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Equivalent Gene Expression Profiles between Glatopa™ and Copaxone®.

D'Alessandro JS, Duffner J, Pradines J, Capila I, Garofalo K, Kaundinya G, Greenberg BM, Kantor D, Ganguly TC - PLoS ONE (2015)

Bottom Line: Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate.No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes.In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

View Article: PubMed Central - PubMed

Affiliation: Momenta Pharmaceuticals, Inc., Cambridge, MA, United States of America.

ABSTRACT
Glatopa™ is a generic glatiramer acetate recently approved for the treatment of patients with relapsing forms of multiple sclerosis. Gene expression profiling was performed as a means to evaluate equivalence of Glatopa and Copaxone®. Microarray analysis containing 39,429 unique probes across the entire genome was performed in murine glatiramer acetate--responsive Th2-polarized T cells, a test system highly relevant to the biology of glatiramer acetate. A closely related but nonequivalent glatiramoid molecule was used as a control to establish assay sensitivity. Multiple probe-level (Student's t-test) and sample-level (principal component analysis, multidimensional scaling, and hierarchical clustering) statistical analyses were utilized to look for differences in gene expression induced by the test articles. The analyses were conducted across all genes measured, as well as across a subset of genes that were shown to be modulated by Copaxone. The following observations were made across multiple statistical analyses: the expression of numerous genes was significantly changed by treatment with Copaxone when compared against media-only control; gene expression profiles induced by Copaxone and Glatopa were not significantly different; and gene expression profiles induced by Copaxone and the nonequivalent glatiramoid were significantly different, underscoring the sensitivity of the test system and the multiple analysis methods. Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate. No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes. In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

No MeSH data available.


Related in: MedlinePlus

Methodology for the generation of murine GA-responsive Th2-polarized T cells.In vivo immunization of naive mice with Copaxone was followed by 13 rounds of ex vivo restimulation of the CD4+ T-cell population over 6 months for development of the Th2-455 line. APCs, antigen-presenting cells; GA, glatiramer acetate; IL-2, interleukin 2; Th, T-helper.
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pone.0140299.g001: Methodology for the generation of murine GA-responsive Th2-polarized T cells.In vivo immunization of naive mice with Copaxone was followed by 13 rounds of ex vivo restimulation of the CD4+ T-cell population over 6 months for development of the Th2-455 line. APCs, antigen-presenting cells; GA, glatiramer acetate; IL-2, interleukin 2; Th, T-helper.

Mentions: Fig 1 summarizes the steps in the generation of Copaxone-specific Th2-polarized T cells using a modification of the methods detailed in Aharoni et al [18]. Briefly, Balb/c mice were immunized with Copaxone 250 μg (lot 538455), and lymph nodes were harvested. CD4+ T cells were isolated from the lymph nodes using negative immunomagnetic isolation (EasySep Mouse CD4+ T Cell Isolation Kit, Stem Cell Technologies, Vancouver, BC, Canada). Splenocytes were isolated from naive (non-immunized) mouse spleens depleted of T cells using positive immunomagnetic isolation. APCs were treated with mitomycin C 50 μg/mL (Calbiochem, EMD Millipore, Billerica, MA, USA) for 25 minutes at 37°C to prevent a proliferative response from residual T cells. CD4+ T cells were rechallenged ex vivo for 3 to 4 days with Copaxone 20 μg/mL presented using the mitomycin C-treated splenocytes as APCs followed by a period of maintenance for 10 days in the presence of 20 ng/mL murine interleukin-2 (mIL-2; PeproTech, Rocky Hill, NJ, USA). The process of Copaxone restimulation and cell expansion followed by maintenance was repeated for 13 rounds using 20% conditioned media from the cells and mIL-2 as just described; this led to production of a Th2-polarized nonclonal T-cell population. The Th2 phenotype of these cells, which were exclusively responsive to Copaxone, was further verified by enzyme-linked immunosorbent assay (ELISA; Meso Scale Discovery, Rockville, MD, USA); mouse Th1/Th2 (9-plex); custom IL-6, IL-13, IL-17 (3-plex), and mouse TGF-β1Quantikine (R&D Systems, Minneapolis, MN, USA) ELISA kits for Th1, Th2, Th17; and other cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17, tumor necrosis factor-alpha [TNF-α], and transforming growth factor-beta 1 [TGF-β1]). These cells exhibited Copaxone-induced dose-dependent release of Th2-specific cytokines (IL-4, IL-5, IL-10, and IL-13), whereas levels of Th1 cytokines (TNF-α and interferon-gamma [IFN-γ]) were negligible. The cell bank of Th2-polarized T cells (designated Th2-455) was created and used for all gene expression studies.


Equivalent Gene Expression Profiles between Glatopa™ and Copaxone®.

D'Alessandro JS, Duffner J, Pradines J, Capila I, Garofalo K, Kaundinya G, Greenberg BM, Kantor D, Ganguly TC - PLoS ONE (2015)

Methodology for the generation of murine GA-responsive Th2-polarized T cells.In vivo immunization of naive mice with Copaxone was followed by 13 rounds of ex vivo restimulation of the CD4+ T-cell population over 6 months for development of the Th2-455 line. APCs, antigen-presenting cells; GA, glatiramer acetate; IL-2, interleukin 2; Th, T-helper.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608686&req=5

pone.0140299.g001: Methodology for the generation of murine GA-responsive Th2-polarized T cells.In vivo immunization of naive mice with Copaxone was followed by 13 rounds of ex vivo restimulation of the CD4+ T-cell population over 6 months for development of the Th2-455 line. APCs, antigen-presenting cells; GA, glatiramer acetate; IL-2, interleukin 2; Th, T-helper.
Mentions: Fig 1 summarizes the steps in the generation of Copaxone-specific Th2-polarized T cells using a modification of the methods detailed in Aharoni et al [18]. Briefly, Balb/c mice were immunized with Copaxone 250 μg (lot 538455), and lymph nodes were harvested. CD4+ T cells were isolated from the lymph nodes using negative immunomagnetic isolation (EasySep Mouse CD4+ T Cell Isolation Kit, Stem Cell Technologies, Vancouver, BC, Canada). Splenocytes were isolated from naive (non-immunized) mouse spleens depleted of T cells using positive immunomagnetic isolation. APCs were treated with mitomycin C 50 μg/mL (Calbiochem, EMD Millipore, Billerica, MA, USA) for 25 minutes at 37°C to prevent a proliferative response from residual T cells. CD4+ T cells were rechallenged ex vivo for 3 to 4 days with Copaxone 20 μg/mL presented using the mitomycin C-treated splenocytes as APCs followed by a period of maintenance for 10 days in the presence of 20 ng/mL murine interleukin-2 (mIL-2; PeproTech, Rocky Hill, NJ, USA). The process of Copaxone restimulation and cell expansion followed by maintenance was repeated for 13 rounds using 20% conditioned media from the cells and mIL-2 as just described; this led to production of a Th2-polarized nonclonal T-cell population. The Th2 phenotype of these cells, which were exclusively responsive to Copaxone, was further verified by enzyme-linked immunosorbent assay (ELISA; Meso Scale Discovery, Rockville, MD, USA); mouse Th1/Th2 (9-plex); custom IL-6, IL-13, IL-17 (3-plex), and mouse TGF-β1Quantikine (R&D Systems, Minneapolis, MN, USA) ELISA kits for Th1, Th2, Th17; and other cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17, tumor necrosis factor-alpha [TNF-α], and transforming growth factor-beta 1 [TGF-β1]). These cells exhibited Copaxone-induced dose-dependent release of Th2-specific cytokines (IL-4, IL-5, IL-10, and IL-13), whereas levels of Th1 cytokines (TNF-α and interferon-gamma [IFN-γ]) were negligible. The cell bank of Th2-polarized T cells (designated Th2-455) was created and used for all gene expression studies.

Bottom Line: Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate.No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes.In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

View Article: PubMed Central - PubMed

Affiliation: Momenta Pharmaceuticals, Inc., Cambridge, MA, United States of America.

ABSTRACT
Glatopa™ is a generic glatiramer acetate recently approved for the treatment of patients with relapsing forms of multiple sclerosis. Gene expression profiling was performed as a means to evaluate equivalence of Glatopa and Copaxone®. Microarray analysis containing 39,429 unique probes across the entire genome was performed in murine glatiramer acetate--responsive Th2-polarized T cells, a test system highly relevant to the biology of glatiramer acetate. A closely related but nonequivalent glatiramoid molecule was used as a control to establish assay sensitivity. Multiple probe-level (Student's t-test) and sample-level (principal component analysis, multidimensional scaling, and hierarchical clustering) statistical analyses were utilized to look for differences in gene expression induced by the test articles. The analyses were conducted across all genes measured, as well as across a subset of genes that were shown to be modulated by Copaxone. The following observations were made across multiple statistical analyses: the expression of numerous genes was significantly changed by treatment with Copaxone when compared against media-only control; gene expression profiles induced by Copaxone and Glatopa were not significantly different; and gene expression profiles induced by Copaxone and the nonequivalent glatiramoid were significantly different, underscoring the sensitivity of the test system and the multiple analysis methods. Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate. No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes. In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

No MeSH data available.


Related in: MedlinePlus