UA126558C2 - Anti-msrv/herv-w env ligand for the prevention and/or treatment of progressive multiple sclerosis - Google Patents

Abstract

The invention relates to an anti-MSRV/HERV-W Env ligand for use in the prevention and/or treatment of progressive multiple sclerosis, wherein said anti-MSRV/HERV-W Env ligand comprises each of the complementarity determining regions (CDRs), and a method of preventing and/or treating progressive multiple sclerosis comprising administering the anti-MSRV/HERV-W Env ligand to a subject.

Description

The invention relates to the anti-M5AM/NENMU-M/Epm ligand for use in the prevention and/or treatment of progressive multiple sclerosis, where the specified anti-M5BM/NEVMU-M/Epu ligand contains each of the sites that determine complementarity (СОН5), and the method prevention and or treatment of progressive multiple sclerosis, which includes the administration of the anti-MOAM/NEVM-M/ Epu ligand to the subject.

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Ha; : MON: WE OZ EY SHOCK - BLAME! x Y y o sh: " Your tires s s NN vo sechi MM hard s MU tagrdy za, V 8. z p y ren v : kah te 7 I Z : y du Z: Ж Fo sh yi 2 zh si

Ou Zha Fo and 77 dija Vo:

Oh look. di mistletoe with - o

There were 5 kilos of solid MU in the roll

In Myky NemMY vody EV st ZEO EMMOEMM

CHUG. WITH

MO - nitrogen oxide

NSKU inducible synthesis of nitric oxide I

TME "tumor necrosis factor 1 - Eterlenkiv

ХВ I'm a comtrap

Field of invention

This invention belongs to innovative compounds and compositions for the prevention and/or treatment of a newly discovered damaging mechanism that blocks the ability to restore endogenous myelin in the mature nervous system (NS) in diseases associated with the expression of the sheath protein (EMM, epmeiore rgoivip) NEVM- M/ (epidodepoiv5 heigomigy5, an endogenous retrovirus of the human MU type), in particular, its subtype MOVU (tyiriye zsiehoviv-azosiaied heigomigyv, a retrovirus associated with multiple sclerosis).

This new therapeutic approach combines the inhibition of upper, pathogenic influences

Epm on oligodendrocyte progenitor cells (ORS»5, oiidodepagosuie rhesig5og seiIv) together with inhibition of downstream pathogenicity effectors of Epm on ORS differentiation (MO-radicals), which, as currently shown, block remyelination in NECM-MU/-associated diseases, affecting the nervous system.

The present invention relates to therapeutic compositions containing (i) at least one anti-NEVMU-M/ Epm ligand, and/or (ii) at least one drug that inhibits free radicals of nitric oxide (MO, Mygis Ohude), used for prevention and/or treatment of remyelination blockade in diseases associated with the expression of the envelope protein (EMM) NEKM-MU, in particular its subtype MB5KM.

A composition in which both a ligand and a drug that inhibits MO are combined affects the upstream Epm inducer as well as the downstream MO effectors in the above-described process that causes blockade of remyelination in NS lesions.

In addition, the synergistic effect of two types of compounds (targeting Epm and MO, respectively) increases the speed and efficiency of therapy in patients with non-remyelinating lesions that are caused by the activation of NECM-MU (in particular, associated with the M5CM-subtype, which is isolated as a particle of virions from cells M5') and the expression of its Epu protein in the nervous system.

Detailed description of the invention

In accordance with the first aspect, the present invention relates to the ligand anti-NENKMU-M/ Epua, which is used for the prevention and/or treatment of blockade of remyelination in diseases that are associated with the expression of the envelope protein (EMM) NEKEM-MU/, in particular its subtype MEM.

In the present invention, the terms "blockade of differentiation" or "blockade of remyelination"

It is used to generalize a recently discovered phenomenon consisting in (i) inhibition of the differentiation of oligodendrocyte progenitor cells (ORS5, oIidodepagosuie rhesigzog seiYi5), which is caused by blanching of the NEKM-M/ membrane (in particular, the M5OKM subtype), (ii) in the final production of MO with

ORSZ5, and (iii) final inhibition of myelin production ORS"5, as described in Example 1.

Diseases associated with the expression of the envelope protein (EMM) of NECM-MU, in particular its subtype M5CM, are defined as NECM-M/-associated diseases affecting the nervous system, which are mainly, but not limited to, multiple sclerosis (M5, ptiirie v5siegov5iv), mainly relapsing remitting multiple sclerosis (KKM5, getichcipd-heIarzipud tiyPirie 5siego5iz), progressive multiple sclerosis, such as secondary progressive multiple sclerosis (5РМ5, zesopaagu rgodgebvzime tiyKirije zsiego5i5) or primary progressive multiple sclerosis (ПРМ5, rgitagu rgodgezzime tiyirie z5siegozi5) , chronic inflammatory demyelinating polyneuropathy (CIOR?, sPpgopis ipPyattayogu etueiipaipud roiupeigoraiiu), mental disorders such as schizophrenia or bipolar disorders, for which myelin damage is also described.

Usually, lesions in multiple sclerosis (M5) are associated with immune-mediated loss of oligodendrocytes and myelin sheaths in axon damage. The effect on immune cells, which is mediated by the envelope protein (EMM) of the retrovirus associated with multiple sclerosis (M5KM, Myirie Zsiegov5i5 azzosiagei heigomiga5) from the NEKM-MU family of endogenous retroviruses, in association with inflammatory lesions of the nervous system (NS), has also been described. However, the direct pathogenicity of M5EM-Epm on glial cells, involving the blockade of remyelination of plaques by Mo5 oligodendrocyte progenitor (ORP) cells, was not previously known and has never been demonstrated.

According to one aspect of this invention, the inhibition of the upper pathogenic effects of Epm on oligodendrocyte progenitor cells (ORS3) is carried out due to the ligand anti-NENM-M/

Ep. The anti-NENU-MU Epm ligand of the present invention is determined by its ability to bind to the Epu protein.

The term "binding" or "attachment" of a ligand means at least a temporary interaction or association with a target antigen, such as an EMM, which includes fragments containing an epitope.

According to the present invention, NEKM-UM EMM means a protein that is encoded by the epm gene of any member of the NEKM-MU family, including the MKM subtype, as determined from the sequences that are determined in the RNA of retroviral particles from M5"» 27,

The ligand according to the present invention can also be defined as the one contained inside the recombinant protein 5cEm (zipdie-spaip itadtepi oi magiarie hediop, single-chain fragment of the variable region), Har-fragments (Hadtepi apiidep Bipaipo, antigen-binding region), antibodies, while this antibody can be polyclonal, monoclonal, oligoclonal, chimerized, synthetic or humanized, or human antibody. In a specific aspect of the invention, the antibody containing the ligand is a humanized or human Id (immunoglobulin) 0, and more preferably Ids1 or Idsai.

More specifically, the ligand according to the present invention contains at least one, and more preferably each of the sites that determine complementarity (SOK5, sotrietepiagu-deieptipipu hediopv), having the amino acid sequence ZEO IU MO: 1, 5EO IU MO: 2, 5EO IO MO: 3, 5EO AND MO: 4, ZEOIIUO MO: 5 and ZEO IYU MO: 6.

The above-mentioned ligand contains at least one and, more preferably, each of the complementarity-determining regions (SOC»5) encoded by ZEO IYUO MO: 25, 5EO IYUO MO: 26, 5EO I

MO: 27, ZEO IU MO: 28, 5EO IU MO: 29, 5EO IO MO: 30.

As mentioned above, blockade of remyelination also leads to the production of MO cells

ORS. According to a further aspect, the present invention also relates to a medicinal product that inhibits the nitric oxide radical, which is used for the prevention and/or treatment of remyelination blockade in diseases that are associated with the expression of the envelope protein (EMM) NEBM-MU, in particular its subtype MB5KM .

Diseases associated with the expression of NECM-MM EMM are defined above.

In a more specific aspect of the invention, a drug that inhibits nitric oxide free radicals or a drug that inhibits MO is any drug that can inhibit the biological effects of MO molecules, also called MO radicals. The drug that inhibits MO is selected from Me-nitro-I-arginine methyl ether (I-MAME), 5-methyl-isothiourea (5MT), fumaric acid and dimethyl fumarate (OME).

In another aspect, the present invention relates to a pharmaceutical composition that combines at least an anti-MONAM/NENM-UU Epm ligand, as defined above, and at least one drug that inhibits the nitric oxide radical, as defined above. The specified composition is used for the prevention and/or treatment of remyelination blockade in diseases associated with the expression of the envelope protein (EMM) NEKM-MU/, in particular its subtype M5KU, the diseases defined above.

A pharmaceutical product containing an anti-NENM-M/Epm ligand, as defined above, and a drug that inhibits nitric oxide, as defined above, is considered as a combination product for simultaneous, separate or time-spaced administration for the prevention and/or treatment of blockade of remyelination in diseases associated with the expression of the envelope protein (EMM) NENMU-MU, in particular its subtype MOKU.

According to the results described below in mouse models of experimental allergic encephalomyelitis (EAE, ehrghitepia!I achoittipe episerpaipotueii5), induced

Epu, a synergistic effect of the combination of the ligand anti-M5AM/NEVNM-UM Epm with at least one of the MO-inhibitory molecules was shown, compared to either of these therapeutic agents used alone. Even when given alone, drugs that inhibit nitric oxide radicals or anti-M5AM/NEVNMU-UM Epm ligands demonstrate therapeutic efficacy in restoring the clinical deficit known to be associated with myelin damage in EAEV. However, a comparison between (ii) a nitric oxide radical-inhibiting drug or an anti-M5AM/NEVU-MU/Epm ligand and (ii) a combination of at least one nitric oxide radical-inhibiting drug and at least one anti-M5BM /NENMU-M/ Epu ligand, demonstrates a great advantage in the use of this combination in the treatment process.

Thus, the absence of inflammatory infiltrates and/or lesions with inflammatory activity (for example, inactive M5 plaques) does not exclude the treatment of remyelination blockade induced by

EPM in patients. This is especially relevant in patients with progressive M5 (primary or secondary progressive form), which is known to lead to multiple lesions without inflammatory activity, which for a long time are not detected by magnetic resonance imaging of the signal with gadolinium contrast in most lesions of the brain and spinal cord.

The present invention relates to a method of prevention and/or treatment of remyelination blockade in diseases associated with the expression of the envelope protein (EMM) NECM-MU, in particular its MCM subtype, which includes the administration of at least one anti-MOVM/NENU-MU Epm ligand to a person .

In a specific aspect, the method further includes the administration of at least one drug that inhibits nitric oxide radicals.

In another aspect, the invention relates to a method of prevention and/or treatment of remyelination blockade in diseases that are associated with the expression of NECM-M/envelope protein (EMM), in particular its M5EM subtype, which includes the administration of at least one drug that inhibits the radical nitric oxide, to a person.

Brief description of graphic materials

Figure 1. Detection of Epm protein and expression of TI K4 receptor in M5 tissues. (A, B) Anti-Epu immunohistochemistry showed the presence of Epu protein (arrows) in MAMUM adjacent to active chronic lesions (B), but not in white matter of healthy brain (A). (B) OiYid2-positive ORS5 in close proximity to Epm-positive microglial cells in the area of chronic active lesion. (G-G") Control healthy brain tissue with ROSEKa (platelet growth factor receptor c)-positive OROS showing co-expression of TI K4 (Toll-like receptor 4) (arrows). (D-D")In ORS5 in MAMUM are close to the active lesion of M5 in patients expressing both TI K4 and ROSEKa (arrows). Scale bar: 50 μm.

Figure 2. Regulation of TI K4 expression during ORS differentiation. (A) Quantitative RT-PCR showing progressive down-regulation of TIIK4 expression during spontaneous differentiation of rat ORS in culture over a period of nine days. SARON was used as a gene for expression control. Data are shown as the mean -/-standard deviation obtained from 3 independent experiments, t-test ("7P<0.001). (B, B) Anti-TI K4 immunostaining showed that upon suppression of TI K4, which mediated small pins

RNA (zPEMA, 5TaiI-Naigrip EMA), transfected ORS»5 were devoid of expression of TI KA (arrow), which indicates the specificity of the antibodies. The ORS shown were recorded three days after transfection, and the transfected cells showed expression of esER (green fluorescent protein). Interestingly, adjacent non-transfected cells were T1K4 positive. (V-G)

Representative examples of galactocerebrosidase (saIS)/T1K4-positive ORS after three days (B, B") and myelin basic protein (MBP)/TIC4-positive ORS5 after six days (G, Г") of spontaneous differentiation in culture Arrows in (G) indicate more highly differentiated ORS only with a reduced level of TI K4 expression, thus reflecting

From the gene expression data shown in (A). Arrows indicate strong expression of less mature cells.

Figure 3. Inflammatory effects of recombinant Epm on rat ORS culture. (A B C)

Stimulation of ORS by recombinant Epm in solution (A, soluble Epm), recombinant surface-bound Epm (B; solid Epm) and membrane-bound Epm (B) expressed in pMUI4Epm transfected 0343 glioblastoma cells, ( Г-Г") results in a strong induction of MOH expression after eight hours of stimulation compared to the respective buffer controls or ORS grown in the presence of rmiI4si transfected 343 glioblastoma cells (Д-Д"). In the sample of recombinant Epm, the level of endotoxin was "5 IU/ml (Epdoiohip Opium 5 in ml) as measured by the G AG test... (H-D") anti-Epm immunostaining of transfected rmi4Epm and rmid4sigi 0343 cells, showing surface Epm expression (E-

(G") Oligodendroglial morphology, as assessed by 04 immunostaining, remains unchanged upon stimulation with surface-bound recombinant Epm, as shown after one day (E, E"), as well as after five days (G, G ") in culture. (3) Indirect spectrometric analysis of cell culture supernatants to measure decay product

MO-nitrite showed a significant increase in dose-dependent MO levels in ORS samples stimulated with soluble recombinant Epm for 24 h. (I-L) Pro-inflammatory cytokines such as TME (tumor necrosis factor-a), II (interleukin)-18 and I1/-6 are up-regulated upon stimulation with surface-bound recombinant Epu. ZARON was used as a control gene. Data are shown as mean values h/- standard deviation, and is 1 of 6 (A), 8 (B), C (C), C (3) and 8 (I-K) independent experiments, respectively, i-test (""P (0.001).Scale bar: 50 µm.

Figure 4. Comparison of sensitivity to NEKM-MU Epm in immature (at the age of one day - ORS) and mature

ORSz" of a rat (at the age of seven days; "mature ORS5" or oligodendrocytes - ОЙ).

Both types of cells were stimulated with 100 ng/ml of soluble recombinant Epu. Despite the significant, compared to buffer controls, activation of iMO5 and cytokines in response to Epm stimulation in OI 5 is less pronounced compared to ORS. The SARON gene was used to control expression. The data are presented in the form of the mean value w/- standard deviation obtained from one of three independent experiments, I-test (Р«0.001, иР«к0.01, Р«О.05).

Figure 5. Epm-dependent reactions of human ORS. (A) Stimulation of cultured human ORS with surface-bound (solid) and soluble recombinant (soluble) Epm leads to a strong induction of IMO5 expression after 24 h. stimulation compared to buffer-treated cells. The SZARON gene was used to control expression. Data are presented as mean values w/- standard deviation obtained from one of three independent experiments, Y-test ("P" 0.001). (B, B") Immunofluorescence staining of cultured CysC positive human ORS revealed a strong signal for TI K4 ( shown after three days in culture). Scale bar: 50 μm.

Figure 6. Experiments on the neutralization of the Epm path. (A) Heat inactivation of recombinant Epmu leads to elimination of the induction of the YMO5 gene compared to native recombinant Epmu after 8 h. ORS stimulation. Two samples, native Epm and denatured Epm (temperature-treated Epm), were applied at a concentration of 1000 ng/ml. (B, C) Decreased expression

YIMO5Z in ORS with pharmacological inhibition of IKAK1/4 and TKIR, respectively. After stage 2 h. after pre-incubation with 3200 nM PKAKT/4 (receptor-associated kinase 1-1) inhibitor or 50 µM TKIR inhibitory peptide ORS was stimulated with surface-bound (solid) recombinant Epm for 8 h. and later lysed. (D) Decreased expression of IMOZ in ORS after antibody-mediated blockade of TI K4 receptors. After stage 2 h. preincubation with an antibody to TI KA at a concentration of 15 mg/ml at 37 "С, ORS was stimulated with solid Epm for 8 hours and then lysed. The SARON gene was used to control expression. The data are presented as mean values h/- standard deviation, and obtained from one of three independent experiments each, Y-test ("P" 0.001, "P" 0.01). (E) Increase in nuclear localization of MEKV after ORS stimulation with surface-bound (solid) recombinant Epm for 8 hours.

Data are presented as mean values x/- standard deviation, and obtained from one of three independent experiments each, i--est ("P<0.001). (E-F") Representative MEKV immunostaining of buffer (control) and stimulated Epm ORS5 . Scale bar: 50 μm.

Figure 7. Generation of Z-nitrotyrosine (3-MT) positive ORS5. (A) Immunofluorescence staining showed that after 24 h. Epm of stimulation (bound to the surface recombinant

Epu) significantly more ORS expressed MO-dependent nitrosyl stress marker 3-MT compared to buffer-treated cells. 5-nitroso-M-acetylpenicillamine (ZMAP), a strong MO donor, was used as a positive control. However, when ORS was incubated with 1-

MAME, yIMO5 inhibitory molecules, at a concentration of 100 μM for 30 min at 37 "С before Epm stimulation, 3-MT positivity was reduced to the control level. Inactive enantiomer O-

MAME cannot eliminate Epm-dependent induction of 3-MT. Data are presented as mean values, x/- standard deviation, and are derived from one of three independent experiments. 1- test (P«0.001). (B-E") Representative anti-3-MT immunostaining of control cells (B,

B"), Epm stimulated ORS" (B, B"), ORS"5 pre-incubated with I! -MAME (G, G), O-MAME (D, D" and

ORS5 subjected to ZMAR (EE, EM. Scale bar: 50 μm. (F-F).

Immunohistofluorescence staining of MAMUM tissue sections of MzZ-patients showed that resident ORS (marked by the expression of their precursor marker ROSEK, arrows) can undergo nitrosative stress, as shown by their 3-MT positivity.

Figure 8. Epm-dependent inhibition of ORS differentiation. (A, B) Immunofluorescence analysis demonstrating that after one to three days of stimulation with surface-bound recombinant Epm, a significantly reduced number of ORS expressed an early myelin marker

SMRavze, also as a late myelin marker of MVRakh compared to control cells. Data are shown as mean values h/- standard deviation, and obtained from one of four independent experiments, i-test ("P"0.001). (8-B") and (G-GU show representative anti-SMRavhe and anti- MVR immunostaining of control and EPM stimulated ORS"5. (D) Incubation

ORS" with 100 μM I-MAME in combination with surface-bound recombinant Epm for three days, results in the cessation of Epu-mediated blockade of ORS differentiation, as shown by anti-MVR immunostaining. Application of O-MAME was unable to rescue myelin expression. The data are presented in the form of mean values w/- standard deviation, and are obtained from one of four independent experiments, y-test (RO, 001, item 5. not significant). (E-3") Representative MVR immunostaining of ORS stimulated with Epm alone, with a combination of Epm and G-MAME, and with a combination of Epm and O-MAME, respectively.

Scale bar: 50 μm.

Figure 9. Expression of ORS of maturation markers after 24 and 72 hours of stimulation in chambers (Iabiekz) pre-coated with M5KM-Epy. The applied MO5KMU-Epm (-Epu-T) induced a decrease in the proportion of SMRase positive ORS after 24 hours. stimulation (A), as well as a decrease in the percentage of MVR of positive ORS after 72 h. stimulation (B), compared to its diluted buffer (buffer).

Data are presented as the mean value -/-standard error of one experiment estimated in 10 replicates (from 410 to 563 counted cells in a group; "p" 0.05 I-test (A) and from 364 to 491 counted cells in a group; " p«0.001 rank test Mann-Whitney (B)).

Figure 10. Expression of SMRase in human ORS after 24 hours of stimulation with MBCM-Epm and ZMBAS1 (humanized monoclonal antibody compared to MKM). (A) Applied M5EKM-Epy causes a decrease in the percentage of SMRase-positive ORS after 24 h. stimulation (ZMBASI1 at 50

NM or 200 nM completely suppresses the effect of M5KM-Epu, with both tested ZMBASI1 treatment protocols (ZMBAS1-A or CMrAS1-B). Data are presented as the mean value w/- standard error from 2 to 8 independent experiments (from 705 to 3878 counted cells per group). (B) ZMBASI (50 pM or 200 pM), applied alone or together with B5A (bovine serum albumin) (1 μg/ml), with both ZMBAS1 treatment protocols tested (ZMBAS1-

A or SMBAS1-B) has no effect on the percentage of SMRase positive OROS. Data are presented as mean value w/- standard error of one experiment estimated in 10 repetitions (399 to 512 counted cells per group; p»0.05; one-way analysis of variance (ANOVA)).

Figure 11. Expression of SMRavze in human ORS culture after 24 hours of stimulation by MOCM-Epm and

SMBAST, which are added to the cultural environment.

M5AM-Epum (3 nM), which is diluted in the cell culture medium, significantly reduces

It will stop expressing after 24 hours. processing SMBACI1 (200 nM) completely inhibited this effect, while it showed no effect on its own. Data are presented as the mean value w/- standard error from 2 to 4 independent experiments (from 927 to 1913 counted cells per group); "p" 0.001 in comparison with the buffer; one-way analysis of variance (AMOMA) followed by the posterior method of Fisher's I 50 (Ie55 zidpiisapse dibiapse, least significant difference).

Figure 12. Expression of MVR in human ORS cultures after 72 hours of stimulation with M5KM-Epm and

FUNNY. (A) Applied MZKM-Epm (Epu-T) induces a decrease in the percentage of MVR of positive ORS after 72 h. stimulation ZMBACI1 at 50 nM partially inhibits the action of MOCM-Epm, and this inhibition is complete at 200 nM, in both tested protocols of the treatment of ZMBACI (ZMBAC1-A or CMBACI1-

IN). (BU M5KMU-Epm (3 nM), added to the cell culture medium, significantly reduces the expression

Ministry of Interior after 72 hours. processing ZMBAST1 (200 nM) completely inhibits this effect (B).

Data are presented as the mean value -/- standard error (A) from 2 to 8 independent experiments (from 924 to 4156 counted cells in a group) "p"0.05; ""p"e0.001

Zo compared to the buffer; fr«0001 compared to Epu-T; one-way analysis of variance (ANOVA) followed by Fisher's 1 5O posterior method, and (B) from 2 to 4 independent experiments (1013 to 1834 counted cells per group; "p" 0.001 compared to buffer; fr" 0.001 compared to Epu- T; one-way analysis of variance (ANOVA) followed by the posterior method of Fisher's I 50).

Figure 13. Expression of ORS maturation markers after 24 and 72 hours of stimulation using M5KM-Epm and SMBAS1 application. Applied M5KM-Epm induces (A) a decrease in the percentage of SMRavze positive human ORS after 24 hours. stimulation, as well as (B) a decrease in the percentage of MVR positive ORS of a person after 72 hours. stimulation (A) OMBAS1 (200 nM) completely inhibited the effect

M5AM-Epu, with both tested protocols of processing SMBAS1T (SMBAS1-A or SMBAS1-B). The data are presented as the mean value w-/- standard error b of independent experiments (from 2805 to 3039 counted cells in a group; "p" 0.05 in comparison with the buffer; fr" 0.05 in comparison with M5KM-Epum; rank criterion Kruskel-Wallis AMOMA-1 followed by Student-Newman-Kale a posteriori analysis and (B) from 5 to b independent experiments (1671 to 2066 counted cells per group; "p" 0.05 compared with buffer; fr" 0.05 in comparison with M5KM-Epm; Kruskel-Wallis AMOMA-1 rank test with further a posteriori analysis of Dunn's test).

Figure 14. M5KM-Epm inhibition of human ORS maturation is completely reversed by ZMBASI. The applied M5AKM-Epm (EMM) induces (A) a decrease in the percentage of SMRase positive human ORS after 24 hours. stimulation, as well as (B) a decrease in the percentage of MVR positive ORS of a person after 72 hours. stimulation zMBACI1 (200 nM) completely inhibits the MBEM-Epmy effect, in both tested protocols of processing of ZMBACI (-ZMBACI A or i«CMrACI1 B).

The results are expressed as the percentage of SMRase (A) or MVR (B) positive cells, and are the mean -7- standard error of 8-14 independent experiments (3600 to 6800 counted cells in group (A) and 2700 to 5800 counted cells in group ( B)) "p"e0.05 in comparison with STEK; tr-"0.05 in comparison with MBEKM-Epm; Kruskel-Wallis AMOMA-1 rank test with further a posteriori analysis of Dunn's test.

Figure 15. Inhibition of maturation of human ORS caused by M5KM-Epm is completely abolished by SOMBASI1 (normalized data). M5AM-Epm was applied to the glass of the culture chambers before ORS inoculation. SMBASI1 (200 nM) was mixed with MOCM-Epm before application (SMBAS1-A) 60 or incubated on culture chambers coated with M5EM-Epm (S.SMBAS1-B). The data are calculated as the percentage of (АХ) SMRase or (B) MVR-positive cells and are expressed as a percentage change in STKI conditions (M5KM-Epm buffer). Results are the mean ± standard error of 8-14 independent experiments (from 3600 to 6800 counted cells in group (A) and from 2700 to 5800 counted cells in group (B)). "p" 0.05 in comparison with STKI, "p" 0.05 in comparison with M5KM-EMM; AMOMA-1 Kruskel-Wallis rank test followed by a posteriori analysis of Dunn's test.

Figure 16. Kinetics of the EAE clinical indicator of Groups A, B, B and E (A - untreated EAE positive controls, B - EAE treated with OMBAs1 200 μg, B - EAE treated with I/-MAME, G-EAE treated with SMBAs 200 μg and I - MAME).

Figure 17. Kinetics of the EAE clinical indicator of Groups A, B, G and F (A - untreated EAE positive controls, B - EAE treated with ZMrBAs1 200 μg, G - EAE treated with 5MT, F - EAE treated

SMrAs 200 mg and TMT).

Figure 18. Kinetics of the clinical indicator of EAE of Groups A, B, D and Z (A - untreated EAE positive controls, B - EAE treated with 200 μg of SMBAsI1, D - EAE treated with UOME, C - EAE treated with 200 μg of ZMBasCiOME).

Figure 19. Kinetics of time on Roiagoay at 23 rpm

X-axis: Days of measured values, according to the protocol. Y-Axis: Increase or decrease of time on Koyagod, relative to the 7th day. A: Groups A, B, "1 injection" and "2 injections" Epm; B: Groups A, B,

YOU ARE; In: Groups А, Б, ГИЖ; D. Groups A, B, Di 3.

Figure 20. Kinetics of time on Koiagoi 26 rpm

X-axis: Days of measured values, according to the protocol. Y-Axis: Increase or decrease of time on Koyagod, relative to the 7th day. A: Groups A, B, "1 injection" and "2 injections" Epm; B: Groups A, B,

YOU ARE; In: Groups А, Б, ГИЖ; D. Groups A, B, Di 3.

Figure 21. Kinetics of time on Koiagoi at 29 rpm

X-axis: Days of measured values, according to the protocol. Y-Axis: Increase or decrease of time on Koyagod, relative to the 7th day. A: Groups A, B, "1 injection" and "2 injections" Epm; B: Groups A, B,

YOU ARE; In: Groups A, B, Gi Z; R: Groups A, B, D 3.

Figure 22. Kinetics of the clinical indicator of EAE Group A, B, VIG (A - EAE false-negative controls, B - untreated EAE positive controls, D - EAE,

Z treated with SMBAs 200 μg, G - EAE treated with SMBAs 500 μg).

Figure 23. Kinetics of the clinical indicator of EAE Group A, B, Di E (A - EAE false-negative controls, B - untreated EAE positive controls, D - EAE treated with TMT, F - EAE treated with SMBAs 200 μg-TMT)

Figure 24. Kinetics of the clinical indicator of EAE of Groups A, B, Z and Z (A - false-negative controls

EAE, B - untreated EAE positive controls, D - EAE treated with OME, C - EAE treated

SMrAs 200 μgOME).

Figure 25. Kinetics of mass increase during the study period (Relative mass increase compared to the day before the first injection of immunogens): Groups A, B, B and G (A - EAE false-negative controls, B - untreated EAE positive controls, B - EAE , treated with SMBAs 200 μg, G - EAE, treated with SMBAs 500 μg).

Figure 26. Kinetics of mass increase during the study period (Relative mass increase compared to the day before the first injection of immunogens): Groups A, B, D and E (A - EAE false-negative controls, B - untreated EAE positive controls, D - EAE , processed

ZMT, Z - EAE, treated with OMBAs 200 μg and 5MT).

Figure 27. Kinetics of mass increase during the study period (Relative mass increase compared to the day before the first injection of immunogens): A, B, Z and Z (A - EAE false-negative controls, B - untreated EAE positive controls, D - EAE , treated with OME,

C - EAE treated with SMBAs 200 μg i-OME).

Figure 28. Kinetics of time on Koiagoi at 16 rpm

X-axis: Days of measured values, according to the protocol. Y-axis: Increase or decrease of time on Koyagod, relative to Day 7. A: Groups A, B, V and G; B: Groups A, B, D and E; A: Groups A, B,

Life

Figure 29. Kinetics of time at Coyagoa 26 rpm

X-axis: Days of measured values, according to the protocol. Y-axis: Increase or decrease in time on Koyagod, relative to Day 7. A: Groups A, B, C, and D; In: Groups A, B, Di E; C: Groups A, B,

Life

Figure 30. Kinetics of time on Koiagoi at 29 rpm

X-axis: Days of measured values, according to the protocol. Y-Axis: Increase or decrease in time on Koyagod, relative to Day 7. A: Groups A, B, C, and D; A: Groups A, B, D and E; C: Groups A, B, 60 / ZhiZ. b

Figure 31. Kinetics of the clinical indicator of EAE of all groups during the study period (A - sleep-negative EAE controls, B - positive controls falsely treated with an antibody isotype, B - EAE treated with OMBAs 500 μg, G - EAE treated with sodium fumarate and D - EAE , treated with OMBAs 500 μg - sodium fumarate).

List of abbreviations used

Abbreviation | 777

The following examples are illustrative and do not limit the invention.

Example 1: Blockade of remyelination induced by NEKMU-M/family bileuome (inhibits the differentiation of oligodendroglial progenitor cells in demyelinated lesions of the nervous system). 111 Materials and methods 1.1.1. Oligodendrocyte progenitor cell culture

Rat oligodendrocyte progenitor cells (ORS5) were purified as previously described??7. ORS were maintained in proliferation medium (Za medium supplemented with 10 ng/ml recombinant human basic fibroblast growth factor and 10 ng/ml recombinant human platelet-derived growth factor-AA; KO Zuzietv5, UMezbradep-Mogaepvsiaii, Germany), while differentiation initiated with medium supplemented with 0.5 95 fetal calf serum. Fetal human oligodendrocyte progenitor cells (HOPCs) and the appropriate medium were obtained from ZN Viotäläisä, Orsåja, Sweden. Recombinant Epm was obtained and purified by means of RH RH-Tpegareshis (Sgepobrier, France) in accordance with the requirements of OS bSeMeigo (Sepema, Switzerland) supplied protein lots. Endotoxin levels were 5 OU/ml as measured by the IGitiish5 amebocyte lysate test (CA). Epm stimulation was carried out using three different approaches using i) recombinant full-length Epu protein diluted in differentiation medium at 10 ng/ml, 100 ng/ml and 1000 ng/ml, and iii) recombinant Epu protein applied to culture plates plates for cells at a surface concentration of 17.53 ng/cm: and iii) by plating ORS on transfected 0343 glioblastoma cells with Epm overexpression, respectively. Control experiments on stimulation were carried out using a buffer of recombinant Epm (20 mM histidine, 5 wt/vol. 9) sucrose, 0.01 wt/vol. 96 polysorbate 20, pH 6.0) with equal dilutions. The MO concentration in the ORS supernatant was measured using a colorimetric kit for the analysis of nitric oxide (Megok-mMiProge/SaIriospet, Baptista, Germany), and the absorbance was determined at 540 nm using a tablet reader Apipo5 2001 (Apipo5 Iaries ipzigitepov, zaiI27bygu, Austria). Evaluation of the morphology of ORS was carried out using anti-0O4 Megesk-

MiPiroge/Spetisop, Oatviavdia, Germany) taking into account the diameter of the cells and the degree of branching

From the process. ICAK-1/4 inhibitor I (1-(-2-(4-morpholinyl)ethyl)-2-(Z-nitrobenzoylamido)benzimidazole, M-(2-morpholinylethyl)-2-(Z-nitrobenzoylamido)benzimidazole; ZidtalL| Iagis, Natbygo, Germany), experiments were performed at a concentration of 2400 nM, TRIIE inhibitory peptide (Ipmimodep, San-

Diego, USA) experiments were performed at a concentration of 50 µM according to the manufacturers' protocols. Epm inactivation was carried out by exposure to recombinant Epm at a temperature of 123 "C for 1 hour. Experiments on blocking the TI receptor with the P4 antibody were carried out at an antibody concentration of 15 μg/ml. Experiments on blocking I-MAME/O-MAME were carried out at a concentration of 100 μM of each; 5-nitroso-M-acetyl-OI-penicillamine (ZMAP) was used at a concentration of 100 ng/ml. 1.1.2 Oligodendrocyte precursors and transfection of 0343 glioblastoma cells

ORS was grown for 24 hours. in the medium for proliferation, and transfection was carried out with the help of the Mapouitse reagent (Megsk-Mijrohe, Darmstadt, Germany) using the encoding 5PEMA TI K4 suppressor vector based on the Ni5H 29 rogR-U-5 vector (Ogisepe, Rockville, Maryland, USA) for proof of the specificity of anti-TI KA antibodies.

For the expression of membrane-bound Epm in SHZ343 glioblastoma cells, the vector expressing citrine for visualization of transfected cells was combined with increased expression of Epm (rm14 vector from 1 to 1629 bp. NEKEU-M/ M5EKM type encodes sequences of the epm gene-sepVapk AEZ331500.1- supplied by Sepeigo ZA, Switzerland) at a ratio of 1:5. The corresponding empty vector was used as a control. Transfection of 0343 glioblastoma cells was carried out using

Proitesiatipe (I be Thesppoiodieh, Darmstadt, Germany). 1.1.3 Staining of multiple sclerosis tissues

For OiD2/Epm double staining, formalin-fixed paraffin-embedded M5 human tissues were cut and 5-μm sections were deparaffinized with xylene and rehydrated by threading through various concentrations of alcohol and distilled water. Endogenous peroxidase activity was inhibited by incubating the slide in a 0.3 95 hydrogen peroxide solution in methanol. Then the glass was washed with distilled water and transferred to 10 mM Tris, a 1 mM solution

EDTA (pH 9) to achieve heat-induced antigen unmasking. Next, the sections were cooled to room temperature, washed in phosphate-buffered saline (PB5) and incubated with anti-OiID2 (1:300; Megsk-Mijiroge) and anti-Epm antibodies ZB2NA (1:500, SeMeigo,

Geneva, Switzerland) overnight at 4 °C. Sections were then incubated with Aexa 488-labeled donkey anti-rabbit antibodies (1:400; Ie Tesppoiodiez/MoiIesschag Rgore5, Darmstadt,

Germany) for the purpose of detection of Oiid2 and AIekha 555-labeled donkey anti-mouse antibody for Epm visualization. For TI K4/ROCEC double staining, sections were prepared from frozen tissue, and 5 μm sections were incubated with anti-TI NA (1:100; Megsk-Mijiroge) and anti-?ROSCase antibody (1:50; eViozsiepse5, San Diego, CA , USA) overnight at 4 °C. TI K4 was detected using AlIeH 488-labeled donkey anti-mouse antibody (1:400; Ihe TespoIodiez/MoiIesshag

Rhobrekh) and ROSEKa with a biotinylated rabbit anti-rat antibody (1:500; Mesiog I aroghaiogye5,

Vigdate, CA, USA) and 647-labeled streptavidin (1:400; Ie Tesppoiodiez/MoiIesshag

Rgorevs), respectively. To stain the nuclei, the sections were incubated with a solution of Noespvi dye (1:100;

Zidta-AiIdgisj, Hamburg, Germany) for 1 minute and covering Mesiazpiviy (mMesiog

And arogaphogesis). Microscopic analysis was carried out on Geisa TSb5 5P 2 AOV5 confocal laser scanning microscope (Iisa Misgozuzie tv, NeideiIregd, Germany). 1.1.4 Staining of cultured cells

Staining of cultured cells fixed in formaldehyde was performed as previously described22. Primary antibodies were diluted as follows: anti-TI K4 antibodies (1/1000;

Matgek-Mijroge, Darmstadt, Germany), antibody to Epm ЗВ2Н4А4 (1/500; SeMeigo, Geneva,

Switzerland), antibody to 2", 33'-cyclic nucleotide 3-phosphodiesterase (SMRase) (1/1000;

Somapse, Princeton, NJ, USA), monoclonal antibody against myelin basic protein (MBP; 1/1000; Somapse, Princeton, NJ, USA), polyclonal antibody against myelin basic protein (1:1000; Miroghe, Schwalbach, Germany), antibodies against

From galactocerebroside (CaIe), anti-04 antibodies (both 1/1000; Megok-MiiProge, Darmstadt,

Germany); anti-3-MT antibody (11000; Areat, Cambridge, MA, USA) and anti-MEKV antibody (111000; Areat, Cambridge, MA, USA). AIeha Risog 488- and AIeha Riog 594-conjugated antibodies (both 1:500) were used for signal visualization. Nuclei were stained with 4,b-diamino-2-phenylindole (SARI; Cospe, Basel, Switzerland). 1.1.5 RNA preparation, cDNA synthesis and quantitative polymerase chain reaction with reverse transcription

Purification of RNA from cultured cells was carried out using the KMease method (Oiadep,

Niidep, Germany). The isolated RNA was subjected to reverse transcription using a high-efficiency cDNA reverse transcription kit (Ie TesppoiIodiez/Arriea

Viosuziet5). Quantitative determination of the level of gene expression was performed on the 7900 NT sequence detection system (I ie Tespoiodiez/Arriiei Viosoueiet5) with the help of Rozheg Zubgogeep ipimegzai! tavhieg tih (Gie TesppoiIodiez/Arriiei Viozuzietv5). The sequences of the primers were determined using the software RgitegEhrgez5 2.0 (Pe TesPpoiodiez/Arried

Viozuzietv) and checked the specificity of the amplicons: pt vm to SOT OT OZ ASA KONYA

TEKYA gu: DOI T TASAASSSTSTOSTSS FAILED Me: VI

CHO vva ST KASSASAVASSS SAAASVIVKO TSO MO

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TMBa in ABOYUSSTOOSTATOAOOVOSSSATOTA VES MM TV

Ta gam SOS OASTSSOTOATOTSTAAS ES S MO U

CIV VU OALASAOSAATOSTOVOVAS ES O Mo TI

Sho U: ААСАСАСООЙUT TOSATOV TO GE NO MO TI

PO VEK STTOTOSAATOZOSAATTSTOACHKO NO MK 19

In pe TOTOASASTOSATSATSOSTO VKKS OO Me: TO

SARON Me: BAASOVOLAOSTSASTOVOS (ZESNO Me: HO)

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OO LK OT OSASVAVOSSAAASATOS BO Me: Ya

OO tem ZTTOSSASA TT OAE AS ZKO Y MOo

SARON and O0C were used as reference genes, and the relative expression levels of the genes were determined according to LACE (I ee Tespoiodiez/Arried Viosubzietv5). Each sample was measured in four replicates; data are presented as mean values w/standard deviation, and the t-test was used to determine statistical significance. 1.2 Results 1. 2. 1 Localization of Epm protein close to chronic active M5 lesions and in the vicinity of resident ORS

Using immunohistological analysis, we studied the localization of Epm protein in a sample of human brain tissue. This data showed a fairly abundant presence of Epm protein in MAMMM close to chronic active MO lesions (SAI., Fig. 1B), as well as in the vicinity of SAI in the immediate vicinity of OD2-positive ORS (Fig. 1D). MAMUM at a greater distance to the lesions does not show noticeable immunoreactivity (Fig. 1A). In order to further prove the potential effect of the Epm protein on resident ORS and, thus, on the appropriate repair of myelin, we additionally looked for TIK4 and platelet-derived growth factor alpha receptors (ROSEKa) - double-positive ORS5. We could detect them in the healthy brain (Fig. 1D-D") and in MAUUM M:5 (Fig. 1D-D").

It has been clearly demonstrated that in the M5 brain, TI K4-positive ORS5 can be detected in close proximity to NECM-M/Epm expressing cells or the corresponding secreted protein, making them acceptable targets of these TI K-4 pathogenic agonists, as described 10 Nevertheless, this is an unexpected finding and opens up unprecedented prospects for application, because TI K4 was not previously known to be expressed under such conditions in ORS and, moreover, as demonstrated later, because NEKM-M/ coat protein, such as M3KM-

Erm, can't, therefore, . be considered for direct interaction with ORS at this particular stage of differentiation with ultimate dramatic effects on their myelination potential. 1.2.2 TI Kk. expression by cultured rat and human ORS

Previous studies, in fact, indicated TI P4-dependent pro-inflammatory effect on monocytes and

From a dendrite cell!?. Apii-TIK4 immunolabeling confirmed the expression of this receptor on the surface of oligodendroglial cells of rat and human precursors (Fig. 28, Fig. 5B). In addition, the specific 5PEMA-mediated knockdown of TI K4 in the ORS of rats substantiated the specificity of the previous immunolabeling (Fig. 2B, B). In order to evaluate the kinetics of receptor expression

TIC4, from young to mature ORS, we co-stained using antibodies to galactocerebroside (Cas) as well as antibodies to myelin basic protein (MBP), a marker for more mature ORS, after three and six days of differentiation in culture (Fig. 28-G").

Unexpectedly, it was found that the expression of the TIC4 receptor gene was repressed after some time in the process of cell differentiation (Fig. 2A), while the specific detection of the TIC K4 protein gave only a weak signal at late time points in morphologically mature rat ORS (Fig. 2D). , compared to younger cells. 1.2.3 Stimulation of EPM ORS by induced expression of pro-inflammatory cytokines and induced

MO synthases Stimulation of rat ORS with recombinant Epm protein dissolved in the medium (soluble Epu; 100 ng/ml; Fig. 3A) applied to cell culture dishes (solid

Umm;. Fig. ЗB) or overexpressing on the surface of transfected glioblastoma 0343 cells (PM14Epu; Fig. C B-D") led to a strong increase in the transcription of inducible nitric oxide synthase (NOS), as well as to the induction of pro-inflammatory cytokines such as TMEa; 1-18 and 11 -6 (Fig.

Z I-L) compared to control (buffer or empty processing vector) conditions. In turn, increased expression of IMO5 leads to an Epm concentration-dependent increase in its nitrosative stress, forming a product of nitric oxide (NO) in cell supernatants (Fig. 3).

Interestingly, the morphology of the rat ORS remained unchanged during Epm stimulation, as shown by 04 staining of oligodendroglia performed after one and five days of exposure to Epm

(Figs. E-G"). CaIS-positive human ORS were also found to express TC4 (Fig. 5B, B) and stimulation with recombinant Epm (both in solution and as applied to the plate surface) similarly induced IMO5 transcription ( (Fig. 5A).Furthermore, we found that Epm stimulation did not affect either the survival of ORS (as shown by TIMEIG and quantification of total cell number; data not shown) or the induced senescence phenotype of ORS (examined by staining for p -galactosidase; data not shown.) Epm stimulation of mature rat ORS (tayuorRc; kept in differentiation medium for six days) resulted in a significantly weaker, pro-inflammatory response, as determined by the level of transcription of YMO5,

TMEa, 1-16 and I/-6, in comparison with the effect on immature ORS (Fig. A). This observation is in accordance with the data on the lower level of expression of TI K4, found in mature ORS (Fig. 2), and confirms the excellent sensitivity of immature cells in relation to Epm, which corresponds to the level of Ti K-4 expression. 1.2.4 Epm, which mediates its effect through TI CA and the pro-inflammatory response includes

IRAQ 1/4, TAE ii MEKV

In order to prove the specificity of Epm to TIC4 and shed additional light on downstream signaling, we conducted a number of control experiments (Fig. 6). Thermoinactivation of recombinant Epm before ORS stimulation leads to a very significant reduction in induction

IMO compared to the control group (Fig. bA). A similar significant reduction of iMO5 transcription can be observed when using an antibody-mediated blockade of TI K4, thereby confirming the specificity of Epm to this receptor and its relevance to the observed downstream pro-inflammatory effects (Fig. 6D). In order to shed light on the intracellular pathways of TI K4 activation by Epm, we investigated the role of two known downstream pathways following TI K4 activation, the Mur88-dependent pathway involving IKAK-1/4 (interleukin receptor 1-associated kinase- 1/4) and mMurv8- an independent pathway involving TKIE (TIC domain-containing adapter inducing interferon-B).

Application of ICAC-1/4 inhibitor I or TKIR inhibitory peptide resulted in a significant decrease in the level of IMO5 expression in the presence of Epm (Fig. 6B, C) demonstrating that Epu-mediated activation of TI K4 leads to the activation of both Mur88-dependent as well as mMurvv - an independent signal transmission path. Both paths can converge on MEKV

Zo (nuclear factor light chain enhancer type K of activated B cells) of nuclear translocation.

Using an anti-MEkKV antibody, we confirmed that Epm stimulation leads to a strong increase in ORS with nuclear localization of MEKV (Fig. 6D-G"), providing an explanation for the observed transcriptional activation of pro-inflammatory cytokines. 1.2.5 Epu-mediated induction of the nitrosative marker nitrotyrosine stress

MO, the level of which was increased after Epm stimulation (see Fig. 3), is the active form of nitrogen (EM5). MO can react with many intracellular molecules, including proteins, nucleic acids and lipids, resulting in the formation of 3-nitrotyrosine residues (3-MT).

We found that the effect of recombinant Epm proteins on rat ORS leads to a strong increase in 3-MT-positive cells compared to buffer controls (Fig. 7B, B" and B, C"), which indicates

MO-mediated induction of nitrosative stress. However, after pre-incubation with H-MAME (I-Me-nitroarginine methyl ester) IMO5 inhibitory molecules, MO production was found to be significantly reduced (data not shown), as was the formation of 3-MT positive

ORS (Fig. 7D, GU. When using O-MAME, an inactive enantiomer of I-MAME, as a negative control, EMM-mediated 3-MT formation was not disturbed (Fig. 7D, DU. On the other hand, the use of ZMAR ( 5-nitroso-M-acetylpenicillamine), a strong MO donor, served as a positive control and resulted in nitrotyrosinylation of almost all ORS (Fig. 7E, E").

It is important to note that Z-nitrotyrosine positive ORS, detected due to the expression of their precursor marker ROSEK, can also be detected in MAMUM brains of patients (Fig. 7 G-

Ж"), which indicates a pathologically similar stressor mechanism in M5. 1.2.6 Epm affects the expression of myelin in ORS

After these findings, we investigated whether the Epm protein can affect the processes of oligodendroglial differentiation, which have been shown to be necessary for the possibility of myelin recovery. For this purpose, Epm-stimulated ORS of rats were evaluated relative to the level of expression of myelin proteins CMRavze (2", 3'- cyclic nucleotide 3-phosphodiesterase) after three days of Epm stimulation and MVR (main myelin protein) after six days of stimulation, respectively.

The results showed that Epm strongly reduced the number of SMRase and MVR-positive cells (Fig. 8A-G") in contrast to unchanged expression of the progenitor marker 04 (Fig. C E-G"). This indicates a significantly disturbed reaction of cell differentiation in the presence of Epu protein. However, addition of II-MAME but not O-MAME in parallel with Epm stimulation can restore expression

MVR (Fig. 8 D-3), which indicates a direct relationship between the formation of 3-MT due to nitrosative stress and a reduced ability to differentiate ORS. 1.3 Analysis of results

Inefficient remyelination of NS in neuroinflammatory demyelinating diseases, such as

M5 is believed to be primarily caused by a decrease in the ability of resident ORS to properly differentiate and remyelinize demyelinating axons!""C, but no major and upstream pathogenic molecule that causes blockade of such remyelination was known. Previous studies have already shown that the Epm envelope protein from NEKM-MU, also called "Multiple sclerosis-associated retroviral element" (MEM), when obtained from the corresponding retroviral particles, has a pro-inflammatory effect on mononuclear cells, activates the innate immune system, which, in turn, they produce the main pro-inflammatory cytokines!"!o. However, a direct relationship between Epm and a decrease in the ability to differentiate ORS has not yet been shown, and could not be assumed. Here we have shown that this Epm protein can be detected in M5-affected NS tissue and that Epm can interfere with the differentiation of ORS due to the previously unknown presence of the Epm receptor, TI K4, on ORS at a certain stage of differentiation.

This harmful effect is due to IMO5. This stress response leads to the formation of nitrotyrosine and directly affects the expression of myelin protein. I wonder what

Epm stimulation did not affect the degree of survival of ORS cells, and cell morphology remained unchanged, suggesting that Epm-mediated signals target cytoskeletal elements. It should be noted and confirmed the novelty of the unexpected results, previous studies claim the absence of TI K4 in oligodendroglial cells in the human brain". However, in contrast to this opinion, we could clearly show that

THRESHING FLOOR. expressed not only on cultured primary ORS (of both rat and human origin), but also on ROSECa-positive resident ORS in M5 human tissue. Such differences may be due to the fact that Lenhardt and his colleagues used the more general marker 04 for the detection of oligodendroglial cells, while we relied instead on the recently accepted precursor marker ROSEK-th. Nevertheless, for the first time, we showed this peculiar expression pattern of TI K4 in ORS, while we did not believe in its existence. In addition, we found a strong inhibition of TI K4 during maturation of ORS, which suggests that the expression of the receptor and, therefore, susceptibility to Epma, is limited to immature cells. Nevertheless, this discovery is pathologically significant, as it makes these cells capable of interacting directly with the Epm protein, which has been shown to be expressed and released in M5 lesions. In the light of these findings, we concluded that MOKM/NEKM-UM Epm is not only an immunopathological component of M5, but can also have a significant negative impact on the endogenous ability to repair myelin. Therefore, it was concluded that the loss of repair capacity, as observed in many 3 M5 patients during the disease, is associated with the activation of MB5KM/NEKM-MM elements2. It should be noted that a previous study describing the experimental pain effect of NEKM-M/Epm encoded by a defective copy (which does not manifest

VT activity, does not form particles, due to the absence of coding sequences in the dad and roi genes) with a stable insertion on chromosome 75. This Epm protein can be expressed by a single encoded gene NEKM-UM 74 element (epm, or EKMM/-E1 locus), has a four-amino acid deletion in the C-terminal part of its surface domain, apparently causing intrinsic cellular routing, and is known to have ip mimo expression at the placental-restricted protein level!" "8, This protein is indeed an example of a "domesticated" NECM protein" , which now plays a physiological role in the formation of syncytiotrophoblastic tissue and is therefore named "Syncytium".

The corresponding fusion domain in NECM-M/ Epum, as well as the TI P4-binding domain, may be more or less conserved between NECM-M/ subtypes (eg, MOCM-Epm or Synticin). their availability as a surface protein or an extracellular protein with a conformational exposure of the active domain is tightly regulated, and the physiological role of syncytin remains within the syncytiotrophoblast and is limited to the presence in the placenta. Thus, the associated pathogenicity is apparently a hallmark of non-physiological activation, for example by certain environmental infectious agents???" in humans, or in experimental transgenic conditions of ip mimo or ip myo. Syncytium is reported to be located in the astroglia of NS in patients with multiple sclerosis (M5), but the use of antibodies in a different combination made it possible to detect NECM- M/ M5AM-Epm subtype, not syncytium.22 The syncytin sequence can, however, be expressed by transgenesis in mouse astrocytes, causing the release of cytokines harmful to oligodendrocytes, but transgenic mice have shown non-viability (personal communication S Rougeg to N. Reitop, Meshigomigoiodu zutrozisht, San Diego, 2007).However, while Apiopou and colleagues found that adenomatous polyposis coli (APS)- positive myelinating oligodendrocytes are vulnerable to syncytin-mediated cytotoxicity in such transgenic mouse brains, we were able to demonstrate that only immature ORS5 are significantly susceptible to Epu-mediated proinflammatory signals, whereas mature cells are not. Thus, the results of this study, apparently, are the results of artifactual experimental conditions, which are related to their expression conditions, which is caused by placental protein transgenesis in brain cells. In contrast to these results, which are derived from artificial conditions, the authors show a damaging mechanism that is aimed at the endogenous repair capacity of the nervous system (NS) of an adult, and not M5 pathology, which clearly shows an immune-mediated loss of oligodendrocytes and myelin sheath when axons are damaged. Indeed, EMM-mediated effects on cells of the immune system in association with M5 inflammatory lesions are now well documented""0.2325 but no previous studies indicate such a direct pathogenicity of NECM-M/Epu on glial cells involved in the blockade of remyelination of MO plaques due to ORS"5. These findings linking M5 brain immunohistology showing strong Epm protein expression in active MO plaques or at the edge of less active lesions now support such a role in the known remyelination defect due to ORS in M5 lesions.

Example 2: Blockade of remyelination, which is induced by the membrane whiteness of the NEKM-UM family, can be effectively treated with an antibody specific to Epm

The envelope protein NECM-M/envelope family (Epuys, in particular, from the multiple sclerosis-associated retrovirus subtype, MOCM-Epu) is a potent inhibitor of the ability of non-myelinating

ORS differentiate into myelin produced by mature oligodendrocytes, which is an important step in the process of remyelination. We investigated the effect of MOCM-Epm protein on the expression of two different oligodendrocyte differentiation markers:

CMRavse (2", 3'-cyclic nucleotide-3'--phosphohydrolase) is 4 95 of the total myelin protein and is present in the cytoplasm of the non-compacted oligodendroglial sheath of axons. CMRavse is the earliest known myelin-specific protein, which is synthesized by the development of oligodendrocytes.

MVR (myelin basic protein) is a total of 30 95 myelin proteins and plays an important role in the compaction of myelin in the central and peripheral nervous system. It appears sequentially after SMRave both ip myo and ip mimo, and is a specific marker of mature oligodendrocytes. The purpose of this study was to determine whether specific antibodies to Epu, such as

SMBASI, a recombinant humanized Ids54 monoclonal antibody to Epum, blocked the inhibition of ORS maturation induced by M5KM-Epu. Indeed, such an effect of SMBASI1 would indicate that the antibodies carry out "anti-blockade of remyelinating cells". In order to test this hypothesis, a primary culture of human ORS5 was incubated with M5EM-Epu, with or without

SMrBASI, or by applying the recombinant protein to the surface of the culture or by adding the protein to the culture medium. The expression of SMRave (as an early marker of maturation) and

MVR (as a late maturation marker) was assessed by immunocytochemistry after one or three days of stimulation, respectively.

In this example, M5EM-EMM belongs to the full-length recombinant M5KM-Epu protein, which contains the intracellular, transmembrane and extracellular domains of the envelope protein

NEVU-MU.

The results presented here show that human ORS specifically express TI CA on their plasma membrane, and demonstrate that recombinant M3EM-Epm protein specifically and significantly reduces the number of SMRase and MVR-positive cells. These observations are in accordance with those given in Example 1 on human and rat ORS. In addition, our experiments show, for an initial time, that the inhibition (blockade) of maturation of human ORS, which is induced by M5KM-Epm, is completely eliminated by SOMBASI1 treatment, demonstrating these new and previously unexpected therapeutic properties. This, in turn, provides an additional feature in the treatment of progressive forms of multiple sclerosis. 2.1 Materials and methods 2.1.1. Materials

Materials used for primary culture of human ORS

11111111 fSupplier (Link |Serial no./:.:/7/ГЗГ9О

Cell incubator TpeptoOsiepiyys /|NerRA Siav5 100 and

Rivpeg sterile hood. . Vioriosk EP MUVN Sotrasi

Zsiepiyis

ZN Vioteaisai 1600 10805

Votedis' complete ORS of ZNO! wow 10887

Everyone!

Environment 1652 (ORSO5 10576 row 0503 (R/5 9846 10010 954270

Tt75 bottle 21488 03412012

And abieekK 8-hole 154534 081711-8-0 081611-8-0 rom in uvte (Poli Vidta-Aitist RA7O7 Navy 607180 11032401 606180 11051101

Materials used to evaluate the expression of maturation markers in human ORS 11111111 (Supplier | Reference | Serial no.:.:СЖ/:/Й 10010 954270

Paraformaldehyde Zidta-Aiagisn 15,812-7 ЗИввВ5309

Those 100 VRI151-100 104484

Fetal 0 bovine calf RU17B-BOOMI 00 ovomenoya serum. . Eigodepies

Apii-SYURavze antibodies ZME91-8 E11vE00277

Apii-MVR antibody ZM99 E12Og00468

Somapse

Apiii-TI K4 antibodies ARMOMA nOoOo07099-MOZ 11293-1H7 n donkey antibodies against) uiiroe ARI2AE I m1688510

Setaga Mepge! | 87257M 0290880

Mesiog pouring medium.

Mesiazpivid- SARI NIzoo X1118

Achiozsore microscope

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Materials used for stimulation of ORS M5KM-Epm, OMBASI and MZKM-Epm with buffer 11111111 | Supplier Links | Serial no..:/(/"CO recombinant M8KMIRHTpegareshisv | EMU-T 110719-1 (22A)

M5VU-Epu-T buffer RHTpegareshisvi 1-2.YUB5B ishi |- 8

СМБАСИ1 СМБАСИ1 T96/рАСИ1/81 2.1.2 Protocols

Incubation of human ORS with MOKM-Epm, MONMU-Epm buffer and SМАС1

Full-length recombinant MZKEM-Epm was obtained and purified by LC "Tpegareshisv (548 ac., 61, 44 kDa) (serial 110719-1). The initial concentration was 0.6 mg/ml (9.76 µm). MZKEM-EPM buffer (20 mM Ttidta-NSII, pH 7.5, 150 mM Mass, 1.5 95 505, 10 mM OT) was provided by RHTPegareshis5. This solution was used as a negative control. ZMBASI1 (Vaisp T96/BAS1/81, production of ZMR) was obtained and purified by RoiIutyp zsiepiis, Vienna, Austria.The initial concentration was 10 mg/ml (68.03 µM).

Human ORS were stimulated in various ways, either by applying solutions to the surface of a cell culture plate before cell seeding, or by adding them to the cell culture medium after cell seeding. In any case, an 8-well Iabiek (Mips) slide chamber was precoated with poly-I-lysine (Zidta-Aiygist) overnight at 376.

Covering the glass of the 8-hole camera I abiek

Different substances were diluted in RVZ in a sterile laminar box (Rizspeg Viobiosk,

France), 8-well Gariec slide chambers (Mips; 250 μl per well) were coated as follows:

M5AM-Epm (1 μg/ml) coated for 2 hours. at 37 "C.

M5AM-Epu buffer (same dilution as M5EM-Epu) coated for 2 h. at 37 "C.

MOAM-Epu-amBASI At: MZ5AM-Epm (1 μg/ml) and EMBASI (7.35 μg/ml - 50 nM) mixture with coating for 2 hours. at 37 "C.

MZAM-Epu-aMBASI Ag: MO5AM-Epm (1 μg/ml) and ZMBASI1 (29.4 μg/ml - 200 nm) mixture coated for 2 h. at 37 "76.

M5AM-Epu-aMBASI1 VI: MOAVU-Epm (1 μg/ml) with coating for 2 hours. at 37 "C, washed

PB5 and then covered with ZMBASTI (7.35 μg/ml - 50 nM) for 1 hour. at 37 "C.

M5AM-Epu-aMBACI1 B2: MOAU-Epm (1 μg/ml) coated for 2 hours. at 37 "C, washed

RVZ5 and then covered with ZMBAST1 (29.4 μg/ml - 200 nM) for 1 hour at 37 "С.

SMBASI A! only: OMBASTI (7.35 μg/ml - 50 nM) with coating for 2 hours. at 37 "C

SMBASI A?2 only: OMBASI (29.4 μg/ml - 200 nM), with coating for 2 hours. at 37 "C.

SMBASI1 VI1 only: RV5 within 2 hours. at 37 "C, then covered with ZMBAST1 (7.35 μg/ml - 50

NM) within 1 hour. at 37 "C.

SMBASTI B2 only: RV5 within 2 hours. at 37 "С, then coated with OMBASI (29.4 μg/ml - 200

NM) within 1 hour. at 37 "C.

VZA (1 μg/ml) with coating for 2 hours. at 37 "C.

VBZAZHOMRASI At: a mixture of VBA (1 μg/ml) and ZMBASI (7.35 μg/ml - 50 nM) coated for 2 hours at 37 "C.

VBZAOMrASI A2: VZA (1 μg/ml) and ZMBAS1 (29.4 μg/ml - 200 nm) mixture coated for 2 h. at 37 "C.

VBZAZHOMRASI B1: VZA (1 μg/ml) with coating for 2 hours. at 37 "C, washed with PB5, and then covered with ZMBAST1 (7.35 μg/ml - 50 nM) for 1 hour at 37 "C.

From VB5AZHOMBASI1 82: VZA (1 μg/ml) with coating for 2 hours. at 37 "C, washed with PB5, and then covered with ZMBASI1 (29.4 μg/ml - 200 nM) for 1 hour at 37 "C.

Progenitor cells of human oligodendrocyte culture

ORS isolated from the human brain were purchased from 5siepSei! Kezeagsi I arogaiogie5 (via Z N Vioteais!I, Sweden). Each vial contains » 1 x 106 cells in 1 ml. Complete ORS culture medium (ORSM) was reconstituted by adding growth solution

ORS (ORSOZ) and penicillin/streptomycin (P/5) in ORSM, according to the manufacturer's recommendation (Zsiepseyi).

The vial containing the human ORS was placed in a water bath at 37 "C and mixed gently by rotation until the contents were completely thawed. The cells were gently resuspended using a pipette (1 ml seeding. They were then diluted with complete ORSM and seeded into a well culture vessel Plates (7000 cells/cm"-10000 cells per well) precoated with poly-i-lysine (0.01 95, bidta) and with (or without) the treatments described above (see 3.2.1.1) in a sterile laminar box ( Richpeg ViobiosK, France). Cultures were incubated at 37 "С; 5956 СО: until further immunocytochemical experiments. The culture medium was changed the next day to remove residual UOM5BO and unattached cells in the cultures, which are treated for 72 h.

Incubation in culture medium with MBEM-Epmu

After 3 hours of pre-cultivation (see 3.2.1.2) of human ORS in 8-well chambers for the cultivation of IabiekK, the cells were incubated at 37 "C for 24 hours or 72 hours in 8-well Iariekz chambers (250 μl/well) with the addition of the following solutions:

M5AM-Epm buffer, which is used as a negative control, was added to the complete medium for cell culture.

M5AM-Epu added at 3 nM (0184 μg/ml) to the complete medium for cell culture.

M5AM-Epu (3 nM) 4 SMBACI1 (200 nM; 29.4 μg/ml) mixture, which was added to complete cell culture medium.

SMrBACI (200 nM) added alone to the complete medium for cell culture.

Expression of TI CA and markers of myelination in ORS ip myo

After 24 or 72 hours incubation, the medium for cell cultivation was removed, and human ORS stimulated by the treatments described above (see 3.2.1) were fixed in a paraformaldehyde solution (4 95 PEA in PB5, 250 μl per chamber) at room temperature for 1 hour. Then they were washed three times with PB5 (3 x 250 μl per chamber), nonspecific binding sites were saturated by incubation with 1095 fetal bovine serum (EB5) in a solution of PB5 containing 0.1 95 Triton X100 (250 μl per chamber) for 1 hour at 37 "C. In the case of TI K4 staining, cells were incubated with 10 95 EB5 in PB5 only (250 μl per chamber). After saturation, ORS cultures were incubated with a solution of the primary antibody overnight at 4 "C, diluted in 10 95 EB5Z in RVZ solution (200 μl per chamber), as follows:

Mouse antibodies to TI KA 1/1000 (after 24 and 72 hours of cultivation)

Mouse antibodies to SMRavze 1/200 (after 24 hours of stimulation)

Mouse antibodies to MVR 1/100 (after 72 hours of stimulation)

Cells were washed three times in PB5 (3 x 250 μl per chamber), and incubated with a solution of secondary antibodies for 1 hour. at 37 "C (250 μl per chamber), as follows:

EITO-conjugated donkey anti-mouse antibodies 1/200.

After 3 washes in RV5 (3 x 300 μl per chamber) at room temperature, the plastic chambers were separated from the glass slide. The slide was then placed in Mesiavsiaiip?f casting medium containing BARI (Mesiazpivei4). Staining was visualized by fluorescence microscopy using an Achiozore microscope (2 eii55).

Data analysis

After 24 hours of stimulation with MOKM-Epu-T (or control), SMRavhe immunoreactivity of the cells was counted in 10 frames randomly selected in the chamber. The percentage of SMRase-positive cells was developed as a measure -- p SARI p SMRase - the number of immunoreactive ORS for SMRase

After 72 hours of stimulation with M5KM-Epu-T (or control), MVR immunoreactivity of the cells was counted in 10 randomly selected frames in the chamber. The percentage of MVR-positive cells was calculated as follows: n SARI n MVR i - the number of immunoreactive ORS for MVR

Data are presented as the mean -/- standard error of the percentage of SMRase or MVR positive cells. Graphs were obtained with the help of SgarpRai ZoiWage, version 5.00 for

UMipdomu5 (San Diego California, USA). Nonparametric statistical tests were performed using 5idta gai (Chicago, IL, USA).

The following results were obtained during three independent series of experiments, each of which was performed with a new ampoule of frozen commercial ORS: ORS ISS 01, ORSISS 02 and

ORS ISS 03. 2.2 Results 2.2.1 Expression of TKA receptors in ORS of human ip migo

TI K4 expression was assessed by immunofluorescence microscopy. The saturation stage, which was performed without any detergents, allowed detecting TI K4 only on the cell surface.

This experiment confirmed the presence of the M5KM-Epm target receptor on the cell surface

ORS of a person after 24 and 72 hours. primary culture (not shown). 2.2.2 Expression of SMRavze and MVR in the ORS of human ip miito

The morphology of human ip myo ORS was visualized using bright-field microscopy.

The ORS represented the cell body and a total of two cell extensions. SMRase and MVR expression were confirmed by immunofluorescence microscopy after 24 h. and 72 hours in culture, respectively (not shown). 2.2.3 Effect of M5KM-Epm with or without SMBASI on the differentiation of human ORS Effect of applied M5KM-Epm on the expression of maturation markers of human ORS in primary culture (ORS ISS 01)

The purpose of this first series of experiments (ORS ISS 01) was to establish conditions for the primary culture of human ORS, as well as protocols for immunostaining of slide cameras of Iabiekv, which are pre-coated with M5KM-Epm and SMRavze and MVR.

The bottom of the culture vessel was covered with 1 μg/ml M5KM-Epm before inoculation of human ORS. As a negative control (buffer), the culture vessels were incubated under the same conditions, only with MZKM-Epu buffer.

The percentage of SMRase positive cells was significantly reduced from 85:24 95 in control conditions (buffer) to 69526 95 in human ORS stimulated for 24 h. in Iabtek5 pre-coated MOCM-Epm ("p"e0.05; I-test) (Fig. 9A).

After 72 hours cultivation, the percentage of MVR-positive cells significantly decreased from 74:27 95 under control conditions (buffer) to 25:23 95 in IiIabek5 pre-coated M5EM-Epm (4Epu-T)

Avg.0001 Mann-Whitney rank test) (Fig. 9B). These results show that MZKM-Epm inhibits the maturation of human ORS in myelinating cells of the ip myago.

Effect of SMBAST on the inhibition of maturation of ORS induced by M5KM-Epm (ORS ISS 02)

The purpose of this second series of experiments (ORS ISS 02) was to confirm the effect of MB5KM-

EMM on the differentiation of human ORS and determining whether MO5KM-Epm can inhibit the differentiation of human ORS when the protein is added directly to the cell culture medium. In addition, we also tested whether SMBASI1 could inhibit the effect

M5VU-Epm in this model.

In the case of stimulation of human ORS in Iarbiek5 pre-coated MOCM-Epm, SMIAC1 treatment was evaluated with two different protocols. First, Iariek culture slides were pre-incubated with a mixture of M5KM-Epm (1 μg/ml) and ZMBASTI (50 nM or 200 nM) before plating

ORS (condition SMBASI1-A).

The second protocol included sequential coating with MOCM-Epm (1 μg/ml) followed by OMBASTI coating (50 nM or 200 nM) before ORS seeding (ZMBASTI1-B condition).

The influence of M5KM-EMM and OMBASI1 on the expression of SMRase in human ORS

After 24 hours stimulation, a significant decrease in the percentage of SMRazte positive cells from 75:22 95 in control conditions (buffer) to 5522 95 was observed in Iabiek5 pre-coated with 1 μg/ml

M5AM-Epm Ср«е0.001 in comparison with the buffer; univariate analysis of variance followed by Fisher's posterior test). In addition, stimulation in Iabiekv5 precoated with 1 μg/ml VZA did not affect CMrabze expression (7152 95), demonstrating that the effect observed with MBCM-Epm is specific, as it was not seen with the control protein ( Fig. 10A).

SMBASI significantly inhibited the reduction of SMRase positive cells induced in Iabiequs pre-coated with M5KM-Epm (Fig. 10A). The effect of SMBAS1 is visible at 50 nM and 200 nM, and in the two treatment protocols tested: (I) SMBAS1-A condition, 50 nM (6954 95), 200 nM (80:52 9); (I) 500 nM (96:53 95) and 200 nM (76:54 95) SMBASI1-B condition (p <0.001, one-way analysis of variance followed by Fisher's post hoc test). In addition, the effect

Of SMBASI, obviously, it depends on the concentration (Fig. 10 A).

Additional control experiments showed that human ORS stimulated for 24 h. in Iabiek5, previously coated with one SMBASI (50 nM or 200 nM), or a mixture

SMBASTI (50 nM or 200 nM) -- B5A (1 μg/ml) has no effect on the expression of SMRase compared to the control condition (buffer) (p»0.05; Opéjau AMOMA) (Fig. 10B) . These observations were made according to two protocols of treatment of SMBASI, which were described earlier (SMBAS1-A 50 nm, SMBAST1 200 nM and OMBAS1-B 50 Nm).

This experiment also shows that even low concentrations of M5KM-Epm in the solution cause a significant decrease in the percentage of CMRache positive cells after 24 h, from 7852 95 in control conditions (buffer) to 5752 95 in the MOKM-Epm group (p«0.001 compared to buffer; univariate analysis of variance followed by Fisher's posterior test) (Fig. 11). In addition, SMBACI1 treatment (200 nM) completely counteracts the decrease in SMRase expression caused by MBAKM-EMM (76bzh2 95; fr«0001 compared to EMM-T; one-way analysis of variance followed by Fisher's post hoc test). It is interesting to note that 200 nM SMBACI1 alone (7422 95) has no effect on the expression of SMPrave compared to the control condition (buffer) (Fig. 11).

The influence of MZKM-Epm and OMBASI on the expression of MVR in human ORS

After 72 hours of stimulation, a significant decrease in the percentage of MVR-positive cells from 76:22 95 in controls (buffer) to 571 95 is observed in cells pre-coated with 1 μg/ml M5EM-

Epm ("p" 0.001 compared with buffer; one-way analysis of variance followed by Fisher's post hoc test) (Fig. 12A). In this experiment, it was shown that the percentage of MVR-positive cells in ORS culture in Iarteks pre-coated

B5A (1 μg/ml), was slightly different from the control (buffer) (692 95). However, VZA also showed a significantly different effect compared to M5KM-Epm (p<0.001 compared to Eph-T; univariate analysis of variance followed by Fisher's post hoc test), and thus indicated that there should be isolated and do not affect only in this series of experiments, since they are not observed in other experiments or with SMRs in the same series.

SMBASI significantly inhibited the decrease of MVR-positive cells that were induced in Iabiequs pre-coated with MZ2KM-Epm (Fig. 12A). A partial effect of SOMBASTI appears at 50 nM, and 60 complete inhibition is achieved at 200 nM, and in both treatment protocols tested: (i) ZMAC1-

A 50 nM (69:2 95) and 200 nM (73:2 95) and (iii) CHMABAS1-B 50 nM (68:52 95) and 200 nM (7452 95) (shr«0001 compared to Eph-T ; univariate analysis of variance followed by Fisher's posterior test). In addition, the effect of SOMBAS1 is obviously concentration-dependent (FIG. 12A).

This experiment also demonstrates a significant decrease in the percentage of MVR-positive cells from 76:22 95 in control conditions (buffer) to 55:22 95 in cells stimulated with the addition of

M5AM-Epm in the culture medium (3 nM) for 72 hours. ("p" 0.001 compared to buffer; one-way analysis of variance followed by Fisher's post hoc test) (Fig. 12B). In addition, treatment with SMBASI (SMBASI 200 nM) completely inhibited the reduction of MVR expression induced by MZEM-Epm (76: 22 95; fshr«0001 compared to Ephm-T; univariate analysis of variance followed by Fisher's posterior test) (Fig. 12B).

Confirmation of SMBASI1 effects on the inhibition of MBEM-induced ORS differentiation

Epm (ORS ISS 03)

Although the results described above were obtained from a significant amount of collected data, the goal of this third series of experiments (ORS ISS 03) was to fully reproduce the effect of OMBASI1 in a completely independent series of experiments.

Effect of M5KM-EMM and OMBASI1 on SMRavze and MVR expression in human ORS culture

After 24 hours stimulation, a significant decrease in the percentage of SMRakhzte positive cells from 9051 95 in control conditions (buffer) to 71:51 95 is observed in Iabiek5 pre-coated with MBCM-

Epm (1 µg/ml) (p<0.05; Kruskal-Wallis rank analysis of variance followed by Student-Newman-Keuls post-hoc analysis) (Figure 13A). As described above, ZMrBAS1 (200 nM) completely reverses the inhibition of SMRase expression caused by MZCM-Epm at two

SMrRASI tested treatment protocols (ZMrBAS1-9022 95, YAMBAS1-B: 914 95) (fr«0.05 compared to Epu-T; Kruskal-Wallis rank analysis of variance followed by Dunn's post hoc analysis) (Fig. 13A).

After 72 hours stimulation, a significant decrease in the percentage of MVR positive cells from 78:22 95 under control conditions (buffer) to 55:53 95 is observed in Iabiek5 previously coated with MBKM-

EMM (1 μg/ml) for 72 hours. ("p" 0.05 in comparison with the buffer; rank analysis of variance

From Kruskal-Wallis followed by Dunn's a posteriori analysis) (Fig. 13B). As described above,

SMBACI1 (200 nM) completely reverses the inhibition of MVR expression caused by M5KM-EMM in two

CMrBACI1 in the tested treatment protocols (CMBAS1-A: 80x2 95, CMBAS1-B: 75:22 95) (MIR"0.05 compared to Eph-T; Kruskal-Wallis rank analysis of variance followed by Dunn's a posteriori analysis) (Fig. 13B ).

Combining the data of experiments ORS ISS 02 and ORS ISS 03

The results obtained in two different series of experiments (ORSISCO2 and ORSISSOZ), presented above, were combined in one analysis. Thus, the data plotted in Fig. 14 are 8 to 14 independent experiments for SMRazve (Fig. 14A) and MVR (Fig. 14B). This analysis shows that the effect of M5KM-Epm and ZMBAS1I on the differentiation of human ORS is highly reproducible and consistent.

In order to normalize the expression of the data, the control condition (buffer) was used as a reference and is considered 100 95. Thus, these results show that the concentrations

M5AM-Epu used in this series inhibited 24 95 number of CMRabze positive cells (STK: 10051 90; EMM: 7651 905) and 27 906 number of MVR-positive cells (Ci: 10052 95;

EMM: 732 90). OMBASI (200 nM) completely reverses the effect of M5KM-EMM on the expression of SMRase (SMBAS1-A: 10251 95; YAMBAS1-B 10151 95) (FIG. 15A), but also the effect on the expression of MVR (SMBAS1-A: 10152 95; YAMBAS1 -B 97: same 2 905) (Fig. 155). 2.3 Analysis of results

Thus, the data from this example show that M5KM Epm induces a strong and highly reproducible decrease in the expression of two different specific markers of oligodendrocyte maturation. Thus, MEM Epm inhibits the differentiation of precursors of human oligodendrocytes into myelinating cells. In addition, we clearly demonstrated here that SMBASI1 completely eliminates this harmful effect of M5KM Epm and restores the normal level of SMRavze and MVR in this model.

These results show that SMBASI1 treats the blockade of ORS-induced differentiation

NEVMU-MU/M5AM-Epu. Thus, these results support the innovative indication

SMrACI1 for the treatment of NECM-M/-Epu-induced demyelinated lesions. These particularly belong to the progressive forms of multiple sclerosis, for which remyelination is critical, but does not originate from the surrounding ORS M5 lesions, due to the retention of Epu-positive cells and secretion, as shown in Example 1.

Example 3: Study of the therapeutic effects of the ZMBASI1 antibody, I-MAME, 5MT, OME or sodium fumarate in Experimental Allergic Encephalomyelitis (EAE) in a mouse model of oligodendrocyte myelin glycoprotein induced (MOSS, Mueyip oiIidodepagosuie dChiusorgoihipe) and MBEM/NEKM-MU sheath protein (Epm ).

Z. A Comparison between individual molecules (mrnotherapy) and associations of anti-Epm antibodies with small molecules inhibiting Epu-induced effectors in ORS blockade (combination therapy).

Z.A.1. Materials

C57VI/6 mice from Spazez Vimeg.

MOa 35-55 EzriKet, 5th (Roiureriide sotrapu); reference: 501272.

SMIAsi1; batch T950111-8 (Anti-MOVM/NEVU-M/ humanized Ids4 antibodies, including each of the complementarity determining regions (CDOs), which are listed in BEO IO Mo: 1, 5EO IU

Mo: 2, BEO IO Mo. Z 5EO IO Mo: 4, 5EO IU Mo 5 and BEO IO Mo 6.

I-MAME (Ma-nitro-I-arginine methyl ether) bidta; reference: M5751-525 batch vСвЕ4375У

TMT (5) Methylisothiourea (5idta); reference: M84445-1002 lot ZTVST10OZM

Dimethyl fumarate (OME) Zidt; reference: 242926-1002 lot 2507UvSVN

Metocel MS 5idta; reference: 64605-1002 lot VSVO9989U

IRA (Zidta) bidta; reference: E5506-10MI. lot: 061M8728

PTH (Pertussis toxin; CaIriospet); reference: 516561 lot 00128881

RVZ (phosphate-salt buffer; I opa); reference 516561; party; 000128881

Epm (R'X Tpegareshis5); production batches of purified protein, tested for the absence of endotoxin (test I ОЙ «5|ЦИ /mt) and bioactive (zepeigo internal OS tests). Coyagoy device (I E8200; Viozeb France)

C3.A.2. Methods

Pathogen-free female C57VI/6 mice (6–8 weeks old) were purchased from Spagpe5 Kimeg

And arogyphogesis and were maintained on animal equipment for one week before immunization. One day before immunization, all mice were weighed and then evaluated in the rotating drum test.

Zo Rotary drum

Testing days: 9 and 8 days before immunization. Mice were transported in their home cages from the room in which the animals were kept to the experimental room. Mice were habituated to the experimental room for 15 min. During the training period, each mouse was placed on a rotating drum at a constant speed (4 rpm), if one mouse fell, it was returned to the rotating drum, until the mouse became comfortable on the rotating drum, the mice ran for 120 s All mice were returned to their cages, the speed was increased to 13 rpm, and the mice were again placed on the rotating drum for 120 s All mice were returned to their cages, the speed was increased to 19 rpm, and the mice were again placed on the rotating drum for 120 s The environment of the experimental room remained constant between test sessions in terms of temperature and light intensity.

Test days: 1 day before each immunization, mice were transported in their cages from the room in which they were kept to the experimental room. The mice were habituated to the experimental room for 15 min.

Mice were then subjected to two trials at 10 increasing levels of speed ranging from 7 to 40 rpm. Individual latencies before falling from the rotating drum (for two trials at each speed level) were recorded up to 60 s. Thus, training and performance in the rotating drum system were determined for each animal.

The results of the rotating drum tests are expressed as the kinetics of the average individual score for each group with the standard error, indicated in bars, for each day of the test and for each speed tested in the necessary range for neuromotor deterioration under the given conditions (from 16 to 29 rpm) .

They are calculated for each individual mouse, for each test day, and for each speed value, as a relative increase or decrease in time on the rotating drum (to fall), compared to the first test day at the same speed (D-7, before starting induction of the disease in the respective groups). The value is calculated as "Time on spinning drum on day X" - "Time on day 7 for the same mouse at the same speed". The figures show the kinetics for different groups for the entire training period, for characteristic discriminative values of speed, with a reliable differentiation of sick from healthy animals in our conditions (for example, too high or too low speed values, or healthy people fall because immediately or sick people are able to maintain slow movement) .

Data on initial weight and performance were self-controlled before the mean and standard deviation were calculated within the group. Different groups of animals were housed uniformly in each cage, according to the relevant information provided before each experimental series.

Clinical assessment

Animals were weighed and clinically evaluated 5 days a week in accordance with the following criteria: 0 - without signs; 1 - paralysis of the tail or hyper-reflexia of the hind limb(s) or unilateral weakness of the hind limbs; 2 - bilateral weakness of the rear or front limbs;

C - plus unilateral paralysis or a large deficit; 4 - complete paralysis of the rear or front limbs; 5 - plus partial paralysis or a large deficiency of opposite limbs; 6 - agony or death. These were taken from the standard criteria to reflect the more rapid induction of brain and cerebellar damage by this model.

Mice were weighed once a week on Mondays, Wednesdays and Fridays during all experiments.

For the series of experiments of Example 3.A, the groups were defined as follows.

All groups of 6 (six) mice were first vaccinated subcutaneously in the neck area on day 0, then on the side of the back on day 7 and 14; 200 μg of MOoS/mouse and 60 μg of M3ZEM-EMM-1EA.

Group A was the positive control group of MZCM-Epu inducing EAE without any treatment.

For the Zh-3 groups, treatment with SOMBACS1, I-MAME, TMT and OME alone or together with CMBASCI1 at the doses indicated below was carried out on the 12th day after immunization, when the progression of clinical symptoms of EAE was already observed. SMBAScI1 humanized antibody was administered once, and I -

MAME or 5MT was administered every 4 days until termination of the study on day 29. As for groups

D and 3 related to the treatment of OME, we determined the amount of daily water consumption for each mouse, and found that (under our conditions), each mouse consumed about 3.5 ml of drinking water per day. Thus, based on these measurements, a dose of 1 mg of OME--0.08 95 MeishShoseyi! was added to 3.5 ml of drinking water per mouse daily. 350 ng of pertussis toxin (PTX) per animal was administered (i.p.) in all groups on the same day after each immunogen injection, and repeated 2 days later as usual in EAE protocols to

To facilitate the migration of lymphocytes through the blood - blood-brain barrier.

Generalized presentation of experimental groups in Example 3.А 1 А1 ИИ 200 |! ї1771111171Г11171С111111171111111171ї1 1716

NeoblenyigAE /|//:/ | i | IA | them | - | - | - /! - / here 020 | And || 11 mm 29 | 9 | tA | (b s | sv g 1mg in a 1 1mg zh d 0.08 96 3.5 ml of drinking water. g smmerinami | 20 0 | A | i be

Z. 1 mg in as 1-1

ZM IR

1 mg zhk 0.08 96 z: Meftos ei in drinking water.

Daily

Z.A.Z Results Clinical observations

Assessment of clinical signs and mass was routinely performed as described above. The calculation of EAE kinetics for the studied period is presented in Fig. 16, 17 and 18.

Regular and permanent development of EAE clinical indicators, such as hyper-reflexia, paralysis of the tail, weakness of the hind limbs, forelimbs and partial paralysis, could be recorded in all mice until the end of the experiments in group A (EAE positive control) and up to 12 days in others groups that were treated on the 12th day with SMBAs1 humanized antibodies (group B) or anti-free radicals (groups B, G, D) separately or groups that were treated simultaneously with SMBAs and anti-free radicals (groups E, G, 3).

Observations after treatment: clinical signs in group B, which was treated only 1 time with

SMBasSI, slightly decreased until day 29. In the groups treated only with G-MAME, 5MT, irregular recovery could be observed, but with a decrease in the progression of EAE.

It should be noted that on the 16th day, the introduction of 5MT was interrupted.

Regarding groups D, G and 3, which were treated simultaneously with ZMBAsi and I-MAME, 5MT and OME, respectively, the recovery was much more significant and in particular in groups G and 3. These results show that the synergistic effects significantly accelerate and increase the symptoms EAE. It is also worth noting that treatment with these substances does not cause any abnormalities in any group of mice (including control mice that were studied separately).

According to the results presented in Fig. 16, 17 and 18, it can be summarized that: the most elevated clinical indicator (with the worst development of the disease) is evidenced in untreated animals (group A). Animals treated only with OMBAST (group B) get better after its administration (starting from the 12th day). groups treated with I-MAME or 5MT alone had lower recovery rates and kinetics than group B. group D treated with OME and group E treated with SMBAst (200 μg single dose) in combination with I-MAME had equivalent recovery by the end of the study. group Z, treated with SMBACI1 antibodies in combination with TMT, and group H, treated

SMBASI antibodies in combination with OME had significantly better recovery at the end of the study and earlier kinetics of clinical improvement than all other groups. This demonstrates a synergistic strengthening of the combination of OMBASI1 and Z5MT or SMBASI and OME, for a significant improvement of the therapeutic result.

Testing on a rotating drum

First of all, it should be clearly stated that groups with the inscription "1 injection." and "2 injek." in Fig. 19 correspond to control animals injected only once (1) or twice (2) with Epm protein and MOS antigen, compared with three injections of Epm protein and MOS antigen required to induce clinical manifestation of EAE, as in all other groups. They are control mice exposed to Epm below the pathogenic threshold and without induction of a clinical picture of the disease. They confirm that a similar pattern of three injections is required for all animals tested under different treatments to induce acute and severe EAE symptoms and gradual change. Thus, the therapeutic effects observed in treated animals are relevant to the effectiveness of treatment in current and progressive diseases.

According to the results presented in Fig. 19, with the test readings on a rotating drum at 23 rpm, we can see that:

Exercise at 23 rpm reaches a sufficient threshold to reveal major sensory and motor dysfunctions in animals: untreated group A has the worst performance and kinetics, while animals with a single or double injection ("1 in' eq. or 2 injections"), have much weaker symptoms and more noticeable changes. The negligible and progressive clinical deficit in the control group B confirms that the diseases and corresponding lesions of M5 were very active with C injection of Epu. Therefore, all other groups with different treatments were subjected to a similar strong induction of Epu disease.

Group B with EAE treated with a single injection of 200 μg ZMBACI recovered similar to control mice without induction of disease with low effect on Epa at the end of the study period.

The most remarkable effect, under the given conditions, was found for group Z, treated with the SMAC1 antibody in combination with 5MT, which seems to have a bright curve of recovery kinetics, which ends with the best indicators of all groups at the end of the study. This demonstrates a synergistic enhancement of the combination of SMBASI and 5MT to significantly improve the therapeutic outcome.

Group G, treated with 5MT, also has only improved kinetics and results (the last point of the study period), although to a lesser extent.

According to the results presented in Fig. 20 obtained with a rotating drum at 26 rpm, we see that:

Physical effort at 26 rpm confirms that these conditions are higher than the threshold necessary in this experiment to detect important sensory and motor dysfunction in animals:

control group A has worse results and kinetics, while animals injected only once or twice ("-1 injection or 2 injections") have significantly weaker symptoms and more noticeable changes (very good parameters at the end of the study period for the "1 injection" group).

Group B with EAE, treated with a single injection of 200 μg of SMBASI, has recovered parameters similar to groups G, G, B, D and C and the "72 injection" control at the end of the study period.

The most remarkable effect, under these conditions, indicated that the treated group E

SMBACI1 antibody in combination with G-MAME apparently has a good recovery kinetics curve, ending with a better result of all groups at the end of the study. Group B, processed only I -

MAME also has improved kinetics and results, albeit to a lesser extent. This demonstrates a synergistic enhancement of the combination of OMBASI1 and Y-MAME for significantly improved therapeutic results.

According to the results presented in Fig. 21 when tested on a rotating drum at 29 rpm we see that:

Physical effort at 29 rpm still confirms that these conditions are above the threshold necessary to initiate major sensory and motor disturbances in animals: control group A has the worst performance and kinetics, while animals injected with only one or twice

C" injection or 2 injection"), have significantly weaker symptoms and more noticeable changes with similarly good results at the end of the study.

Group B with EAE treated with a single injection of 200 μg OMBACI1 has recovered scores similar to control mice without disease induction with low effect on Epm at the end of the study period.

The most notable effect, under these conditions, is that group E, treated with SMBACI1 antibodies in combination with Y-MAME, which still seems to have a good recovery kinetics curve, ending with the best result of all groups (end of study). Group B processed I -

MAME had improved kinetics and results, albeit to a lesser extent.

Another general notable result is the systematic improvement of mice from group B treated only with ZMBASI, which always shows significantly stabilized and/or improved performance compared to untreated animals (group A) in all conditions (from 16 to 29 rpm tests).

However, this apparent efficacy appears to be significantly enhanced by combining SMBAsI1 with G-MAME or 5MT. It was also improved by combining with UOME, but with slower kinetics. In any case, this does not exclude improvement with optimized doses and for a longer period of treatment.

Thus, in all testing conditions (including clinical indicators) the combination of TMT, I -

MAME or OME with SMBAs1 provided a significant advantage for clinical and functional recovery compared with the use of either therapeutic molecule tested alone. By themselves, small molecules apparently have lower and rather transient effects, while OMBAs1 has long-term effects, providing better recovery at the end of the study compared to these small drugs, thus showing long-lasting efficacy.

The kinetics and amplitude of final recovery, having been significantly improved by combinations, now show the advantage of using a combination of these therapeutic agents for their synergistic and targeted effects on functional recovery in the setting of ip miigo. This demonstrates indication for early and more potent clinical efficacy in human disease. 3.B. Comparison of the therapeutic efficacy of two doses of anti-Epm antibody given alone as monotherapy or in combination with anti-MO drugs. 3.B.1 Materials and methods:

Unless otherwise noted, all materials and methods are as described in Example ZA.

Experimental groups

Generalized presentation of experimental groups of Example 3.B μl μl NG ey | || Ah riyayu somy | | | tA) from paradise vm | || A paradise vm | 311 I I GO 13 1 Hn

All mice received three injections subcutaneously on the neck in the OO, on the dorsal side with D7 and D14. Here, mice from group A were administered only MY 200 μg/mouse and IRA without Epma, which is the negative control group without EAE in this series (syp-). The positive control group with untreated EAE in this series is represented by group B (sipk). The other group was injected with 200 μg MOoC/mouse - M5AM-EMM (60 μg) w- IRA, with the procedures as indicated in the above presentation. Treatment was initiated on day 12 post-immunization in their respective groups.

Groups B, D, Z received 200 μg of SpBASsI1 at L 2; Groups G, E, C received 500 μg of sprAs1 at

L2; Group B received only SOMBAs1 buffer solution.

Groups D and E received an IR injection of 5MT (200 mg/mouse) twice a week at 12, 914, 919, 321, 926, and 928.

For groups G and 3 receiving UOME, we preliminarily determined the daily water intake of each mouse, and determined that (under our conditions) each mouse consumed about 3.5 mL of drinking water per day. Thus, based on these measurements, a dose of 2 mg of OMEzh0.08 95 metocel per mouse was added to 3.5 ml of drinking water daily.

For each test speed of the rotating drum, the results were confirmed when the curves of the corresponding untreated mice with EMM-induced EAE (group B, which is a control with untreated EAE) showed a significant deficit compared to sham controls (group A, which were immunized with MOS diluted in IRA without Epum protein). The results were considered "non-interpretable" whenever such criteria were not met as appropriate technical and experimental conditions, therefore, as not passing quality control based on a given significant difference in the curves of "Positive versus negative EAE" groups.

After day 20, an intravenous (IM) injection of 20 μg of MeCM-Epm in saline was made to create a systemic acute challenge with M5EM-Epm protein in all mice, when a significant clinical improvement (remission-like) was obtained with most treatments in EAE mice. This was done on day D21 for mice of groups B, G, E, and 3, or on day D22, for mice of groups A, B, D, and Z, this was done in order to study the response of different groups to acute Epm antigenemia, and to evaluate possible differences in the protective effectiveness of various treatment methods. 3.B.2 Results Clinical observations

Clinical parameters and weight were routinely assessed as described above. The kinetics of clinical indicators of EAE during the study period are presented in Fig. 22, 23 and 24. The mass curves indicating the relative mass compared to the day before the first injection of immunogens are presented in Fig. 25, 26 and 27.

Testing on a rotating drum

The results of research on a rotating drum are presented at different speeds (16; 26; 29 rpm) in Fig. 28, 29 and 30.

This series of experiments concerns the effect of medium (200 μg) in comparison with a high concentration (500 μg) of the therapeutic SMBASI antibody as monotherapy, and similar comparisons were also made when combined with 5MT and OME.

A global comparison of the clinical rate curves over the study period was justified because (I) untreated Epu-induced EAEE (group B) had the most adverse changes and (II) sham-immunized controls without EAE (group A) had no clinical signs over the period , which precedes the intravenous injection (IM) of Epm on the 20th day.

Before intravenous injection on day 20, all treated groups were significantly different from untreated Epu-induced EAE. On day 20, treated groups with different disease kinetics during the study period had comparable scores with a mean value below 0.5, indicating a period of remission in all treated groups. This confirms the effectiveness of all antibody doses and all tested combinations with both OME and 5MT.

A further challenge with an intravenous (IM) injection of Epm protein on day 20 mimicked the peak expression of Epm and its release into the blood, as expected, during the initiation of a new and severe relapse, a naturally occurring disease. In Fig. 22, 23 and 24, significant differences in the therapeutic activity of different products and doses are explained: 1) SOMBASI1 showed high efficiency in increasing the stability of this peak of Epm antigenemia at a dosage of 500 μg (group G), but temporary clinical disorders were detected at a dose of 200 μg ( group B). 2) In case of combination with 5MT, no significant variations of clinical parameters were observed at two doses of SMBASI (groups D and E). Thus, 5MT conferred increased resistance to MI M5KM-Epu, for mice that received a low dose of SOMBASTI (200 μg) compared to mice that were treated only with the antibody at this lower dose (group B). 3) OME-treated mice showed clinical resistance to this antigenemia peak when also treated with a higher dose of SMBACI1 (group 3), but failed to prevent clinical impairment at the low dose (group G). OME, thus, does not provide increased resistance to MI M5KM-Epm in mice treated with the lowest dosage of ZMBAS1 (200 μg). Mice treated with OME and higher doses of ZMBAS1 (500 μg, in group 3) showed comparative resistance compared to mice treated with only ZMBASI at the same dose (group D). 4) Curves showing weight gain during the study period in all groups unexpectedly showed that group G treated with ZMBASI1 at 500 μg had a better final

From the point 5) OMBASI at 200 μg (group B) or OME with OMBASI but at a higher dose (Group 3), gave endpoints equivalent to sham controls.

This indicates that, in addition to the relative effectiveness of various methods of treatment, which are shown with the indicator of clinical kinetics:

CMpRASI antibodies alone in high doses (500 μg) demonstrate better clinical efficacy. They also had a positive effect on the general health of the animals, as shown by the dynamics of mass growth.

In combination with ZMBASI at a lower dose (200 μg), 5MT showed significantly greater efficacy for the treatment, with complete resistance to the residual problem of MOCM-Epm MI.

This indicates that SMBACI shows the strongest treatment, as it provides better results at the higher dose (500 μg), but also at its low dose (200 μg), it is significantly enhanced in combination with TMT, which effectively prevents clinical exacerbations after peak

Epm of antigenemia created by IM injection at the end of the study.

In addition, these results also show that higher doses of ZMBASI, alone or in combination with given doses of OME, provide a significant increase in therapeutic efficacy in terms of overall health (according to weight gain curves).

However, the possible synergy of 5MT or OME with SMBASI at higher doses, if any, is not possible, since the antibody itself already provides maximal therapeutic efficacy under the given conditions of disease induction and activity in EAE in mice.

In parallel, the rotating drum test objectively confirms, at all relevant speeds (Figs. 28A, 29A and 30A), that the higher dosage of ZMBASI (500 μg/mouse) is the most effective product (administered separately) in improving the neurological deficit of treated Epmu , which are induced by EAE, mice.

Testing on a rotating drum also confirms the synergistic effects of TMT with

SMrBASI, while showing neurological improvement in animals treated with low doses of SMBASI in combination with MT, showing significant neuromotor improvement at 26 and 29 rpm (Fig. 29B and ZOB). At 16 rpm (Figure 28B), the low speed did not seem to provide enough discriminative power to confirm this clinical improvement over the study period.

Finally, results on the rotating drum show an improvement in Epu-induced EAEs in OME mice at both doses of OMBASI, but this only becomes clearly significant at 29 rpm (Fig. 30

IN).

Therefore, taking into account the results of this objective and the automated tests evaluating the neurological performance of the animals, ZMBASI1 is an apparently reliable treatment when given as monotherapy, as shown in Example 3.B. Synergistic effects with anti-MO molecules, as seen with SMBASI at a lower dose with MT, are also clearly evidenced and suggestive of potent therapeutic enhancement. This should be at different dosages of SMBAST, but cannot be detected under current conditions with already maximum effects of 500 mcg with OMBAST1. 3.8. Comparison between anti-Epm antibody or sodium fumarate alone (monotherapy) and anti-Epm antibody with sodium fumarate (combination therapy). 3. In 1 Materials and methods

Material 1111 MrAsT | about (bepvyu)// | 77777771 17111 toBOT8U sSimApegivoureSst | (77777771 7777717171717171717171111119110280

Experimental groups

Isotype (mcg) no. sodium of their antibodies

Ace! 200 | - | IRA / i | RiasevroY.//- | - | 5 500 mcg 200 I IHA t | riasebo/ 7

SN IR

Ma? | 200 | with IA | i | Riasero VZoml o) 4 |Ma2 Fumag

Fum of drinking water until the end of the study every day drinking water until the end of the study every day 3. B.2 Results Clinical observations

Assessment of clinical signs and mass was routinely performed as described above. The EAE kinetics indicator for the studied period is presented in Fig. 31 31.

Group A, representing negative controls injected with IRA, MOS without Epm protein, and injection of placebo antibodies containing its buffer solvent, did not show significant clinical symptoms during the study period.

A gradual change in EAE clinical indicators can be recorded up to the 12th day in the groups that were treated on the 12th day with SOMBas1 humanized antibodies (group B) or with sodium fumarate, OME-linked anti-nitric oxide free radicals (anti- MO), alone in group G or simultaneously with SMBASI in group E.

It is interesting here that only the groups that received high doses of SMBASI antibodies (B and D) showed a significant return of their clinical curve indicators and a significantly improved end point at the end of the study. Group G treated with sodium fumarate alone did not show any significant clinical improvement compared to group B treated with an ineffective isotype control antibody (Overlapping error bars on the curves). Regular and constant development of EAE clinical indicators can be recorded in all mice until the end of the experiment in the group

B (Epu-induced EAE), which was treated with an isotype control antibody without specificity for the Epu protein. This indicates that the therapeutic effects observed with SMBACI1 are specific for this particular antibody and are not relevant to the injection of any humanized antibody with the same isotype that, for example, would not target the same epitope .

Thus, this last example shows that:

Control group A without injection of Epm protein behaves like healthy controls without clinical signs, despite injections of all other components necessary to nullify Epm induction of EAE (IRA and MOSS), as well as to nullify antibody injections (Solvent antibodies); false delivery of water-soluble sodium fumarate, which is implicitly provided at the expense of normal drinking water.

The most elevated clinical indicator (with the worst development of the disease) was registered in animals that were treated with an irrelevant isotype of the control antibody (Group

B). This injection of false isotype antibodies in the presence of Epm protein thus demonstrates a positive control of EAE with a typical gradual change of EAE.

Group G, treated only with sodium fumarate, had an adverse outcome at the end of the study that was similar to group B, which was treated with ineffective control antibodies.

Groups B and D, treated with high doses of ZMBAS1 antibodies alone or in combination with sodium fumarate, had equivalent and significantly better recovery at the end of the study. Clinical improvement appeared on the 15th day after the start of treatment.

Here, since sodium fumarate alone had no effect and since the curves of dynamics of groups B and D are equivalent, the therapeutic effect observed in these two groups must be entirely and exclusively caused by SMBASI, while sodium fumarate showed ineffectiveness. 3.D Analysis of the general results of Example C

In conclusion, alone or in combination, treatment with anti-Epim specific antibodies, such as SMBAsCI and antibodies compared to free radical nitric oxide (anti-NO) compounds, such as OME, TMT or I-MAME, are preferred, due to treatment and prevention of blockade of potential remyelination activity caused by NEKM-MU membrane protein, in particular,

M5VM-Epm, ip mio and ip mimo.

SMBASI at a high dosage (500 mcg/mouse about 20-25 g) also showed efficacy compared to the low dose (200 mcg) given as monotherapy in these examples.

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Hail Mary! Asai 5si O5 A. 1997; 94: 7583-8. 2. Retop N, Septi V, Vegpaga S, ei a Nytap epdodepoi5 geigomigy5 iuure M/ epmeiIore ehrgezviop ip Biosa apa bBgaip sei rgomide5 pemu/ ipvidnov ipio tiyirie zsiegoziz dizease. Mai! Association 201218: 1721-36. 3. Vhypo 5, Segsidpapi M apa Vop MA. M/nye tatseg arpoptaiiiez ip Biroiag disogaeg: a mohei!- razes aiyiviop iepzog itadipd zishau. Viroiag Oizoga. 2008; 10: 460-8. 4. Sbodnagi MM, Vepyt K, Sapieg S5 apa MasOropaii AM/. Yes! (piskpe55 az a miipegariyu ipadisayog tog 5spihorngepia. Vg U Rzuspiatgu. 2007; 191: 229-33. 5. Biemep5 UV. Mepygoraipoiodo oi zspi2orNngepia. Agsy Sep Rzusniaigu. 1982; 39:11 131-9. b. Reitop N, Nataapi M , Raisaga RN, eyi aYi.

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NENU-MU, a pem/ pitap epdodepoiz geigomigiv Tatiyu. In Moscow. 1999; 73: 1 175-85. 19. Mi 5, G ee X, Her X, ei a). 5upsuynp with a sariime geigomiga! epmeIore rgoivip ipmoimey ip pitap riasepia! togrpodepezviv have 2000; 403: 785-9. 20. Virgesni K, Obroi|ez K, UUepayei! In, her aI. Veashaiyop oi pPytap epdodepoiv geigomigiv M rgoivip ehrgevviop Bu Pegre5 5itrieh migy5 yure 1: itriisayope Tog tyyPiriye 5siegov5i5. In Meshhomigoi. 2006; 12:65-71. 21. Airgesti K apa Reitop N. Ekhrozyge yuyu ipiapi v5ibiipd5 digipd eapu She apa tgizK oyi tipPiriye 5siegovi5. CHAMA 2005; 293: 2089; Ashpog Geriu-90. 22. VoerkKe S, ani 5, I asheg S, yei ai. Ap M-IettpipaiIu ipopsaied epuieiIore rgoivip epsodeid Bu a

Ppitap epdodepoiz heiyomigy5 M Iosy5 op spgotozote Khadg2.3. NeigomigoIodu. 2010; 7: 69. 23. Retop N, Septi V, Vegpaga S, Siagsia-Mopisio M, Oeyeshep S apa Ragipeyi S. Nytap

Epdodepoiv Neigomigivz iTure MU EpmeIore ehrgezviop ip riosa apa bgaip seiI5 rgomide5 pem/ ipvidnov ipto Miyiriye Zsiegoviv disease. Miyirie bsiegoviv (oigpa!. 2012; BO: 10.1177/1352458512441381. 24. Reitop N apa H apa A. Pe pitap epdodepoizh geigomigy5 PpK Beim'yeep depez apa epmigoptepi ip tiyirie zsiegoviv apa ip tiyiasiogia! div Yeazev5 Azzosiaipud PeshgoipPattaiiiop Siip Wem AIPShegau

And so on! 2010; 39: 51-61. 25. Nigoi?gi V, NoiYapa A, Misnei! M, her aI. Miyiriye 5siegov5i5-azzosiaieai geigomigiv5 rapisie5 saivze T

Iutrposuie-derepaepi deaiy m/yp Bgaip petoitpade ip pitapigei ZSIYUO tise todei. In Meigomigoi. 2003; 9: 79-93. "110" Genero "1205 COMPOUNDS FOR THE TREATMENT OF BLOCKADE OF REMYELINIZATION IN DISEASES WHICH

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From "222" (51)...(51) "223" pea,s, 9, both "400" 29 vspEtpasne stragaspny pEdpaZSpVet Khazuzauezea agoKuVn you in B "210" 30 "2115 21 -212" DNA 213" Artificial sequence "2020" "223" Coding sequence for SOK "2020" "221" tivzs Teashte "222" (3)...(3) "г2З3"пеа,с, 9, or и "2020" "221" tivzs Teashte "222" (6)...(6) "223" пеа,с, 9, both "2020" "221" tivzs Teashte "222" (9)...(9) "223" пеа,с, 9, or "2020" "221" Tivzs Teashte "222" (12). .(18) "223" p ea,s, 9, both

"4005" 30 SUCCEEDS IN 81

Claims (5)

FORMULA OF THE INVENTION 1. Anti-M5VM/NENU-M/Epm ligand for use in the prevention and/or treatment of progressive multiple sclerosis, where said anti-M5AVM/NENU-M/Epm ligand contains each of the regions that determine complementarity (SOMK5) specified in 5EO IYU MO: 1, 5EO I MO: 2, ZEO IYU MO: 3, 5EO IYUO MO: 4, BEO MO: 5 and ZEO IYU MO: 6. 2. Anti-MOAM/NENM-MM Epm ligand for use according to claim 1, which is characterized by the fact that the specified anti-NENM-UM Epm ligand is 5cEm, a Rab fragment or an antibody, preferably a chimeric antibody, a synthetic or humanized antibody, and more preferably Ids, such as ЯДО1 or so far. 3. Anti-MOAM/NEVM-M/ Epm ligand for use according to claim 1 or 2, which is characterized by the fact that the progressive multiple sclerosis is secondary progressive multiple sclerosis (PSM5) or primary progressive multiple sclerosis (PRM5). 4. A method of prevention and/or treatment of progressive multiple sclerosis, which includes the administration to the subject of the anti-MONAM/NENM-MM ligand Epm, which contains each of the areas that determine complementarity (SOK5), specified in the ZEO IU MO: 1, 5EO IO MO: 2, 5EO IO MO: Z, zEO IUO MO: 4, BEO IU MO: 5 and ZEO IU MO: 6. 5. The method of prevention and/or treatment of progressive multiple sclerosis according to claim 4, which is characterized by the fact that the indicated anti-MOAM/NEVM-M/ ligand is a ssEm, a Bar fragment or an antibody, preferably a chimeric antibody, a synthetic or humanized antibody, and more preferably Ids, such as Ids1 or Idsai. 6. 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M sho : : Ki Eze yi yi I "o oz cho kh ho yad z kh o yi ya DU TUMUHUMUUUUUUU MUKU KKU KKU KY ХK KUKY 7 " і Z Her 3 x Eh yak і 3 Ko SCHO Re et KTK TI ACT KK KK ЖК ТК КАТ КК ИНК AK Ке КЕЙ Pekty eto ekeeyayaya і і мух і : 3 і і 1 і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і і K dyh і і і І Же і і KK і o -k skh her --k - sk kh z Z kh o sfzhkovm kh y - V KU r I і ih Же. ХЕ З і Es : жо : З -х і Ж і 5. E shk i Z He x Z 5. : Ko i : ko : And I «a av yi because I and Her ShK ef u kA KK KK Kf ok dk Kon zhk ee hIZH Z Na i u Va x shcha i E ee 5 i i i i B i I i x 5 i MMK. Kaya s z h i : i Zh obu IC so: Zh ja ShK hv zneisyakh ZK gH "DMK im i t Km i nn ke X x i ne os KZ bek 1 DK rimy kny 7 7 PTKS Ma KK in zh ke i yi I: ZKU ZE Zk eh E Zh a M, yi yi "Za YAK, Z izh : i OK shcha 3 Ye x X ee ". KU M t ak "I am the whole of me. Because of them the same I and ZK z i ZE. schi TE o i in NN in V i m MO MU i zu Z X skee i ВH z u Zhe i B as VO z "de" х х их З Ko З и І МОЖ is i In the 3rd AS za Kk. Z che x v v Z. i z schi x fresh Y y i i . eh shcha KKU. "Kfkhkukkkkyuk ik oo i 7 ЖЖ yu Т ЖЕ ях 3 Gays t -i: ZE: і t yo. MU. Zk і є і I ХХ KOH . 3 scha і і ah scha 3 x "ik sg TSO " i roi VU KE g TV ZKU V ik ik KK KK ХК ХК КК ХХ КК КК ХК КК Али КН КК КК ХК ХК КК ХКккік»: t TIMUKTUueKH MO ok yuyu Kvi ii cha SHO v ii ii A their KU ii ii ii : their 5 their 1 ii ii Kk their ii і і і Me ki kv : і з ШЯ З ШМ di КК КК КК КК КК КАК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК КК Se VK ih ah i i se . Fig. 25