|HAI LALA AT TATUUS009758577B2 WIELU NA MAMA HATI (12 ) United States Patent ( 10 ) Patent No. : US 9 , 758 ,577 B2 Schurpf et al. (45 ) Date of Patent: * Sep . 12 , 2017 (54 ) COMPOSITIONS AND METHODS FOR (56 ) References Cited GROWTH FACTOR MODULATION U . S . PATENT DOCUMENTS @( 71 ) Applicant: SCHOLAR ROCK , INC ., Cambridge , 5 , 972 ,335 A 10 / 1999 Ferguson et al. MA (US ) 6 , 096 , 506 A 8 / 2000 Lee et al . 6 , 368 , 597 B14 / 2002 Strassmann et al. @( 72 ) Inventors : Thomas Schurpf , Cambridge , MA 6 , 468 , 535 B1 . 10 / 2002 Lee et al. ( US ) ; Nagesh K . Mahanthappa , 6 ,656 ,475 B1 12 / 2003 Lee et al. Cambridge , MA (US ) ; Michelle Marie 7 ,070 , 784 B1 7 / 2006 Halkier et al . 7 , 241 ,444 B2 7 / 2007 Goetsch et al . Straub , Brighton , MA (US ) 7 ,316 , 998 B2 1 /2008 Knopf et al. 7 ,320 ,789 B2 * 1 /2008 Aghajanian et al. 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OTHER PUBLICATIONS CO7K 16 / 00 ( 2006 .01 ) Stockis, J . et al. “ Membrane protein GARP is a receptor for latent CO7K 16 / 18 ( 2006 . 01 ) TGF -beta on the surface of activated human Treg. ” Eur J . Immunol. CO7K 16 / 22 ( 2006 .01 ) Dec . 2009; 39 ( 12 ) :3315 - 22 . CO7K 14 /495 ( 2006 . 01 ) (Continued ) COZK 16 / 28 ( 2006 .01 ) C12N 15 / 10 (2006 .01 ) A61K 38 /00 ( 2006 .01 ) Primary Examiner - Elizabeth C Kemmerer (52 ) U . S . CI. CPC . .. .. .. .. .. CO7K 16 / 22 ( 2013 .01 ) ; CO7K 14 /495 ( 74 ) Attorney , Agent, or Firm — McCarter & English , (2013 . 01 ) ; CO7K 16 /005 (2013 .01 ) ; CO7K LLP ; Marcie B . Clarke ; Maria Laccotripe Zacharakis 16 / 2863 ( 2013 . 01 ) ; C12N 15 / 1037 ( 2013 .01 ) ; A61K 38 / 00 ( 2013 .01 ) ; CO7K 2317 / 21 (57 ) ABSTRACT ( 2013 .01 ) ; COZK 2317 / 24 ( 2013 .01 ) ; COOK 2317 / 31 ( 2013 .01 ) ; CO7K 2317 /55 (2013 . 01 ) ; Provided herein are proteins, antibodies, assays and methods CO7K 2317 / 565 ( 2013 . 01 ) ; CO7K 2317 / 76 useful for modulating growth factor levels and /or activities . ( 2013 .01 ) ; CO7K 2317 /92 ( 2013 .01 ) ; CO7K In some embodiments , such growth factors are members of 2317 / 94 ( 2013 .01 ) ; GOIN 2333/ 71 (2013 .01 ) the TGF- B superfamily of proteins . ( 58 ) Field of Classification Search None See application file for complete search history . 16 Claims, 23 Drawing Sheets US 9 , 758 , 577 B2 Page 2 ( 56 ) References Cited European Search Report for EP application No . 13853885. 5 dated May 13 , 2016 . U . S . 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Histopathologic features of phorbol WO 2007 /050793 A2 5 / 2007 myristate acetate - induced lung injury . Lab Invest. 52 ( 1 ) :61 - 70 . WO 2008 /060371 5 / 2008 Studnicka , G . M . et al .( 1994 ) , Human - engineered monoclonal anti WO 2009 / 114702 A2 9 / 2009 bodies retain full specific binding activity by preserving non -CDR WO 2009 / 144457 A2 12 / 2009 complementarity -modulating residues. Protein Eng . Jun . WO 2014 /093531 6 / 2014 1994 ; 7 (6 ): 805 - 14 . WO 2015 /015003 2 / 2015 McFarlane , Craig , et al . Proteolytic processing of myostatin is auto -regulated during myogensis , Developmental Biology , Mar . 23 , OTHER PUBLICATIONS 2005 , pp . 58 -69 , AgResearch , East Street , Hamilton , New Zealand , Department of Biological Sciences , University of Waikato , Hamil Shi, Minlong , et al . “ Latent TGF - B structure and activation ” , ton , New Zealand . Nature , vol. 474 , No . 7351, Jun . 15 , 2011 ( Jun . 15 , 2011 ) , pp . 343 - 349 . * cited by examiner U . S . Patent Sep. 12 , 2017 Sheet 1 of 23 US 9 ,758 ,577 B2 Figure 1 TGFB1 BMP , GDF10 TGFB2 BMPIS / / TGFB3 // myostatin (GOF8 ) wwwwwwwwwwwwwwwwwww BMP8A , 8 bin - 3A BMPO Inhibin -BB inhibin - BC BMPS murosawa Inhibin -BE BMP4 Lefty1 mm 1300 $ Lafty2 Nodalmumamman Anti BMPO (GDF2 ) ories Mullerian BMP10 homone GOFT GOES - UIQIYU- D GOFOGpF OF3 U . S . Patent Sep. 12 , 2017 Sheet 2 of 23 US 9 ,758 ,577 B2 Figure 2 Prodomain Stext ? C }eatae site signalpeptide Growth factor U . S . Patent Sep. 12 , 2017 Sheet 3 of 23 US 9 ,758 ,577 B2 Figure 3 Growth Factor- Prodomain Complex (GPC ) Free latency Associated Peptide (LAP ) Dimer Free Growth Factor Dimer 1 U . S . Patent Sep. 12 , 2017 Sheet 4 of 23 US 9 ,758 ,577 B2 Figure 4 Free latency Associated Peptide (LAP ) Dimer Trigger loop. man Bowtie region Alpha 2 helix mana Arm region Fastener region Latency loop ( also refer?d to as t?? latency lasso ) Amman Alpha 1 helix Residues for extracellular protein asscaths Free Growth Factor Dimer -~ ~ get egin U . S . Patent Sep. 12 , 2017 Sheet 5 of 23 US 9 ,758 ,577 B2 Figure 5 An embodiment of a recombinant GPC 34fde ??d C -terminus maeneo Growth factor domain Proprotein convertase cleavage site Latency - associated peptide (LAP ) Cys residue - HSM SH N - terminus Histidine tags U . S . Patent Sep. 12 , 2017 Sheet 6 of 23 US 9 ,758 ,577 B2 Figure 6 An embodiment of a proprotein convertase cleavage site mutant ( e . g . RXXR - RXG ; D2G ) HSG LSH An embodiment of an N -terminal cysteine mutant ( e . g . C45: Cys4 - Ser ) ????? U . S . Patent Sep. 12 , 2017 Sheet 7 of 23 US 9 ,758 ,577 B2 Figure 7 Embodiments of recombinantproteins Hobok LTBA GARDE prOTGF- B proprotein prOTGF - 6 proprotein proTGF- B proprotein convertase cleavage conv?rtaså c { e \ ????? convertase cleavage site mutant with N site mutant complexed site mutant complexed terminal cysteine with LTBP with GARP mutation H00M TGF- B LAP with N TGF- B LAP complexed terminal cysteine with GARP mutation U . S . Patent Sep. 12 , 2017 Sheet 8 of 23 US 9 ,758 ,577 B2 Figure 8A at Heix GFBIHU some generaltownWWWWWWWWxwwwwwwwwwwwwwwwwww * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Howto bietetsocialemediergenerelt W * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * SUS X00 aime * AGPAAAAAAAAAARROVOCHOSSRBARSVAPOPO CHVCVXRON 46 ous * A8CPAAAAAAAAAAAGVOORRSSRPAPSAPDOPO CHVCVXROX $ point * * * * * * * * * * * * * * * * * * * * * * * * *XXSCOXUY * BXEGLCXICOXRON 2 in * * * * * * * * wwwww * * * * * www ENSBOKBW * XX10NACEYRON