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(12) United States Patent (10) Patent No.: US 7.282,360 B2 Meyers Et Al

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(12) United States Patent (10) Patent No.: US 7.282,360 B2 Meyers Et Al USOO7282,360B2 (12) United States Patent (10) Patent No.: US 7.282,360 B2 Meyers et al. (45) Date of Patent: Oct. 16, 2007 (54) HUMAN PROTEIN KINASE, PHOSPHATASE, provisional application No. 60/197.508, filed on Apr. AND PROTEASE FAMILY MEMBERS AND 18, 2000, provisional application No. 60/187,454, USES THEREOF filed on Mar. 7, 2000, provisional application No. 60/187.420, filed on Mar. 7, 2000, provisional appli (75) Inventors: Rachel E. Meyers, Newton, MA (US); cation No. 60/186,061, filed on Feb. 29, 2000. Peter J. Olandt, Newton, MA (US); Rosana Kapeller-Libermann, Chestnut (51) Int. Cl. Hill, MA (US); Rory A. J. Curtis, CI2N 9/20 (2006.01) Framingham, MA (US); Mark CI2N IS/00 (2006.01) Williamson, Saugus, MA (US); Nadine CI2N L/20 (2006.01) Weich, Brookline, MA (US) CI2O I/68 (2006.01) C7H 2L/04 (2006.01) (73) Assignee: Millennium Pharmaceuticals, Inc., (52) U.S. Cl. ................ 435/194; 435/320.1; 435/252.3: Cambridge, MA (US) 435/6:536/23.2 (58) Field of Classification Search ................ 435/194, *) Notice: Subject to anyy disclaimer, the term of this 435/6, 252.3, 320.1; 536/23.2 patent is extended or adjusted under 35 See application file for complete search history. U.S.C. 154(b) by 0 days. (56) References Cited (21) Appl. No.: 11/636,948 U.S. PATENT DOCUMENTS (22) Filed: Dec. 11, 2006 6,476,210 B2 * 1 1/2002 Turner et al. .............. 536,232 2004.0034.888 A1 2/2004 Liu et al. (65) Prior Publication Data US 2007/OO99274 A1 May 3, 2007 FOREIGN PATENT DOCUMENTS WO WOOOf73469 A2 12/2000 Related U.S. Application Data (60) Division of application No. 11/151,601, filed on Jun. * cited by examiner 13, 2005, now Pat. No. 7,198.930, which is a division Primary Examiner Maryam Monshipouri of application No. 10/170,789, filed on Jun. 13, 2002, now Pat. No. 7,070.947, and a continuation-in-part of (57) ABSTRACT application No. 10/045,367, filed on Nov. 7, 2001, now abandoned, and a continuation-in-part of appli The invention provides isolated nucleic acids molecules, cation No. 09/961,721, filed on Sep. 24, 2001, now designated 53070, 15985, 26583, 21953, m32404, 14089, abandoned, and a continuation-in-part of application and 23436 nucleic acid molecules, which encode novel No. 09/934.406, filed on Aug. 21, 2001, now aban human protein kinase family members, serine/threonine doned, and a continuation-in-part of application No. protein kinase family members, serine/threonine phos 09/882,166, filed on Jun. 15, 2001, now abandoned, phatase family members, prolyl oligopeptidase family mem and a continuation-in-part of application No. 09/861, bers, trypsin family members, trypsin serine protease family 801, filed on May 21, 2001, now abandoned, and a members, and ubiquitin protease family members. The continuation-in-part of application No. 09/829,671, invention also provides antisense nucleic acid molecules, filed on Apr. 10, 2001, now abandoned, and a con recombinant expression vectors containing 53070, 15985, tinuation-in-part of application No. 09/801.267, filed 26583, 21953, m32404, 14089, or 23436 nucleic acid mol on Mar. 6, 2001, now abandoned, and a continuation ecules, host cells into which the expression vectors have in-part of application No. 09/801.275, filed on Mar. 6, been introduced, and nonhuman transgenic animals in which 2001, now abandoned, which is a continuation-in-part a 53070, 15985, 26583, 21953, m32404, 14089, or 23436 of application No. 09/797,039, filed on Feb. 28, 2001, gene has been introduced or disrupted. The invention still now Pat. No. 6,730,491. further provides isolated 53070, 15985, 26583, 21953, (60) Provisional application No. 60/246,561, filed on Nov. m32404, 14089, or 23436 proteins, fusion proteins, anti 7, 2000, provisional application No. 60/235,023, filed genic peptides and anti-53070, 15985, 26583, 21953, on Sep. 25, 2000, provisional application No. 60/226, m32404, 14089, or 23436 antibodies. Diagnostic methods 740, filed on Aug. 21, 2000, provisional application utilizing compositions of the invention are also provided. No. 60/212,078, filed on Jun. 15, 2000, provisional application No. 60/205,508, filed on May 19, 2000, 15 Claims, 35 Drawing Sheets U.S. Patent US 7.282,360 B2 #ºwºº..),\ 3___________ ^^^^^^^^^A, Z98.18Z10€.|Z|??7 FIGURE 1 U.S. Patent Oct. 16, 2007 Sheet 4 of 35 US 7.282,360 B2 |,'! FIGURE 4 U.S. Patent Oct. 16, 2007 Sheet 9 Of 35 US 7.282,360 B2 PROTEINBOSPAASE pROTEN prospas 2C 4 DOMAIN 2C DOMAN 4, 8, 12 16l 20, 24l 28, 32 36l 4O 44 48 52 FIGURE 9 U.S. Patent Oct. 16, 2007 Sheet 12 Of 35 US 7.282,360 B2 SSS COLONMETSSo COLONTSs. ECOLONT Sea SECOLONT 32S ECOLONT Son ECOLONT SSN ECOLONT SS COONT &so EcotoNN &es Scoton N. Sssa EUNGT SSS Esu GT SSS SS UNGT SSS EUNG N SS EUNGN also S. REAST T s SEESS. B RE AST N BREASTN s 8 & Š S. s 3 & O C FIGURE 1. E. U.S. Patent Oct. 16, 2007 Sheet 13 Of 35 US 7.282,360 B2 3. s FIGURE 12 U.S. Patent Oct. 16, 2007 Sheet 17 of 35 US 7.282,360 B2 A| "Aliyah'Nwa f i is WMNAN , , R. A. Mr. An Cys I Ngly 1. 80 160 240 320 400 480 552. FIGURE 15 U.S. Patent Oct. 16, 2007 Sheet 22 of 35 US 7.282,360 B2 Trypsin Domain 1 40 8O 120 160 200 24 FIGURE 8 U.S. Patent US 7.282,360 B2 TVN?ISHOSHINDRIAGINTHASASV?LOHà//(8)LLOW(8)LAHL(II)d?RIL(68%)z66d9*< RIOJLOVHGHNH0IJLTÍTWXTIWVHNICHLOHJOOXTONGIÐOW?Z(HSVTORIOLAH 99z=q??šuºT FIGURE 20A U.S. Patent Oct. 16, 2007 Sheet 29 of 35 US 7.282,360 B2 VPA48HRCONTP4 VPA48HRCONTP42 VPA50 g/ml. 48HRLP141 VPA50pg/ml 48HRLP142 VPA100ug/ml 48HRLP14 VPA 100 g/ml. 48HRLP42 VPA d6 CONTP26 WPA dOCONTP26 VPA 50 g/ml d6 LP126 29.8 SSS VPA 100 g/ml d6 LP126 20. VPA 50 g/mldOuP126 25.2255 VPA 100 g/ml dOP126 7.6 SVPAd6 CONTLP130 VPAdOCONTLP130 30.2&SVPA50g/mld6 LP130 VPA50 g/ml dOP30 l545iS VPA00 g/ml dolp130 VPAdOCONTLP134 445 SSSSSSSSS VPA 50 g/ml d6 LP134 2.33i:3 VPA100 g/ml d6 LP134 VPA 50 g/mldOP134 VPA100 g/ml dOLP134 3. TGFBLP15548 HR CONT 60.8 SSSSSSSSS 65.25: GRBCONT 48HRLP159 GB50g/mLP159 GFB100 g/ml LP159 C O O C O O C C C C g g s S S S S S 9 o RELATIVE EXPRESSION FIGURE 23 U.S. Patent Oct. 16, 2007 Sheet 30 Of 35 US 7.282,360 B2 TGFB100 g/ml LP159 GFB50g/ml LP159 GFBCONT48HRLP159 TGFB50 g/ml LP55 TGFB50g/ml LP55 CONT BRU d7-3EPOLPOA BRUd7-3EPOP8 BFU d7 LP79 VPA100 g/ml dOP134 VPA100 g/ml dOP130 VPA100 g/mldO LP126 VPA 50g/mtdIOLPI34 VPA 50 g/ml d10 LP126 VPA100 g/m d6P134 VPA100 g/ml d6 LP30 MPA100 g/ml d6 LP126 VPA 50 g/mld6 LP30 MPA 50 g/mld6P126 VPA100 g/ml 48HRLP42 VPAlOOug/ml. 48HRP4 VPA 50 g/ml 48HRP42 iszVPA50 g/ml48HRLP41 SSVPAdOCONTLP3A SVPA dOCONTLP130 VPAdOCONLP126 PAEES NEUTROPH d4 LP3-6 NEUROPH dALF78 NEUROPH dAF/ NEUROP-d3LF78 NEUROPHL d2 LP26B EUROPHL di LF78 NEUROPHL d8 LF78 NEUTROPHL d7 PA-3 NEUROPHL d6 P78 NEUTROPHL déP7 EUTROPHILod6 LP27 NEUTROPHILd6 LF26 NEUTROPHL d4 F78 NEUROPHL d4 F7 3 8 woO C C O RELAIME EXPRESSION (NTCUSED AS REFERENCESAMPLE FIGURE 24 U.S. Patent Oct. 16, 2007 Sheet 31 of 35 US 7,282,360 B2 HEP3bHMP 254.2074393 EP3bNORM270523795 MOA 23690248 NC 40 40 NK562 2382A3789 NBMGPA 274926.340 NERYTH 23.204276 scord BLOOD 293723.97237 s Y PBMCREST 260,759 29 CD4 25,486,267 hoo466h 2568.758 sufnDRO79 27032266 s LMERNDR1928.52500 MERCHT33934,99230303 PASSEL 2922447 Easis NHDFTGF 27722.89356 NtAMER 24.7283277 UNG 27.288.259 3 & 8 5 & 5 . RELATIVE EXPRESSION (NTCUSED AS REFERENCE SAMPLE) FIGURE 25 U.S. Patent Oct. 16, 2007 Sheet 32 of 35 US 7,282,360 B2 MEGd4P3-5 2549042 MEGd2F35 24209.68 MEGd2F02 272985 6 MEGd7P32 2589753 MEG48RF790 270323,335 MEG48HRF76 29842476296 Eyd4GPAP3-4 12624,949.93 Eyd2F240 239357 Eydt2F23-8 2.84943974 sEydop25-4 2443982409 3:55 Eyd P24-5 2409.20.8665 EydéP3 24872O4346 Ey48HRF76 26679.8589 mBMCD5CDbP52126958.2324 mBMCD5P5 270682222 BMGPAoP82 25842082308. CORDBOODCD34+P228.620527 BMCD3438 25.2878960 mPBCD34+P52 2524.84389 nPBCD34+F70 2906205328 mBMNCLP139 2523.72840 SKMUSCLEMP67 29.827.458 tAMERMP425 255223.0756 MERNDR200 32320508 KDNEYMP58 2593.99660 SCOLONMP60 UNG 2683778. s & S S & RELATIVE EXPRESSION (NTC USED AS REFERENCE SAMPLE FIGURE 26 U.S. Patent Oct. 16, 2007 Sheet 33 Of 35 US 7.282,360 B2 SeSYS-Ysary 5 sassassraisasaksstrasaesarsayersssssstrasasters WSX assassassraesareer Sess Sassists SSSSSSSSSSSSSSafeSSSSSSSassasssssssssssss See SafeS.5se 3 S S 3 3 S 3 iss s ast SS FIGURE 27 U.S. Patent Oct. 16, 2007 Sheet 34 of 35 US 7.282,360 B2 g CN S.SES. sists SšSSéSSSSSSS EHepG2-B sists c C 323E3&g 2.5- Cneur sts SSSSSSSS S W SSSSsea HepG2-A MEEisigles SMAoO RSS E. at 90 f - WO au o un o un d d S. co cy. CN N - F O c RELATIVE EXPRESSION (HepC+519 USED AS REFERENCE SAMPLE) FIGURE 28 U.S. Patent Oct. 16, 2007 Sheet 35 of 35 US 7,282,360 B2 BFUd73Epo 263820239 BRUd3Epo 2642,252 BRUd779 27.262473 NEyd4GRAP34 28239.6726 NByd2F3 2706268240 NEyd2F24-O 2465943.274 NEyd2F238 23057852.72 NByd8F3 2604243409 NEydt P24-5 24.91927202 NEyd3LP3NByd6F3.
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