.
`(cid:14)
`Journal of Neuroimmunology 92 1998 113–121
`
`w /
`(cid:15)
`Treatment of multiple sclerosis with Copolymer-1 Copaxone :
`implicating mechanisms of Th1 to Th2rTh3 immune-deviation
`Ariel Miller a,b,c,), Sarah Shapiro a,d, Rosa Gershtein a, Amalia Kinarty d,
`Hanna Rawashdeh a,b, Silvia Honigman b,c, Nitza Lahat d,c
`a Neuroimmunology Research Unit and Multiple Sclerosis Center, Lady Da˝is Carmel Medical Center, 7 Michal Street, 34362 Haifa, Israel
`b Department of Neurology, Lady Da˝is Carmel Medical Center, 7 Michal Street, 34362 Haifa, Israel
`c Faculty of Medicine and Rappaport Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel
`d Molecular Immunology Research Unit, Lady Da˝is Carmel Medical Center, 7 Michal Street, 34362 Haifa, Israel
`
`Received 19 May 1998; revised 28 July 1998; accepted 28 July 1998
`
`Abstract
`
`.
`(cid:14)
`w
`The synthetic polypeptide copolymer-1 Cop-1; Copaxone ; Glatiramer Acetate has been recently approved as an effective treatment
`(cid:14)
`.
`in relapsing multiple sclerosis MS . A large body of evidence demonstrates that Cop-1 induces active suppression of CNS-inflammatory
`disease in animal models. However, Cop-1-mediated suppressor mechanisms have not yet been elucidated in humans. A 12-month open
`study following clinical and immunological parameters of ten relapsing MS patients treated with Cop-1 is presented. Relapse rates and
`(cid:14)
`.
`disability scores EDSS were evaluated prior to and after 12 months of treatment. The immunological parameters assessed prior to and at
`(cid:14)
`.
`3 months’ interval during treatment included serum levels of soluble IL-2 receptor sIL-2R and IL-10 as well as leukocyte cytokine
`mRNA expression of TNFa, IL-4 and TGF-b. Copaxone treatment was found to lead to a significant reduction in the mean annual
`(cid:14)
`.
`relapse rate from 1.4 prior to treatment to 0.6 during treatment and stabilization of disability in 90% of the patients. The treatment was
`accompanied by an elevation of serum IL-10 levels, suppression of the pro-inflammatory cytokine TNFa mRNA, and an elevation of the
`anti-inflammatory cytokines TGF-b and IL-4 mRNAs in PBLs. These results suggest that the beneficial clinical effects of Copaxone in
`MS patients may be attributed to changes in activation of T cell subsets and a shift from Th1 to Th2rTh3 cytokine profile, probably
`leading to Cop-1-driven mechanisms of bystander suppression. q 1998 Elsevier Science B.V. All rights reserved.
`
`Keywords: Multiple sclerosis; Immunomodulation; Copolymer-1; Th1rTh2; Cytokines; Bystander suppression
`
`1. Introduction
`
`.
`(cid:14)
`Multiple sclerosis MS is an inflammatory disease of
`(cid:14)
`.
`the central nervous system CNS of suspected autoim-
`mune origin. Studies in MS and its animal model, experi-
`(cid:14)
`.
`mental autoimmune encephalomyelitis EAE , suggest that
`MS results from immune-dysregulation and aberrant acti-
`vation, whereby CNS myelin proteins serve as autoanti-
`
`Abbreviations: CNS, central nervous system; Cop-1, Copolymer-1;
`EAE, experimental autoimmune encephalomyelitis; IL, interleukin; MS,
`multiple sclerosis; MBP, myelin basic protein; PBL, peripheral blood
`leukocytes; sIL-2R, soluble IL-2 receptor; TGF,
`transforming growth
`factor; Th, T helper cells; TNF, tumor necrosis factor
`Corresponding author. Tel.: q972 4 8250851; fax: q972 4 8250909;
`)
`e-mail: millera@tx.technion.ac.il
`
`gens leading to a T cell-driven inflammatory and demyeli-
`(cid:14)
`nating process Martin et al., 1992; Steinman et al., 1994;
`.
`Hafler and Weiner, 1995 . The immune-dysregulation in
`MS involves both cellular and humoral arms of the im-
`mune response and can be identified in the peripheral
`(cid:14)
`.
`blood, cerebrospinal fluid CSF and CNS. These include:
`(cid:14)
`.
`defective immune-suppressor responses Antel et al., 1986 ;
`elevated T cell reactivity against various myelin antigens,
`(cid:14)
`.
`such as myelin basic protein MBP , proteolipid protein
`(cid:14)
`.
`(cid:14)
`.
`PLP or myelin oligodendrocyte glycoprotein MOG
`(cid:14)
`.
`Steinman et al., 1995 ;
`increased expression of MHC
`(cid:14)
`.
`class II molecules on antigen presenting cells APCs ,
`monocytes, endothelial as well as glial cells; elevated
`circulating memory T cells CD4 rIL-
`q
`(cid:14)
`levels of
`2R rCD45RO
`q
`q. (cid:14)
`Martin et al., 1992; Steinman et al.,
`.
`1994; Hafler and Weiner, 1995; Inobe et al., 1996 ; raised
`
`0165-5728r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved.
`(cid:14)
`.
`PII: S 0 1 6 5 - 5 7 2 8 9 8 0 0 1 9 1 - X
`
`Page 1 of 9
`
`YEDA EXHIBIT NO. 2067
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`114
`
`A. Miller et al.rJournal of Neuroimmunology 92 1998 113–121
`)
`(
`
`levels of cell surface adhesion molecules on T cells and
`(cid:14)
`macrophages Antel and Owens, 1993; Cannella and Raine,
`.
`1995; Weller et al., 1996 . The elevated levels of adhesion
`molecules correlate with the degree of blood brain barrier
`(cid:14)
`.
`(cid:14)
`BBB eruption Sharief et al., 1993; Rieckmann et al.,
`.
`1994 . MS is also characterized by elevated levels of
`pro-inflammatory cytokines such as tumor necrosis factor-a
`(cid:14)
`.
`(cid:14)
`.
`(cid:14)
`.
`TNFa , interleukin IL -1, IL-2 and interferon IFN -g, in
`(cid:14)
`the peripheral blood Sharief and Hentges, 1991; Trotter et
`.
`(cid:14)
`al., 1991; Hartung et al., 1995 , CSF Hauser et al., 1990;
`.
`(cid:14)
`Rudick and Ransohoff, 1992 and in brain lesions Hof-
`.
`mann et al., 1980 . The association of these inflammatory
`(cid:14)
`cytokines with disease activity Hauser et al., 1990; Sharief
`and Hentges, 1991; Rudick and Ransohoff, 1992; Hartung
`q
`.
`et al., 1995 implies that CD4 T cells of the T helper type
`(cid:14)
`. (cid:14)
`.
`1 Th1 Voshuhl et al., 1993; Windhagen et al., 1996 and
`(cid:14)
`.
`macrophages Cua et al., 1995; van der Laan et al., 1996 ,
`play a pivotal role in the immunopathogenesis of the CNS
`demyelinating disease. Moreover, the relative low levels
`and defective production of IL-10, IL-4 and TGF-b in
`(cid:14)
`patients with active multiple sclerosis Mokhtarian et al.,
`(cid:14)
`.
`1994 , and their protective role in EAE Johns et al., 1991;
`Kuruvilla et al., 1991; Racke et al., 1991; Kennedy et al.,
`1992; Van der Veen and Stohlman, 1993; Fabry et al.,
`.
`1995; Falcone and Bloom, 1997 , suggest that T cells of
`the Th2 and Th3 phenotypes, and their characteristic cy-
`tokine products may be involved in induction of remission
`and in suppression of the disease process.
`Increased understanding of the animal model and the
`human demyelinating disease has recently led to the imple-
`mentation of a number of immunomodulatory strategies in
`(cid:14)
`the treatment of MS. These include: IFN-b-1b IFNb
`.
`multiple sclerosis study group, 1993; Paty, 1993 , IFN-b-1a
`(cid:14)
`.
`Jacobs et al., 1994; Pozzilli et al., 1996 , as well as the
`(cid:14)
`w
`synthetic polypeptide copolymer-1 Cop-1; Copaxone ;
`.
`Glatiramer Acetate , originally synthesized with the aim of
`mimicking the myelin antigen MBP. Cop-1 treatment sup-
`(cid:14)
`presses EAE induced in a variety of species Teitelbaum et
`.
`al., 1997 , and has been demonstrated to reduce clinical
`disease activity in humans with relapsing–remitting MS
`(cid:14)Abramsky et al., 1977; Bornstein et al., 1987; Johnson et
`.
`(cid:14) .
`al., 1995 . The mechanism s of Cop-1 action are not fully
`elucidated. It has been suggested that following binding to
`(cid:14)
`.
`MHC class II molecules on antigen presenting cells APCs ,
`Cop-1 competes with myelin antigens for presentation by
`APC,
`thus preventing myelin-specific T cell activation
`(cid:14)Racke et al., 1992; Teitelbaum et al., 1992; Fridkis-Hareli
`.
`et al., 1994; Teitelbaum et al., 1997 . An additional mech-
`anism suggested to be involved in Cop-1 immunomodula-
`tory activity is the induction of specific suppressor cells
`(cid:14)
`.
`Teitelbaum et al., 1997 . However, Cop-1-driven suppres-
`sor mechanisms have not yet been clarified in patients. To
`further evaluate Cop-1 immunomodulatory activities, we
`conducted a clinical and immunological study in relapsing
`remitting MS patients treated with Copaxonew for 12
`months in an open trial.
`
`2. Materials and methods
`
`2.1. Patients and treatment
`
`.
`(cid:14)
`Ten patients nine female and one male , age range
`(cid:14)
`.
`21–58 years mean, 37.5 "11.94 years , with clinically
`definite and laboratory supported relapsing remitting MS
`(cid:14)
`.
`according to the criteria of Poser et al. Poser et al., 1983
`the MS Center, Lady Davis Carmel
`were followed at
`Hospital, Haifa, Israel,
`in an open trial. Mean disease
`duration was 9.4 years. Inclusion criteria included a history
`of at least two clearly identified and documented relapses
`in the 2 years prior to study entry; all patients were
`ambulatory, defined by an expanded disability status scale
`(cid:14)
`. (cid:14)
`.
`EDSS Kurtzke, 1993 of 0 through 5.0; patients had not
`been corticosteroid treated for at least 3 months prior to
`trial. Patients were excluded if they had ever received
`immunosuppressive
`therapy with cytotoxic
`activity
`.
`(cid:14)
`azathiorine, cyclophosphamide or cyclosporine or lym-
`phoid irradiation. Additional exclusion criteria included
`pregnancy or lactation, and all women were required to use
`an adequate contraceptive method. Signed informed con-
`sent was obtained from patients and the study was ap-
`proved by the Ethical Committee, Lady Davis Carmel
`Medical Center. Age and sex matched controls ns6
`(cid:14)
`.
`were included in all immunological assays. Copaxonew, 20
`(cid:14)mg supplied by Teva Pharmaceutical Industries, Petach
`.
`Tiqva, Israel , was daily self administered, subcutaneously,
`under a protocol approved by the Israeli Health Adminis-
`tration.
`Clinical outcome measures were: annual relapse rate,
`the absolute changes in EDSS score and the proportion of
`patients with ‘improved’, ‘stable’ or ‘worsened’ clinical
`disability, by the end of the treatment. Relapse was defined
`as the appearance or reappearance of one or more neuro-
`logical abnormalities that persisted for at least 24 h, and
`had been preceded by a stable or improving neurological
`state of at least 30 days.
`
`2.2. Serum le˝els of cytokines
`
`.
`(cid:14)
`The IFN-g, IL-2 Endogen, MA, USA , sIL-2R, IL-4
`(cid:14)
`.
`and IL-10 high sensitivity, R&D Systems, CA serum
`levels were determined by commercial ELISA kits. Data
`was calculated from duplicate samples on the basis of
`titration curves obtained from standards supplied by the
`manufactures.
`
`2.3. Peripheral blood leukocytes
`
`.
`(cid:14)
`Peripheral blood 10 ml was collected in EDTA and
`(cid:14)
`.
`immediately placed in ice. Red blood cells RBC were
`(cid:14)
`.
`spun down at 100=g, 48C and leukocytes PBL were
`then pelleted from the plasma by centrifugation at 650=g,
`
`Page 2 of 9
`
`YEDA EXHIBIT NO. 2067
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`A. Miller et al.rJournal of Neuroimmunology 92 1998 113–121
`)
`(
`
`115
`
`Fig. 1. Copolymer-1 treatment leads to reduced annual relapse rate in MS
`patients. The number of relapses for each patient during the 2 years prior
`(cid:14)
`.
`(cid:14)
`pre to treatment and during 1 year of treatment are presented figure on
`left; › designates start of treatment . Figure on the right shows mean
`.
`annual relapse rate prior to and after 1 year of treatment ps0.001 .
`(cid:14)
`.
`
`Fig. 3. Copolymer-1 treatment is associated with reduction in serum IL-2
`(cid:14)
`.
`soluble receptor
`sIL-2R . Serum sIL-2R levels were determined by
`ELISA in MS patients, prior to and during Cop-1 treatment, and in
`healthy controls. The values presented were calculated using a standard
`titration curve and represent mean values for each group"standard error
`of mean.
`
`48C, for 10 min. Residual RBC were lysed with TE buffer
`(cid:14)
`.
`10 mM Tris–HCl and 1 mM EDTA, pH 8.0 . RNA was
`extracted from either fresh or liquid nitrogen frozen PBL.
`
`2.4. RT-PCR
`
`Total cellular RNA was isolated from PBL with Tri-Re-
`(cid:14)
`.
`agent Medical Research Center, OH according to the
`manufacturer’s instructions. RNA samples were washed
`twice in 75% ethanol, air dried and suspended in 10 ml
`RNase free TE buffer. RNA quantity and quality were
`determined by spectrophotometric absorbance at 260r280
`nm. RNA samples were stored at y708C. Complementary
`(cid:14)
`.
`DNA cDNA was prepared by reverse transcription at
`378C for 60 min in 50 ml reaction mixture containing 2 mg
`RNA, 400 U Moloney Murine leukemia virus reverse
`(cid:14)
`.
`transcriptase Amersham, OH in the presence of RNA-
`(cid:14)
`.
`(cid:14)
`guard Pharmacia, Germany and oligo-hexamers Phar-
`.
`macia, NJ . The enzyme was than inactivated and the
`integrity of the RNA was assessed by amplifying 1 ml
`(cid:14)
`.
`cDNA using G3PDH specific primers Clontech, CA . The
`(cid:14)
`.
`(cid:14)
`.
`polymerase chain reaction PCR mix 25 ml volume
`(cid:14)
`included reaction buffer 10 mM Tris–HCl, 50 mM KCl,
`.
`1.5 mM MgCl , 0.1% Triton X-100 0.2 mM each dNTP
`2
`
`X
`X
`.
`(cid:14)
`Sigma, MO , 0.4 mM 5 and 3 primers and 1U DNA Taq
`(cid:14)
`.
`polymerase Appligene, France . Thirty-five cycles of PCR
`amplification were performed, each consisting of denatura-
`tion at 948C, annealing at 688C, and extension at 728C in a
`(cid:14)
`.
`thermal cycler MJ Research, MA, USA . PCR products
`were visualized by UV following electrophoresis of prod-
`(cid:14)
`.
`ucts in ethidium bromide stained 3% agarose gels Sigma .
`(cid:14)
`Standard molecular size markers, negative controls PCR
`.
`(cid:14)
`mix without sample cDNA and positive controls standard
`.
`cDNA supplied by manufacturer, Clontech were run with
`each PCR assay.
`
`2.5. Semi-quantitation of cytokine mRNA
`
`(cid:14)
`Semi-quantitative assessment of cytokine IL-4, TNFa,
`.
`and TGF-b mRNA was performed by parallel PCR using
`(cid:14)
`.
`cytokine specific primers and G3PDH primers Clontech .
`To ensure that amplification was being performed at linear
`(cid:14)
`phase, three different quantities of cDNA 2, 1, and 0.5 ml
`cDNA for G3PDH amplification; 4, 2, and 1 ml cDNA for
`TNFa and TGF-b amplification; 6, 4, and 2 ml for IL-4
`.
`amplification, determined in preliminary experiments were
`utilized in all assays. PCR was performed as above using
`
`Fig. 2. Copolymer-1 treatment leads to stabilization of clinical disability.
`Disability of patients, assessed and graded according to Kurtzke’s ex-
`(cid:14)
`.
`tended disability status scale EDSS scores , were evaluated prior to
`(cid:14)
`.
`(cid:14)
`.
`treatment pre and after 1 year of Cop-1 treatment 12 m .
`
`Fig. 4. Copolymer-1 treatment leads to elevation in serum IL-10. Serum
`IL-10 levels were determined in MS patients, prior to and during Cop-1
`treatment, and in healthy controls by ELISA. The values presented were
`calculated using a standard titration curve and represent mean values for
`each group"standard error of mean.
`
`Page 3 of 9
`
`YEDA EXHIBIT NO. 2067
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`116
`
`A. Miller et al.rJournal of Neuroimmunology 92 1998 113–121
`)
`(
`
`.
`(cid:14)
`appropriate annealing temperatures 60–658C for each
`cytokine. Resulting PCR products were visualized by UV
`following electrophoresis, intensity of bands was measured
`
`(cid:14)
`by video densitometry Bio Imaging Systems, Applitec,
`.
`Israel and the ratio between sample to G3PDH products
`was calculated.
`
`Fig. 5. Copolymer-1 treatment leads to modulation of cytokine mRNA expression in peripheral blood leukocytes. Semi-quantitative assessment of cytokine
`(cid:14)
`.
`(cid:14)
`.
`TNFa, A; IL-4, B; TGF-b, C mRNA from PBL was performed by RT-PCR using three concentrations of cDNA for each time period as described in
`(cid:14)
`.
`(cid:14)
`.
`Section 2. Housekeeping gene, G3PDH, was amplified in parallel. Histogram upper panel shows mean ratio "standard error between densitometric
`intensity of specific cytokine to G3PDH PCR products for 10 treated MS patients at each time period examined and for four controls. Results of
`representative samples obtained at three different times presprior to treatment; 3 and 6 months; NCsnegative and PCspositive controls for PCR
`(cid:14)
`.
`amplification of Cop-1 treatment, are shown in bottom panel of figures.
`
`Page 4 of 9
`
`YEDA EXHIBIT NO. 2067
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`A. Miller et al.rJournal of Neuroimmunology 92 1998 113–121
`)
`(
`
`117
`
`2.6. Statistical e˝aluation
`
`Mean relapse rate and changes from baseline according
`to EDSS, were assessed using repeated-measures ANOVA.
`Statistical significance of data was determined using Stu-
`dent’s t-test for paired analysis of parameters with normal
`distribution when comparing a single parameter at differ-
`ent times and Ranked Signed Wilcoxon for comparison of
`groups. P values of -0.05 were considered significant.
`
`3. Results
`
`3.1. Copolymer-1 treatment leads to reduced annual re-
`lapse rate
`
`During the 1-year follow-up the Cop-1 treated patients
`(cid:14)
`.
`had six confirmed relapses Fig. 1 . The mean annual
`relapse rates was 0.6 during the treatment as compared to
`1.4 prior to Cop-1 therapy, a 57% reduction, which was
`ps0.001 . Five 50% MS pa-
`(cid:14)
`.
`(cid:14)
`.
`statistically significant
`tients receiving Cop-1 were relapse-free throughout the
`trial.
`
`3.2. Stabilization of neurological disability by Copolymer-1
`treatment
`
`Among the 10 patients receiving Cop-1, neurological
`disability improved in four, was stable in five, and wors-
`(cid:14)
`.
`ened in only one patient Fig. 2 . Thus, 90% of the patients
`treated with Cop-1 were free of disease progression. No
`clinically significant adverse event was noted. The most
`commonly recognized adverse event during treatment was
`a localized injection-site reaction consisting of mild ery-
`themia and induration, which sometimes persisted for sev-
`eral days.
`
`3.3. Modulation of serum sIL-2R and IL-10
`
`(cid:14)
`The levels of sIL-2R in the sera were higher
`though
`not of statistical significance, ps0.7 in pretreated MS
`.
`(cid:14)
`.
`patients compared to healthy controls Fig. 3 . Slight
`changes, including first a significant elevation at 3 months
`ps0.04 and eventually a slight decrease in the mean
`(cid:14)
`.
`value of this receptor at 12 months of treatment ps0.43;
`(cid:14)
`.
`pre vs. 12 months were observed. At all times a wide
`variability in the level of sIL-2R was found amongst the
`patients.
`The mean level of serum IL-10 was slightly lower,
`though not significant, in pre-treated MS patients, com-
`ps0.4
`(cid:14)
`. (cid:14)
`.
`pared to healthy controls
`Fig. 4 . A 2-fold
`ps0.04 increase in the mean level of this
`(cid:14)
`.
`significant
`cytokine was observed at 3 months of treatment, as com-
`pared to baseline levels. However,
`the increment was
`attenuated at 6 and 12 months of treatment ps0.16 . It
`(cid:14)
`.
`should be noted that no detectable levels of IFN-g, IL-2
`and IL-4 were observed in these serum samples.
`
`3.4. Cytokine mRNA expression of TNFa, IL-4 and TGF-b
`in PBL
`
`Profound differences in baseline cytokine mRNAs ex-
`pression were observed in PBL derived from MS patients
`as compared to controls and marked changes were identi-
`(cid:14)
`.
`fied in patients during Cop-1 treatment Fig. 5 . Elevated
`mRNA expression levels of the pro-inflammatory cytokine
`TNFa were observed in all MS patients prior to treatment,
`whereas no detectable levels were observed in healthy
`controls. The TNFa levels decreased in nine of the 10 MS
`patients following Cop-1 treatment and after 12 months of
`(cid:14)
`treatment there was a 4-fold, statistically significant p -
`.
`0.004 , decrease in the mean mRNA level of this cytokine
`(cid:14)
`.
`compared to the mean level prior to treatment Fig. 5A .
`The changes in TNFa mRNA were accompanied by
`enhanced mRNA expression of the anti-inflammatory cy-
`(cid:14)
`.
`tokines IL-4 and TGF-b Fig. 5B–C . In 70% of patients
`no mRNA expression for IL-4 was observed prior to
`Cop-1 administration. After 12 months of treatment, seven
`of the 10 patients showed IL-4 mRNA expression in PBL
`(cid:14)
`.
`Fig. 5B . Following 3 and 6 months of treatment, the
`expression of this cytokine was significantly elevated in
`comparison to pre-treatment levels ps0.05 and ps0.03,
`(cid:14)
`.
`respectively . Although still higher than baseline or control
`levels, after 12 months of treatment the levels of IL-4
`mRNA were more attenuated. TGF-b mRNA expression
`was observed in 80% of MS patients prior to Cop-1
`(cid:14)
`.
`treatment Fig. 5C . However a 5-fold, statistically signifi-
`cant ps0.03 increase in the mean level of this cytokine
`(cid:14)
`.
`was observed after 6 months of treatment. At 12 months of
`treatment, the differences between MS treated patients and
`ps
`(cid:14)
`healthy controls were still statistically significant
`ps0.38 com-
`.
`(cid:14)
`.
`0.02 though they were not significant
`pared to pre-treatment values.
`
`4. Discussion
`
`The immunological follow-up carried out in the present
`study adds additional insight into the mechanisms of Cop-1
`immunomodulatory activities in MS patients and is, to the
`best of our knowledge, the first report of regulatory mech-
`anisms of Cop-1 in humans. Although only 10 patients
`were studied in an open trial, our results demonstrating
`that Cop-1 treatment leads to a significant reduction of the
`mean relapse rate as well as stabilization of disability,
`(cid:14)
`confirm previous published clinical studies Abramsky et
`.
`al., 1977; Bornstein et al., 1987; Johnson et al., 1995 . The
`association of the clinical reduction of disease activity with
`the shift of cytokine profile, observed in the present work,
`may contribute to our understanding of the immunomodu-
`latory effects mediated by Cop-1.
`It has been proposed that the predominant mechanism
`underlying the immunomodulatory activities of Cop-1 re-
`
`Page 5 of 9
`
`YEDA EXHIBIT NO. 2067
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`118
`
`A. Miller et al.rJournal of Neuroimmunology 92 1998 113–121
`)
`(
`
`sides in the immunological cross-reactivity of Cop-1 with
`MBP and its competition with MBP for MHC class II
`presentation. This may result in the inhibition of MBP-
`(cid:14)
`specific T cell activation Racke et al., 1992; Teitelbaum et
`al., 1992; Fridkis-Hareli et al., 1994; Teitelbaum et al.,
`.
`1997 . Nevertheless, molecular mimicry to MBP and
`blocking of MBP-specific autoimmune T cells, does not
`explain recent observations suggesting that the effect of
`Cop-1 in EAE is not restricted to a particular encephalito-
`gen, but rather suppresses disease induced by various
`(cid:14)
`.
`myelin antigens such as PLP Teitelbaum et al., 1996 and
`.
`(cid:14)
`MOG Ben-Nun et al., 1996 . The suppressive activity of
`Cop-1 has however, been shown to be organ-specific and
`limited to inflammatory and autoimmune diseases involv-
`ing CNS-myelin, such as EAE, while no effect was
`(cid:14)
`demonstrated in SLE or other autoimmune diseases Arnon
`.
`et al., 1996 . Studies in experimental animals have indeed
`suggested that the effectiveness of Cop-1 in preventing
`EAE results also from the induction of antigen-specific
`(cid:14)
`suppressor T cells that cross-react with MBP Teitelbaum
`.
`et al., 1997 . Cop-1 specific suppressor T cells isolated
`form rodents showed polarized secretion of Th2 type
`(cid:14)
`.
`cytokines Aharoni et al., 1997 . However, active suppres-
`sion as a possible mechanism for the beneficial effect of
`Cop-1 in humans with MS has not yet been described.
`q
`(cid:14)
`.
`CD4 T helper Th cells can be divided into subsets
`based on their characteristic cytokine secretion patterns
`(cid:14)
`and effector functions Mosmann and Coffman, 1987;
`.
`Street et al., 1990 . Cytokines produced by Th1 cells
`(cid:14)
`.
`IL-12, IFN-g, IL-2 and TNFa are inflammatory media-
`tors of various autoimmune processes, including autoim-
`mune demyelinating diseases where oligodendrocytes are a
`(cid:14)
`target for immune attack Voshuhl et al., 1993; Windhagen
`.
`(cid:14)
`et al., 1996 . Cytokines produced by Th2 cells IL-4 and
`.
`(cid:14)
`.
`IL-10 or Th3 cells TGF-b mediate antibody production,
`anti-inflammatory cascade and resolution of inflammatory
`(cid:14)
`and autoimmune processes Johns et al., 1991; Kuruvilla et
`al., 1991; Racke et al., 1991; Kennedy et al., 1992; Van
`der Veen and Stohlman, 1993; Fabry et al., 1995; Falcone
`.
`and Bloom, 1997 . Our present study demonstrates that
`treatment of MS patients with Cop-1 leads to modulation
`in serum sIL-2R and in TNFa mRNA expression in PBL,
`and at the same time an elevation of serum IL-10 as well
`as IL-4 and TGF-b mRNA expression. sIL-2R, generally a
`marker of T cell activation, binds secreted IL-2,
`thus
`probably leading eventually to the inhibition of T cell
`activation. This inhibition as well as the shift from Th1 to
`Th2rTh3 type immune response implies that mechanism s
`(cid:14) .
`of active suppression mediated by regulatory cytokines
`may be involved in suppression of MS by Cop-1. Of note
`is the more prominent induction of Th2rTh3 cytokines
`during the first 6 months of Cop-1 treatment which was
`later attenuated. This finding may be due to the stabiliza-
`tion of autoreactivity and maintained suppression of the
`Th1 response, manifested by the continued low levels of
`TNFa during the treatment.
`
`Modulation of an immune response from one dominated
`by excessive Th1 activity to one dominated by the protec-
`tive cytokines produced by Th2rTh3 cells, has been
`demonstrated as an effective immunomodulatory and ther-
`apeutic strategy in certain experimental autoimmune dis-
`eases. For example, IL-4 treatment in EAE resulted in
`amelioration of clinical disease, inhibition of the synthesis
`of inflammatory cytokines, the induction of MBP-specific
`TGF-b-producing Th2rTh3 cells, and diminished CNS
`(cid:14)
`.
`demyelination Van der Veen and Stohlman, 1993 . Simi-
`larly, induction of IL-10 mRNA expression was reported
`to correlate with recovery in EAE. Although in some
`(cid:14)
`reports IL-10 failed to suppress EAE Cannella et al.,
`.
`1996 , others have demonstrated that Th2 cells producing
`IL-10 inhibit encephalitogenic Th1 cells and suppress EAE
`(cid:14)Van der Veen and Stohlman, 1993; Windhagen et al.,
`.
`1996 . The induction of TGF-b demonstrated in our study,
`in association with the clinical efficacy of Cop-1 treatment
`is in-line with recent reports regarding the importance of
`this cytokine in down-regulating autoimmunity. TGF-b, a
`multifunctional anti-inflammatory cytokine with immune-
`(cid:14)
`.
`suppressive effects on T cells in vitro Merrill et al., 1989 ,
`was successfully implemented as treatment of both acute
`(cid:14)
`and chronic EAE Johns et al., 1991; Kuruvilla et al.,
`.
`1991; Racke et al., 1991 .
`Reversal of the autoimmune response from Th1 to Th2
`appears to be a common denominator of a number of novel
`immune-therapeutic strategies, both in EAE and in MS.
`(cid:14)
`.
`Cytokine-mediated immune-deviation Racke et al., 1994
`has been demonstrated also in humans during IFN-b treat-
`(cid:14)
`ment, the first treatment found to be effective in MS IFNb
`.
`multiple sclerosis study group, 1993; Paty, 1993 . Cy-
`tokine switching was also demonstrated during vaccination
`(cid:14)
`.
`with naked DNA encoding T-cell receptor TCR Vb 8.2
`(cid:14)
`.
`Waisman et al., 1996 , using Retinoid for the treatment of
`(cid:14)
`.
`EAE Racke et al., 1995 , Pentoxifilline in relapsing–re-
`(cid:14)
`.
`mitting MS Rieckmann et al., 1996 and Linomide in
`(cid:14)
`.
`progressive MS Karussis et al., 1996 . It is of interest that
`some of the beneficial effects of glucocorticosteroids in the
`treatment of acute exacerbation, appear to involve inhibi-
`tion of TNFa and lymphocyte adhesion as well as the
`induction of anti-inflammatory Th2rTh3 cytokines such as
`(cid:14)
`.
`TGF-b Pitzalis et al., 1997 .
`Cop-1 is a prototype of antigen-driven immune-inter-
`vention for the treatment of organ-specific autoimmunity.
`In addition to Cop-1, antigen-driven immune-modulation
`may be elicited by other therapeutic strategies such as oral
`tolerance, altered peptide ligands, and vaccination with
`(cid:14)
`.
`T-cell receptor TCR peptides. In all these approaches,
`including Cop-1, as shown previously in the EAE model
`and in the present study, there is evidence for the involve-
`ment of
`immune deviation and bystander suppression
`(cid:14)
`mechanisms Miller et al., 1991; Karin et al., 1994; Van-
`denbark et al., 1996; Aharoni et al., 1997; Teitelbaum et
`.
`al., 1997; Weiner, 1997 . Extensive studies in oral toler-
`(cid:14)
`.
`ance Weiner, 1997 , have shown that
`it may not be
`
`Page 6 of 9
`
`YEDA EXHIBIT NO. 2067
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`A. Miller et al.rJournal of Neuroimmunology 92 1998 113–121
`)
`(
`
`119
`
`necessary to identify the target autoantigen to suppress an
`organ-specific autoimmune disease via antigen-driven tol-
`erance; it is necessary only to administer a protein capable
`of inducing regulatory cells that secrete suppressive cy-
`tokines upon encountering the autoantigen at the target
`organ. This characteristic phenomenon of suppressor cy-
`tokines,
`that may shut-down the inflammatory process
`independently of
`the primary autoantigen or of
`the
`antigen-specificity of the autoimmune cells, has been de-
`(cid:14)
`fined as antigen-driven bystander suppression Miller et
`.
`al., 1991 . Furthermore, therapeutic strategies implicating
`mechanisms of bystander suppression may solve the prob-
`lem of spreading autoimmunity to additional autoantigens
`(cid:14)
`during the course of the ongoing disease Lehmann et al.,
`the Th1 to Th2rTh3 immune-deviation
`.
`1992 . Thus,
`demonstrated in our study,
`implicating mechanisms of
`bystander suppression, may explain the effects of Cop-1 on
`(cid:14)
`PLP and MOG-induced experimental diseases Ben-Nun et
`.
`al., 1996; Teitelbaum et al., 1996 . Immune-reactivity to
`these myelin antigens as well as spreading autoimmunity
`to other putative neuro-antigens may be downregulated by
`the anti-inflammatory mediators released at the microenvi-
`ronment of the target organ. It should be noted, however,
`that a shift from a Th1 to a Th2 cytokine production
`phenotype may carry the risk of elicitation of the humoral
`arm of the immune response in a dysregulated manner
`with potential aggravation of the autoimmune process.
`(cid:14)
`.
`Lafaille et al. Lafaille et al., 1997 recently reported that
`MBP-specific Th2 cells cause EAE in immune-deficient
`hosts rather than protect them from the disease. Similarly,
`Genain et al. have demonstrated that enhanced titers of
`autoantibodies to MOG may mediate the late complica-
`(cid:14)
`tions of immune-deviation therapy in EAE Genain et al.,
`.
`1996 . Hence, careful follow-up, both clinical as well as
`immunological parameters may be required in patients
`treated with therapeutic strategies implicating immune-de-
`viation, in order to identify such possible delayed compli-
`cations.
`In conclusion, the beneficial clinical effects of Copa-
`xonew in MS patients, demonstrated also in the present
`study, may be attributed to the induction of Cop-1-specific
`regulatory Th2rTh3 cells
`(cid:14)
`.
`cross-reactive with MBP
`which, upon encountering MBP at the target organ, secret
`anti-inflammatory cytokines, leading to bystander suppres-
`sion. Further understanding of the mechanisms implicated
`in the immune-modulatory effects of Cop-1 as well as
`other immunoregulatory drugs, may contribute to the de-
`sign of future therapeutic cocktails and combination thera-
`(cid:14)
`.
`pies Milo and Panitch, 1995 .
`
`Acknowledgements
`
`We thank Drs. Rivka Riven-Kreitman and Nora Tarcic
`for helpful discussions. This work was supported by a
`(cid:14)
`.
`grant from Teva Pharmaceutical Industries, Israel and the
`
`combined research grant from the Ministry of Health,
`Carmel Medical Center and the Technion, Israel Institute
`of Technology, Israel.
`
`References
`
`Abramsky, O., Teitelbaum, D., Arnon, R., 1977. Effect of a synthetic
`(cid:14)
`.
`polypeptide Copolymer 1 on patients with multiple sclerosis and
`acute disseminated encephalomyelitis: preliminary report. J. Neurol.
`Sci. 31, 433–438.
`Aharoni, R., Teitelbaum, D., Sela, M., Arnon, R., 1997. Copolymer 1
`induces T cells of the T helper type 2 that crossreact with myelin
`basic protein and suppress experimental autoimmune encephalomyeli-
`tis. Proc. Natl. Acad. Sci. USA 94, 10821–10826.
`Antel, J.P., Owens, T., 1993. The attraction of adhesion molecules. Ann.
`w
`x
`Neurol. 34, 123–124, editorial .
`Antel, J.P., Bania, M.B., Reder, A., Cashman, N., 1986. Activated
`suppressor cell dysfunction in progressive multiple sclerosis. J. Im-
`munol. 137, 137–141.
`Arnon, R., Sela, M., Teitelbaum, D., 1996. New insights into the
`mechanism of action of copolymer 1 in experimental allergic en-
`cephalomyelitis and multiple sclerosis. J. Neurol. 243, S8–13.
`Ben-Nun, A., Mendel, I., Bakimer, R., Fridkis-Hareli, M., Teitelbaum,
`D., Arnon, R., Sela, M., Kerlero de Rosbo, N., 1996. The autoim-
`(cid:14)
`.
`mune reactivity to myelin oligodendrocyte glycoprotein MOG in
`multiple sclerosis is potentially pathogenic: effect of copolymer 1 on
`MOG induced disease. J. Neurol. 243, S14–22.
`Bornstein, M.B., Miller, A., Slagle, S., Weitzman, M., Crystal, H.,
`Drexler, Keilson, M., Merria, M., Wassertheil-Smollerer, S., Spada,
`V., Arnon, R., Jackobsohn, I., Teitelbaum, D., Sela, M