Journal of the Neurological Sciences 201 (2002) 71 – 77
`
`www.elsevier.com/locate/jns
`
`Sustained immunological effects of Glatiramer acetate in patients with
`multiple sclerosis treated for over 6 years
`
`M. Chen a, K. Conway a, K.P. Johnson a, R. Martin b, S. Dhib-Jalbut a,c,*
`
`aUniversity of Maryland School of Medicine, Baltimore, MD 21201, USA
`bNeuroimmunology Branch, NINDS, National Institutes of Health, Bethesda, MD 20892, USA
`cBaltimore VA Medical Center, Baltimore, MD 21201, USA
`
`Received 6 February 2002; received in revised form 9 April 2002; accepted 10 June 2002
`
`Abstract
`
`The availability of a group of multiple sclerosis (MS) patients at the University of Maryland, who had participated in the pivotal
`CopaxoneR trial in the early 1990s, provided an opportunity to examine the long-term immunologic effects of Glatiramer acetate (GA)
`treatment in MS. Forty-eight GA-reactive T-cell lines (TCL) were generated from 10 MS patients who have been receiving GA treatment for
`6 – 9 years. Proliferative responses, cytokine production, and cross-reactivity with myelin basic protein (MBP) and the MBP im-
`munodominant peptide 83 – 99 were compared to responses obtained from 10 MS patients who were tested pretreatment and after a shorter
`period of treatment ranging from 1 to 10 months. The results indicate that while long-term treatment with GA results in a 2.9-fold decrease in
`the estimated precursor frequency of GA-reactive T-cells, the sustained response to GA remains Th2-biased and in part cross-reactive with
`MBP and MBP (83 – 99) as measured by proliferation and cytokine release assays. The results indicate that despite a drop in the precursor
`frequency of GA-reactive T-cells with long-term treatment, the sustained response remains predominantly Th2-biased and cross-reactive with
`MBP, which is consistent with the anti-inflammatory effects of the drug and bystander suppression.
`D 2002 Elsevier Science B.V. All rights reserved.
`
`Keywords: Multiple sclerosis; Glatiramer acetate; CopaxoneR; Immune deviation; Immunotherapy
`
`1. Introduction
`
`Glatiramer acetate (GA) (CopaxoneR), formerly known
`as Copolymer-1, is a synthetic amino acid copolymer that
`consists of four amino acids: L-alanine, L-lysine, L-glutamic
`acid, and L-tyrosine in fixed molar ratio and an average
`molecular weight of 4.7 – 11 kDa. The molecule was found
`to inhibit experimental allergic encephalomyelitis (EAE),
`and was subsequently developed for treating patients with
`relapsing – remitting multiple sclerosis (MS) [1 – 4]. When
`given subcutaneously in daily doses of 20 mg, the drug
`reduces the relapse rate, slows the accumulation of disabil-
`ity, and reduces MRI disease activity approximately 6
`months after treatment is initiated [5 – 8].
`
`* Corresponding author. Department of Neurology, University of
`Maryland Hospital, Rm. N4W46, 22 S. Greene Street, Baltimore, MD
`21201, USA. Tel.: +1-410-706-4216; fax: +1-410-706-0186.
`E-mail address: sjalbut@umaryland.edu (S. Dhib-Jalbut).
`
`Although the mechanism of action of GA is not fully
`understood, a number of studies both in EAE and in MS
`point to the induction of a GA-reactive T-cell repertoire with
`a protective Th2 anti-inflammatory phenotype as a likely
`mechanism of action [9 – 16]. The GA-reactive T-cells may
`exert their protective action by entering the CNS compart-
`ment and the production of anti-inflammatory cytokines in
`response to cross-recognition of myelin basic protein (MBP;
`bystander suppression) [17]. Additional mechanisms have
`been proposed, as a result of the ability of GA to bind
`promiscuously to HLA class II molecules associated with
`MS, including DR2 and DR4, on antigen presenting cells
`[18 – 20]. This includes inhibition of presentation of several
`myelin antigens to T-cells [4,11,15,21 – 24], induction of
`anergy or deletion of a population of GA-reactive T-cells
`[15,26].
`Although peripheral blood mononuclear cells (PBMC)
`from the majority of MS patients proliferate in response to
`GA, this response declines approximately 6 months after
`treatment is initiated [13,25]. The surviving GA-reactive T-
`
`0022-510X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
`PII: S 0 0 2 2 - 5 1 0 X ( 0 2 ) 0 0 2 0 1 - 0
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`cells demonstrate a high degree of degeneracy, with a Th2-
`polarized response characterized by IL-5 and IL-13 secre-
`tion [13]. In view of the decline in the proliferative response
`to GA with time, we investigated whether MS patients who
`had received the drug for 6 – 9 years, continue to generate
`the anti-inflammatory GA-reactive Th2 cells associated with
`the therapeutic effect of the drug, and whether these cells
`have the characteristics of bystander suppression.
`
`2. Methods
`
`2.1. Antigens
`
`Glatiramer acetate (Copaxone lots 119135 and 119137)
`was from Teva Pharmaceutical Ind., Israel. Guinea pig MBP
`was purchased from Sigma (St. Louis, MO, USA). MBP
`peptide (83 – 99) (purity > 95%) was synthesized by Mixture
`Sciences (San Diego, CA, USA) using the simultaneous
`multiple synthesis methods [27]. Tetanus toxoid (TT) was
`obtained from Pasteur Merieux Connaught (North York,
`Ontario, Canada).
`
`2.2. Subjects
`
`Ten patients with clinically definite MS, who participated
`in the Copolymer-1 pivotal multicenter trial [7,8] and
`followed up at the Maryland Center for MS, were included
`in this study. Those patients received Glatiramer acetate
`treatment for 6 – 9 years and are termed the long-term
`treatment group. The clinical characteristics of the patients
`are presented in Table 1. Results were compared to those
`from another group of 10 MS patients who were studied 1 –
`10 months after they have been on Glatiramer acetate
`treatment (short-term treatment group) and on whom we
`have reported earlier [16]. The study was approved by the
`University of Maryland Institutional Review Board, and the
`subjects’ consent was obtained.
`
`Table 1
`Proliferative responses of GA – TCL from the long-term treatment group
`Relapsesa EDSSb
`
`Patient
`
`Duration of
`GA treatment
`(year)
`
`Precursor
`No.
`GA – TCL frequency
`
`Entry End
`
`2.3. Cells
`
`Approximately 100 cc of heparinized blood was obtained
`by venipuncture from the 10 MS patients at each time point.
`Peripheral blood mononuclear cells (PBMC) were prepared
`using a Ficoll-Hypaque gradient as described in the suppli-
`er’s protocol (ICN Biomedicals, OH, USA). For the gen-
`eration of antigen specific T-cell lines (TCL), cells were
`cultured without prior freezing.
`
`2.4. Generation of antigen-specific T-cell lines
`
`Antigen-specific TCL were generated by the split-well
`assay as described previously [28]. Briefly, PBMC were
`seeded in complete medium RPMI 1640 (Biofluid, Rock-
`ville, MD, USA) containing 5% human AB serum (Sigma),
`2 mM L-glutamine, 50 Ag/ml gentamicin, 100 U/ml pen-
`icillin/streptomycin, at 1 105 cells/well into 96-well U-
`bottom microtiter plates (Nunc, Roskilde, Denmark) and
`stimulated with antigens (20 Ag/ml of GA or 5 Ag/ml of TT).
`Thirty microtiter wells were prepared for each antigen.
`Human recombinant IL-2 (Biosource International Cama-
`rillo, CA, USA) was added to the culture on day 8 at a final
`concentration of 20 U/ml. On day 15, 50 Al of the cell
`suspension were transferred into each of two adjacent wells
`of a separate 96-well U-bottom microtiter plate, and 150 Al
`of complete medium containing 1 105 autologous irradi-
`ated PBMC (3000 rad) was added. One well was stimulated
`with 20 Ag/ml GA and the other with medium only. After 48
`h, 1 ACi/well of 3H-thymidine (Amersham Pharmacia Bio-
`tech, Piscataway, NJ, USA) was added. Eighteen hours later,
`cells were harvested on an automated cell harvester (Tom-
`tec, Hamden, CT, USA) and 3H-thymidine incorporation
`was measured using a Betaplate counter (Wallac, Gaithers-
`burg, MD, USA). Wells that showed stimulation index (SI,
`cpm of cells with antigen/cpm of cells without antigen) of
`>2, and background counts >200 cpm were further
`expanded and characterized.
`
`2.5. Precursor frequency estimation
`
`The precursor frequency of GA-reactive T-cells was
`estimated following the first stimulation cycle based on
`the number of proliferating microtiter wells and seeding
`105 cells/well; precursor frequency = number of GA-reactive
`TCL/number of wells plated 105.
`
`2.6. Cytokine production
`
`IFN-g and IL-5 were measured as markers of Th-1 and
`Th-2 phenotypes, respectively. Cells from GA-reactive
`wells identified after the first stimulation were restimulated
`with antigen, feeders, and IL-2 for 7 – 10 days. These cells
`were then harvested, washed, and cultured at 1 105 cells/
`well with 1 105/well of irradiated autologous PBMC with
`or without antigen for 48 h. Supernatants were harvested
`
`1
`1
`1
`7
`3
`7
`0
`1
`2
`1
`
`MS 12(503) 9
`MS 13(504) 6
`MS 14(525) 8
`MS 15(513) 6
`MS 16(524) 8
`MS 17(517) 8
`MS 18(518) 8
`MS 19(502) 9
`MS 20(519) 6
`MS 21(501) 6
`Total
`a Relapses during treatment period.
`b EDSS: expanded disability status scale at entry and end of the
`reporting period.
`
`1.5
`1.5
`2.5
`1.5
`5.0
`1.5
`1.5
`2.0
`2.5
`1.5
`
`1.0
`1.5
`2.5
`4.5
`4.0
`4.0
`1.0
`2.0
`2.5
`1.0
`
`5
`7
`4
`1
`17
`6
`4
`2
`2
`2
`48
`
`1.56/106
`1.09/106
`1.25/106
`0.33/106
`5.31/106
`1.87/106
`1.33/106
`0.67/106
`0.26/106
`0.33/106
`1.40/106
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`and stored at 70 jC. IL-5 and IFN-g were measured by
`ELISA according to the manufacturer’s protocol (Bio-
`source). The sensitivity of the ELISA was < 4 pg/ml for
`both IL-5 and IFN-g. The GA-specific TCL were classified
`as Th0-, Th1- or Th2-biased based on the ratio of IFN-g/IL-
`5 secretion. A ratio >2 was arbitrarily defined as Th1 bias,
`< 0.5 as Th2 bias, and a value between 0.5 and 2 as Th0.
`
`2.7. Cross-reactivity studies
`
`Cross-reactivity of GA – TCL with MBP and MBP pep-
`tide 83 – 99 were examined using 10 Ag/ml of the antigens.
`These antigen concentrations had previously been found to
`give at least half-maximal stimulation in the vast majority of
`TCL [15]. Cross-reactivity of GA-reactive T-cells with MBP
`was determined by the split-well technique as described
`z
`above. Proliferative responses
`2 with a background of
`200 cpm or higher were considered cross-reactive. Cross-
`reactivity by cytokine release was performed as follows:
`1 105 cells/well of GA-reactive TCL were seeded in 96-
`well U-bottom microtiter plate together with 1 105 irradi-
`ated autologous PBMC in the absence of antigen or in the
`presence of GA, MBP, or MBP peptide 83 – 99 in triplicate
`wells. After cells were cultured for 48 h, 100-Al supernatants
`were removed from each well for cytokine level determi-
`nations. Cross-reactivity as measured by cytokine production
`was determined by pooling supernatants from triplicate wells
`and subsequent measurements of IL-5 and IFN-g levels.
`
`2.8. Statistical analysis
`
`A software package (Graphpad, Prismk) was used in the
`statistical analysis. Differences in lymphoproliferative
`responses and cytokine production between groups were
`compared using the Student’s t-test. A p-value < 0.05 was
`considered significant.
`
`3. Results
`
`3.1. Frequency of GA-reactive T-cells
`
`Forty-eight GA-reactive TLC were generated from 10
`MS patients who have been receiving GA treatment for 6 – 9
`
`Fig. 1. Regression lines for changes in the precursor frequency of GA and
`TT reactive T-cells with increasing GA treatment duration in MS patients.
`GA responses were obtained from 18 MS patients and TT responses from
`nine. The numbers adjacent to the regression lines indicate slopes (S) and p-
`values ( P).
`
`years (long-term treatment group; MS 12-21). The estimated
`precursor frequency of the GA-reactive T-cells ranged from
`0.26 to 5.31 in 1 million (Table 1). Precursor frequencies of
`GA-reactive T-cells generated from the long-term treatment
`group were compared to those generated from 10 MS
`patients who were studied pretreatment (81 TCL) and 1 –
`10 months after initiation of treatment with GA (130 TCL)
`(short-term treatment group) and on whom we have reported
`previously [16] (Table 2). The mean estimated precursor
`frequency of the GA-reactive TCL was 1.4/106 in the long-
`term treatment group, significantly lower than that of the
`short-term treatment group (4.03/106) ( p = 0.027). Regres-
`sion lines for the estimated precursor frequency of GA-
`reactive T-cells generated for the short-term treatment group
`z
`(1 – 6 months; n = 8) and long-term treatment group (
`6
`years; n = 10) demonstrated a significant drop in the esti-
`mated precursor frequency of GA – TCL with increasing
`treatment duration ( P = 0.010). In contrast, the estimated
`precursor frequency for the TT – TCL did not change sig-
`nificantly with long-term treatment (Fig. 1).
`
`3.2. GA-reactive TCL maintain a Th2 phenotype bias during
`long-term treatment with GA
`
`We and others have previously demonstrated that GA
`treatment induced an immune deviation in MS patients,
`characterized by a cytokine profile shift from Th1 pretreat-
`ment to Th2 during GA treatment [16]. In this study, we
`examined the cytokine secretion profile of 34 GA-reactive
`TCL generated from the long-term treatment group. A
`comparison of the average levels of IL-5 and IFN-g for
`GA and TT-reactive TCL from short- and long-term treat-
`ment groups is presented in Fig. 2. Mean IL-5 level was
`47.14 pg/ml pretreatment, 90.78 pg/ml for the short-term
`treatment group, and 94.69 pg/ml for the long-term treat-
`ment group. Mean IFN-g level was 171.2 pg/ml pretreat-
`
`Table 2
`Comparison of the estimated precursor frequency of GA-reactive T cells in
`the long-term and short-term treatment groups
`Precursor frequency/106
`Mean F S.D.
`2.66 F 3.32
`4.03 F 2.96
`1.40 F 1.48
`
`Range
`
`0.31 – 9.0
`0.94 – 9.67
`0.26 – 5.31
`
`P
`
`0.027
`
`GA – TCL
`
`Pre-Rx
`S.T. Rx
`L.T. Rx
`
`S.D.: standard deviation; Rx: treatment; S.T.: short-term; L.T: long-term;
`P: student t-test p-value.
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`Fig. 2. Mean IL-5 (A and C) and IFN-g (B and D) produced by the GA-reactive (A and B) and TT-reactive (C and D) TCL pretreatment with GA, during short-
`term and long-term treatments.
`
`ment, 16.99 pg/ml for short-term treatment, and 31.19 pg/ml
`for long-term GA treatment. Both treatment groups had
`IFN-g levels significantly lower than pretreatment levels. In
`contrast, cytokine levels produced by TT-reactive TCL did
`not change significantly in both treatment groups compared
`to pretreatment levels.
`
`the Th1 and Th2 phenotype bias of the GA-
`Next,
`reactive TCL generated from the MS patients was analyzed.
`GA-reactive TCL producing IL-5 and IFN-g above the
`detection limits of the ELISA were classified as Th0-,
`Th1- or Th2-biased based on the ratio of IFN-g/IL-5
`secretion as described in Methods (Fig. 3). The mean ratio
`
`Fig. 3. Ratios of IFN-g to IL-5 levels for each of the GA-reactive T-cell lines pretreatment and in both treatment groups. The insert figure shows the mean ratios
`from the three groups.
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`Table 3
`Comparison of cross-reactivity of GA – TCL with MBP in short-term and long-term treated groups
`
`MS patient
`
`Cross-reactive by proliferation
`
`Cross-reactive by cytokine secretion
`
`No. TCL
`
`tested
`
`No. TCL
`
`No. TCL
`
`No. TCL cross-reactive (%)
`
`cross-reactive (%)
`
`tested
`
`IL-5
`
`IFN-g
`
`Pre-Rx (n = 10)
`81
`10 (12.34%)
`51
`5 (9.80)
`4 (7.84)
`S.T. Rx (n = 10)
`130
`21 (16.15%)
`74
`23 (31.08)
`9 (12.16)
`L.T. Rx (n = 10)
`52
`6 (11.54%)
`32
`10 (31.25)
`2 (6.25)
`a Some TCL were cross-reactive for both IL-5 and IFN-g secretion, and therefore were counted as a single TCL in the ‘‘Total’’ column.
`
`Total
`
`8 (15.69)a
`24 (32.4)a
`12 (37.5)
`
`of IFN-g/IL-5 of GA-reactive TCL was 1.15 and 0.32 for
`long-term and short-term GA treatments, respectively; both
`significantly lower than the corresponding pretreatment
`ratio (2.76). It
`is also noteworthy that
`this ratio was
`significantly different between the two treatment groups
`with less Th2 bias in the long-term treatment group. The
`percentages of GA-reactive TCL classified as Th1, Th0, or
`Th2 were 20.59%, 23.53%, and 55.88% for the long-term
`treatment compared to 8%, 9%, and 83% for the short-term
`treatment group. In contrast, the Th1/Th0/Th2 distribution
`of TT-reactive TCL did not shift post-treatment in either
`group (data not shown).
`
`3.3. Cross-reactivity of GA-reactive TCL with MBP and
`MBP peptide 83 – 99
`
`We have previously reported cross-reactivity of GA-
`reactive TCL with MBP and MBP peptide 83 – 99 in the
`short-term treatment group [16]. Cross-reactivity was
`observed by proliferation for some GA – TCL and by
`cytokine secretion for others. In this study, we determined
`whether GA – TCL generated from the long-term treatment
`
`group manifest cross-reactivity with MBP and MBP (83 –
`99), since such cross-reactivity is a prerequisite for by-
`stander suppression. Cross-reactivity by proliferation
`(SI>2.0) was observed in 3 of the 10 patients, and in
`approximately 12% of the 52 GA – TCL examined (Table
`3). Cross-reactivity of the GA – TCL as measured by cyto-
`kine secretion was examined in 32 and 24 GA – TCL for
`MBP and MBP (83 – 99), respectively. Cytokine secretion
`(>50% over background levels) in response to MBP or MBP
`(83 – 99) was considered a cross-reactive response. Approx-
`imately 25% of the GA – TCL cross-reacted with MBP, and
`33% cross-reacted with MBP (83 – 99). The cytokines
`secreted by the cross-reactive GA – TCL in response to
`MBP or MBP (83 – 99) were biased in favor of the Th2
`phenotype (Fig. 4). Comparison of the cross-reactive
`responses in the long-term treatment group with that for
`the short-term treatment group is presented in Table 3.
`While the percentage of cross-reactive T-cell lines as meas-
`ured by proliferation was lower with long-term treatment,
`cross-reactivity as measured by cytokine secretion was not
`significantly different between the two groups. Interestingly,
`while 12% of the cross-reactive GA – TCL in the short-term
`
`Fig. 4. IL-5 and IFN-g levels produced by the MBP cross-reactive (A and B) and the MBP 83-99 cross-reactive (C and D) GA – TCL. A and C show the levels
`for each cross-reactive TCL. B and D show the mean levels for the cross-reactive TCL.
`
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`treatment group produced IFN-g, only 6% did in the long-
`term treatment group, indicating a sustained Th2-polarized
`cross-reactive response in the long-term treatment group.
`
`4. Discussion
`
`A number of laboratories including ours reported on the
`generation of GA-reactive T-cells with a Th2-biased phe-
`notype as early as 1 month after initiation of GA treatment
`in MS patients [9,10,13 – 17,29]. Currently, it is believed
`that these cells mediate the therapeutic effect of GA in MS.
`Of some concern, however, is the observation that the robust
`proliferative response to GA observed early on during
`treatment declines after 6 months [13,25]. Therefore, the
`objective of this study was to examine the characteristics of
`the immune response to GA in patients who have been on
`the drug for several years and compare the findings to those
`from a group of MS patients who were studied in our
`laboratory before and after they had been on the drug for
`a shorter period (1 – 10 months). The availability of a group
`of MS patients at the University of Maryland who had
`participated in the pivotal CopaxoneR trial
`in the early
`1990s provided an opportunity to examine the long-term
`immunologic effects of Glatiramer acetate treatment in MS
`[30].
`First, we observed that the estimated precursor frequency
`of GA-reactive T-cells declined 2.9-fold compared to that
`during the first 10 months of treatment. Although the
`method we used is not a precise measure of precursor
`frequency, regression analysis supports such a decline with
`time. These observations are consistent with the findings of
`Ragheb et al. [26]. Since the peptide composition of GA is
`heterogeneous, these results may suggest that chronic stim-
`ulation with GA could result in the selection of a more
`restricted T-cell repertoire compared to that during early
`treatment. Alternatively, chronic administration of GA may
`result
`in the induction of anergy or deletion of highly
`reactive T-cells [15,26].
`The second notable observation is that the Th2 pheno-
`type of the GA-reactive T-cells remains dominant after
`many years of treatment. This is supported by the fact that
`significantly lower levels of IFN-g and higher IL-5 levels
`are produced by GA – TCL from both short-term and long-
`term treatment groups compared to pretreatment. Similarly,
`the IFN-g/IL-5 ratio remained Th2-biased in both groups.
`Therefore, despite a drop in the precursor frequency of GA-
`reactive T-cells with prolonged treatment,
`the sustained
`GA – T-cell response remains Th2-biased and therefore in
`favor of an anti-inflammatory response. The finding that
`long-term treatment with GA did not significantly affect the
`response to TT indicate that GA does not cause generalized
`immune suppression in MS patients. Of interest
`is the
`observation that some GA – TCL were Th1-biased despite
`prolonged treatment. One possible explanation is that in
`some individuals, GA induces CD8 + T-cells that produce
`
`IFN [25,31]. Another possibility is that GA constantly
`stimulates subpopulations of naı¨ve T-cells that
`initially
`produce IFN-g although our earlier studies indicate that
`the GA-reactive T-cells examined 1 week after the first
`
`/RO + ) [15]. It is
`stimulation are memory T-cells (CD45
`also conceivable that GA stimulates Th1-committed mem-
`ory cells with a low activation threshold in some donors and
`that these cells are expanded or at least perpetuated over
`time. The observation that the IFN-g/IL-5 ratio was signifi-
`cantly higher in the long-term treatment group (Fig. 3)
`seems to indicate that progressive deletion of Th1 cells as
`a result of chronic GA treatment is not a universal mech-
`anisms that accounts for the Th2-biased response. It is quite
`possible that the Th phenotype response is heterogeneous
`and is influenced by the genetic background of the patient
`including genes encoding for the HLA and T-cell receptor.
`Thirdly, cross-reactivity of some GA – TCL with MBP is
`observed after long-term treatment and the magnitude of this
`cross-reactivity is not compromised. More importantly, the
`cytokines released in response to the cross-reactive antigen
`remain Th2-biased. These findings are relevant
`to the
`mechanism of action of GA in MS as it is currently believed
`that GA-reactive T-cells cross the blood – brain barrier,
`cross-react with myelin antigens and consequently produce
`bystander suppression through the secretion of anti-inflam-
`matory cytokines.
`trial, who remained
`Patients enrolled in the pivotal
`clinically stable, continued participation in the open label
`phase which included the 10 patients examined in this study.
`Therefore, the patient population we examined is biased in
`favor of clinical responders as reflected by the stability of
`the EDSS scores over at least 6 years of treatment. In spite
`of the rather uniform clinical stability among the 10 patients,
`we observed heterogeneity in the precursor frequency and
`the phenotype of the GA – TCL response, and in cross-
`reactivity with myelin antigens. The two patients who had
`relatively higher relapse rate during the course of treatment
`(Table 1, cases MS 15 and MS 17) did not have a unique
`immunologic profile that distinguished them from the rest of
`the group. Currently, it is not known whether the immuno-
`logic responses to GA correlate with the clinical response.
`Although this study was not intended to address this issue
`because of the small sample size, the fact that there is
`substantial heterogeneity in the immune responses to GA
`warrants further investigations in a larger patient population,
`in order to determine if the immunologic parameters corre-
`late with the clinical response, and whether they can be used
`as surrogate markers of treatment response.
`
`Acknowledgements
`
`This work was supported by grants from TEVA
`pharmaceuticals, The National Institute of Neurological
`Disorders and Stroke (K-24-NS02082), and the Department
`of Veteran’s Affairs.
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`Page 7 of 7
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`YEDA EXHIBIT NO. 2126
`MYLAN PHARM. v YEDA
`IPR2015-00644