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WO2018053109A1 - Caractérisation d'expression génique d'un produit médicamenteux associé à l'acétate de glatiramère dans des cellules humaines et de mammifères - Google Patents

Caractérisation d'expression génique d'un produit médicamenteux associé à l'acétate de glatiramère dans des cellules humaines et de mammifères Download PDF

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Publication number
WO2018053109A1
WO2018053109A1 PCT/US2017/051534 US2017051534W WO2018053109A1 WO 2018053109 A1 WO2018053109 A1 WO 2018053109A1 US 2017051534 W US2017051534 W US 2017051534W WO 2018053109 A1 WO2018053109 A1 WO 2018053109A1
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WIPO (PCT)
Prior art keywords
genes
activity
acetate related
expression
product
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English (en)
Inventor
Iris Grossman
Tal Hasson
Michael Hayden
Daphna LAIFENFELD
Shlomo BAKSHI
Attila Konya
Kevin D. FOWLER
Sarah Elisabeth KOLITZ
Benjamin James ZESKIND
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Teva Pharmaceutical Industries Ltd
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Teva Pharmaceutical Industries Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • MS Multiple sclerosis
  • CNS central nervous system
  • RRMS relapsmg-reniitting
  • SP secondary progressive
  • PP primary progressive
  • MS is the most common cause of chronic neurological disability in young adults (Bjartmar et al. 2002; Fleming 2002).
  • Anderson et al. (1992) estimated that there were about 350,000 physician-diagnosed patients with MS in the United States in 195)0 (approx. 140 per 100,000 population). It is estimated that about 2.5 million individuals are affected worldwide (Compston et al. 2006). I general, there has been a trend toward an increasing prevalence and incidence of MS worldwide, but the reasons for this trend are not folly understood ( Anderson et al. 1992).
  • Copaxone* (leva Pharmaceutical dusfnes Ltd.) is a glatiramer acetate drag product approved for treatment of patients w t relapsmg-reiinttmg multiple sclerosis (RRMS) and clinically isolated -syndrome (CIS) (Copaxone, Food and Dra Administratio Approved Labeling (Reference ID: 3443331) [online], TEVA Pharmaceutical Industries Ltd., 2014 [retrieved on December 24, 2014], Retrieved from the Internet: ⁇ L!RL: mv .a.ccessdat,a.fda.gov?3 ⁇ 4 Glatiramer acetate drag substance (GA), the active substance of Copaxone ® , is a complex mixture of polypeptides and is the first member of the gjatiraniokl class: i.e., a complex mixture of synthetic polypeptides of varying sizes assembled from four naturally occurring amino acids: L-glutamic acid, L-alanine, L-
  • GA elicits anti-inflammatory as well as neuiOprotective effects in various animal models of chronic inilarnmatoiy and neurodegenerative diseases (Filippi M, et al. (2001); ipnis J. Schwartz M (2002); Teitelbaimi D et al. (1988); Putheti et al. (2003)) and has been shown to be safe and effective in reducing relapses and delaying neurologic disability in MS patients following long-term treatment (Ford et al. 2010).
  • GA appears to act as an altered peptide ligand (APL) of encephalitogenic epitopes within myelin basic protein (MBP) (Aharoni et al. 1999) and demonstrates cross-reactivity with MBP at the humoral and cellular levels (Anion R. Aharoni R (2004); Teitelbaimi et al. (1991): Webb et al. (1973): Duda et al. (2000); Brenner T et al. (2001); Aharoni et al. (1997)).
  • APL peptide ligand
  • MBP myelin basic protein
  • the unique antigenic sequences of the GA polypeptide mixture compete with myelin antigens for binding to MHC class II molecules on antigen presenting cells (APCs) and presentation to the T cell receptor (TCR). resulting in the induction of anergy or deletion of autoreactive MBP -reactive T cells and proliferation of GA-reaciive T cells.
  • APCs antigen presenting cells
  • TCR T cell receptor
  • Copaxone ® also increases the number and suppressive capacity of CD4+CD25+FOXP3+ regulatory T cells, which are functionally impaired in MS patients (Venken et al. (2008); Haas et al. (2009); Hong et al. (2005)). Furthermore, treatment leads to antigen-nonspecific modulation of APC function. Copaxone ® treatment promotes development of anti-inflammatory type ⁇ monocytes characterized by an increase in mterleiikin (IL)-IO and tiansfonnmg growth factor-beta (TGF- ⁇ ) and decreased production of IL-12 md tansor /necrosis factor (I (Weber et ai, 2007),
  • IL mterleiikin
  • TGF- ⁇ tiansfonnmg growth factor-beta
  • the .present, invention provi des a process for efen'aeterizing a. gl atimiier acetate related drug subs tance or drug product comprising, the steps of
  • step (b) contacting mamm li ceils with an .amount of the gia&ainer acetate related drug substance or drug product of step (a):
  • lnacrornolecule metabolic process (00:0044259), multicellular organismal metabolic process (00:0044236), myeloid leukocyte activation (GO:0002274), neutrophil chemotaxis (GO:OQ30593), phagocytosis, engulfinent (GO:0006911), positive regulation of cellular component organization (GO:00S1130), positive regulation of endocytosis (00:0045807), positive regulation of foam cell differentiation (GO;0010744),.
  • Sterol metabolic process (GO-.0016125), Sterol biosyndietic process (GO:0016126), Hematopoietic cell lineage (mmu04640).
  • Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (00:0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (GO;0045885), Hematopoietic cell lineage (mmu04640) or Metabolism, of xenobioties by cytochrome P450 (airauO09SQ); (ix) determining the level of expression of at least one gene iavo!yed in one or more pathway of Intrinsic to membrane receptor activity.
  • Immune response Leukocyte migration, Cseffiokine receptor binding, Cytokine-eyto ne receptor interaction. Cytokine activity, lak- ST.AT signaling pathway, Sterol bipsynthetie process. Steroid biosyuthetic process. Ceil surface. Cytokine binding. Cholesterol metabolic process, Cheniokine 'activity.
  • Negative regulation of hormone secretion, Xsoprenoid biosyntlietic process or Positive regulation of peptidyl-serine phosphorylation (x) determining the level of expression of at least one gene involved in one or more pathway of Glucose metabolic process, Glycolysis, Gluconeogenesis, Hexose catabolic process, Glucose catabolic process, Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol catabolic process. Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor metabolites and energy. Phagocytosis. Leukocyte migration. Leukocyte chemotaxis.
  • Fructose and niannose metabolism (rnmuOOOSl) or Galactose metabolism (mmuO0O52); (xiii) determining the level of expression of at least one gene involved in one or more pathway of RIG-I-like receptor signaling pathway or Adenylyltransferase activity; or (xiv) determining the level of expression of at least one gene of Cxcl3 t Clec4a3, UgtlalO, Ugtla6b, UgtlaSa, Ugtla7c. Ugtla9, UgtlaS, Ugtlal, Ugiia2, Clec4n, Tgfti, Nov.
  • Gchn SlOOaS, Ptplad2, Clec4a2.
  • Clec4bl Marcks, CsEra, Fcerlg, Clec4a2, Ppic, Cd302.
  • Gstml McptS, Cd3001f, Csf2rb, Bnip3, Ak4.
  • step (a) (a) obtaining a batch of the glatiramer acetate related drug substance or drug product: (b) contacting mammalian cells with an amount of the glatiramer . acetate related drug substance .or drug product of step (a): and
  • step (a) thereby characterizing the glatirainer acetate related .drug substance or drag product of step (a).
  • the present invention also provides a process for discriminating ' between gl atlraraer acetate related drug substances or drug products comprising the steps of:
  • step (b) comparing tlie characteristics of the giatiranier acetate related drug substances or drag products obtained in step (a),
  • the present invention also provides a process for producing or releasing a drug product comprisin a glatiramer acetate related drag substance, the improvement comprising the steps of:
  • Csilr, I r2 or Tbx21 is not substantially identical to the level of expression by the same type of cells in the presence of the gla tiramer acetate drag substance under the same conditions; (ii) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the level of expression of one or more genes of Aheyl2, Cln5, Cxcr6, Dhrs3, Egr2 t Fabp5, Ptp4al ?
  • Gprl83, Lanipl, Ncoa4, Ptger4, Qk, Rragd, Sec24a f Se ll Sestdl , Slc30a4, Soatl or Strip2 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; (iii) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in tlie drug product if the level of expression of one or more genes of Fam212a, Gzma, Itprl or Zbtb32 is no substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance vmder the same conditions: (iv) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion hi the drug product if the level of expression of one or more genes of Gzmb, Fami a3, Nfil3, Gzma, Eaf2.
  • Carl or St? is downregulated or substantially identical to the level of expression by the same type of ceils in the absence of the glatiramer acetate related drug substance under the same conditions; (v) discarding the batch of the glatiramer ace ate-related-dnig-sabstaoee a,s unacceptable for inclusion to;t3 ⁇ 4eito3 ⁇ 4 r9di «; if ib leveiof $q»e8&taii of one or more genes of Crisp3, Apoe, Dapii, Pde5a, Scg5, StSsiaS, 4 30426DQ5Rik or Pgapl is, upregulated.
  • Sterol metabolic process (00:0016125), Sterol biosynthetic process (GO:0016126), Hematopoietic cell lineage (mmiiG4540), Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (GO:0045884), Regulation of leukocyte proliferation (00:0070663), Positive regulation of survival gene product expression (00 0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) discardin the batch of the giatiramer acetate related dmg substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of mtrinsie to membrane receptor activity.
  • Immune response Leukocyte migration, Cheniokine receptor binding. Cytokine-cytokine receptor interaction, Cytokine activity, Jak-STAT signaling pathway. Sterol biosynthetic process. Steroid biosynthetic process. Cell surface. Cytokine binding, Cholesterol metabolic process, Chemokine activity, Negative regulation of hormone secretion, Isoprenoid brosyrnhetic process or Positive regulation of peptidyi-se iBepiiosp!iorylatipii; fx) discarding the batch of the glatiranier acetate related; drug substance as unacceptable for inclusion in fee drug pr oduc t if th set of differentially expressed genes indicates emich eut of one or more pathway of Gluco se metabolic process.
  • Glycolysis GlueoneogeHesis, Hexose esiaooiie process, Glucose eatabolic process, Monosacdiaride eatabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate eatabolic process, Alcohol eatabolic process. Carbohydrate eatabolic process. Carbohydrate bmding, Generation of precursor metabolites and energy. Phagocytosis, Leukocyte migr ation. Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway, Galactose metabolism. Carbohydrate kinase activity.
  • Myeloid leukocyte activation Chemokine receptor binding. Regulation of type I hypersensitivity. Defense response to Gram-negative bacteriuin or Positive regulation of foam cell differentiation; (xi) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of leukocyte migration (00:0050900), cell chemotaxis (00:0060326), leukocyte chemotaxis (00:0030595), phagocytosis (GO:0006909), positive regulation of phagocytosis (GO: 0050766), cell motion (GO:0006928), regulation of phagocytosis (00:0050764), behavior (00:0007610), membrane invagination (00:0010324), endocytosis (00:0006897), positive regulation of endocytosis (GO:0045807), phagocytosis, engulfment (GO:00069i I), defense response to bacterium (00:0042742), membrane organization
  • Ciec4n Tgfbi, Nov, Ltf Ccl6, Mmpl2, Ms4a.6d, Lpl, Mmp8, Cdl77, Tfec, Ciec7a, Mpo Ccl9, Ccrl, Gpninb, Marl, Ms4a6d. Lyz2, CtsL Thbsl, Dab2, Arg2, Ccl6, Thbsl, Paldl, Clec5a, Cfp, Ifitm6, Lyzl, 01fin4, Rnfl28, Ms4a3, Clec4d, S100a4, Ctsg, Prtn3, gp, F10, Hal, Cci2, Lyzl.
  • Thbsl Anxal, LgalsS S100a6, Nfaml, Chil3, Chil4, Sppl. Stfa211, Ptgrl, Ilif9. Cd68, Olfrnl, Fcgr3, Hlr2, Cregl, Dmxl2, Sirpa, 116. Elane. Csflr, Lcn2, CdSOGlb, AioxSap.
  • Histlirfb Hist.lh4a, Hist4h4, Histlh4i Histlh4d, Histlh4k, Histlh4j, Histlli4i, Hist h4h, Histlli4n, Histlh4m, Hist2h4, Dstn, IgsfS, Miff. Csf3r, Pf4, Pygl, Pclpn. S100a9, Fcgr2b, Ml, Emrl, Cd9, Cebpd, Pla2g7, Arg2, F10, Gclm, S100a8.
  • the present Invention also provides a process fo identifying suboptima! activity of a glatiranier acetate related drug substance or ding product comprising the steps of:
  • FegrS C ' siSr, Hlr2 or Tbx21 is not substantially identical to flie level of expression b the sarae type of cells in the presence of the .glatiramer acetate related drug subs.tsn.ee or drug product under the same conditions; (ii) identifying the glatirainer acetate related drug subs.tsn.ee.
  • Immune response Leukocyte migration, Chemokine receptor binding, Cytokine- cytokine receptor interaction, Cytokine activity, Jak-STAT signaling pathway.
  • Sterol biosynthetic process Steroid biosynthetic process. Cell surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity.
  • Pha ocytosis Leukocyte migration, Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway. Galactose metabolism. Carbohydrate kinase activity, Myeloid leukocyte activation, Chemokine receptor binding.
  • endocytosis (00:0045807), phagocytosis, eiigulfmeiit (GO:0006911), defense response to bacterium (00:0042742), membrane organization (00:0016044), neutrophil chemotaxis (00:0030593), defense response to Gram-negative bacterium (00:0050829), regulation of endocytosis (GO:003Gi00), leukocyte mediated immunity (00:0002443), cell migration (GO:0016477), regulation of vesicle- mediated transport (GO:0060627), localization of cell (00:0051674), cell motility (00:0048870), collages .
  • seriue-type peptidase activity (G0:OOO8236), serihe-type endopeptidase activity (60:0004252), cytokine activity (00:0005125), calcium ion binding (GO:0005509), immtmoglobulin binding (GO.-0019865), chemokine activity (00:0008009), chemokine receptor binding (GO:0042379), peptidase activity, acting on L-animo acid peptides (GO:0070011), peptidase activity (GO:G008233), IgG binding (00:0019864), protein complex binding (00:0032403) or enzyme inhibitor activity (GO:0004857); (xii) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of glucose metabolic process (00:0006006), glycolysis (00:0006096), hexose metabolic process (GO:00i9318),
  • carbohydrate kinase activity (GO:0019200), Glycolysis / Gluconeogenesis (mmuOOOlO). Pentose phosphate pathway (mmu00030). Fructose and mannose metabolism (mmuO0O51) or Galactose metabolism (nimu00052); (xiii) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of RIG-I-like receptor signaling pathway or Adenylyltransferase activity; or (xiv) determining the level of expression of at least one gene of Cxcl3, Clec4a3, UgtlalO, Ugtla6b, Ugtla6a.
  • TritoS a PreiidL Gapdli, Sdc3, BC06225S, LOC 102643247, 2310022B05Rik.
  • the present invention also provides a process for producing or releasing a drag product comprising a glatirarner acetate related drag substance, the improvement comprising the steps of:
  • step (a) ( ⁇ contacting a first gr oup of niarrmiahan cells with an amount of the glatiramer acetate related drag substance or drag product of step (a);
  • step (b) relative to genes expressed by the second group after the contacting of step (c);
  • ineinbrane it aginatioH (GO:O0iO324), membrane organization (GO:O016044), multicellular organismal maaOnioIeeul metabolic process. (00:0044259), multicellular organisnral metabolic process (00:0044236), myeloid leukocyte activation (00:0002274), neutrophil chemotaxi (GO:0030593), phagocytosis, engulfinent. (00:0006911)., positive regulation, of cellular component organization.
  • Jak-STAT signaling pathway (mmu04630), Sterol metaboli process (00:0016125), Sterol hiosynthetic process (00:0016126), Hematopoietic cell lineage (mmuG4540), Myeloid cell apoptosis (GO: 003 028), Regulation of survival gene product expression (GO:0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (GO:00458S5), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) discarding the batch of the giatirainer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of Intrinsic to membrane receptor activity.
  • Immune response Leukocyte migration, Chemokine receptor binding, Cytokine-cytokine receptor interaction. Cytokine activity, Jak-STAT signaling pathway. Sterol biosynthetic process, Steroid biosynthetic process. Cell surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serrne phosphorylation; (x) discarding the batch of the giatirainer acetate related h ug substance as unacceptable for inclusion in the dr ug product if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process.
  • phagocytosis positive regulation of phagocytosis (GO:0Q50766), cell motion (GO:0006928), regulation of phagocytosis (00:0050764), behavior (00:0007610), membrane invagination (GO:0010324), endocytosis (GO:0006897), positive regulation of endocytosis (GO:0045807), phagocytosis, engulfinent (00:0006911), defense response to bacterium (GO:0042742), membrane organization (00:0016044), neutrophil chemotaxis (00:0030593), defense response to Gram-negative bacterium (GO-.0050829), regulation of endocytosis (GO:0030100), leukocyte mediated inimunity (00:0002443), cell migration (GO:0016477), regulation of vesicle-mediated transport (GO:0060627), locaiization of cell (00:0051674), cell motility (GO:0048870), collagen metabolic process (GO:0032963),
  • Thbsl Anxal, LgalsS, SI00a6, NfamL CMS, ChiR Sppl, Stfa21L Ptgrl, III ⁇ , Cd68, Olfinl, Fegr3, Hlr2, Cregl. Drnxl2, Sirpa, 316, Eiane, Csflr, Lcn2, CdSOOIb, AloxSap, Him.
  • Histlirfb Hist.lh4a, H_st4h4, Histlh4f, Histlh4d, Histlh4k, HistlMj, Histih4i. Histlb4h, Hisiih4n, Histlh4m, Hist2h4, Dsto, IgsfS, Mirf, Csf3r, Pf4, Pygl, Pdpn, S100a9, Fcgr2b, Ml, Emrl, Cd9, Cebpd, Pla2g7, Arg2, F10, Gclin, SlOOaS, Ptpiad2, Clec4a2, Clec4bl, Marcks, Csf2ra, Fcerlg, Clec4a2, Ppic, Cd3G2, Gstml, McptS.
  • Grtapr Cxcll 1, Rasa4, Ndrgl, Faml62a, Pkm, Eglnl, Aidoa, Gml0480, Bnip3L Galkl, Pgaml, Pgkl, Mil Enolb, Enoi, Pdxp, PpplrSb, Stomll, Gzma, Sed2.
  • the present invention also provides a process for producing or releasing a drug product comprising a glatiranier acetate related drug substance, the improvement comprising the steps of:
  • step (b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a);
  • step (d) detemiining a set of genes differentially expressed by the first group after the contacting of step (b) relative to genes expressed by the second group after the contacting of step (c);
  • Tlie present invention also pro ides a process for identifying xuboptimal activity of a glatiramer acetate related drug substance or drug product comprising the steps of :
  • step (b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a) ;
  • step (d) determining a set of genes differentially expressed by the first group after the contacting of step (b) rela tive to genes expressed by the second group after the contacting of step (c);
  • the set of differentially expressed genes mezes one or more genes of Fam212a,. Gzma, Itprl or Zbtb32; (iv) identifying the glatiramer acetate related drug substance or drag product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Gzmb, Faml9a3, Nfii3, Gzma,.
  • cytokine activity (00:0005125), ion homeostasis (GO:0050801) or adenyly!transferase activity (GO:0070566); (viii) identifying the giatiramer acetate related drag substance or drag product as having a suboptimai activity if the set of differentially expressed genes indicate no substantial enrichment of one or more pathways of Cytokine- cytokine receptor interaction (mmu04060), Jak-STAT signaling pathway (mmu04630), Sterol metabolic process (00:0016125), Sterol biosynthetic process (00:0016126), Hematopoietic cell lineage (mniuG4640), Myeloid ceil apoptosis (GO: 0033028), Regulation of survival gene product expression (GO:0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (GO:0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of
  • Immune response Leukocyte migration, Chemokine receptor binding, Cytokine- cytokine receptor interaction. Cytokine activity, Jak-STAT signaling pathway. Sterol biosynthetic process. Steroid biosynthetic process. Cell surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serine phosphorylation; (x) identifying the glatiramer acetate related drug substance or drug product as having a suboptimai activity if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process.
  • Gfil Pail, Ldha, Eroll, 3000002C10Rik t Hk2, Slc2a3, Chchd6, P4hal , Eroll, Gpil , Gimap7, Oas l, Gml 1 i 10, Syce2, Ndrgl, Ptgs2, Trappc6a, Freiidi, Ifi2712a, Zbtb32, TriinSOa, Prelidl, Gapdh, Sdc3, BC062258, LOC 02643247, 2310022B05Rik.
  • the present invention also provides a process for identifying suboptimal activity of a glatiramer acetate related drag substance or drag product comprising the steps of:
  • step (b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drag product of step (a);
  • step (d) determining a set of genes differentially expressed by the first group after the contacting of step (b) relative to genes expressed by the second group after the contacting of step (c);
  • the present mvention also provides a process for producing a drug product comprising a glatiramer acetate related drug substance which involves an array of testing, the improvement comprising including in the array of testing the steps of: (a characterizing the glatiramer acetate; related drag substance according to the process of the; present invention; and
  • the present invention also provides a process for releasing a drug product comprising a glatiramer acetate related drug substance, which process involves an array of testing, the improvement comprising including in the array of testing the steps of:
  • Dhrs3 Egr2, FabpS, Pt.p4al. Gpi S3, LampL Ncoa4, Ptger4, Qk, Rragd, Sec24a, Sehll, Sestdl, Slc30a4, Soatl or Steip2 is substantially identical to me level of expression by the same type of cells in the presence of the glatiranier acetate drag substance under the same conditions; (lii) including the batch of the glatiiame acetate related drug substance in the prodiieiioa of the drag produc t if the level of expression of one or more genes of Fani212a, Gzma, Itprl or Z b32 is substantially identical to the level of expression b the sa me t ype of cells .in . The presence of the glatiramer acetate thug substance underthe same conditions; (iv) including the batch of the glatiramer acetate related drug substance in.
  • Figure I Expression Levels of 116 i (left) Microarray Data and (right) Confirmation by KT-PCR.
  • Fig, 3. Gene expression levels for control (maanitol). treatment, Copaxone ® freairaeni, and GLATOPA treatment for key genes relevant to inflarnaiatoiy processes and Copaxone ® mechanism of action. Levels of anti-inflammatory 1110(A), 114(B) and Foxp3(C) are increased relative to control by both Copaxone 8' and GLATOPA. Levels of pro-iiiflammatoiy H12a(D) are reduced relative to control by both Copaxone* and GLATOPA. Treatment of splenoeytes from Copaxone ® -immunized mice; similar results obtained with GLATOPA-iinniimized mice. Fold Change (FC) and adjusted p values are relative to mannitol control.
  • FC Fold Change
  • Figure 4 Pathways enriched among top probesets differentially expressed in response to Copaxone ® treatment in Copaxone s' -iimimnized mice.
  • the dashed line represents a significance level ofBenjamini- eorreeied value 0.05.
  • GLATOPA and Copaxone ® differentially modulate a significant subset of probesets (including approximately 7% of probesets modulated by Copaxone ® ), a) Copaxone®-mimunized samples; b) GLATOPA-mimiinized samples.
  • Figure 7 Increased expression of Clec4n with GLATOPA versus Copaxone ® treatment, under bom immunization conditions, for multiple probesets.
  • Figure 8 Increased expression of Fcei g with GLATOPA versus Copaxone ® treatment, under both immunization conditions.
  • FIG. 9 Expression of cytokine gene Cxcl3 is increased with GLATOPA treatment relative to Copaxone ® treatment, under both immunization conditions.
  • Figure 10 Pathways enriched among top probesets differing between GLATOPA and Copaxone ® (mannitol-coiiected comparison) under both immunization conditions.
  • the dashed line represents a significance level of Benjammi-corrected p value 0.05.
  • FIG. 11 Volcano plot showing probesets driving enrichment of the cytokine -cytokine receptor interaction pathway. Light dots correspond to Copaxone ® immunization, and dark dots correspond to GLATOPA immunization. Multiple dots of the same color for a given gene represent separate probesets, adding robustness to the observed results. . Figure 12. Volcano plot of probeseis driving enrichment of the pathway: regulation of adaptive; immune response based on somatic recombination of tmiiuiie receptors built Sam himiimoglobulin supeiianiily domains. Dark and light dots represent data from GLATOPA and Copaxone ® iminiierizatioti conditions, respectively.
  • FIG. 13 Multiple immune genes were differentially expressed between GLATOPA d Copaxone ® in the mouse splenocyte model system, in both nifcroarray and qRT-PCR studies. Shown here are Cel2 (A), Ciec4n (B), Fcgr3 (C), and C ' sBr (D).
  • FIG. 15 Volcano plot of probeseis (hiving enrichment of the pathway: regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfaniily domains. Dark and light dots represent data from the Hungary and Israel studies, respectively. Multiple dote of the same color for a given gene represent separate probeseis, adding robustness to the observed results.
  • FIG. 16 Volcano plot of probesete in the GSEA leading edge for the membrane-dependent cytokine- cytokine receptor interaction pathway and significantly differentially expressed (FDR ⁇ 0.05) in the comparison between GLATOPA and Copaxone* in THP-i cells. Dark and light dots represent data from the Hungary and Israel studies, respectively. Multiple dots of the same color for a given gene represent separate probeseis, adding robustness to the observed results.
  • Figure 19 Design of reciprocal internal control mouse splenocyte study using Copaxone ® and POLIMUNOL.
  • Figure 20 Effects of Copaxone® versus mannitol control tr eatment on splenocytes from Copaxone£>- immunized mice.
  • Figure 22 Copaxone®-immunized samples. Relative coloring scheme according to the row minimum and row maximum. Figure shows ihe 223 present probeseis with adj p ⁇ 0.05 for POLIMUNOL vs Copaxone's* (Mamiiiol-correeted; CopaxoneC-hnmimized). . Figure 23.
  • the present invention provides a process lo characterizing a glatff&rner acetate related drag substance or drag prodoet comprising the steps of:
  • step (b) contacting inaiimiahan cells with an amount of the glatiramer acetate related drug substance or drag product of step (a);
  • (c) (i) detenniiiing the level of expression of at. least one gene of Mmpl2, Tgfbi Clec4n. Clec4a3, Fcerlg, Fcgr2b, Fcgr3, Csf3r, I!Ir2 or Tbx2i; (ii) determining the level of expression of at least one gene of Alicyi2, Cln5, CxenS, Dnrs3, Egr2, FabpS, Ptp4al, Gprl83, Lampl, Ncoa4, Ptger4, Qkjagd, Sec24a, SehlL Sestdl .
  • Slc30a4, Soatl or Strip2 (iii) deteiiniiiing the level of expression of at least one gene of Fam212a, Gzma, Itprl orZbtb32; (iv) determining the level of expression of at least one gene of Gzmb, Fanii9a3 Nfil3, Gzma, Eaf2, Carl or St7; (v) detemiining t e level of expression of at least one gene of Crisp3, Apoe, Dapll, PdeSa, Scg5, St8sia6, 4930426D05Rik or Pgapi: (vi) deterniining the level of expression of at least one gene involved in one or more pathway of collagen metabolic process (00:0032963), defense response to bacterium (GO:0042742) 5 defense response to Gram- negative bacterium (GO:00S0829), endocytosis (GO:0006897).
  • leukocyte mediated immunity (00:0002443), membrane invagination (GO:0010324), membrane organization (GO: 0016044), multicellular organismal macromolecrate metabolic process (GO:Q044259), multicellular organismal metabolic process (GO:Q044236), myeloid leukocyte activation (00:0002274), neutrophil chemotaxis (00:0030593), phagocytosis,, engulfhient (00:0006911),.
  • positive regulation of cellular component organization (GO;005 i 130) encompass positive regulation of endocytosis (GO:0045807), positive regulation of foam cell differentiation (GO:0010744), positive regulation of phagocytosis (00:0050766), regulation of endocytosis (GO:0030100), regulation of foam cell differentiation (GO: 0010743), regulation of phagocytosis (00:0050764), regulation of ty e I hypersensitivity (GO:0001810), regulation of vesicle- mediated transport (00:0060627) or response to yeast (GO:000i S78); (vii) detennining the level of expression of at least one gene involved in one or more pathwa of nucleotide binding (00:0000166), double-stranded RNA binding (00:0003725), NAD+ ADP-ribosyltransferase activity (00:0003950), transferase activity, transferring pentosyi groups (00:0016763), transmission of nerve impulse (GO:0019226), regulation of
  • Jak-STAT signaling pathway (mmu04630). Sterol metabolic process (00:0016125), Sterol biosynthetic process (00:0016126), Hematopoietic cell lineage (mmu04640). Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product, expressio (00:0045884), Regnkfion of leukocyte ⁇ proliferation .(00:0970663), Positive regulation of.
  • survival gene prodiief expression (00:0045885), Hematopoietic cell lineage (inimi94640) or Metabolism of xenobioiies by cytochrome P450 (nnnii009S0): (ix) deiennhiing the level of expression of at least one gene involved in one or more pathway of Intrinsic to membrane receptor activity, itniimne response.
  • Leukocyte migration C einokine receptor bmdmg, Cytoknie-cytokine receptor iniei3 ⁇ 4ction. Cytokine activity. Jafc- STAT signaling pathway.
  • Sterol biosynmetic process Steroid biosynt etic process. Ceil surface. Cytokine binding.
  • Cholesterol metabolic process Ciiemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serine phosphorylation; (x) deiei niniiig the level of expression of at least one gene involved in one or more pathway of Glucose metabolic process. Glycolysis, Glueoneogenesis, Hexose catabolic process. Glucose catabolic process. Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol catabolic process. Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor metabolites and energy.
  • Phagocytosis Leukocyte migration. Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway. Galactose metabolism. Carbohydrate kinase activity. Myeloid leukocyte activation, Ciiemokine receptor binding. Regulation of type I hypersensitivity.
  • At least one gene involved in one or more 'pathway of glucose metabolic process (GO; 0006006), glycolysis ⁇ GO:OOO6096 ⁇ , hexose metabolic process GOrOO 19318), monosacdiaiide metabolic process: (00:0005996), .hexose catabolic process (GQ:0019320), glucose catabolic process (00:0006007), monosaccharide catabolic process (000046365).
  • Fructose and mamiose metabolism (tmimOOGSl) or Galactose metabolism (mmuOO052); (xiii) determining the level of expression of at least one gene involved in one or more pathway of RIG-I-like receptor signaling pathway or Adenylyitransferase activity; (xiv) determining the level of expression of at least one gene of Cxcl3, Clee4a3, UgtlalO, Ugtla6b, Ugtla6a, Ugtla7c, Ugtla9, UgtlaS, Ugtlal, Ugtla2, Clec4n, TgfbL Nov, Ltf Ccl6, mpl2, Ms4a6d.
  • Pdxp, Ppplr3b Stomll, Gzina, Scd2, Ccng2, Higdla, Eno2, Gfil, Pinl, Ldha, Era II, 3000002C10Rik, Hk2, SIc2a3, Chchd6, P4hal, Eroli, Gpil, Gimap7, Oasll, Gml l llO, Syce2, Ndrgl, Ptgs2, Trappc6a, Prelidl, Ifi2712a, Zbtb32, TrimSOa, Prelidl, Gapdh, Sdc3, BC062258, LOC 102643247, 2310022B05Rik, Ms4a4c, I112rbi, Mtbldll, Sniagp, Nampt, Fasl, Pafahlb3, Preiid2, Gngl2, IrfS, Kdni3a, Aif3, TnfsrS,
  • step (a) thereby characterizing the glatiramer acetate related ding substance or drag product of step (a).
  • the present, invention also provides a process for characterizin a glatiranier acetate related dru substance or drug product comprising the steps of:
  • step (b) contacting mammalian cells with an amount of the glatiranier acetate related drug substance or drug product of step (a):
  • step (a) thereby characterizing the glatiranier acetate related drug substance or drug product of step (a).
  • step (c)(i) further comprises determining the level of expression of at least one gene of Mmp9, H7r. 116, Ilib, ILK), IL4, Foxp3, 1112a, MmpS, Cxcl3 (GRO-1), Csflr, Ccl2. Cxcl9, Cd40, Cel5, IliOra or TnfsflSb.
  • step (c)(ii) ftuther comprises determining the level of expression of at least one gene of Ifi271i, Lonrfl or Pdlim4.
  • s tep (e)(iii) ftuther comprises determining the level of expression of at least one gene of AI607873, Daxx, Epstil , Ftsjd2(RniS), Hcfc, Ifi44, Ifitl, IiippSb, Mxl, Nmrall , Oasll, Pyhinl, Sgcb, Tdrd7, Tor3a. Zcchc2 or Zfyve26.
  • step (c)(iv) ftuther comprises determining the level of expression of at least one gene of 114, Ccl3, H6, Tnfisf9, Cish, CD9, IL7R, RAB27B, SLAMF8 or FGL2.
  • ste (c)(v) further comprises determining the level of expression of at least one gene of I17r, Gpnmb, BMP8B, SERPI B10, DEPTOR ⁇ DEPDC6 ⁇ or KCNQ4.
  • step (c) fiirther comprises deiemiining the level of expression of at least one gene of Mxl , Ifhg, Rsad2, Eif2ak2, IfihL Irf7, Oaslaor Gm9706 (IsglS).
  • steps (c)(vi) - step (c)(xiv) or step (c) of comprises determining the level of expression of all genes involved in one or more pathways, two or more pathways, three or more pathways, torn' or more pathways, five or more pathways or six or more pathways.
  • steps (c)(vi) - step (c)(xiv) or step (c) comprises determhiing the level of expression of at least two genes, at least three genes, at least four genes, at least five genes or at least six genes involved in the same pathway.
  • step (c)(viii) or step (c)(ix) or step (c) ftuther comprises determining the level of expression for all genes involved in one or more additional pathway, two or more additional pathways, three or more additional pathways, four or more additional pathways, five or more additional pathways or six or more additional pathways set forth in Table 3 or Table 10.
  • step (c)(yrii) or step (c)(ix) or. step (c) ftirtlier comprises detemiimng the lew! of expression for at least one gene involved, in one or more additional padiway set forth in Table 3 or Table 10,
  • step (c ⁇ fvm) or step (e3 ⁇ 4is) or step (c) fiirtlier comprises detenniaing the level of expression for at least one gene, at least t o genes, at least three genes, at least four genes, at least five genes or at least six genes involved- in one additional pathway set. forth, in Table 3 or Table 10.
  • step (c)(x), step (c)(xi), step (c)(xii) or step (c)(xiii) or step (c) ftirtlier comprises deteriiihiing the level of expression for all genes involved in one or more additional pathway, two or more additional pathways, three or more additional pathways, four or more additional pathways, five or more additional pathways or six or more additional pathways set forth in Table 5, Table 7, Table 8, Table 14 or Table 15.
  • step (c)(xi), step (c)(xii) or step (c)(xiii) or step (c) further comprises deterniining the level of expression for at least one gene involved in one or more additional pathway set form in Table 5, Table 7, Table 8, Table 14 or Table 15.
  • step (c ⁇ (vi), step (c) ⁇ vii ⁇ t step (c) ⁇ x), step (c)(xi), step (c)(xii) or step (e)(xiii) or step (c) further comprises detemiinhig the level of expression for a leas one gene, at least two genes, at least diree genes, at least four genes, at least five genes or at least six genes involved in one additional pathway set form in Table 5, Table 7, Table 8, Table 14 or Table 15,
  • contacting the mammalian cells in ste (b) comprises i) administering to a mammal a predetermined amount of glatiramer acetate related drag substance or drug product of step (a), or ii) incubating the cells with an amoimt of the glatiramer acetate related drug substance or drug product of ste (a), or a combination thereof.
  • contacting the mammalian cells hi ste (b) comprises i) administering to a mammal a predetermined amount of glatiramer acetat related drug substance or drug product of ste (a), and ii) obtaining cells from the mamma! at one or more predetermined time points.
  • contacting the mammalian cells in ste (b) comprises incubating monocytic cell line cells wit an amount of the glatiramer aceta te rela ted drug substance or drag product of step (a).
  • the mammalian cells are THP-1 cells.
  • contacting the mammalian cells in step (b) comprises (i) immunizing a mammal with a predetermined amount of glatiramer acetate related drug substance or drug product, (ii) preparing a culture of cells from the mammal of step (i) at one or more predetermined time points after immunization, and (iii) incubating cells from the culture of cells obtained from the mammal with an amoimt of the glatiramer acetate related drug substance or drug product of step (a).
  • the glaiiramer acetate rela ted dra substance or drag product of step (iii) is the same gktirsaier acetate related, dru substance or drug product of step (i).
  • the. giatiramer acetate related drug, substance or drag product of step ' (iii) is a different glatiramer acetate related drug substance or drag product of step ( ' i).
  • fee mciibation is for about 24 hours, for about 1 hoars * or for about 6 hours.
  • the predetenniiied rime point, after immunization is 3 days.
  • the contacting of step (b) is in a cell culture.
  • the culture is a primary culture.
  • the contacting of step (b) is in a mammal.
  • the mammal is a rodent or human.
  • the giatiramer acetate related drag substance or drug product is othe than giatiramer acetate drug substance or drag product.
  • the cell is of a type of FoxP3+ T cells, regulatory T cells, natural killer T cells, T helper 2 cells.
  • the present invention also provides a process for discriminating between giatiramer acetate related drug substances or drug products comprising the steps of:
  • step (b) comparing the characteristics of the giatiramer acetate related drug substances or drag products obtained in step (a),
  • the present invention also provides a process for producing a drag product comprising a giatiramer acetate related drag substance, the improvement comprising the steps of:
  • Fcgr3, Csf3r, Hlr2 or Tbx21 is not substantially identical to the level of expression by the same type of cells in the presence of the giatiramer acetate drug substance under the same conditions; (ii) discarding the batch of the giatiramer acetate related drag substance as unacceptable for inclusion in the drag product if the level of expression of one or more genes of Ahcyl2, Chi5, Cxcr6, Dhrs3, Egr2, Fabp5, Ptp4al, Gprl83, Lampl, Ncoa4, Ptger4, Qk, Rragd, Sec24a, Sehll, $estdl, Slc30a4, Soatl or Strip?
  • leukocyte mediated immunity (00:0002443),. membrane invagination (GO:0010324), membrane organization (GO:0016044), multicellular organismal macromolecule metabolic, process (00:0044259). multicellular organismal metabolic process (00:0044236).
  • Cytokine-cytokine receptor interaction Cytokine activity, iak-STAT signaling pathway. Sterol biosynthetic process. Steroid biosynthetic process, Cell surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity,. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive reg lation of peptidyl-serine phosphorylation; (x) discarding the batch of the g!atiramer acetate related drug substance as unacceptable for inclusion hi the drug prodiict if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process. Glycolysis, Gluconeogenesis, Hexose catabolic process.
  • Glucose catabolic process Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol cataboli process. Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor metabolites and energy. Phagocytosis, Leukocyte migration. Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway. Galactose metabolism. Carbohydrate kinase activity, Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity.
  • leukocyte chemotaxis GO;0030595
  • phagocytosis GO;0006909
  • positive regulation of phagocytosis GQ:0050766
  • cell motion 00:0006928
  • regulation of phagocytosis GO:0050764
  • behavior GG: 0007610
  • membrane invagination GO:0010324
  • endocytosis GO:0G06897 ⁇
  • positive regulation of endocytosis GO:0045807
  • phagocytosis eiigiilfment
  • defense response to bacterium GO:0042742
  • membrane organization 00:0016044
  • neutrophil chemotaxis (00:0030593
  • defense response to Gram-negative bacteiium GO:0050829)
  • regulation of endocytosis GO:0030100
  • leukocyte mediated immunity GO:0002443
  • cell migration GO;0016477
  • regulation of vesicie-mediated transport (00:00
  • macromolecnle metabolic process (00:0044259), positive regulation of cellular component organization (GO:005i 130), myeloid leukocyte activation (GO:G002274), positive regulation of foam cell differentiation (GO:0010744), multicellular organismal metabolic process (GO:0Q44236), response to yeast (GO;0001878>, regulation of type I hypersensitivity (GO:000i 810), regulation of foam cell differentiation (00:0010743), sugar' binding (00:0005529), eiidopepiidase activity (00:0004175), serine hydrolase activity (00:0017!
  • oxidoreductase activity acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen (00:0016702), oxidoreduciase activity, acting on single donors with incorporation of molecular oxygen (00:0016701), cytokine activity (00:0005125), carbohydrate kinase activity (00:0019200), Glycolysis / Gluconeogenesis (mmuOOOlO), Penrose phosphate pathway (mniiiOOOSO), Fructose and mannose metabolism (nmiu00051) or Galactose metabolism (mniu00052); (xiii) discarding me batch of the glatirarner acetate related dmg substance as unacceptable tor inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of R
  • Histlh4c Histlh4b, Hisilh4a, Hist4h4, Histlh4f, Histlh4d, Histlh4k, Histlhlj, Histih4i. Histlh4h, Histlh4n, Histlh4m, Hisf2h4, Dstii, Igsf6, Mitf, Csf3r, Pf4. Pygl, Pdpn.
  • Grhpr Cxcll 1, Rasa4, Ndrgl, Faml62a, Pkm, Eglnl, Aidoa, Gml0480, Bnip3i. Gaikl, Pgaml, Pgki, Mif, Enolb, Enoi, Pdxp, PpplrSb, Stomll, Gzma, Sed2. Ccng2, Higdla, Eno2, Gfil. Piiii.
  • the present, invention also provides a process for releasing a drag product comprising a glatiramer acetate related drug substance, the improvement comprising the steps of;
  • Soatl or Sfiip2 is not substantially identical to the level of expression b the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; (iii) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion hi the drag product if the level of expression of one or more genes ofFam212a, Gzma, Itpi or Zbtb32 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; (iv) discaidmg the batch of the glatiranier acetate related drag substance as unacceptable for inclusion in the drag product if the level of expression of one or more genes of Gzmb, Faml9a3, fil3 5 Gzma, Eaf2, Carl or St7 is downregiilated or substantially identical to the level of expression by the same type of cells in the absence of the glatiramer
  • multicellular organismal macromolecule metabolic process (GO:0044259), multicellular organismal metabolic process (00:0044236), myeloid leukocyte activation (00:0002274), neutrophil chemotaxis (00:0030593), phagocytosis, engulfmeiit (GO;OO069!J), positive; regulation of ' cellular, component organization (00:0051130), positive regulation of endocytosis (GO;004S807), positive regulation of foam: cell differentiation (GO;00i6 ' 744 , positive regulation of phagocytosis (00:0050766), regulation of endocytosis (00:0030100), regulation of foam cell differentiatioii (GO:0010743), regulation of phagocytosis (GO:0 €50764j, regulation of type I hypersensitivity ' (Op: 0001810), regulation of vesicle- mediated transport (00:0060627) or response to yeast • (GO-tOOOl 878): (vii) discarding the batch of the giat
  • the iset of differentially expressed genes indicates enrichment of one or more pathway of nucleotide binding (GO.-0000166), double-stranded RNA binding (GO.O0O3725), NAD+ ADP-ribosyltransferase activity (00:0003950), transferase activity, transfening pentosyi groups (GO:0016763), transmission of nerve impulse (GO:0019226), regulation of membrane potential (GO:0042391), cytokine activity (GO-.0005125), ion homeostasis (GO:0050801) or adenyiyltransferase activity (00:0070566); (viii) discarding the batch of the giatiramer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicate no substantia!
  • Steroid biosynthetic process Cell surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serine phosphorylation; (x) discarding the batch of the giatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process.
  • Glycolysis Gluconeogenesis, Hexose catabolic process.
  • Glucose catabolic process Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process.
  • Cellular carbohydrate catabolic process Alcohol catabolic process.
  • Carbohydrate catabolic process Carbohydrate binding, Generation of precursor metabolites and energy.
  • Phagocytosis Leukocyte migration, Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway, Galactose metabolism.
  • Carbohydrate kinase activity Myeloid leukocyte activation, Chemokine receptor binding.
  • phagocytosis engulftnent ⁇ ;0 ⁇ 69 ⁇ ).,, defense response to bacterium (GQ;00 2742). ? . membrane organization (GO.O016044), neutrophil chemotaxis (GO:0030593), defense response to Gram-negative bacterium (GO:0050829), regulation of endoeytosis (GO:0030100), leukocyte mediated mimunify (GO:0002443), cell migration (00:0016477),.
  • vesicle-mediated transport GO: 0060627), localization of cell (GO:0051674), ceil motility (GO:0048870), collagen metabolic process (GO:0032963) t multicellular organismal macromolecule metabolic process (GO:Q044259), positive regulation of cellular component organization (00:0051130), myeloid leukocyte activation (GO:0002274), positive regulation of foam eel! differentiation (GO: 0010744), multicellular organismal metabolic process (00:0044236), response to yeast (00:0001878),.
  • regiiiation of type I hypersensitivity (00:0001810), regiiiation of foam cell differentiation (GO:0010743), sugar binding (00:0005529), endopeptidase activit (GO:0004175), serine hydrolase activity (00:0017171).
  • Pfcxd 1 Aldoc, Rgsll, Ankrd37, Pfkl, Pdkl , Cxcl9, Tbx21 , Pgfcl, Egm3, Ddit4, Pfkp, Pgm2, P4hal, Sicl6a3, Ubd, Steal, 5330426P16Rik, Grhpr, Cxcil 1, Rasa4, Ndigi, Faini62a, Pkm, Eglnl, AMoa, Gml04S0, Bnip3L Galkl, Pgaml, Pgkl, Mil Enolb, Enol, Pdxp, Fpplr3b, Stonill, Gzrna, Scd2, Ccng2, Higdla Eno2 Gfil, Prol, Ldha, ErolL 3000002C10Rik, Hk2, Sic2a3, Chc 6.
  • P4hal ErolL Gpil, Gimap7, Gasll, Grai l 110, Syce2, Ndrgl, Ptgs2, Trappc6a, Prelidi, Ifi27i2a, Zbtb32, TrimSOa, Prelidl, Gapdh, Sdc3, BC062258, LOC102643247, 2310022B05Rik, Ms4a4c, U12rbi, Mttrfdll, Smagp, Narnpt, Fasl Pafahib3, Prelid2, Gngl2, IrfS, Kdm3a, Atf3 Tnfsf9.
  • the present invention also provides a process for identifying suboptimal activity of a glarirainer acetate related drug substance or drug product comprising the steps of:
  • Fcgr3, Csf3r, Hlr2 or Tbx21 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate related drag substance or drug product under the same conditions; (ii) identifying the glatiramer acetate related drag substance or drug product as having a suboptimai activity if the level of expression of one or more genes of Ahcyl2, Cm5, Cxer6, Dhrs3, Egr2 FabpS, Ptp4al, GprlS3, Lampl, Ncoa4, Ptger4, QL Rragd. Sec24a, Sehll, Sestdl.
  • Soatl or Stiip2 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate related drug substance or drug product under the same conditions: (iii) identifying the glatirainer acetate related drag substance or drag product as having a.
  • transferase activity transferring pentosyl groups (00:0016763), transmission of nerve impulse (GO-.0019226), regulation of membrane potential (GG.0042391), cytokine activity (00:0005125), ion homeostasis (00:0050801) or adenylyitransfera.se activity 7 (00:0070566); (viii) identifying the glatii'amer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicate no substantial enrichment of one or more pathways of Cytokine- cytokine receptor interaction (mmu04060), Jak-STAT signaling pathway (mmu04630).
  • Sterol metabolic process (GO:0016125), Steroi biosynthetic process (GO:0016126), Hematopoietic cell lineage (mmu04640).
  • Myeloid ceil apoptosis (GO; 0033028), Regulation of survival gene product expression (00:0045884), Regulation of leukocyte proliferation (00:0070663), Positive regulation of survival gene product expression (00:0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of .xenobiotics fay cyt ch o e P450 (nimu0u980); ix) identifying the glatiramer acetate related drug substance or drug .product -as having- a sutopiiiiai activity if the set of differentially expressed genes indicate no substantial enrichinent of one or more pathway of ' tiinsic to membrane receptor activity,.
  • Iinmtme response ' Leukocyte migration, Chemokine receptor binding, Cytokine- cytokine receptor ' interaction. Cytokine activity, Jak-STAT signaling pathway. Sterol biosynmeti.c process. Steroid biosyntheiic process. Ceil surface, /Cytokine binding.
  • Cholesterol metabolic process Chemokine activity, Negative regulation of hormone secretion, Isopreaoid biosyntheiic process o Positive regulation of peptidyl-serine phosphorylation; (x) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates one or more patliway of enrichment of Glucose metabolic process, Glycolysis, Glueoneogenesis, Hexose catabolic process. Glucose catabolic process. Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process.
  • Alcohol catabolic process Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor metabolites and energy.
  • Pha ocytosis Leukocyte migration, Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway.
  • Galactose metabolism Carbohydrate kinase activity, Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity.
  • Ndrgl Faml62a, Pkm, Eglnl, Aldoa, Gml0480. Bnip31. Galkl , Pgaml, Pgkl, Mif, Enolb. Enol, Pdxp. Ppplr3b, Stomll , Gziiia, Sed2.
  • the mammai is a rodent or human.
  • the level of expression is determined in hematological cells. In. some embodiments, the level of expression is determined in splenoeytes. In some embodiments, the level of expression is. determine in monocytes. In some embodiments, the monocytes ar THP- 1.
  • the human has previously been treated, with a glatiiamer acetate related drag subs.Ssn.ee. or drag .product,
  • the human is a naive human.
  • the human is a g!atiramoid naive human.
  • the human is afflicted with RRMS.
  • the rodent is a mouse.
  • the mouse is a female (SJL X BALB/C) Fl mouse.
  • the mouse is about 8 to 12 weeks old.
  • the primary culture is a cuitee of spleen cells.
  • the primary culture is a culture of lymph node cells.
  • the primary culture of spleen cells is prepared about 3 days after immunization.
  • the glatiiamer acetate related drag substance is a glatiramoid or wherein the glatiramer acetate related drug product comprises a glatiranioid.
  • the glatiramer acetate related drag substance is a glatiramoid other than glatiramer acetate drag substance, or wherein the glatiramer acetate related drag product comprises a glatiramoid other than glatiramer acetate drug substance.
  • the present invention also provides a process for producing a drug product comprising a glatiramer acetate related drag substance, the improvement comprising the steps of:
  • step (b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drag product of step (a);
  • step (d) determining a set of genes differen tiall expressed by the first group after the contacting of step (b) rela tive to genes expressed by the second group after the contacting of step (c);
  • Sterol metabolic process (00:0016125), Sterol biosynthetic process (00:0016126), Hematopoietic cell lineage (mmu04640).
  • Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (GO:0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (00:0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobioti.cs by cytochrome P450 (mmu00980); (ix) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway ofmtrinsic to membrane receptor activity.
  • Immune response Leukocyte migration, Chemokine receptor binding. Cytokine-eyt.okine receptor interaction, Cytokine; activity, lak-STAT signaling pathway. Sterol biosy3 ⁇ 4theiie process. Steroid biosytithetic process.
  • Glycolysis, Ghiconeogenesis Hexose cafabolic process.
  • Glucose catabolic process Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process.
  • Cellular carbohydrate catabolic process Alcohol cataboli process.
  • Carbohydrate catabolic process Carbohydrate binding, Generation of precursor metabolites and energy.
  • Phagocytosis Leukocyte migi3 ⁇ 4iion.
  • Leukocyte clieniotaxis Positive regulation of phagocytosis.
  • Pentose phosphate pathway Galactose metabolism.
  • Carbohydrate kinase activity Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity.
  • leukocyte chemotaxis (00:0030595), phagocytosis (00:0006909), positive regulation of phagocytosis (00 0050766), cell motion (00:0006928), regulation of phagocytosis (00:0050764), behavior (GO: 0007610), membrane invagination (GO:0010324), endocytosis (GO:0006897), positive regulation of endocytosis (GO.O045807), phagocytosis, eiigivlfmeiit (GO:0006911), defense response to bacterium (00:0042742).
  • serine hydrolase activity (GO:0017i7I), serine-type peptidase activity (GO.-0008236), serine-type endopeptidase activity (00:0004252), cytokine activity (00:0005125), calcium son binding (00:0005509), immunoglobulin binding (00:0019865), clieniokine activity (00:0008009), chemokine receptor binding (00:0042379), peptidase activity, acting on L-amino acid peptides (GO:0070011), peptidase activity (GO:0008233).
  • IgG binding (GO:0019S64), protein complex binding (00:0032403) or enzyme inhibitor activity (00:0004857): (xii) discarding the batch of the glatiramer acetate related dmg substance as unacceptable for inclusion in the dmg product if the set of differentially expressed genes indicates enrichment of one or more path wa of glucose metabolic process (GO:0006OO6), glycolysis (GO: 9006096), hexose metabolic process: (GQ:G0193I8), monosaccharide metabolic process (00:0005996), hexose cafabobc process (00:0019320), ghjeose eatabojic process (00:0006007); monosaccharide catabolic process (00:0046365), cellular carbohydrate catabolic process (G ⁇ :0O44275), alcohol catabolic process (00:0046164), carbohydrate catabolic process (00:0016052) generation of precrasor metabolites and energy (00:0006091 oxidoreduct
  • UgtlaS Ugtlal .
  • Lyz2 Ctsl. Thbsl, Dab2, Arg2, Ccl6, Thbsl, Paldl, ClecSa, Cfp. Ifitm6.
  • Lyzl 01fm4, Rnfl28, Ms4a3, Clec4d, Si00a4, Ctsg, Prtn3, Ngp, F10, Hal, Ccl2, Lyzl, Thbsl, Anxal, Lgals3, S100a6, Nfami, C 3, C 4, Sppi, Stfa211 t Ptgrl, Ill 9, Cd68, Olfinl, Fegr3. Iiii2, Cregl. Dmxl2, Sirpa, 116, Elane, Csflr, Lcn2. Cd3Q01b AloxSap, lilm, Slcl lal, Tyrobp, CdSOOa, Pira2, Piral.
  • Histlh4c Histili4b, Histlh4a, Hist4h4, Histlh4£ Histlh4d, Histlh4k, Histih4j, Histlh4i. Histlh4h, Histih4ii. Histlh4m. Hist:2h4, Dst , Igsf6, Miff, Csi3r, Pf4. Pygl, Pdpn.
  • Grhpr Cxcil 1, Rasa4, Ndrgl , Faml62a, Pkm, Eghil, Aldoa, Giiil0480, BnipSL Galfcl, Pgaml , Pgkl, Mi£ Enolb, Enol, Pdxp, Ppplr3b, Stonill, Gzma, Scd2, Ccng2, Higdla, Eno2, Gfil, Pail, Ldlia, ErolL 3OO0OO2CiORik, Hk2, Sk2a3, Chchd6.
  • the present invention also provides a process for producing a drug product comprising a glatiramer acetate related drug substance, the improvement comprising the steps of: (a obtaining a batch of the glatirame acetate related drug substance or drag, product:
  • step (b) contacting a. first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a) :
  • step (d) detemiiniiig a set of genes differed ialJ expressed by the first group after the contactiBg of step (b) relative to genes expressed by the second group after the contacting of step (c);
  • the present invention also provides a process for releasing a ding product comprising a glatiiamer acetate related drug substance, the improvement comprising the steps of:
  • step (b) contacting a first gr oup of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a);
  • step (d) detennining a set of genes differentially expressed by the first group after the contacting of step (b) relative to genes expressed by the second group after the contacting of step (c);
  • endocytosis ⁇ (30:0006897), leukocyte mediated imiifflity GQ;fl002443)., membrane invagination (00:0010324), membrane organization (GO:0O16044), multicellular organismal macroinolecule metabolic process (GO:0044259), multicellular organismal metabolic process (00:0044236), myeloid leukocyte activation (00:0002274), neutrophil cheniotaxis (00:0030593), phagocytosis, engulftnent (GO:0006911), positive regulation of cellular component organization (GO:0051130), positive regulation of endocytosis (00:0045807), positive regulation of foam cell differentiation (GO:0010744), positive regulation of phagocytosis (00:0050766), regulation of endocytosis (GO:0030i00), regulation of foam cell differentiation (GO: 0010743), regulation of phagocytosis (00:0050764), regiilaiion of type I hypersensitivity (00:0001810), regiil
  • Sterol metabolic process (00:0016125), Sterol biosynthetie process (00:0016126), Hematopoietic cell lineage (mniu04640).
  • Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (GO:0045884), Regulation of leukocyte proliferation (00:0070663), Positive regulation of survival gene product expression (00:0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) discardin the batch of the glath amer acetate related drag substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of mtrinsic to membrane receptor activity.
  • Immune response Leukocyte migration, Cheinokine receptor binding. Cytokine-cytokine receptor interaction, Cytokine activity, Jak-STAT signaling pathway. Sterol biosynthetie process. Steroid biosynthetie process. Ceil surface, Cytokine binding. Cholesterol metabolic process, Cheinokine activity'.
  • Alcohol eatabolic process Carbohydrate eatabolic process. Carbohydrate binding, Generation of precursor metabolites and energy.
  • Phagocytosis Leukocyte ' migration.
  • Leukocyte chemotaxis Positive regulation of phagocytosis.
  • Pentose phosphate pathway Galactose metabolism.
  • Carbohydrate kinase activity Myeloid leukocyte activation, Cheniokine receptor binding. Regulation of type I hypersensitivity.
  • atoms of oxygen (GO;0016702), oxidoredoctase activity, actin on single donors: with. iiicor oralioH of molecular oxygen (GO .0016701), cytokine activity (GO • .0005125), carbohydrate kina.se activity (GO; 0019200), Glycolysis Glucoheogenesis (mnajOOOlQ), Pentose phosphate pathway (miiruOOO30), Fructose and niamiose metabolism (nraiuOOOS i) or Galactose metabolism (mniii00052); (xiii) discarding the batch of the glatirarner acetate related drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of RIG-I-like receptor signaling pathway or Adeny yltransferase activity; or (xiv) determining the level of expression of at least one gene of CxcI
  • the present invention also provides a process for releasing a drug product comprising a glatiramer acetate related drag substance, the improvement comprising the steps of:
  • step (b) contacting a first group of mammalian ceils with an amount of the glatirarne acetate related drug substance or drug product of step (a); (c ) contacting a second group of mammalian cells of the same type with an amount of a reference standard;
  • step (d) determining a set of genes differentially expressed by the first group after th contacting of s tep (b) relative to genes expressed by the second group after ' the contacting of step (e);
  • the present invention also provides a process for identifying suboptimal activity of a glatiramer acetate related ding substance or ding product comprising the steps of :
  • step (b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a);
  • step (d) determining a set of genes differentially expressed by the first group after the contacting of step (b) relative to genes expressed by the second group after the contac ting of step (c);
  • (e) (i) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Mmpl2, Tgfbi, Cleoto, Ciec4a3, Fcerlg, Fcgr2b, FcgrS, Csf3r, Illr2 or Tbx21; (ii) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Ahcyl2, ClnS, Cxcr6, Dhrs3, Egr2.
  • RNA binding (00:0003725), NAEH- ADP-ribosyitransferase activity (00:0003950), transferase activity, transferring pentosyl groups (GO:0016763), transmission of nerve impulse (00:0019226), regulatio of membrane potential (GO:0042391) , cytokine activity (00:0005125), ion homeostasis (GO:0050801) or adenylyl transferase activity (00:0070566); (viii) identifying the glatiramer acetate related drag substance or drag product as having a suboptimal activity if the set of differentially expressed genes indicate no substantial enrichment of one or more pathways of Cytokine- cytokine receptor interaction (mmu04060), Jak-STAT signaling pathway (mmu04630), Sterol metabolic process (00:001 125), Sterol biosynthetic process (00:0016126), Hematopoietic cell lineage (mmu04640).
  • Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (GO: 0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (GO:0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) ideffiifying the glatiramer acetate related drug substance or drag product as having a suboptimai activity- if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of Intrinsic to membrane receptor activity. Immune response, Leukocyte migration, Cheinokine receptor binding. Cytokine- cytokine receptor interaction.
  • Cytokine activity Jak-STAT signaling pathway.
  • Sterol biosyntheti process Steroid biosynthetic process. Ceil surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serine phosphorylation; (x) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process. Glycolysis, Gluconeogenesis, Hexose catabolic process. Glucose catabolic process.
  • Monosaccharide catabolic process Hexose metabolic process.
  • Monosaccharide metabolic process Cellular carbohydrate catabolic process.
  • Alcohol catabolic process Carbohydrate catabolic process.
  • Carbohydrate binding Generation of precursor ' metabolites ami energy.
  • Phagocytosis .
  • Leukocyte migration, Leukocyte ehemotaxis.
  • Positive regulation of phagocytosis Pentose phosphate pathway, Galactose metabolism. Carbohydrat kinase activity.
  • leukocyte mediated immunity (00:0002443), cell migration (GO:0016477), regulation of vesicle- mediated transport (GO:0060627). localization of ceil (GO:0051674), cell motility (00:0048870), collagen metabolic process (00:0032963). muiticellular organismal macromolecule metabolic process (GO:0Q44259), positive regulation of cellular component organization (00:0051130), myeloid leukocyte activation (00:0002274), positive regulation of foam cell differentiation (GO:0010744), multicellular organismal metabolic process (GO:0044236), response to yeast (GO:0001878), reguiation of type I hypersensitivity (00:0001810). regulation of foam cell differentiation (00:0010743).
  • the present inventio also provides a process for identifying suboptimal activity of a glatiramer acetate related hug substance or drug product comprising the steps of:
  • step (b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a);
  • step (d) determining a set of genes differentially expressed by the first group after the contac ting of s te (b) relative to genes expressed by the second group after the contac ting of step (c);
  • the referenee standard is gktiranier acetate related, drug substance or drag product in some embodiments, the referenee standard is mamiitQ.!. hi some embodiments, the reference standard is medium.
  • the determining step (d) comprises comparing the expression of genes expressed by the first group to the expression of genes expressed by the second group. In some embodiments, the determining step (d) comprises comparing the expression of genes by the first group of cells and by the second group of cells to expression of the genes by the same type of ceils exposed to mannitol or medium.
  • the present invention also provides a process for producing a drug product comprising a glatiramer acetate related drug substance which involves an array of testing, the improvement comprising including in the array of testing the steps of:
  • Fabp5, Ptp4al, Gprl83, Lampl, Ncoa4, Ptger4, Qk, Rragd, Sec24a, SehlL Sestdl, Slc30a4, Soati or Strip2 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drag substance under the same conditions; (in) including the batch of the glatiramer acetate related drug substance in the production of the ding product if the level of expression of one or more genes of Fam212a, Gzrna, Itprl or Zbtb32 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drag substance under the same conditions; (iv) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes of Gzmb, Fanil9a3.
  • NJ313, Gzma, Eaf2, Carl or St7 is upregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drag substance under the same conditions; or (v) including the batch of the glatiramer acetate related drug substance in the production of the drag product if the level of expression of one or more genes of Crisp3, Apoe, Dapll, PdeSa, Scg5, StSsiafS. 4930426D05Rik or Pgap is downi'egulated relative to the level of expression by the same type of cells in the absence of the giatirainer acetate related drag substance under the same conditions.
  • the present invention also provides a process for releasing a dru product comprising a giaiaarner acetate related drug substa ce:, which process involves an array of testing, the .improvement comprising: including in die array of testing the steps of
  • a "naive human” is a human that has not been treated with any multiple sclerosis drug.
  • a "glatiramoid naive human” is a hitman that has not been treated with any glatiramoid drug.
  • a glatiiamoid naive human could have been treated with another multiple sclerosis drug.
  • '3 ⁇ 4 the blood of is represented by peripheral blood mononuclear cells (PBMCs), lymphocytes, monocytes, macrophages, basophils, dendritic cells or oilier cells derived from a mammal' s blood.
  • PBMCs peripheral blood mononuclear cells
  • under the same conditions means that when two or more substances are tested and eornpared, variables or conditions that are not. tested are controlled to allow eoiicltisions to be made regarding the tested variables.
  • oilier variables should be adjusted, if necessary, so as to be comparable, at the discretion of the person skilled in the art - including, for example, the amount of the cell culture, the amount of each tested substance, and/or the type, components and additions to medium.
  • the variables that should be controlled may be established according to the knowledge and experience of the person skilled in the art.
  • in the absence of the glatiramer acetate related drug substance means in the presense of a negative control, e.g. mannitoi or medium only.
  • a negative control e.g. mannitoi or medium only.
  • the presence of a different glatiramer acetate related drug substance or drug product, such a glatiramer acetate drug substance or drug product does not fall under the definition of "in the absence of the glatiramer acetate related drug substance” or "in the absence of the glatiramer acetate related drug product.”
  • a "reference standard” is a sample or value which serves as a point of comparison for another sample or value which differs from the reference standard with respect to one or more variables.
  • a “reference standard” is a value or range of values that characterizes a defined population in a defined state of health.
  • a reference standard can characterize a healthy human or a human afflicted with multiple sclerosis, and when the hmnan is afflicted with multiple sclerosis the human can be naive or having received glatiramer acetate drag substance.
  • Glatiramer acetate the active ingredient of Copaxone®, consists of the acetate salts of synthetic polypeptides, containing four naturally occurring amino acids: L-ghitamic acid, L-alanine, L-fyroshie, and L-lysine with an average molar fraction of 0.141 , 0.427, 0.05)5, and 0.338, respectively.
  • the peak molecular weight of glatiramer acetate is between 5,000 and 9,000 Daltons (Copaxone, Food and Drug Admimstration Approved Labeling (Reference ID; 3443331) [online], TEVA Pharmaceutical Industries Ltd..
  • glatiramer acetate is designated L-glutamk acid polymer with L-alanine, L-lysine and L- tyrosine, acetate (salt). Its structural formula is: (Gl «Ala,Lys J x)x. CH3COOH
  • CopaxonetHs a clear, colorless to slighdy yellow, sterile, itorrpyrogeme solution for suixitfaneou iBjeetios
  • Each I niL- o Copaxorie® solution contains 20mg or 40aig of GA, the active ingredient, and 40mg of HiaranioL
  • the pH of the solutions is appi3 ⁇ 4xunatel 5.5 to 7,0.
  • Co axone*!; 20ffigmrL in a prefvetted syringe (PFS) is an approved product indicated for the treatment of patients with relapsing forms of multiple sclerosis, the safety and efficacy of winch are supported by over two decades of clinical research and over a decade of post-marketing experience.
  • Copaxone's; 20mg/iiiL is administered daily.
  • Copaxone's* 40mg/iiiL in a PFS was developed as a new formulation of the active ingredient GA.
  • Copaxone® 40mg/mL is a prescription medicine indicated for the treatment of patients with relapsing forms of multiple sclerosis (Copaxone, Food and Drug Administration Approved Labeling (Reference ID: 3443331) [online], TEVA Pharmaceutical Industries Ltd., 2014 [retrieved on December 24, 2014], Retrieved from the Internet ⁇ URL: mvw.accessdata.fda.gov ihiigsatfda ⁇ Copaxone® 4Qing/mL is administered three times per week.
  • glatiramer acetate related drug substance is intended to include polypeptides with a predetermined sequence as well as mixtures of polypeptides assembled from the four amino acids glutamic acid (E), alanine (A), lysine (K), and tyrosine (Y); fi'om any three of the amino acids Y, E, A and K, i.e. YAK, YEK, YEA or EAK; or from three of the amino acids Y, E, A and and a fourth amino acid.
  • glariramer acetate related drug substances are iiisclosed in U.S.
  • Glatiramer acetate related substances include glatiramoids.
  • a "glatiramer acetate related drug product” contains a glatiramer acetate related drug substance.
  • glatiramer acetate related drug substance or drug product is a glatiramer acetate related drug substance or a glatiramer acetate related drug product.
  • glatiramoid is a c omplex mixture of synthetic proteins and polypeptide s of varying sizes assembled from four naturally occurring amino acids: L-glutamic acid, L-aianme, L-lysine, and L-tyrosme.
  • glatiramoids include glatiramer acetate dr ug substance (the active ingredient in Copaxone'*) as well the active ingredients in other products, e.g. GA-Natco, POLIMUNOL and GLATOPA.
  • a glahramer acetate drug substanc “ (GADS) is glatiramer acetate produced by leva Pharmaceutical Industries, Ltd, and is the active .ingredient in a giatiranier acetate drug product. .
  • a "giatiranier acetate drag product” contains a glatiramer. acetate drug substance produced by Teva Pharmaceutical Industries, Ltd.
  • a ⁇ giatiranier: acetate drug substance o drag product is a giatiranier acetate drug subslan.ee. or a, glatiramer acetate drug product
  • glatiramer acetate reference standard is or contains the drag substance found in a glatiramer acetate drug product.
  • suboptimal activity refers to a negative response or to a response which is less than the response to giatiranier acetate drag substance or glatirame acetate drag product produced by leva Pharmaceutical Industries, Ltd.
  • release of a drag product refers to making the product available to consumers.
  • about 100 mg therefore includes the range 90- 110 rag and therefore also includes 90, 91, 2, 93, 4, 95 96, 97, 98, 99, 100, 101, 102. 103, 104, 105, 106, 107, 108, 109 and 1 10 mg. Accordingly, about 100 nig includes, in an embodiment 100 mg.
  • hematological cell comprises neutrophils, erythrocytes, basophils, monocytes, eosinophils, platelets, lyrnphoc-ytes, and spienocytes.
  • an "array of testing" for a. glatiramer acetate related drag substance or drag product includes, but is not limited to, any analytical method test such as in vitro tests or molecular weight tests, biological assays such as the ex vivo tests and clinical efficacy tests which characterize the GARDS or GA DP, or clinical trials.
  • Examples of testing for a giatiranier acetate related drug substance or drag product are disclosed in U.S. Patent Application Publication Nos. US 2012-0309671 and US 2011- 0230413, and hi PCT International Application Publication Nos. WO 2000/018794, WO 2012/051106, WO 2013/055683, WO 2014/058976, the disclosures of which are hereby incorporated by reference in their entireties.
  • 0.2-5 nig is a disclosure of 0.2 mg, 0.21 mg. 0.22 mg, 0.23 mg etc. up to 0.3 mg. 0.31 mg, 0.32 mg, 0.33 mg etc. up to 0.4 mg, 0.5 mg, 0.6 mg etc. up to 5.0 mg.
  • characierization or “characterizmg” is understood, to include obtaining information which was produced, in the. same location .or country, or a different location or country from where any remaining steps of the raerfiod are performed.
  • mice in each treatment group were injected with 100 uL of a 2.5 mg/mL solution of either Copaxone® (GA drug product, Teva Pharmaceutical Industries, Petach Tikva, Israel) or POLIMUNOL (Synthon, Nijmegen, Netherlands) in phosphate-buffered saline (250 pg GARS per mouse).
  • Copaxone® G drug product, Teva Pharmaceutical Industries, Petach Tikva, Israel
  • POLIMUNOL Synthon, Nijmegen, Netherlands
  • splenocytes from the same immunization grou were pulled and resuspended to a final eoiieenhation of 10 x 10° cells niL in defined, ceil culture media (BCCMl) (Biological Industries, Beit Haexaek, Israel ⁇ (96.7% v/v) enriched with L-ghiiaBiine 2 inM (1 v/v), MEM Non-E-ssential Amino Adds 2 niM (3 ⁇ 4 -v/v), sodium pyruvate 1.
  • BCCMl ceil culture media
  • Splenocytes were treated with activator samples diluted in medium (125 L per well of 80 iig/ L solutions, final concentration in the well after addition of cells: 40 pg/mL) of: i) 3 different batches of GA drug product manufactured by Tev ii) one batch of POLIMUNOL (purported generic GA).
  • POLIMUNOL ⁇ purported generic GA is a product marketed as generic GA and manufactured by a company other than Teva (i.e. Synthon).
  • the activator samples or marmitol the nonactive excipient in Copaxone ® ) were added to 96-weU tissue culture plates (three wells pe sample).
  • Splenocytes (125 uL 10 x If SPL cell mL suspension) were added to the activator solutions. Each activator sample was loaded in two different plates. One tor the cells from mice immunized with GA and one for cells from mice immunized with proposed generic. Plates were incubated for 24 h at 37°C. Cells were collected from the wells and were centrifuged at 300g for 5 minutes. Supernatants were aspirated and cell pellets were resuspended in RLT buffer (from RNEASY MINI KIT OF QIAGEN, Cat # 74106) for cell lysis. The cell lysates were centrifuged and supematants were collected and frozen at -70°C. Samples were sent for further processing. Figures 19 and 22,
  • mice were immunized with either Copaxone* or .
  • POLIMUNOL purported generic GA
  • splenocytes were isolated and treated ex vivo with Copaxone ® or POLIMUNOL (purported generic GA)
  • RNA was extracted and expression profiled across the entire genome using the AFFYMETRT MOUSE GENOME 430 2 CHIP.
  • Outlier samples were identified using the R package ArrayQCMetrics and excluded from further data processing steps. A sample was considered an outlier if it failed more than half of the included tests either before or after RMA normalization. Data were RMA normalized using the Affy R package. .Batch correction.
  • ConiBat is an empirical Bay siaa approach using location and scale metrics across several genes to adjust for batch effects in datasets, even datasets containing small sample sizes... Date of nheroarray experiment wa used as batch, and t e combination o treatment and immunization w3 ⁇ 4s used as covariaie.
  • Principal Component Analysis (a multivariate approach) showed thai the main effect in die first principal component remained due to treatment effects after batcii correction.
  • Probesets were filtered by MAS5 calls of presence on the chi (to be considered present, a probeset was required to have on average a call of present or marginal across samples), Probesets were mapped to genes using the annotation available for the Mouse 4302 chip from Afrymetrix. Unless otherwise noted, FDR adjusted p values reported for genes represent the lowest FDR adjusted p value for present probesets for that gene. For the comparison of immunization and activation with GA versus immunization and activation with POLIMUNOL (purported generic GA), an additional correction for run order, performed using LIMMA (Smyth, 2004), was evaluated (as described in Hasson et al, 2016).
  • Upregulaied and dowmegulaied probesets were analyzed separately for pathway enrichment, using DAVID (Huang et al. Nucleic Acids Res 2009). Pathway enrichment results were visualized using volcano plots, plotting either -log adjusted p values or untraiisformed adjusted p values versus enrichment scores for the pathways.
  • upregulaied or downregulated probesets with FDR-adjusted p values ⁇ 0.05 and ibid changes (FC) with absolute value greater than 1.2, or for further analyses greater than 1.4. were used for pathway enrichment.
  • upregulaied or downregulated probesets with FDR-adjusted p values ⁇ 0.05 and FC with absolute value greater than 2 were used for pathway enrichment. DAVID runs were conducted in December 2014, January 2015, and March-April 2016.
  • Cells, from a human monocyte eel! line (THP-1) were Slsniiilated ' with .either branded glatiiamer acetate - Copaxone ® , of purported generic PQLIMt!NOL, or vehicle control (mannitol) f r 6 hours based OB prior observations of GA effects in this model system ( olitz et al . , . 2015), KNA was extracted and expression profiled i a blinded fashion across the entire genome f using the iymetrix Human Genome TJ133 Plus: 2.0 e p, mteifogamig a total of over 47,000 transcripts.
  • Outlier samples were identified using the R package ArrayQCMetrics and excluded from further data processing steps. A sample was considered an outlier if it tailed more than half of the included tests either before or after RMA normalization. Data were RMA normalized using the Affy R package.
  • Bayesian approach utilizing location and scale metrics across several genes to adjust for batch effects in datasets, even datasets containing small sample sizes. Date of experiment was utilized as batch. Treatment labels were added as covariates to the batch correction in order to preserve relevant treatment effects. Principal Component Analysis (a multivariate approach) showed that the main effect in the first principal component remained due to treatment effects after batch correction.
  • probeset was required to have on average a call of presen or marginal across samples.
  • An di tk l QC step wax performed to remove probesets determined to be highly variable between mi iple THP-i datasefs, as follows; a probeset was deemed highly variable if across three THP-i studies to date, that probeset was observed to be upregulaied, downregulated, and not modulated by Copaxone ® across the three studies. This criterion resulted in filtering out 216 probesets. Probesets were mapped to genes using the annotation available for die U133 Phis 2.0 chip from Affymetiix. FDR adjusted p values reported for genes represent the lowest FDR adjusted p value for present probesets for that gene.
  • Upregulated and dowmeguiated probesets were analyzed separately for pathway enrichment,, using DAVID (Huang et al. Nucleic Acids Res 2009). Pathway enrichment results were visualized using volcano plots, plotting -log p values versus enrichment scores.
  • upregulated or downregulated probesets with FDR-adjusted p values ⁇ 0.05 and fold changes (FC) with absolute value greater than 1.1 were used for pathway enrichment.
  • FDR-adjusted p values ⁇ 0.05 and fold changes (FC) with absolute value greater than 1.1 were used for pathway enrichment.
  • Copaxone ® and niamiitoi upregulated or downregulated probesets with FDR- adjusted p values ⁇ 0.05 ami FC with absolute value greater than 1.3 were used for pathway enrichment.
  • mice 8- to 12-week-old female (Balb c X SJL) Fl mice (Janvier, France) were used. Mice were kept at 21 ⁇ 3°C; the relative humidity was 30-70%. the light/dark cycle was 12/12 li. Animals were maintained on a standard rodent pellet diet and sterile filtered tap water available ad libitum.
  • mice in each treatment group were injected with 100 uL of a 2.5 mg/niL solution of either Copaxone® (GA drug product) or GLATOPA'D in phosphate-buffered saline (250 pg GAper mouse). Mice were housed for 3 days after iinmtmizatiom mice were then sacrificed and their spleens were aseptically removed and placed on ice in RPMI 1640. A single cell suspension was prepared.
  • DCCM1 defined cell culture medium
  • Splenoeytes. were treated wife activator samples diluted hvrnedkim (125 pL per well of 80 ig/iiiL solutions, final, concentration in me well after addi tion of ike cells: 40 jig/niL.
  • Six different batches of Copaxone® and six different batches of GLATOPA were used for treatment.
  • the activator samples or mannitoi (the nonactive excipient in Copaxone® and GLATOPA, used as contra! in fee experiment) in medium were added to 96-weU tissue culture plates. Ail batches were tested in 6 replicates, each replicate in 3 wells. The study was performed twice resulting in 12 replicates for each batch.
  • Splenocyt.es (125 ⁇ iL 10 x 106 SPL ce!l/mL suspension) were added to the activator solutions. Each activator sample was loaded in two different plates, one for the cells from mice immunized with GA and one for cells from mice immunized with GLATOPA. Plates were incubated for 24 h at 37 a 'C in 5% C02 atmosphere. Cells were collected from the wells and were centrifuged at 300g for 5 minutes. Supernatants were aspirated and cell pellets were resuspended in RLT buffer (from R easy mini kit of Qiagen, Cat # 74106) for cell lysis. The cell lysates were centiifuged and supernatants were collected and frozen at -70°C. Samples were sent fo further processing.
  • THP-1 cells human monocytic cell line
  • RPMI 1640 medium supplemented with 10% Fetal bovine serum, ImM sodium-pyruvate, 0.025mg ml D-glucose. lOmM Hepes, 0.05mM 2- mercaptoeihano!.
  • IniL of THPi cell suspension containing 2x106 cells/mL was added to each well of 6 well plates.
  • ImL of lOOpg/ml Copaxone® or GLATOPA batches were added to the wells. Five different batches of Copaxone® and five different batches of GLATOPA were used tor treatment.
  • Mannitoi (the nonactive excipient in Copaxone® and GLATOPA) used as control in the experiment. Ail batches were tested in 10 replicates. Plates were incubated for 6 h at 37*C in 5% C02 atmosphere. Cells were collected from the wells and were centrifuged at 300g for 5 minutes. Supernatants were aspirated and cell pellets were resuspended in RLT butler (from RNeasy mini kit of Qiagen, Cat # 74106) for cell lysis. The cell lysates were centiifuged and supernatants were collected and frozen at.-?0 ⁇ C, Samples were sent for furthe processing.
  • Outlier samples were identified using the R package ArrayQCMetri.cs and excluded from further data processing steps. A sample was considered an outlier if it failed more than half of ' the included ' tests either befo e or after -RMA normalization. Daia were RMA normalized using the Aff R package.
  • Bayesian approach utilizing location and scale metrics across several genes to adjust tor batch effects in datasets, eve datasets containing small sample sizes.
  • ⁇ -1 dataset ftora the Israel lab. day of lab experiment and date of microarray run were combined for use as batch with treatment as covariate.
  • THP-1 dataset from the Hungary lab no batch correction was needed tor date of experiment or array run.
  • mouse splenocyte dataset date of experiment combined with date of microarray ran was used as batch, and the combination of tieatment and immunization was used as covariate. Principal Component Analysis showed that the main effect in the first principal component remained due to treatment effects after batch correction. Differential expression analysis
  • probesets were identified across conditions using linear models for microarray data (LIMMA: Smyth, G. K. (2004)), a standard R Bioconductor package. To compare Copaxone®' and purported generic, comparisons were corrected to compare each treatment rela tive to mannitol control (e.g., [Copaxone3 ⁇ 4> vs mannitol] was compared via LIMMA to [GLATOPA vs mannitol]). Probesets were filtered by MAS5 calls of presence on the chip (to be considered present, a probeset was required to ha ve on a verage a call of present or marginal across samples).
  • Probesets were mapped to genes using the annotation available for the Mouse 430 2 chip from Affymetiix in the case of the splenocyte study, and tire HG U133 Plus 2 for the THP-1 study.
  • run order from the microarray run was used as covariate in the analysis.
  • FDR adjusted p values reported for genes represent the lowest FDR adjusted p value for present probesets for that gene.
  • FDR in this context refers to the Benjamini Hochberg method, as implemented in the LIMMA R package.
  • FDR-adjusted p values ⁇ 0.05 and fold changes (FC) with absolute value greater than 1 ,1 under both an uitiz tion conditions (splenocyte data) or FDR-adjtisted p values 0.05 hi both laboratories (THP-1) were used, for pathway eniichnient.
  • FDR-adjusted p values ⁇ 0.05 and FC with absolute value greater tha 2 were used for pathway enrichment. DAVID runs were conducted during March- May 2016.
  • GSEA Gene Set Enrichment Analysis
  • the intact material Upon injection, the intact material is hydrolyzed locally through complex mechanisms, at unknown dynamics and at unknown preferential cleavage sites.
  • the fragments are the taken up by antigen presenting cells.
  • Antigen presenting cells present specific, yet undefined, epitopes to T-cells of die immune system, modifying the delicate balance between proinflammatory, anti-inilammatory and regulatory immune cells (Thl, Th2 and Treg, respectively) in the brain. Because ' o a one® is hydro yzed locally, there are no known pharmacokinetics. In addition, there are aq validated phaniiaeodynarnics markers.
  • Copaxone® is an antigen that participates in the immunological triad, along with antigen presenting cells and T cells.
  • a mouse spienocyte system and human monocyte (THP-1) cell line were utilized. Unbiased genome-wide gene expression studies were conducted to examine effects of treatment with Copaxone® or GLATOPA in each model system.
  • Copaxone® treatment modulated thousands of probesets versus mannitoi control in splenocytes from CopaxoneC-immunized mice (Table 1), Genes consistent with prior literature about Copaxone® mechanism of action were modulated, including prominent immunomodulatory (Fig, 3A-D): 1110, the gene for anti-inflammatoiy cytokine IL-10, was upregulated with Copaxone® treatmen (FC 1.54, ad] p ⁇ 1.8e-16). IL-10 is expressed by monocytes and lymphocytes, and has multiple immunological functions, including downregulation of Thl cytokine expression, and enhancement of B cell survival and antibody production.
  • Table 2 Shown in Table 2 are top genes modulated by Copaxone® treatment relative to niamiitol spienocytes fiom CopaxQiie®-iminiBiized mice.
  • Extracellular region ( €30:0005576) .
  • Myeloid ceil apoptosis (GO: 0033028)
  • the top egg pathway enriched among probesets upregulated by Copaxone® treatment was the cytokine-cytokine receptor interaction pathway (adj p ⁇ 5e-5). This observation is consisten with prior observations for Copaxone® treatment in this system, as well as other model systems (e.g. THP-1) ( oiitz et a , 2015: Hasson et al, 2016).
  • FC 2.3, adj p ⁇ 6.5e-i3
  • a sigmfk3 ⁇ 4it ⁇ subset of probesets incl « ⁇ fcig approximately 7% of probesets affected by Copaxone® treatment ⁇ was modula d to differing degrees by GLATOPA and. Copaxone®, as illustrated in Figure 5.
  • 1.Q58 probesets (6.02 up, 456 down) were significantly differentially expressed (FDR. ⁇ 0. ⁇ 5 betwee GLATOPA and Copaxone® (Jnaamtol-corfected) under Copaxone!? kmmHrization, The range of observed fold change difference among these probesets: as -1.56 to 1.46..
  • GLATOPA ⁇ Cepaxoue Differentially expressed genes common to both . Immunization conditions
  • This subset represents the most robust signature of dissimilarities, regardless of sequence of switching, thus reflecting a conservative assessment of inherent biological dissimilarities.
  • MMP9 blood brain barrier
  • Mmp9 functionally relevant genes and pathways are specifically encoded within this subset of probesets: for instance, expression levels of several matiix metal!oproteinases were higher with GLATOPA versus Copaxone® treatment, including Mmp9, MmpS, and Mmpl.2.
  • the MMP protein is key to blood brain barrier (BBB) disruption, in particular increasing access of immune cells (including autoimmune T cells) to the CNS.
  • BBB blood brain barrier
  • High MMP9 levels have been consistently associated with MS (Rosenberg 2005; Romme Christensen et al 2013; Kouwenhoven et al 2002), as well as in MRI active patients with gadohnitim-eiAaiiciiig lesions versus patients without (Waubant 2006).
  • MMP9 has been proposed as a biomarker for MS diagnosis and progression (Milward et al 2008). GA has been reported to inhibit expression ofMMP9 in healthy human peripheral blood mononuclear cells (Kiiop 2013). Consistent with these data, Copaxone® treatment in the splenocyte model lowered MMP expression relative to maimitol control to a greater degree tha did GLATOPA. This was the case under both immunization conditions, and for both probesets for Mmp9 present on the chip. This gene has been previously observed to differ in level between Copaxone® and other purported generics, including POLIMUNQL (Hasson et al, 2016) and PROBIOGLAT (Kolitz et ai, 2015).
  • Tgfbi gene (coding for transforming growth factor beta induced protein) was increased in level between GLATOPA and Copaxone® (adj p ⁇ 5.3e-10, FC 1.34: Figure 6). That is. relative to maimitol control, GLATOPA treatment decreased levels of this gene less than Copaxone® treatment. As shown in Figure 6, this result was observed for both immunization conditions. Four probesets were present on the chip for this gene, and all four showed the same pattern, lending additional support to the differential expression of this gene. Tgfbr lias roles in adhesion a d.
  • iiifiaiimiafion and is induced by transfoniiing growth factor beta, a iactor with cpatext-depeadeat effects on T cell polarization that can induce Thl 7 differentiation (Khnura and KislHiaoto, 2010; Giiicber efc al, 2011).
  • C!ec4n was also increased in expression with GLATOPA relative to GopaxoneC>ti"eatnient (Figia 7); that is, Copaxone® treatment decreased the expression level of this gene relative to control to a greate extent than GL ATOPA treatment This was true under both immunization conditions., and for both probesets for Clec4s present o the array, A related, mouse gene, Ciec4a3, also shewed similar differences.
  • the Ckc4n gene representing the mouse homolog of human CLEC6A, codes for the antigen-presenting cell lectin-like receptor complex APLEC, also known as dendritic cell inhibitory receptor 3, which allows transduction of signaling through Fc receptor gamma chain FCER1G (SwissProf). Interestingly and consistent with this functional activity,.
  • Fcei is itself modulated differentially by GLATOPA and Copaxone® ( Figure 8), along with several related genes, Fcgr2b and Fcgr3 (each increased with GL ATOPA relative to Copaxone® treatment).
  • AM four probesets tor Fcgr2b tha were called present on the array showed the same result.
  • Fcgr2b encodes a receptor for the Fc region of eomplexed or aggregated immunoglobulins gamma, involved in a range of effector and regulatory functions including modulation of antibody production by B-cells. Fcgr2b receptor binding is reported to lead to downregulatioii of existing cell activation that had been caused by antigen receptors on B-cells (BCR), T-cells (TCR) or by another Fc receptor (UniProf).
  • BCR B-cells
  • TCR T-cells
  • UniProf another Fc receptor
  • Cxcl3 also known as GRQ-1.
  • a cliemokme that participates in recruitment of neutrophils (Figure 9; FC 1.46, adj p ⁇ 3.9e-6 and FC 1.48, adj p ⁇ 3.2e-5 for Copaxone®and GLATOPA immunization, respectively): Csf r (FC 1.13. adj p ⁇ 1.0e-5 and FC 1.2, adj p ⁇ 9.8e-l l for Copaxoiie®and GLATOPA immunization, respectively) and Csf3r (FC 1.12, adj p
  • GLATOPA versus Copaxone! 1 Differentially expressed pathways imder both Immuiiizatioi ⁇ conditions
  • probesets differentially expressed between Copaxone® and GLATOPA treatment under both immunization conditions were also examined tor imdeiiying pathway enrichment.
  • this subset of probesets was filtered using a FC cutoff of LI, to yield 1 6 probesets (representing 112 genes) in the up and 1 17 probesets (representing 7 g aes) in. the ' .down direction.
  • probesets were subjected to pathway enrichment using the NTH DAVID platform tor pathways in GO (Gene Ontology: Ashbumer et al, 2000) and Kegg ( anehisa et al, 2000) ( Figure 10 and Table 5). 72 pathways were significantly enriched (adj p ⁇ 0.05) among probesets increased in expression, and 19 pathways among probesers decreased in expression with GLATOPA relative to Copaxone® treatment. These pathway enrichments lend further robustness and biological relevance to the observed differences.
  • Pathways enriched among probesets increased in expression with GLATOPA relative to Copaxone® included immunological pathways such as cytokine-cytokine receptor interaction (adj p 2.066-4), inflammatory response (adj p ⁇ 7.78e-5), hematopoietic cell lineage (adj p ⁇ 3.75e-4), regulation of adaptive immune response based on somatic recombination of immune receptors built from iminimogiobiilm sitpeifamily domains (adj p ⁇ 0.048), and leukocyte migration (adj p ⁇ 2.82e-7), as well as immunoglobuli binding (adj p ⁇ S.9e-4) and regulation of type I hypersensitivity (adj p ⁇ 0.025).
  • immunological pathways such as cytokine-cytokine receptor interaction (adj p 2.066-4), inflammatory response (adj p ⁇ 7.78e-5), hematopoietic cell lineage (adj p ⁇ 3.
  • Figure i 1 shows the probesets imdeiiyiiig the enrichment of the cytokine-cytokine receptor interaction pathway among probesets differing between GLATOPA and Copaxone 1 !', of particular interest since this pathway is modulated by Copaxone® and relevant for its mechanism of action.
  • Figure 12 shows probesets underlying enrichment of the pathway: regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfaniily domains.
  • Pathways enriched among probesets having lower expression with GLATOPA versus Copaxone® included glycolysis (adj p ⁇ 1.7e-37) and multiple additional metabolism-related pathways.
  • Thl7 related genes 116, Illb, and il7r were confirmed as significant in the qRT- PCR data.
  • many of the genes tested participated in pathways of interest that were enriched among differences between GLATOPA and Copaxone® in the microarray studies.
  • Cxcl3 and Ccl2 encode chemokmes relevant for leukocyte accumulation during inflammation. These genes participate in the chemokine receptor binding pathway, enriched among probesets increased in expression with GLATOPA relative to Copaxone® (adj p ⁇ 0.002).
  • GLATOPA increases Ccl2 expression, while Copaxone® does not.
  • Fig. 22B Cc!2 is increased by GLATOPA (FC 1.20, adj p ⁇ 0.012) but not Copaxone ® (adj p>0;74) vs control and is up with GLATOPA relative to Copaxone ® (FC 1.17, adj p ⁇ 0.0003) in Copaxone ® immunization array data; similar for GLATOPA immunization. It has been shown that positively charged nanopartides significantly stimulate chemokine (C-C Motif) Ligand Ccl2 expression (a chernoattractant involved in recruitment of immune cells), while negatively charged ones do not (Fromen CA, et al. 2016)).
  • Fig. 22A Positive surface charge distribution is mechanistically linked to imiiHiomodiilation and cytotoxicity according to extensive literature, potentially linking fee observed immunomodulation signature of GLATOPA to its higher: positivity in surface charge.
  • Csf 1 r 5 ;Csf3r, and B lr2 each encode cytokine receptors, and participate in the cytokme-cytokme receptor interaction pathway discussed above, as well as the cytokine, binding pathway, also enriched among such differences between GLATOPA and Copaxone® (adj p 0.009).
  • Fcgr3, Fcgr2b, and Fcerlg. ifiummogiobulm receptor genes, participate in die similarly enriched imnaHioglohii!iii binding pathwa (adj p ⁇ S.9e-4), Ciec4n (dendritic cell inhibitory receptor 3), as discussed above, is known to allow signaling through Fcerlg.
  • the three irnmunoglobu!in receptor genes also participate in the regulation of type I hypersensitivity pathway, also enriched among GLATOPA-Copaxone®differences (adj p ⁇ 0.025). These immunoglobulin receptor genes, as well as 116, also participate in the regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains pathway described above.
  • Mmp9 matrix metalloproieinase genes were confirmed as differentially expressed, including Mmp9, Mm l2, and Mmp8.
  • Mmp9 also diifered m expression level between GLATOPA and Copaxone® treatment in the THP-1 experiment, as well as between Copaxone® and additional purported generics POLIMUNOL and PROBIOGLAT in prior studies ( olitz et al, 2015; Hasson et al, 2016).
  • MMP9 is reported to increase immune cell access to the CNS, and high levels have been associated with MS ⁇ Kouweiihoven et al. 2012).
  • MmpS plays roles in the regulation of innate immunity (Tester et al, 2007), as well as in macrophage differentiation and polarization (Wen et al, 2015), and Mrn l2 may have a role in antiviral immunity (Marchant et al, 2014).
  • Tbx21 was less highly expressed with GLATOPA relative to Copaxone® treatment.
  • This gene encodes transcription factor T-bet, which can control mteiferon-gamma expression and development of Thl- polarized T cells (ITniProt).
  • Cxcl9 is an inflammatory cytokine that was also lower in expression with GLATOPA relative to Copaxone®.
  • the effect of Copaxone® was to increase the level of expression relative to mannitol control, whereas GLATOPA increased the expression level less than did Copaxone®.
  • GLATOPA increased the expression level less than did Copaxone®.
  • the THP-1 human monocyte cell line primarily comprised of Antigen Presenting Cells capable of interacting with T and B cells loaded with (auto)anti ens (Chanput et al, 2014), was used for in vitro experiments to characterize unbiased genome-wide expression profiles of treatment, with GL ATOPA and .Copaxone®.
  • the robustness of this model, system to diaracTerizafiou of Copaxone® mode of action and u orted generic differentiation has been studied ' extensively, including validation in . primary human monocytes (Kolitz ei al, 2015). In order: to ensure robustness and. repeatability of the herein reported results, two independent experiments were earned out in. two independent labora tories, one in Israel and one in Hungary.
  • IL7R which was also seen to differ between Copaxone® and GLATOPA in the splenoeyte study, was also expressed more highly with GLATOPA treatment relative to Copaxone® treatment in both of the THP-1 studies, for multiple IL7R probesets. As shown in Figure 14, the same was true for MMP9, for the single probeset present on the chip.
  • a targeted Gene Set Enrichment Analysis was also performed to assess emichment of the cytokine-cytokine receptor interaction pathway observed to be enriched in the splenoeyte studies in the comparison between GLATOPA and Copaxone® in each study separately. This pathway was significantly enriched (p ⁇ 0.001) inborn studies. The significantiy modulated probesets participating in the leading edge of this pathway from each study are illustrated in Figure 16. Both the adaptive immune response athwa s and the cytokme-cyto iie receptor interaction pathway as well as genes. CD40, CCL5, TL 1 OA, TNFSF 13B and MMP9 were enriched in comparing; Copaxone's. 1 with other purported generics, nicluding : POLI U OL and PR.OBIOGLAT (Kolitz et . al 2915; Hassoa ef a 2016) .
  • Multiple probesets differed between Copaxone® and GLATOPA for many of the differentially expressed genes. This means mat in cases where they mieroarray chip has two different "detectors" for a given gene, both detectors (i.e. probesets) frequently identified the same genes as differing between Copaxone® and GLATOPA - an observation that further reduces the likelihood of any spurious signals.
  • Pathway enrichment The probesets that differed between Copaxone® and GLATOPA were significantly enriched for biologically relevant pathways. In the unlikely event that the hundreds of genes passing the strict p-value threshold above were simply a random set of genes (i.e. noise), they would be scattered across the genome. Instead, the differences fell non-randonily into specific pathways with a strict threshold of adj. p ⁇ 0.05 statistical significance applied also to the pathway enrichments. The results consistently indicated differences in expression of immunological genes and pathways between Copaxone® and GLATOPA treatment. The consistent pathways and directionality both support the robustness of the observed differences.
  • a mouse splenocyte model and a human monocytic cell line (THP-1) were used to model each of these interdependent cell types in validated model systems.
  • An unbiased genome-wide approach was used to assess effects of Copaxone® and GLATOPA.
  • fn splenocytes, both gk iramoids modulated thousands of genes relative to controls, including genes known to be associated with Copaxone® 's mechanism of action, and demonstrated in prior publications, providing validation of the model systems.
  • Copaxone® and GLATOPA differed significantly in then effect on hundreds of genes.
  • 7% of genes modulated by Copaxone® were differentially modulated by GLATOPA re!ative to Copaxone®, and specifically multiple immunologically-relevant gene and pathway differences were observed (Table 7 and Table 8, Figures 1 and 2).
  • mice splenocyte system and human monocyte (THP-1) cell liae were utilized. Unbiased genome-wide gene expression studies were conducted to examine effects of treatment with Copaxone-® or POLIMLI OL in each model system.
  • Comparison for immunization labeled "corresponding to treatment” refers to treatment and immunization with POLIMUNOL versus treatment and immunization with Copaxone®.
  • Adj p adjusted p value, collected for multiple hypothesis testing using Benjamin! Hochberg.
  • probesets were upreguiated by Copaxone® relative to niarsnitol (in Copaxone® immunized mice). These probesets enriched for 76 pathways, many of which are immunologically relevant, and include relevant aspects of Co a one's MoA such' as .the eyiokine-eytofcme receptor interaction pathway identified also. in the ⁇ -l data described, below.
  • Copaxone® treatment iipregulated key anti-inflammatory cytokine genes 114, as well as markers of regulatory T cells, Foxp3. Complementarity, Copaxone® downregiilated proinflammatory cytokine genes such as 1112a ( Figure 21).
  • Gefte expression profiles from splenocytes of Copa one#-immoiiized mice treated ex vivo with Co a one® were compared with gene expression profiles ironi splenocytes of POLIMUNOL- HBffiUuized mic treated ex vivo Willi POLJMUNOL.
  • probesets were difiereHtiaiiy expressed after imposing a fold change filter of jFCj > 1.2 and correction lor multiple .hypothesis ' testing (FDR p value b 0.05); 206 of those were upregmated and 30 were dowiiregnlared in fee POLIM.U GL- iiiamiiiized mice ⁇ treated e vivo with POIJ O OL relative to Copaxone#-in3 ⁇ 4nioiized mice treated ex vivo with Copaxone®.
  • Table 12 below shows top probesets significantly downregukied by POLIMUNOL-inammized mice treated with POLIMUNOL relative to Copaxone ⁇ -immumzed mice treated with CopaxonetXTab!e 12. Top probesets significantly dowmegalated by POLIMLTNOL-imnmiiized mice treated with POLIMUNOL relative to Copaxoiie®-raim «iiized mice treated with Copaxone®.
  • Table 13 below shows top probesets significantly upregulated by POLIMIjNOL-ii «maiiized mice treated with POLIM0 OL relative to CopaxoasiMnimunieed miee treated with Copaxone®,
  • Kegg and GO Biological Process pathways significantly enriched among probesets more highly expressed (adj p ⁇ 0.05, FC>1.4) by POLIMUNOL relative to Copaxone® included immunological pathways such as immune response (adj p ⁇ 6.8e-9), response to virus (adj p ⁇ 2.3e-8) come defense response (adj p ⁇ 0.017), cytokine activity (adj p ⁇ 0.03), RIG-I-like receptor signaling pathway (adj p ⁇ 9.2e-5), and cytokine-cytokine receptor interaction (adj p 0.044).
  • immunological pathways such as immune response (adj p ⁇ 6.8e-9), response to virus (adj p ⁇ 2.3e-8) come defense response (adj p ⁇ 0.017), cytokine activity (adj p ⁇ 0.03), RIG-I-like receptor signaling pathway (adj p ⁇ 9.2e-5), and cyto
  • probesets in the "response to virus" pathway differing in expression (FC>1.4) between POLIMUNQL and Copaxone 1 !' include MxL Img t Rsad2, Eif2ak2, Ifihl, 7, Oasla, Gni9706 flsglS).
  • FC>1.4 Cost to virus
  • Table 14 Significant pathway earkiii «i*iif among probesets more highly expressed with POLI IINQL relative to Co axone®,
  • Table 15 below shows a list of pathways enriched among probesets significantly modulated by POLIMU OL relative to Copaxone®.
  • Table 15 List of pathways enriched among probesets stgBiflcautly niodtiiated by P LEVftJ OL relative to Copaxone®.

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Abstract

La présente invention concerne un procédé de caractérisation d'une substance médicamenteuse ou d'un produit médicamenteux associés à l'acétate de glatiramère comprenant les étapes consistant à : (a) obtenir un lot de la substance médicamenteuse ou du produit médicamenteux associés à l'acétate de glatiramère ; (b) mettre en contact des cellules de mammifères avec une quantité de la substance médicamenteuse ou du produit médicamenteux associés à l'acétate de glatiramère de l'étape (a) ; et (c) (i) déterminer le niveau d'expression d'au moins un gène parmi Mmp 12, TTgfbi Clec4n, Clec4a3, Fcer1g, Fcgr2b, Fcgr3, Csf3r, Il1r2 et Tbx21 ; (ii) déterminer le niveau d'expression d'au moins un gène parmi Ahcyl2. Cln5, Cxcr6. Dhrs3, Egr2, Fabp5. Ptp4al, Gpr183, Lamp1, Ncoa4, Ptger4, Qk, Rragd, Sec24a, Seh1l, Sestd1, Slc30a4, Soat1 et Strip2 ; ou (iii) déterminer le niveau d'expression d'au moins un gène parmi Fam212a, Gzma, Itpr1 et Zbtb32, ce qui permet de caractériser la substance médicamenteuse ou le produit médicamenteux associés à l'acétate de glatiramère de l'étape (a).
PCT/US2017/051534 2016-09-14 2017-09-14 Caractérisation d'expression génique d'un produit médicamenteux associé à l'acétate de glatiramère dans des cellules humaines et de mammifères Ceased WO2018053109A1 (fr)

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CN111372449A (zh) * 2018-09-27 2020-07-03 公益财团法人实验动物中央研究所 免疫缺陷小鼠

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