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    US2011263831A1 - Nik 2

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    US2011263831A1 - Nik 2

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    US2011263831A1- nik 2

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    9 views35 pages

    US2011263831A1 - Nik 2

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    US2011263831A1- nik 2

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    1102638; (NON A US 201 S1AL «) United States «2 Patent Application Publication (10) Pub. No.: US 2011/0263831 Al KAPLOW et al. (4) Pub. Date: Oct. 27, 2011 (S4)_ NUCLEAR FACTOR KAPPAB INDUCING G0) Foreign Application Priority Data FACTOR Ju, 26,2000 (GB) 00183079 (75) Inventors: June KAPLOW, Doylestown, PA (US); Thomas HAWS, Wilaingtoa, DE (US); Marie ROSIER, Antony (51) Int.Cl Publication Classification (FR); Patrice DENEFL CO7K 1628 (2006.01), Maur (FR) CO7K 1708 (2006.01), (2) US.CL 53013879; 530/350 (73) Assignee: AVENTIS - PHARMACEUTICALS INC, on ABSTRACT Bridgewater, NJ (US) The present invention is directed to nuclear factor KB (NFe8)-nducing factor polypeptides (NFIF polypeptides) (1) AppLNos — rans7aIs ‘which ar capable of indoing NFxB. The presen imenton inches withnis cope FTF polypeptides, incloing NFTF- (22) Fite Jun, 10,2011 I and NFIF-Ta, DNA, ineliding EDNA, encoding these polypeptides and expression vectors capable of expresin Related US. Application Data NEF polypeptides, Also include! are metbods sn eompo- 7 sions for increasing NFxB indoetion ia a patent, methods (60). Division of application No, 17388560, led on Feb. ap compositions fr lering NFB indton ina pale, 15, 2006, now Pat. No. 7.972.844, which is a continu ppethods for inhibiting inflammation, and methods for manu- ation of application No. 09/823,119, filed on Mar. 30, facture of a medicament intended for the treatment and/or 2001, now Pat. No. 7,081 49 prevention of an NFxB-regulated inflammatory response. In Eckition, melbods for detemining wheter atest compound (60). Pvisiona application No, 6/198, 905, filed on Mar. inhibits or enhances the activity of NFIF polypeptides are 31, 2000, provided. Patent Application Publication Oct. 27, 2011 Sheet 1 of 9 Plasmid: NEIFL4B Amino Acids: 453 MALVRALVCCLLTAWHCRSG LGLPVAPAGGRNPPPAIGOP WHVTDLHLDPTYAITDDATK VCASSKGANASNPGPEGDVL CDSPYQLILSAFDFIKI EASEMIWTGDSPPAVPVPEL STDIVINVITNY TTIQSLE PNLQVFPALGNHDYWPQDQL SVVISKVYNAVANLWKPHLD EEAISTLRKGGFYSQKVTIN PNLRIISLNTNLYYGPNIME LNKTDPANQFEWLESTLNNS IKEKVYT [AHVPVGYLPS: GON SQNITAMREYYNEXLI .GOFYGHTHRDSIM VLSDKKGSPVNSLEVAPAVE PYKSVLEXQTNNEGIRLFOY DPRDYKLLDMLQYYLNLTEA NUKGESIWKLEYILTOTYDI EDLQPESLYGLAXQFTILDS KQFIRYYNYFFVSYDSSVIC DKTCKAFQICAIMNLDNISY al CLAQLYTKHNY 100 400 420 440 465 US 2011/0263831 AI FIG. 1 Patent Application Publication Plasmid: NE Amino Acids: 364 MALVRALVCCLLTAWHCRSG LGLPVAPAGGRNPPPAIGOF WHVTDLEL PTYHITDDHTK VCASSKGANASNPGPFGDVL CDSPYQLILSAFDFIKNSGO EASFMIWIGDSPPHVPVPEL STDIVINVITNMTTTIQSLF PNLOQVFPALGNHDYWPQVYI TAHVPVGYLPSSQNITAMRE YYNEKLIDIFOKYSDVIAGQ FYGHTHRDSIMVLSDKKGSP VNSLFV2 .VIPVKSVLEKQ TNNPGIRLFOQYDPRDYKLLD MLOYYLNLTEANLKGESIWK LEYILTQTYDIEDLQPESLY GLAKQFTILDSKQFIKYYNY FFVSYDSSVICDKTCKAFOI CRIMNLDNISYADCLKQLYI KHNY Oct. 27,2011 Sheet 2 of 9 US 2011/0263831 AI FIG. 2 Patent Application Publication Oct. 27, 2011 Sheet 3 of 9 US 2011/0263831 AI NPIPLAB COGCTCCAGCCRCGSECTSCOCCTCECCOCCECASCCCGCAGGAATCET \GGCGATAGGACAGTNITGGCAY GGCGRTAGGACAGETTTOGCATSRCR C1 SCCCTTTIGGAG! coc "TAGATGARGRAGCTA, NFIELIB NFIFIA BAAAGPRCAGIG 534 851 AGAGACAGCATTAS NFIFL4B 584 AIGICATIGCASGACA n c BFIFIA 301, e 634 Patent Application Publication CIGGAAGOTGEACTATATCCTS: ACAIGI CACARTTACTAG CACARTIACTAG SUATCRGACTGYTTCAGTATEATCCTCY ABAGPTEATREGEA AAGCAGTTTATAAART SAGCAGTTTATAAARTACTACAATIAC?? ‘TOTARCATGIGATAY Oct. 27,2011 Sheet 4 of 9 CEGGAACCTGGAGTATANCOTGACCEAG: RCARETAC FIG. 3 (CONT'D) TGTGRGTTATGRCAGCAG NF IGIGAGTTATGACAGCAG M1 US 2011/0263831 AI CCAGTCAAGRGTS! RC \GIGAAGAGTGTTTTAGRAAAACAGACCAACAATCCTG "TICAGEATOATCCTCGION MADTGGATARG COIGAPERTRAATPATNGGRTATS STATTACTTGRATCTGACAGAGGCGAATCTAAAGGGAGAGICCAT NFIFL4B GARICT SARTCTAAAGGGRGAGTCCAT | NFIEVA PCCTSI NFIPLAB NPIETA AGOCGGMAGTPIAEATOGADTAGCTAAKCAATTTACARTOCTAGACAGT -NELEL4B. A PPACUTARACAATTUACARICOTRGECAGD — NPIFT 3 IETS |AGRCRTGTAAGGC! 'GIGCARTIANGA NEIFL¢B “AAGACATGTAAGGCOTTICAGATTIGNGCAATIATGA NPIPTA VPOPIGATAATATETCCEATCCAGATIGCCTCAAACAGCTTTATATARAG —NEYET43 “YCCEATGCAGATISC AGCIPIANATARAG NET NFIPLGB Patent Application Publication Oct. 27, 2011 Sheet 5 of 9 US 2011/0263831 A1 800 S00 1000 1100 1200 1300 740 200 300 «4400 «500 600 700 472 FIG. 4 100 14b NFIF NFIF 7a Oct. 27, 2011 Sheet 6 of 9 US 2011/0263831 A1 Patent Application Publication NFeB Reporter with NFIF 962 #1 ALIN si-Dhw—U Le yNgod SUE Pb ALIN SIH~2Ku—u Ye yNaod ¥L LIN siH-odu—u yg yNGod -96€ VE ALIN SIH-okw-U Le yNaod 1g VZ SIN SIH-2hw~u YE yNed Ve JIN SH~>4u-U Le yNgod sH-okw—w VE yNODd g 8 g BEE SEEe EE” 1 Se FIG. 5 Oct. 27, 2011 Sheet 7 of 9 US 2011/0263831 A1 Patent Application Publication NFeB Reporter with NFIF 22000 96E Ph SIN Sih Le WNGOd SIS PL alaN SiH-Ohw—U PE WNGOd Yh ALIN siH-ah—u 1g yNged 968 YE ALIN StH-OAW-U YE YNGEd LE YL ALN SIH-2Kw—U be yNCEd I si4~ofwv Ye yNCOd 88ssse8882828° BES28 88 898 seat FIG. 6 Patent Application Publication Oct. 27, 2011 Sheet 8 of 9 US 2011/0263831 A1 SKGANASNPGPFGDV FIG. 7 Patent Application Publication Oct. 27, 2011 Sheet 9 of 9 US 2011/0263831 Al 2.0K> yueepid ‘SauQSajuy jjows an Aaupy, FIG. 8 uegds snutig ‘wope ‘apsnuy joyajays yoay 41039 US 2011/0263831 Al NUCLEAR FACTOR KAPPAB INDUCING FACTOR, FIELD OF THE INVENTION {0001} Nuclear fictor xB (NPB) comprises a family of cukaryoie transcription facies involved in regulation of genes involved in immune espoases and othe cellar fane- tions, In some instances, NERB atvation lead oan name ratory response which ultimately result in acsase stato “Accoringly, it would be desirable to develop means for controling induction of NFB. The preset invention pro- ‘les polypeptides which are involved in the induction of [NEB and which may he wsed to eahance NFB expression ‘or ativaton oo identi ail prepare inhibitors of NFKB. expression or setiaton REPORTED DEVELOPMENTS. {0002]_ Nucl fictor KB (NFB) comprises family of teanserpton factors found in almost all cokaroti coll NF plas roles te regulationaf genes ivolvedin sue inlammation,celllr proliferation, and cellular diferent tion [0003] A umberof studies ave examined the ratinship between disease states and expression of the subunit eoeias ‘which comprise NFxB or activation of NFXB already present ina cll. Li etal, studied the regulation of NFxB by the LITLVG1 Tx poten demonstrating thatthe ability of Tax to activate the NPxB pathway plays an essential role in HTL = ‘induced cellular transformation (Li eta, Gene Bsr, 7, 45, 233-245 (1999), Lensch and Wart sted the ative tion and ezolaton of NFxB during acute inflammation and cescribed the relationship between NFB and the inhibiting proteins ofthe [Kappa family Centsch et Cin, Chem: ‘Lab Med, 3,3, 208-208 (1999) Visconti eta. investigated the role of NFB in thyroid carcinogenesis by analyzing tym carcinoma celine. Theistadies indicated that ate ‘ation ofthe NFB complex by overexpression ofthe p6S protein played exited role inthe process of thyroid cell ‘eansfomation (Visconti eal, Oncogene, 15,16, 1987-1994 (1997) Makbopadhyay etal investigated the enression of to p50 subunit of NFB tanscripion factor complex in onal cell hing carcinoma tissues demonstrating that $81%offesh non-small cll ang cancer tisues expres rom two to twentysfold higher levels of the pS0 subunit than sonal lng sve (Mukhopadiyey’et al, Oncogene, 1,5, 9991003195), Khaled etl. targeted p50 gene expression ‘with specific antisense 3” phosphorothicate modified ant cose oligadconynueleotides an were abe to reduce NPD expresion, Their rests demonstrated p50 antisense mol- ecules couk! redce NFRB expression and coud downre- late the immune response providing possible testmeat lor autoimmune disorders (Khaled et a, Clin. nmunol fa ropathol, 83,3, 284-263 (1997). SUMMARY OF THE INVENTION [0004] In accordance with the present invention, thee are provided mucleic aid sequences encoding nuclear factor KB. inducing factor (NFIF) 1db and 7a. Included also areeDNAS encoding NFIP-14b and NFIF-Ta and isolated and purified [NFIF-14b and NFIF-7a polypeptides which induce NFKB. ‘The present invention includes also methods of inducing [NFB comprising introducing into the body of a patient 3 composition that sctvates NFB induetoa, including for Oct. 27, 2011 example, expression vectors that express NFIF-I4b and [NFIF-Ta polypeptides, Examples of preferred expression vectors are retroviral vetors, adenoviral vectors, ademas ovate viral vetos, herpes, herpesviral vectors and naked DNA vectors. The present invention also provides methods ‘wherein the composition ofthe present invention comprise an [NFIF-I4b or NFIF-Ta polypeptige and a pharmoceuticaly acceptable carr [0003] _ Another aspect ofthe present invention are compo sitions fr lowering the expression ofthe NFIF gene compeis- ing an antisense nucleic aid. Sill nother aspect of the ‘resent invention sa composition fr lowering the activity of an NFIF polypeptide comprising ncutalizing antibody that binds to an NFIF polypeptide and lowers its activity {0006} Yet nother embodiment ofthe present invention is «composition for lowering the expression of NFIF in a patent comprising a ribozyme that cus RNA encoding an [NFIF polypeptide. {0007} ‘The present invention provides methods fr evalne ‘ting whether a est compound is effective in inhibiting the ativity of NEIF-14bpolypetidescomprsing(A) comparing te evel of NFxB-reguated gene expression ina fist sample comprising: (1) NFIF-14b; (2) the NFxD-egulated reporter sgene:and (3) hetestcompound withthe evel of gene expres sina second sample comprising (4) NFIF-4b:and (5) he [NFB regulated reporter gene and (B) determining whether the expression of the reporter genes lower inthe fis sample relative tothe second sample [0008] Sill another aspect ofthe present invention provides ‘methods for evaluating Whether atest componnd is effective ‘ninhbiting the wetivity of NFIF-Ta polypeptides comprising (4) comparing the level of NFxB-repulte gene expression ina fist sample comprising: (1) NFIF-7a; 2) the NFxB- regulated reporter gene; and (3) the test compound with the level of gene expression ina second sample comprising (4) [NFIF-7a; and (S) the NFxB regulated reporter gene; and (B)

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