|HAO WANATATALITATUS009850297B2 DE LA MULT MAI MULT (12 ) United States Patent (10 ) Patent No. : US 9 ,850 , 297 B2 Majeti et al. ( 45) Date of Patent: Dec . 26 , 2017 ( 54 ) SECRETED FRIZZLE - RELATED PROTEIN 5 C12N 15 / 10 ( 2006 .01 ) (SFRP5 ) BINDING PROTEINS C12N 15 / 11 ( 2006 .01 ) A61K 39 / 00 (2006 .01 ) (71 ) Applicant: AMGEN INC . , Thousand Oaks , CA (52 ) U . S . CI. (US ) CPC .. C07K 16 / 18 (2013 .01 ) ; A61K 2039/ 505 (72 ) Inventors : Jiangwen Majeti , Palo Alto , CA ( US) ; ( 2013. 01 ) ; CO7K 2317 /14 (2013 . 01 ) ; CO7K YuMei Xiong, Palo Alto , CA (US ) ; 2317 /51 ( 2013 . 01 ) ; CO7K 2317 / 515 (2013 . 01 ) ; Wen -Chen Yeh , Belmont, CA (US ) ; CO7K 2317 / 565 (2013 .01 ) ; CO7K 2317 /76 Alykhan S . Motani, San Francisco , CA ( 2013 .01 ) ; CO7K 2317/ 92 ( 2013 .01 ) (US ) ; Yang Li, Mountain View , CA (58 ) Field of Classification Search (US ) None See application file for complete search history . (73 ) Assignee : Amgen Inc ., Thousand Oaks , CA (US ) (56 ) References Cited ( * ) Notice : Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U . S . C . 154 (b ) by 105 days . PUBLICATIONS MacCallum et al. , Antibody -antigen interactions: contact analysis (21 ) Appl. No .: 14 / 776 , 012 and binding site topography, J . Mol. Biol. 262: 732 -745 , 1996 . * Lamminmaki et al. , Chrystal structure of a recombinant anti (22 ) PCT Filed : Mar. 14 , 2014 estradiol Fab fragment in complex with the 17beta -estradiol , J . Blol . Chem . 276 : 36687 - 94 , 2001 . * ( 86 ) PCT No . : PCT/ US2014 /029015 Chen et al ., Enahncement and destruction of antibody function by somatic mutation : unequal occurrence is controlled by V gene $ 371 ( C ) ( 1 ) , combinatorial associations, EMBO J. , 14 ( 12 ) : 2784 -2794 , 1995 . * Sep . 14 , 2015 Blumenthal et al. ; Blood , American society of Hematology ; 2006 ; 108 : 3 ; 965 - 973 . ( 87) PCT Pub . No. : WO2014 /144553 (Continued ) PCT Pub . Date : Sep . 18 , 2014 Primary Examiner - Claire Kaufman (65 ) Prior Publication Data US 2016 /0024196 A1 Jan . 28 , 2016 (57 ) ABSTRACT Methods of treating metabolic diseases and disorders using Related U . S . Application Data an antigen binding protein specific for the SFRP5 polypep (60 ) Provisional application No . 61 / 799, 619 , filed on Mar. tide are provided . In various embodiments the metabolic 15 , 2013 . disease or disorder is type 2 diabetes, obesity , dyslipidemia elevated glucose levels , elevated insulin levels and diabetic (51 ) Int . Cl. nephropathy. COZK 16 / 18 (2006 .01 ) C12N 5 / 10 ( 2006 .01 ) 11 Claims, 16 Drawing Sheets 2000 , A 24C18 . 3 ? 12E6 . 2 mAb,ug/ml A14 A 24P2 . 2 50075 HEM D Fata Ao OyO 50 100 150 200 Time post- injection (hrs ) US 9 ,850 , 297 B2 Page 2 (56 ) References Cited PUBLICATIONS He, et al ., Science ; A member of the Frizzled protein family mediating axis induction by Wnt -5A ; 1997 ; 275 : 1652 - 1654 . International Search Report for International application No . PCT/ US2014 /029015 , dated Aug . 5 , 2014 . Li et al . ; Genes & Development; 2008 ; Sfrp5 coordinates foregut specification and morphogenesis by antagonizing both canonical and non -canonical Wnt11 signaling ; 22 : 21 ; 3050 - 3063 . Maury et al . ; Molecular and Cellular Endocrinology ; Elsevier Ireland ; 2010 ; 314 : 1 - 16 . Mori et al. ; J Clinical Investigation ; Secreted frizzled - related protein 5 suppresses adipocyte mitochondrial metabolism through WNT inhibition ; 2012 ; 122 : 7 ; 2405 - 2415 . Oh et al .; WNT fans the flames in obesity ; Science ; 2010 ; 329 : 397 - 398 . Ouchi et al. , Science ; Supporting online material for Sfrp5 is an anti - inflammatory adipokine that modulates metabolic dysfunction in obesity ; 2010 ; 329 : 1 - 18 . Rauch et al ., J Clinical Invest .; Lighting the fat furnace without SFRP5 ; 2012 ; 122 : 7 ; 2349 - 2352 . * cited by examiner U . S . Patent Dec . 26 , 2017 Sheet 1 of 16 US 9 ,850 , 297 B2 Figure 1 • rhwnt- 3a OrhWnt- 3a + rhsFRP5 (6ug /ml ) A Vehicle 60 ,000 RelativeLucunits 2 ,000 0 , 000 0 __ . .. 2 -04 20 1 2 3 4 Log (ng / ml ) B rmSFRP5 -hemiFc 40000 W 0 ug /ml ? 0 . 0067 ug/ ml 30000 0 . 02 ug /ml AbsoluteLucunits(cps) XX 0 . 061 ug/ ml 20000 VZ 0 . 185 ug /ml 10.55 ug /ml 10000 1 .67 ug /ml UIIIIIIIIIIZ WITHIN HHH 3 5 ug /ml OPTI- MEM only rmWnt- 3a Treatment group U . S . Patent Dec . 26 , 2017 Sheet 2 of 16 US 9 ,850 , 297 B2 . + + . Neg. -. + . .- +. Ab-111220 . - . 1 1 20ugimiMABw/sfrp5+Wnt-3a . ZOLI 1 Bing3 . Ab-s 10H . Bin#2 Ab-5 .. A5-19 24P2 60ugimiMABwisfrp5+Wnt-3a Ab-1518 2019 Ab-14 Bin#1 . 1403 AbgA5-10 360ug/mlMABonly+Wnt-3a Ab-2 . 1614 . wnt-3a+Abri sfrp5 . NOMABadded . only . cells Figure2 . the on one Thousands ) cps( Units Luc Average U . S . Patent Dec . 26 , 2017 Sheet 3 of 16 US 9 ,850 , 297 B2 168h 96h 72h 24C18.3 20200 blood collection 24P22 .212E6 n=3foreachtimepoint 48h € 160 24h 100 hours SFRP5Ab 01h3h6h i.pdosing VVVV Figure3 )ugimi ( mAb A ? U . S . Patent Dec . 26 , 2017 Sheet 4 of 16 US 9 ,850 , 297 B2 Figure 4 A 8007 . IgG Control F anti- SFRP5 24P2. 2 anti- SFRP5 12E6 . 2 Bloodglucose(mg/dl) anti- SFRP5 24C18 . 3 À AEanti- SFRP5 1702 . 1 zl 60 min o IgG Control 30mpk p . anti- SFRP5 24P2. 2 30mpk A anti- SFRP5 12E6 . 2 30mpk Glucose(ml/dl) 6 anti -SFRP5 24C18 . 3 30mpk C- - - - - - - anti - SFRP5 1702. 1 30mpk * min U . S . Patent Dec . 26 , 2017 Sheet 5 of 16 US 9 ,850 , 297 B2 Figure 5 Fasting blood glucose over time o IgG Control anti - SFRP5 24P2 . 2 glucose,mg/dL A anti- SFRP5 12E6 . 2 - * * Z anti- SFRP5 24C18 . 3 HIZINIZ* *INUN anti- SFRP5 1702 . 1 IT MMMM T7I7/IINIA baseline WK1 WK2' WK3 WK4 Fasting serum Insulin = * o IgG Control = *TWITTTTTTTT anti - SFRP5 24P2. 2 A anti- SFRP5 12E6 . 2 Z anti - SFRP5 24C18 . 3 ng/dl - anti - SFRP5 1702. 1 ?????????? VIINIL W baselineTULO VILTIZO WK2 WK4 U . S . Patent Dec . 26 , 2017 Sheet 6 of 16 US 9 ,850 , 297 B2 Figure 5 (continued ) riguas continuesBody Weight O gG Control anti - SFRP5 24P2. 2 W anti - SFRP5 12E6 . 2 Bodyweight(g) 2 anti- SFRP5 24C18 . 3 ölö | anti- SFRP5 1702 . 1 ö ç ZITO WK1 WK2 ZIIIIIIIIIIIIZA, Fasting triglycerides O IgG Control w anti- SFRP5 24P2 . 2 anti- SFRP5 12E6 . 2 anti - SFRP5 24C18 . 3 Vioww anti- SFRP5 1702. 1 WINT KUL ww WIMMILTITULO WK1 WK4MUNIKA osobaseline U . S . Patent Dec . 26 , 2017 Sheet 7 of 16 US 9 ,850 , 297 B2 24P2.2SFRP5anti- anti-SFRP512E6.2 anti-SFRP524C18.3 anti-SFRP51702.1 IgGControl o ? * * * C-peptide Terminalfastingserum Figure6 15000, 5000 pmol10000 , peptide- C U . S . Patent Dec . 26 , 2017 Sheet 8 of 16 US 9 ,850 , 297 B2 oemptyvector oemptyvector SFRP5ASFRP5 BSFRP5 * * * s * * WK6 * * * Timepost-injection(min) +a CO- ©153060 WK4 . ---- WK2 OL ? ? ? ? baseline w dl/ mg , glucose blood o ,weightg body Oemptyvector oemptyvector BSFRP5 ESERP5 * -- -- * Timepost-injection(min) * - Timepost-injection(min) --- uö153060 - ©153060 --- 7Figure OLT cpFRES/bw '?so?n 6 pooja dlo/ mg ,glucose blood U . S . Patent Dec . 26 , 2017 Sheet 9 of 16 US 9 ,850 , 297 B2 oemptyvector ESFRP5 * * * * WK6 WH4 SFRP5emptyvector WK2 8 6 8 9 baseline 15000, 000 5000 TI mL/ ng , insulin I units arbitrary vectorempty vectoroempty SFRP5 ESFRP5 o ES * * * - -0- WK6 injection(min)Timepost- .. o204060 WK4 * ---- 1 * continued)Figure7( WK2 baseline de site ce ?i ? w pow ' ?soon 6 pooja dl/ mg , glucose blood USU . S . PatentPatent maisonDec . 26 , 2017 Sheet 10 of 16 US 9 ,850 ,297 B2 emptyvector SFRP5 * * SFRP5vectorempty baseline2WK 400007 30000 20000 ö õ units arbitrary g , weight body emptyvector ASFRP5 oemptyvector SFRP5 * :*00 ** Timepost-injection(min) Timepost-injection(min) ----- ó204060 * KIO ** oó204060 *& Q ** Figure8 ? ? 200- D dl/ mg , glucose blood dl /mg , glucose blood U . S . Patent Dec . 26 , 2017 Sheet 11 of 16 US 9 ,850 ,297 B2 Oemptyvector SFRP5 II II * 1 IT * baseline2WK * SFRP5 ?? 0 vectorempty mL/ ng , insulin I pmol , proinsulin Oemptyvector SFRP5 * * * * baseline2WK SFRP5 Figure8(continued) vectorempty § § ñ ñ § 5000, 00 . m dl/ mg , glucose blood O pmol , insulin U . S . Patent Dec . 26 , 2017 Sheet 12 of 16 US 9 ,850 ,297 B2 anti-SFRP5mAb(ug/ml) ood -0.512 60ug/ml w/noz6.7ug/ml anti-SFRP5mAb 52.2ug/ml N0.74ug/ml OOug/ml 4x104, 104 ) CPS ( Luminescence RETTITUNTER MMMMMM 10 24P2.2 VM . rmSFRP5(ug/ml) recombinantmouseSFRP5(ug/ml) 3 7777777777777777777 10.011 LETTMT MTTEITIM ????? DUNIIHINNAPITIA 1090.0110 12E6.2 1x1004 5x105. W ) CPS( Luminescence 1 6 .. TITUTI H - - " - - - - - - - - - - - - - . - 09 12. 19 2 . no 50100150200 rmWNT3a(ng/ml) 24C18.3 JUL ( O M XXXXXXXXXX * WWW 09 4x104, x104 2x104 1x104 Figure9 -c0 2x105 oderno 11) CPS( Luminescence ) CPS( Luminescence atent Dec . 26 , 2017 Sheet 13 of 16 US 9 ,850 ,297 B2 12E6.2 A24C18.3 A24P2.2 5 Timepost-injection(hrs) KA 050100150200 HH Figure10 19946 š iš500 ml/ ug , mAb U . S . Patent Dec. 26 , 2017 Sheet 14 of 16 US 9 ,850 ,297 B2 24C18.3 24C18.3 IgGControl A12E6.2 V24P2.2 IgGControl A12E6.2 #24P2.2 WK4 Timepost-injection(min) ó153060 WK2 . WK1 o og dl/ mg , glucose blood o ,gweight body baseline 24C18.324C18.3 24C18.3 24P2.2 IgGControl 12E6.2A V24P2.2 IgGControl A12E6.2 TO Timepost-injection(min) Timepost-injection(min) ö153060 to153060 11Figure § 8 8 8 8 x dl/ mg , glucose blood 0 dl/ mg ,glucose blood U . S . Patent Dec . 26 , 2017 Sheet 15 of 16 US 9 ,850 ,297 B2 Figure 11 (continued ) bloodglucose,mg/dlm IgG Control 24C18 . 3 lei TOIR A 12E6 . 2 * * * 24P2 . 2 * * * * baseline 1WK 2WK7 4 WK 71 on insulin/proinsulin NW– IgGControl 24C18 .3 12E6 .2 24P2 .2 * * * * * * * * * * * C-peptideproinsulino/ IgGControl 24C18 .312E6 .24 24P2 .