WO 2013/111897 A 1
Contents
1.2. RhoGAP RhoGAP RhoGAP ARHGAP1 1A 2 A D A B
3. s G2M HT MM M2 3 G1 sjG2 M HO RhoeTP RhoGAP ARHeAP11A S G2 M G1 10 ARHGAP 11A GenBank Access ion No NM 014783 3 1 GenBank Access ion No NM 199357 1 2 mRNA ARHeAP 11A Ge nBank Access ion No NMO 14783 3
4. ARHGAP11A mRNA MAT VRR RhoGAP 1 EFI 1 R2 Fucci WO 2013 111897 9 PCT JP2013 051733 FACS 2 3INOD SCID
5. RhnoGAP UBAHE RR RRACE T 2 3 4 ulcc mm ARHGAP11A RhoGAP T 2 3 4 WO 2013 111897 16 PCT JP2013 051733 0028 0029 0030 RhoGAP
6. HER2 h EGFR cML Bcr Ab L B CD20 AML CD33 EGFR VEGF nd Bortezom ib
7. S G2 M ct S G2 M G1 ARHGAP1 1A S G2 M shRNA RNA ARHGAP1 1A NOD SCID WO 2013 111897 11 PCT JP2013 051733 00 15 00 16 00 17 ARHeA1 1A siRNA RNA in vivo
8. RhoGAPp RnoGAp ARHeAP11A WO 2013 111897 29 PCT JP2013 051733 RhoGAP ARHGAP1 1A mRNA
9. PCT 1SA 2 10 1 2 2009 7 PCT JP2013 051733 1 3 RhoGTP PCT 5 ARHGAP11A RX k shRNA POCT 6 RhoGTP 1 3 PCT 6 RhoGTP
10. CHS AGS
11. Rho GAP HH BAW ER L BARU RhoGAP RnoGAP RhoGAP ARHeAP11A
12. ARHGAP1 1A R shRNA siRNA microRNA A RhoGAP ARHGAP1 1A B RhoGAP WO 2013 111897 17 PCT JP2013 051733 0031 0032 0033 0034 C A ROB RhoGAP KOE D A
13. 1 1 1 14 1 1 14 PCT 6 4 a 2 3 Sh 1 3
14. RhoGAP DNA DNA ARHGAP1 1A TXSsC 0 SDSJ 6 5X SSC 0 1 SDS D 1Xssc 0 1 SDSJ EF 37 42 55 65 RhoGTP Rho GTP ase act ivating prote in RhoGAP RhoGAP
15. LAF ALKRR ED WO 2013 111897 7 PCT JP2013 051733 1 ARHGAP 625 BNA 1 6 5 L T L 5 L aaatttgaa5 L T L 5 L c 1 0 2 ARHGAP 969 BNA 1 6 T L 5 L 5 L gaaaaagcc5 L T L T L c 1 3 3 ARHGAP 1 344 BNA 1 6 T L 5 L T L tttcatgtc5 L T L T L c 1 6 4 ARHGAP 1447 BNA 1 6 T L 5 L 5 L aggataaaaT L 5 L T L g 17 5 ARHGAP 1 748 BNA 1 6 5 L T L T L gatggactt5 L 5 L T L t 19 6 ARHGAP 1 931 BNA 1 6 T L T L T L gcctgcaatT L 5 L T L t 2 1 7 ARHGAP 2032 BNA 1 6 5 L 5 L T L agattgaatT L T L 5 L a 2 2 8 ARHGAPv1 3484 BNA 1 6 T L T L 5 L gagggtaacT L 5 L 5 L a 530 9 ARHGAPv2 221 5 BNA 1 6 5 L T L 5 L taacagtagT L A L T L g 3 4 1 0 ARHGAPv2 2285 BNA 1 6 T L 5 L T L agaacagtaA L A L T L t 3 5 1 1 ARHGAPv2 2306 BNA 1 6 T L T L 5 L aaacatgaa5 L T L T L t 3 6 1 2 ARHGAPv2 2355 BNA 1 6 T L 5 L 5 L caattgttgA L T L A L g 3 7 1 3 ARHGAPv2 2404 BNA 1 6 T L T L T L taacataagA L A L T L g 3 8 N L BNA 5 L mC
16. Geminin 99 non cancer Gy BRUM RS S G2 M 2 10 WO 2013 111897 3 rz 2 mRNA mS oF mAG mKO2 PCT JP2013 051733 a3 a 30 5 EE a a 2 10 w H w S 9 Be 0 Giff S G2 M mAG nmKO2 3 10 WO 2013 111897 PCT JP2013 051733 5 A B 6 ARHGP11A A A ant E2F1 antibody 1 5 1 0 0 5 0 0 ae oe gt Ka a 4 10 WO 2013 111897 PCT JP2013 051733 e17 A 4 SH ARHGAP11a relative mitotic ratio B 8 ARHGAP11A ened 18 eo n EEE sas a sjRNA 16 os b SRF ARBIY OIE ose c Y MO 34 ARHGAP11A siRNA in vivo siRNA A See _ 2 mm Co 8 two way ANOVA a B 0 000
17. RhoGAP ARHGAP1 1A RhoGAP ARHGAP1 1A PCR CTC Ci WO 2013 111897 18 PCT JP2013 051733 0035 0036 0037 0038 rcuLating tumor ceLL
18. 5 ARHGAP 11A BNA BNA 3 5 BNA HcTt1e ARHeAP 11A 4 3 8 s 5 BNA 3 9 4 1 LAF ARHGAP 11A GenBank Access ion No NM O14783 3 1 GenBank Access ion No NM 199357 1 2 mRNA 4 2 3 BNA 1 2 2 4 3 3 BNA 1
19. RNA ARHGAP1 1A SERE 2 ARHGAP1 1A ARHGAP1 1A 14 200 1 3 1 13 4 1 ARHGAP 625 BNA 1 6 5 L T L 5 L aaatttgaa5 L T L 5 L c E WES 10 2 ARHGAP 969 BNA 1 6 T L 5 L 5 L gaaaaagcc5 L T L T L c 13 3 ARHGAP 1 344 BNA 1 6 T L 5 L T L tttcatgtc5 L T L T L c 16 4 ARHGAP 1447 BNA 1 6 T L 5 L 5 L aggataaaaT L 5 L T L g 17 5
20. dsRNA ARHeAP 11A DNA BH AR HGAP11A ARHoG WO 2013 111897 14 PCT JP2013 051733 0022 0023 AP11A dsRNA 1e 49 1e so 19 21 dsRNA dgsRNA RhoGAP ARHGAP11A AR HGAP 1 1A shRNA siRNA microRNA
21. 1 RhoGTP RhoGAP 2 RnoGAP ARHeAP 11A 1 3 ARHGAP 11A ARHGAP 11A RNA ARHeAP 11aA 2 4 ARHGAP 11A ARHeAP 11A WO 2013 111897 14 200 PCT JP2013 051733 1 3
22. S G2 M ei EFFI 2 41S G2 M mAG ei mK02 mRNA U ARHeAP 11A 2 5 ARHGAP1 1A 3 6 ARHGAP11A 4 RI 7JARHGAP1 1A shRNA Shi hl 1 8 NOD SCID ARHGA1 1A shRNA wl 2 9 E mRNA ARHeAP 11A 3 10 ARHeAP11A n 38
23. Bul GeneNo te GeneCa rds ID GC15P032907 Th ARHGA WO 2013 111897 21 PCT JP2013 051733 0043 0044 0045 P11A ARHGAPI 1A 1 ARHeAP1 1A RhoGAP ARHoAP1 1A 1 2 2 shRNA SH nc T116 ARHeAP1 1A GHH SH 2 3
24. 1 AteLoGene 5 Koken cj 4 ARHeA P11A shRNA 8 3 ARHeAP 11A Lase r microdissec tion L mRNA TARGA 11A A RHGAP 11A ARHeAP 11A
25. 3 4 3 8 BNA 2 WO 2013 111897 24 PCT JP2013 051733 N L BNA 5 5 mC 5 1_ L mC BNA ARHGAP 47 BNA 16 ARHGAP 126 BNA 16 ARHGAP 203 BNA 16 ARHGAP 382 BNA 16 ARHGAP 406 BNA 16 ARHGAP 591 BNA 16 ARHGAP 625 BNA 16 ARHGAP 720 BNA 1 6 ARHGAP 843 BNA 1 6 ARHGAP 969 BNA 1 6 ARHGAP 1 006 BNA 1 4 ARHGAP 1 162 BNA 1 5 ARHGAP 1 344 BNA 1 ARHGAP 1447 BNA 1 7 ARHGAP 1 538 BNA 1 8 T L 5 L 5 L gcccccagcT L 5 L 5 L 6 6 6 6 5 L T L 5 L ccccatcag5 L 5 L T L g 5 L T L 5 L aggcaactcT L T L 5 L c T L 5 L T L attgccagaT L T L 5 L 5 L T L T L tctgattctT L T L 5 L g 5 L 5 L 5 L gtctggcacT L T L 5 L 5 L T L 5 L aaatttgaa5 L T L 5 L c T L 5 L T L gatcccacaT L T L 5 L c T L T L T L taccccctaT L T L T L c T L 5 L 5 L gaaaaagcc5 L T L T L c T L T L 5 L tttagtgctT L T L T L a T L T L T L tcctctgtg5 L 5 L T L a T L 5 L T L tttca
26. gsRNA DpDNA o dsRNA Rho GAP RhoGAP ARHeAP 11A shRNA siRN WO 2013 111897 15 PCT JP2013 051733 0024 0025 0026 0027 A microRNA RADE osRNA
27. ARHeAP11A ARHGAP11A 10 ARHeAP11A 1
28. ARHGAP11A MEGK amp 5 6 PCT 1SA 2 10 2009 7
29. ARHeAP 11A CED pNA RNA mRNA 1 4 2oo 1s so RNAi RNA interference RNA dsRNA RNA si RNA shRNA microRNA dsRNA ARHeAP 11A LE
30. MAAT IPA RhoGAP RhoGAP ARH GAP1 1A mRNA RhoGAP ARHeGAP1 1A WO 2013 111897 30 PCT JP2013 051733 1 2 3 4 5 RhoGTP RhoGTP AR HGAP1 1A 1 ARHGAP1 1A ARHGAP1 1A
31. R1 B44 3 Eo2F ARHeAP 11A EF ARHGAP 11A ch r15 3290769 1 20 28b ch r15 329076 63 3290767 1 EzFs ARHeAP 11A E2F 5A Ch IP ARHGAP 11A 5B WO 2013 111897 20 PCT JP2013 051733 0042 R 1 Rho GAPs Sene Fc AmHeAPiiA 1490 ar AReAPtiA 11 03 4 ARHGAP 11A ARH
32. 1 AFA BSH REFA F 1 ARHGAP1 5 ARHGAP1 1A ARHGAP1 1A RNA ARHGAP1 LE ARHGAP1 1A 1A ARHGAP1 1A 14 200 1 1 7 oF RTOS KA 1 8 1A
33. T 1 UCC TNM T 2 T 3 T 4 BQ WO 2013 111897 23 PCT JP2013 051733 0048 0049 0050 o 4 Lase r microdissec tion mRNA ARHeAP 11A ARHeAP 11A n 38 n 26 0 ARHeAP 11A B10 ARHeAP 11A 3 4
34. ARHGAP1 1A ARHeAP1 1A WO 2013 111897 13 PCT JP2013 051733 0020 002 1 ARHGAP 11A ARHeAP 11A RhoocApPp RNAi RNA interference RNA shRNA siRNA MicroRNA BH RAMS BBs DNA ME OTH IC PNA Pept ide Nuc Leic Acid BNA Bridged Nuc Leic Acid BNA eridged Nuc Leic Acid
35. IO IP SA JP 100 8915 4 3 PCT SA 2 10 2 of the cytoskeleton Nature an oncogenic 1 4 7 10 Reviews Cancer 2004 5 6 MP Ti ABHA KX ITY 1 I amp TERE EROS OME 358 1 34 5 2
36. ARHeAP1 1A B ARHGAP1 1A C A ROB ARHGAP1 1A D A B WO 2013 111897 T2 lt Al HF Nl N PCT JP2013 051733 RhoGAP RhoGAP ARHGAP1 3 RhoGAP RhoGAP fA 4 qT 1A 1
37. BNA ARHGAP 1748 BNA 16 1748 in vivo L HT1080 nr1080 Lu KRY DAICHT1080 Luc 5xi0eceLl Hr1 080Luc 14 gmgikg BN A10mgkg 7 15omgkg HT 1080 LUC B16 R F n 3 BNA n 3 17 BNA 1 8 BNA
38. S A e2 M Gi S ei resting state Go A G1 R LHS WO 2013 111897 2 PCT JP2013 051733 0005 0006
39. BNA 1 748 14 FITC 8 161 1 BNA 1748 EHA o 171 ABNA 1 748 9 181 BNA 1748 ie slg RhoGAP RhoGAP ARHGAP1 1A Rho GTPase act ivat ing prote in 11A
40. 1 2 A L RNA CCGGCGGTATCAGTTCACATCGAT CCGGCCTTCTATTACACCTCAAGA 1 6 1 WO 2013 111897 32 PCT JP2013 051733 8 9 10 11 12 13 14 RhoGTP RhoGTP ARHGAP1 1A 8 A D 9 A ARHGAP1 1A B ARHGAP1 1A C A
41. HcT1 16 ARHGAP1 1A GH 1 SH 2 DNA 6 2 BrdU S DNA BrdU Bp BioCoat WTA ARHeAP1 1A GH 1 SH 2 7 B ARHGAP1 1A 7A B ARHeAP1 1A ARHGAP1 1A
42. BNA TIL A WO 2013 111897 8 PCT JP2013 051733 001 1 001 2 2 0 ARHGAP1 1A RNA 14 15 17 1 4 ARHGAP1 1A 1 TRCN0000047281 CCGGCGGTATCAGTTCACATCGATACTCGAG TATCGATGTGAACTGATACCGTTTTTG 1 1 5 ARHGAP1 1A 2 TRCN0000047282 CCGGCCTTCTATTACACCTCAAGAACTCGAG TTCTTGAGGTGTAATAGAAGGTTTTTG 2 2 1 1 7 RhoGAP ARHeAP1 1A
43. RhoGA P RhoGAP RhoGAP RhoGAP RhoGAP RhoGAP RhoGAP RhoGAP mRNA RNN RhoGAP RhoGAP RNAi RNA interference RNA Eh RNA SiRNA MicroRNA RhoGAP ARHGAP1 1A ARHGAP1 1A ARH GAP1 1A ARHeAP1 1A ARHGAP1 1A
44. GAPDH 1 LE 10 13 16 17 19 2 1 2 2 30 34 35 3 8 BNA AR HGAP1 1A 1 1 6 BNA ARHGAP1 1A 5 ARHGAP1 1A BNA ARHGAP1 1A WO 2013 111897 27 PCT JP2013 051733 0054 0055 BNA ARHGAP 625 BNA 16 625 1 0 ARHGAP 969 BNA 16 969 1 3 ARHGAP 1344 BNA 16 1344 1 6 ARHGAP 1447 BNA 16 1447 1 7 ARHGAP 1748 BNA 16 1748 1 9 ARHGAP 1931 BNA 16 1931 2 1 ARHGAP 2032 BNA 16 2032 2 2 4 0 ARHoGAP 1931 BNA 16 con t12 Bg NN wt DLD
45. 1 3 A ARHGAP 1748 BNA 16 13 B 5 BNA in vivo BNA ARHGAP 1748 BNA 16 1748 in vivo NoD Scip 2 HcT116 FnITc ARHGAP 1748 BNA WO 2013 111897 28 PCT JP2013 051733 0056 0057 16 2 AteLoGene P Koken ARHGAP 1748 BNA 16 1 4 ARHGAP 1748 BNA 16 14 GY brain Uver ARAB spleen Lung B kidney intestine Firc ARHGAP 1748 BNA 16 ARHGAP 1748 BNA 16 15 9 BNA in vivo
46. WO 2013 111897 3 PCT JP2013 051733 0007 0008 0009 00 10 4S SF OC BBR Nk4 HGF an tagon istang iogenes is inhibitor in cance r bioL ogy and therapeu tics Mat sumo to K Nakamu ra T 2003 Cance r Science 94 32 1 327 RhocT P Rho GTPase activat ing protein RhoGAP rRhoGAP RhoGAP
47. n 26 A 11 35 Y ABNA HCT116 ARHeAP1 1A 5 12 5 ABNA ARHeAP1 1A HC R13 A BNA 1748 HcT1 16 ARHGAP1 1A B HcT1 16 BNA 1748 FITC fluoresce in isoth iocyanate WO 2013 111897 10 PCT JP2013 051733 001 3 0014 BNA 174 She Fl 7 14 NOD SCID FITC BNA 1 748 in vivo ie hls 15 NOD SCID FITC
48. Fuc i G1 mKO2 s G2 M mAco G1 cqt1 s G2 M cGeminin Fuc i FAcSAria II BD sjG2M HO if mac G1 mko2 Fuc i H CcT116 s e2 M M2 s eGzMu G1 M3 o RE DA Ssje2M cG1 mRNA 2 1ese 1 RnoeAp ARHeAP 11A s G2 M
49. Rnoeep RhoGAP KE EO RNA BB Inhibition of SH ARHGAP11a gNA Ge EE After 24 hours e RhoGTP CRE LO SMH ER AD 5 WO 2013 111897 A1 WIANMINUNUNNN NUNI NORM ON TAT ATA OAPI BF BJ CF CG Cl CM GA GN GQ GW ML 84 RAROBURW SCTOBBOLAR MR NE SN TD TG ARIPO BW GH GM KE LR LS MW M 3 a f Z NA RW SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ RU TJ TM A lt AL AT BE BG CH CY CZ DE DK EE ES F RWB 213R 3 FR GB GR HR HU IE IS IT LT LU LV MC MK _ MT NL N
50. NMJ 99357 1 RnoeTP 0 DARHGAP 11A ARHGAP 11A ARHeAP 11A GenBank Access ion No NM O 14783 3 NMJ 99357 1 mRNA DpNA DNA BY GenBank Access ion No NM O 14783 3 NMJ 99357 1 RnocAp WO 2013 111897 12 PCT JP2013 051733 001 8 001 9 DNNA DNA
51. ceminin s G2 M Geminin SG2M A B C B eeminin lt S G2 M 1 2 HcT11e S G2 M G1 ARHeAP 11A Ss G2 M NODISCID ucc i Fluorescen t Ub WO 2013 111897 19 PCT JP2013 051733 0039 0040 0041 iquitinat ion based CeLL Cyc Le Indicat or HcT1t16e HCT116
52. shRNA DNA shRNA target Sequence Strand 5 3 1 ARHGAP1 1A 1 TRCN0000047281 CCGGCGGTATCAGTTCACATCGATACTCGAGTA TCGATGTGAACTGATACCGTTTTTG 1 2 ARHGAP1 1A 2 TRCNO000047282 CCGGCCTTCTATTACACCTCAAGAACTCGAGTT WO 2013 111897 22 PCT JP2013 051733 0046 0047 CTTGAGGTGTAATAGAAGGTTTTTG 2 3 SHC002 CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCT TCATCTTGTTGTTTTT 3 2 ARHGAP 11A in vivo ARHeAP 11A NODSCID HcT116 1X IO06ceLL 1 1 2 shR NA SH SH 1 SH 2 in Vivo siRNA AteLoGene R Koken siRNA a CULT a 1 3 sh RNA shRNA b
53. B B A RhoGAP A B RhoGAP ARHeAP1 1A PA Big A RMR AA BRYA RAP ASKART SMHS HcT1 16 RhoGAP ARHGAP1 1A RT PCR
54. 1101 AR 200 9 7 BARBARA PcTYJIP2013 051733 C Y Homo sapiens Rho GTPase activating protein 11A ARHGAPIIA A transcript variant 1 raRNA DATABASE NCBI Nucleotide online accession No NM_014783 2011 11 27 URL lt http fiwww ncbi nim nih gov nuccore 197333716 sat 5 amp sat kev 6322659 gt Y Homo sapiens Rho GTPase activating protein 11A ARHGAPIIA 1 4 7 10 A transcript variant 2 raRNA DATABASE NCBI Nucleotide online 5 6 accession No 199357 2011 11 19 URL lt http 7www ricbi nim nih gow nuccore 407880 172sa t 15a satk ey 6242735 gt X wo 2008 111464 Al A 2008 09 18 0008 0023 64 amp US 2010 0021918 Al amp EP 2136209 Al 0008 0023 64 H PCT 1SA 2 10 2 2009 7 PCT JP2013 051733 1 2 8 3 PCT 17 2 a
55. 5 1 13 4 1 ARHGAP 625 BNA 1 6 0 2 ARHGAP 969 BNA 1 1 3 3 ARHGAP 1 344 BNA 1 6 1 6 4 ARHGAP 1447 BNA 1 6 17 5 ARHGAP 1 748 BNA 1 6 19 6 ARHGAP 1 931 BNA 1 6 2 1 7 ARHGAP 2032 BNA 1 6 2 2 8 ARHGAPv1 3484 BNA 1 530 9 ARHGAPv2 221 5 BNA 1 534 1 0 ARHGAPv2 2285 BNA 1 3 5 1 1 ARHGAPv2 2306 BNA 1 3 6 1 2 ARHGAPv2 2355 BNA 1 T L 5 L T L 5 L T L 5 L aaatttgaa5 L T L 5 L c 6 T L 5 L 5 L gaaaaagcc5 L T L T L c ttcatgtc5 L T L T L c T L 5 L 5 L aggataaaaT L 5 L T L g 5 L T L T L gatggactt5 L 5 L T L T L T L T L gcctgcaatT L 5 L T L 5 L 5 L T L agattgaatT L T L 5 L a 6 T L T L 5 L gagggtaacT L 5 L 5 L a 6 5 L T L 5 L taacagtagT L A L T L g 6 T L 5 L T L agaacagtaA L A L T L 6 T L T L 5 L aaacatgaa5 L T L T L 6 T L 5 L 5 L caattgttgA L T L A L g 1 t t t t E WO 2013 111897 5 PCT JP2013 051733 3 7 1 3 ARHGAPv2 2404 BNA 1 6 T L T L T L taacataagA L A L T L g 538
56. ARHGAP 1 748 BNA 1 6 5 L T L T L gatggactt5 L 5 L T L t 19 WO 2013 111897 6 7 6 ARHGAP 1 931 BNA 1 6 2 1 7 ARHGAP 2032 BNA 1 6 2 2 8 ARHGAPv1 3484 BNA 1 3 0 9 ARHGAPv2 221 5 BNA 1 3 4 1 0 ARHGAPv2 2285 BNA 1 3 5 1 1 ARHGAPv2 2306 BNA 1 3 6 1 2 ARHGAPv2 2355 BNA 1 3 7 1 3 ARHGAPv2 2404 BNA 1 3 8 31 T L T L T L gcctgcaatT L 5 L T L 5 L 5 L T L agattgaatT L T L 5 L a 6 T L T L 5 L gagggtaacT L 5 L 5 L a 6 5 L T L 5 L taacagtagT L A L T L g 6 T L 5 L T L agaacagtaA L A L T L 6 T L T L 5 L aaacatgaa5 L T L T L 6 T L 5 L 5 L caattgttgA L T L A L g 6 T L T L T L taacataagA L A L T L g PCT JP2013 051733 t N L BNA 5 LmC BNA L ARHGAP1 1A 1 4 ARHGAP1 1A 1 TRCNO000047281 ACTCGAGTATCGATGTGAACTGATACCGTTTTTG 1 5 ARHGAP1 1A 2 ACTCGAGTTCTTGAGGTGTAATAGAAGGTTTTTG 14 TRCN0000047282 1 5 3
57. N L BNA 5 L mC BNA TL A L 6 ARHGAP1 1A RNA 14 15 3 1 4 ARHGAP1 1A 1 TRCN0000047281 CCGGCGGTATCAGTTCACATCGATACTCGAG TATCGATGTGAACTGATACCGTTTTTG 1 1 5 ARHGAP1 1A 2 TRCN0000047282 CCGGCCTTCTATTACACCTCAAGAACTCGAG TTCTTGAGGTGTAATAGAAGGTTTTTG 2 7 1 6 1 8 RhoGAP 9 RhoGAP ARHGAP1 1A 8 10 A D 9 A
58. DHARMAcON http des ign dha rmacon com Ambion htt p www ambion comt ech Lib m isc s iRNA finder htmL http www taka ra b io co jp rnai intro htm dsRNA dsRNA DNA siRNA dsRNA EB dsRNA
59. ROB ARHGAP1 1A D A B RhoGTP RhoGTP ARHeAP1 1A 1 1 1 1 12 1 1 13 1 1 10 WO 2013 111897 PCT JP2013 051733 1
60. an DEVS sams NNNN RN 8ErdYSHSV Senge ee NNN OZdVDHBV aia i FNN NN 9ZI dVSHHV SSRN WA Li dVOHUY ar HSN COUNEIS NRN TYNN Uo A de N O ir g wy Q ws O uy fee ify ita m m VTTJVSHHY 40 HOiSSS CXS 9AHS1 8 10 WO 2013 111897 PCT JP2013 051733 12 ARHGAP11A wt Cont 625 969 1344 1447 1748 1931 2032 DLD3 ARHGAPIIA B HT29 ARRGAPIIA B PANCI ARHGAPLIA B PSNi ARHGAPIIA N N SN 13 HCT116 A Control 1748 FITC FITC Control 1748 ARHGAP11A B B 9 10 WO 2013 111897 PCT JP2013 051733 14 1000 P lt 0 05 800 x 600 BNA 1748 hu p 400 ug wa 200 ET 0 7 0 7 14 28 ARHGAP 1748 BNA 16 A 15 tumor 1 o 16 HEO80 L pe 8mg kg E 5x 10 BNA 1748 10mg kg 150me ke ae ERGEN 0B 148 2
61. 1 HTr29 Pane PSN1 4 BNA 5 ARHeAP 11A BNA ARHeAP 11A ARHGAP 1748 BNA 16 1748 1 9 12 7 BNA in vitro Firc BNA ARHGAP 1748 BNA 16 1748 HCcT116 o 1 4 0 ARHGAP 1748 BNA 16 ARHeAP 11A
62. 14include invent ion s wherei n a medi cal docto r diagno ses adi sease onthe basis of obtained informati on without stati ng about the inevitable judgment of the pre sence or absence of the di sease using a speci fic examinat ion step or judgment criteria or a step for forecasting the extent of the progre ss and progno sis of the di sease Therefore the se invent ions pertain todiagnosticmethods tobepract i ced on the human body and there fore rel ate to a subj ect matter which thi s Internati onal Searchi ng Autho rity is not requi red to search Form PCT ISA 210 extra sheet July 2009 INTERNATIONAL SEARCH REPORT International application No PCT JP2013 051733 Claims 1to 3 relate to a novel ant itumo r agent which compri ses as the active ingredient a compound defined by a desired property a sub stance capable of inhibi ting a protei n with RhoGTPase act ivity Although the se cl aims include any compounds havi ng the above prope rty onl y an ant i sense oli gonucl eoti de to a gene encodi ng ARHGAPIIA and an shRNA are exclu sive y di sclo sed under the provi sion of Art icle 5 of the PCT Therefore it appears that the se claims are not supported by the descripti on under the provi sion of Art icle 6 of the PCT Even though the common techni cal knowl edge at the poi nt of the appl i cati on is taken into consideration it cannot be recogni zed that the scope of compounds havi ng the above prope rty as a substance capa
63. 18 WO 2013 111897 17 10 10 PCT JP2013 051733 Sme ke 0 16mg body 18 Body weight mg P 0 9720 Lung weight mg 10mg kg 0 2mg body P 0 0299 2 5 2 0 1 5 1 0 0 5 0 0 EN V amp v INTERNATIONAL SEARCH REPORT International application No PCT JP2013 051733 A CLASSIFICATION OF SUBJECT MATTER A61K45 0 0 2006 01 i A61K31 7088 2006 01 i A61K31 711 2006 01 i A61K480 0 2006 01 i A61P350 0 2006 01 i C12N1 5 113 2010 01 i C12Q1 8 2006 01 i G01N33 1 5 2006 01 GO1N335 0 2006 01 1 GO1N33 5 74 2006 01 i According to International Patent Classification IPC or to both national classification and IPC B FIELDS SEARCHED Minimum documentation searched classification system followed by classification symbols A61K45 00 A61K3 1 7088 A61K31 711 A61K48 00 A61P35 00 C12N1 5 113 C12Q1 68 GO1N3 3 15 GO1N3 3 50 GO1N3 3 574 Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Jitsuyo Shinan Koho 1922 1 996 Jitsuyo Shinan Toroku Koho 1996 2013 Kokai Jitsuyo Shinan Koho 1971 2013 Toroku Jitsuyo Shinan Koho 1994 2013 Electronic data base consulted during the international search name of data base and where practicable search terms used MEDLINE CAplu s EMBASE BIOS IS STN JST Plus JME DPlu s JST7580 JDreaml I REGI STRY STN GenBank EMBL D
64. 1N33 15 2006 01 i A61P35 00 C12N1571 13 C12Q1 68 GO1N33715 GO01N33 50 MEDLINE CAplus EMBASE BIOSIS GenBank EMBL DDBJ GeneSeq STN JSTPlus JMEDPlus JST7580 JDreaml REGISTRY STN C X P190 RhoGTP RhoGTPase ME ZDA LERTA Y 2007 Vol 47 A pp 9 10 No 3 9 10 2 et al Regulation function for CDK innibitors Vol 4 No 2 pp 948 955 950 BAM 1 2 R Box Besson A C IA TE
65. 2 9 L I 11BeB116Be G 9 91 YNd ZE0Z dV9HyV L 1 1 5 13828982 ooB m L m m 91 VNa g6 L dVOHyV 1 1 L S 1 S9e61e6e61 J s 1 1 A 0 S 9 6 8 E 6 1 1 9 G449266326 9 1 1 G 9 8 dV9HyV EzTS0 EI0zdf X d 88 Z68EEDEEOZ OM WO 2013 111897 26 PCT JP2013 051733 0051 0052 0053 ARHGAPv1 51 94 BNA 1 6 T L 5 L T L aacaaccaaT L 5 L T L 3 3 ARHGAPv2 221 5 BNA 1 6 5 L T L 5 L taacagtagT L A L T L g 3 4 ARHGAPv2 2285 BNA 1 6 T L 5 L T L agaacagtaA L A L T L t 3 5 ARHGAPv2 2306 BNA 1 6 T L T L 5 L aaacatgaa5 L T L T L t 3 6 ARHGAPv2 2355 BNA 1 6 T L 5 L 5 L caattgttgA L T L A L g 3 7 ARHGAPv2 2404 BNA 1 6 T L T L T L taacataagA L A L T L g 3 8 ARHGAP 1 931 BNA 1 6 cont1 T L tT L gecT L gcaaT L t5 L T L t 3 9 ARHGAP 1 931 BNA 1 6 cont2 T L tgT L ccT L gcT L aat5 L T L t 4 0 ARHGAP 1 931 BNA 1 6 cont3 T L 5 L T L caatgcctgT L T L T L t 541 BNA HCT1 16 ARHGAP1 1A mRNA BNA qPCR GAPDH
66. 2013 111897 1 MUNDNIN UATU MINDE N OT OUN TVU ON OIT UNT 51 21 22 25 26 30 71 72 54 54 12 19 c C 43 2013 8 1 01 08 2013 45 00 2006 01 31 7088 2006 01 317711 2006 01 48 00 2006 01 35 00 2006 01 A61K A61K A61K A61K A61P C12N C12Q GO1N GO1N GO1N Z M TO EL EO IK AA TAY RA IN IWA Hl A LM 10 WO 2013 111897 A1 W PO IPCT T 56508071 i 1 15 113 2010 01 osaa VP A MoRu 1 68 2006 01 Masaki Tseso871 1 1 33 15 2006 01 osaka up BH 33 50 2006 01 ISHII HideshD T 5650871 33 574 2006 01 Bais osaka JP PCTJP2013705 1733 74 6Ho Takashi et al 7 53201 1 6 amp ES 2013 28 28 01 2013 oe WO FS oae im BR le vor e oe J T HE A
67. 4 Pe siRNAs BBV 4IXZOAR Day 7 O 7 TE 624 l i i 0 7 14 21 ERA CHER UREA CMTE 5 10 PCT JP2013 051733 WO 2013 111897 9 CN IN ON LGCL 0 d Vb l zl KHEWREHEOVTTIdV9SHYY 6 10 WO 2013 111897 PCT JP2013 051733 6410 Relapse free survival 100 low n 38 2 80 T 8 70 high n 26 D a 60 50 0 200 400 600 800 1000 1200 1400 Days 7 10 WO 2013 111897 PCT JP2013 051733 11 NNN OT TAEV OHYV scum LERENA SEHEN seai PES RIGS POLS TAGVOHUY ccamnen OPTS TASWOHUY cenama BSEb tAGVDRHW Tess ae TAdY DKY Y ocHRKE SLTE TAAVOHNY ez Senate seam BORTTASVOHHY STAES 9Z9Z TAGVRHNW orem TTESTAGVOHNY Sease 9 Z TAGVDHHW reagi S 9ZTZ dVOHBYV ete Ni LCEOECHVSHN Zz Shem Es AA y OCS BSS Mu E TERY aT Swami acm ease NN NsssN Ba COTE d ORRY Tt Hs GOOE HVSNHV rtea oa sama cr8 dYOHNV ITSEN He a VORYY TT SSB OSE 0 BR i ON I i amp f A a II Te GC9 dVOHHV amp orem II ARHGAP11AOMRNARAHHAROE AN as SN T6S d HUY SHES SS
68. DB J GeneS eq C DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document with indication where appropriate of the relevant passages Relevant to claim No XIY IA Toshi yuki KUSAMA et al PI90 RhoGT P ni yoru 1 7 2 4 RhoGT Pase Sogai o Oyo shita Sui Gan Saibo no 8 10 5 6 Ten i Shi njun Yokus ei ni Kan suru Kento Seijinbyo 2007 vol 47 no 3 page s 9 to 10 summary page 9 left column page 10 left column 2nd paragraph Y A Besson A et al Regulati on of the 4 7 1075 6 cyto skeleton an oncogeni c funct ion for CDK i nhibitor s Nature Reviews Cancer 2004 Vol 4 No 2 pp 948 955 page 950 left column st to 2nd paragraphs Boxl Further documents are listed in the continuation of Box C LO See patent family annex Special categories of cited documents T later document published after the international filing date or priority document defining the general state of the art which isnot considered date and not in conflict with the application but cited to understand to be of particular relevance the principle or theory underlying tiie invention earlier application or patent but published on or after the international document of particular relevance the claimed invention cannot be filing date considered novel or cannot be considered to involve an inventive document which may throw doubts on priority claim s or which is step when the document istaken alone cited to establish the publica
69. E AG AL AM AO AT AU AZ BA A BB BG BH a BR BW BY BZ CA CH CL CN 43 BA 2012 015982 2012 1 27 H 27 01 2012 JP geht See GUR KARIDE Dry Ons ee Ce aC tae cot Fl GB GD GE GH GM GT HN HR HU ID IL IN osAKA UNIVER IS JP KE KG KM KN KP KR KZ LA LC LK LR SITY PP T5650871 1 LS LT LU LY MA MD ME MG MK MN MW MX 1 Osaka JP MY MZ NA NG NI NO NZ OM PA PE PG PH _ PL PT QA RO RS RU RW SC SD SE SG SK SL Bsn _ Masaru T 5650871 SM ST SV SY TH TJ TM TN TR TT TZ UA UG 1 1 US UZ VC VN ZA ZM ZW Osaka JP B JII KAGAWA Yoshinori Title NOVEL ANTITUMOR AGENT AND METHOD FOR SCREENING SAME 57 Abstract The present invention provides a novel antitumor agent with a novel action mechanism The novel antitumor agent comprises as the active ingredient a Ad SHARHGAP 1a yn substance capable of inhibiting a cell cycle dependent Rho GTPase activating protein 2 4 BB RhoGAP The Rho G p which depends on the cell cycle plays an important role in ig E 3 the course of the acquisition of metastatic ability and or invasive ability by c
70. O PL PT RO RS SE SI SK SM TR 5 2 a WO 2013 111897 1 PCT JP2013 051733 0001 0002 0003 0004 2 0 12_ 0 15982 ci gap SHA DNA synthes is G2 gap2 M mitos i s ei lt o ci S R restriction point H R
71. ancer gm s cells By targeting the RhoGAP the invasion and or metastasis of cancer cells can be F 9 2 controlled A s the substance capable of inhibiting the cell cycle dependent RhoGAP AA Bt an antisense oligonucleotide against an RhoGAP encoding gene an oligonucleotide inducing RNA interference etc can be cited An oligonucleotide containing a synthet a amp ie ae ic nucleic acid such as BNA is preferred because of the high stability thereoi The present invention also provides a screening method for selecting an antitumor agent which is capable of suppressing the invasion and or metastasis of cancer cells by a B novel mechanism This method for screening a novel antitumor agent is characterized ce in that a substance capable of inhibiting a cell cycle dependent Rho GTPase activating protein is selected thereby ey ARR MAX HI AA CK VERT SMT A Rnocrp Rho er a Pase activating protein RhoGAP A F Rnoca ns RhocAp
72. ble of inhibit ing a prote in with RhoGTPase act ivity could be speci fied Thus claims 1to 3 also fai to sati sfy the requi rement of clearne ss as defined in Art icle 6 of the PCT Such being the case the search was conducted exclus ivel y on the rel ationship between a protei n with RhoGTPase act ivity or ARHGAPI IA and an ant itumor agent and the ant itumor agent s which compri se as the active ingredient the compounds that are part icul arly stated in the descripti on and specified in claims 5 and 6 Form PCT ISA 210 extra sheet July 2009 A 1 PC A61K45 00 2006 01 i A61K31 7088 2006 01 i A61P35 00 2006 01 i C12N1571 13 2010 01 i G01N33 50 2006 01 i GO1N33 574 2006 01 i Int Cl B A61K31771 I PC Int Cl A61K45 00 A61K31 7088 G01N33 574 9 9 9 9 A61K3177 1 C12Q1 68 2006 01 i 1 A61K48 00 22 1996 71 2013 96 2013 94 2013 PCT JP2013 051733 1 2006 01 i A61K48 00 2006 01 i GO
73. eAP 11A HcCT116 Hepe2 Human Lymphat ic Fibrob Lasts HLF PSN1 MiaPaca2 Pane 1 AR HGAP 11A PcR 1 ARHeAP 11A HCT116 PSN 1 ARHeAP 11A 6 GeneNo te GeneCa rds 1D GC15P032907 ARHeAP 11A
74. inuation of item 3 of first sheet This International Searching Authority found multiple inventions in this international application as follows A sall required additional search fees were timely paid by the applicant this international search report covers all searchable claims A sall searchable claims could be searched without effort justifying additional fees this Authority did not invite payment of additional fees A sonly some of the required additional search fees were timely paid by the applicant this international search report covers only those claims for which fees were paid specifically claims Nos No required additional search fees were timely paid by the applicant Consequently this international search report is restricted to the invention first mentioned in the claims it is covered by claims Nos Remark on Protest L The additional search fees were accompanied by the applicant s protest and where applicable the payment of a protest fee L The additional search fees were accompanied by the applicant s protest but the applicable protest fee was not paid within the time limit specified in the invitation LI No protest accompanied the payment of additional search fees Form PCT ISA 210 continuation of first sheet 2 uly 2009 INTERNATIONAL SEARCH REPORT International application No PCT JP2013 051733 Conti nuat ion of Box No IMA of conti nuation of first sheet 2 The i nventi onsinclaims 11to
75. sat key 6322659 gt Homo sapi ens Rho GTPase activati ng prote in 11A 1 4 7 10 5 6 ARHGAP IA tran script vari ant 2 mRNA DATABASE NCBI Nucleot ide onl ine acce ssion No 199357 2011 11 19 URL lt http www ncbi_ nim nih gov nucco re 40788017 sat 1 5 amp satkey 6242735 gt wo 2008 111464 Al Ei sai R amp D Management Co Ltd 18 Septembe r 2008 18 09 2008 paragraph s 0008 to 0023 page 64 amp US 2010 0021918 Al amp EP 2136209 Al paragraph s 0008 to 0023 page 64 Form PCT ISA 2 10 continuation of second sheet July 2009 INTERNATIONAL SEARCH REPORT International application No PCT UPZ013 051 733 Observations where certain claims were found unsearchable Continuation of item 2 of first sheet This international search report has not been established in respect of certain claims under Article 17 2 a for the following reasons 1 Claims Nos 11 14 because they relate to subject matter not required to be searched by this Authority namely See ext ra sheet 2 L Claims Nos because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out specifically Claims Nos because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6 4 a Observations where unity of invention is lacking Cont
76. tgtc5 L T L T L T L 5 L 5 L aggataaaaT L 5 L T L g T L T L 5 L accaggagtT L T L 5 L a TL T A BNA t 4 5 6 t 7 8 t 9 10 1 1 12 13 1 1 16 1 1 Z Elige gig Rig Elige Rig Yo FE 1g 0H a84 a gig gR Z gg Z E Elg Z Sge z E Elg L amp x i e 1 01 9 1 SREIET 9499 J 1 1 1 9 DL L I 9442899396 9 1 G e 1 9 1 9 1 Loeey666e6 7 9 6 4 1 9 7 16436628363 7 G G 9 9 1 1 4148389389 7 G D LDL 1 1 1 S 1 re 991161e9 Ld L 9 S1e1966619 S S VTL TCD 1498292896 VLDL 9 9 1 163332828366 9 G 1 G D L 5 8 91 VNd OVLG LAdVOHyV LE 91 VNd 66 p LAdV9HyV o 9 VN3 t78 dv HYV 6z 91 VNd 8 ZE LAdVOHyV 8 Z 9 h YN 6 Z k dV5HYV Lz 9 L VNa 8083 LAdV9HyV 9 z 9 8ZHAdV Vv G2 9LVNa IEZ IAdV9HyV 9 VN3 Z9 AdVOHMv 6 S Leoeeoteo J L L t 9 VNa 9 IZ dVQHYV
77. tion date of another citation or other document of particular relevance the claimed invention cannot be special reason as specified considered to involve an inventive step when the document is document referring to an oral disclosure use exhibnion or other means combined with one or more other such documents such combination document published prior to the international filing date but later than being obvious to aperson skilled in the art the priority date claimed document member of the same patent family Date of the actual completion of the international search Date of mailing of the international search report 25 February 2013 25 02 13 05 March 2013 05 03 13 Name and mailing address of the ISA Authorized officer Japane se Patent Of fi ce Facsimile No Telephone No Form PCT ISA 210 second sheet July 2009 INTERNATIONAL SEARCH REPORT International application No PCT JP2 013 051733 C Continuation DOCUMENTS CONSIDERED TO BE RELEVANT C Continuation DOCUMENTS CONSIDERED TO BE RELEVANT DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document with indication where appropriate of the relevant passages Relevant to claim No Y A Homo sapi ens Rho GTPase activati ng prote in 11A 1 4 7 10 5 6 ARHGAP I IA tran script vari ant 1 mRNA DATABASE NCBI Nucleot ide onl i ne acce ssion No 014783 2011 11 27 URL lt http www ncbi nlIm nih gov nucco re 197333716 sat 15 amp
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