disease* 

myelin basic protein peptides by serum autoantibodies: novel biomarker for multiple sclerosis. Journal of immunology. Vol.180, No.2 (January 2008) ISSN 1258-

Deisenhammer, F. & Reindl, M. (2003) Anti-myelin antibodies as a predictor of clinically definite multiple sclerosis after a first demyelinating event. The New

remyelination and the restoration of previously lost glia functions in MS.

will have considerable practical potential in many different medical applications, i.e., would form the frame of medical abzymology.

Medical abzymology as a novel trend in medical immunology and enzymology, and a new avenue in the clinical practice appears to demonstrate a revolutionary growth today. The antibody catalysis as itself and regardless to a defined field of medical application appears to stress a new area of medical research and a novel field of medical application that provoke considerable interest for medical investigators and clinical practitioners. (Suchkov et al., 2006; Suchkov et al., 2001)

Autoimmune diseases actually represent a challenging frontier in contemporary medical research and clinical practice, and, thus, in areas overlapped with medical abzymology. The proper relation of clinical autoimmunity to the generation of natural catalytic antibody response is absolutely evident, and, in general terms, the phenomenon of autoantibody catalysis can potentially be applied to isolate efficient catalytic domains directed against autoimmune epitopes pathogenically and clinically relevant. This can be done by exposing the autoimmune repertoire to identify autoantigens or their mimicking counterparts capable of recruiting the germ line genes encoding the catalytic site.
