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|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. . C07K 16 / 22 @(73 ) Assignee : Scholar Rock , Inc. , Cambridge, MA 424 / 130 . 1 (US ) 7 , 371 , 726 B2 5 / 2008 Junker et al. 7 , 432 , 079 B2 10 / 2008 Junker et al. @( * ) Notice : Subject to any disclaimer , the term of this 7 , 632, 499 B2 12 / 2009 Davies et al. patent is extended or adjusted under 35 7 , 635 , 760 B2 12 / 2009 Han et al. 7 , 750 , 128 B2 7 / 2010 Gegg et al . U . S . C . 154 ( b ) by 0 days . 7 , 785 , 587 B28 / 2010 Whittemore et al. 7 ,807 ,159 B2 10 /2010 Chin et al. This patent is subject to a terminal dis 7 , 888, 486 B2 2 / 2011 Walsh et al . claimer . 8 , 063 , 188 B2 11/ 2011 Sayers et al . 8 ,178 , 488 B2 5 /2012 Knopf et al. ( 21 ) Appl. No. : 15 / 187 ,278 8 , 420 , 082 B2 4 /2013 Veldman et al. 8 , 455 , 448 B2 6 / 2013 Jeanplong et al . (22 ) Filed : Jun . 20 , 2016 8 , 710, 016 B2 4 /2014 Seehra et al . 8 , 840, 894 B2 9 / 2014 Stitt et al . (65 ) Prior Publication Data 8 , 871 , 209 B2 10 / 2014 Stitt et al. 8, 933 ,199 B2 1/ 2015 Cload et al. US 2016 /0289318 A1 Oct. 6 , 2016 8 ,992 , 913 B2 3 /2015 Mader et al. 8, 999 ,343 B2 4 /2015 Han et al. 2003 /0129171 AL 7 /2003 Grobet et al. Related U .S . Application Data 2005 /0250936 A1 11/ 2005 Oppermann et al. 2006 / 0034831 A1 2 / 2006 Tobin ( 60 ) Division of application No . 14 /795 , 033 , filed on Jul. 2006 /0263354 A1 * 11/ 2006 Chin CO7K 16 / 22 9 , 2015 , now Pat . No. 9, 399, 676 , which is a 424 / 133 . 1 continuation of application No . PCT/ US2014 /036933 , filed on May 6 , 2014 . (Continued ) FOREIGN PATENT DOCUMENTS ( 60 ) Provisional application No . 61/ 819 , 840 , filed on May EP 0404097 12 / 1990 6 , 2013 , provisional application No . 61/ 823 , 552 , filed EP 2422811 2 / 2012 on May 15 , 2013 , provisional application No . ( Continued ) 61/ 900, 438 , filed on Nov . 6 , 2013 . (51 ) Int. Cl. 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 . PATENT DOCUMENTS Japanese Office Action for JP application No . 2015 - 540878 dated Apr . 26 , 2016 . 2007 /0178095 A18 / 2007 Smith et al . 2008 /0206219 AL 8 / 2008 Coussens et al. Notification of Transmittal of the International Search Report and 2008 / 0279834 AL 11/ 2008 Garaczi et al . the Written Opinion of the International Searching Authority , or the 2009 /0136481 AL 5 / 2009 Kambadur et al. Declaration , from International Application No . PCT /US2013 / 2010 /0322925 Al 12 / 2010 Knopf et al . 68613 , dated May 7, 2014 . 2011/ 0129469 A1 6 / 2011 Koncarevic et al . Battaglia et al. The Tregs ' world according to GARP. Eur J 2011/ 0250198 A 10 / 2011 Wolfman et al. Immunol. Dec . 2009 ; 39 ( 12 ) : 3296 - 300 . 2011/ 0256132 Al 10 / 2011 Ashman et al. Tran DQ . TGF - B : the sword , the wand , and the shield of FOXP3 ( = ) regulatory T cells. J Mol Cell Biol. 2012 (Feb ; 4 ( 1) : 29 -37 . FOREIGN PATENT DOCUMENTS Cuende , Julia et al . “ Monoclonal antibodies against GARP / TGF -B1 complexes inhibit the immunosuppresive activity of human regu ?? 2832747 2 / 2015 WO 92 / 00330 AL 1 / 1992 latory T cells in vivo ” , Sci . Transl. Med ., Apr. 22 , 2015 , vol. 7, Issue WO 94 /09812 A1 5 / 1994 284 , pp . 1 - 12 . WO 00 / 43781 7 / 2000 Cuende, Julia et al. “ Supplemental Materials for Monoclonal anti WO 00 /66620 11 / 2000 bodies against GARP / TGF -B1 complexes inhibit the immunosup WO 01/ 12777 2 / 2001 presive activity of human regulatory T cells in vivo ” , Sci . Transl. WO 02 / 09641 2 / 2002 Med ., Apr. 22 , 2015 , vol. 7 , Issue 284 , pp . 1 - 10 . WO 2005 /010049 2 / 2005 International Preliminary Report on Patentability , International WO 2006 / 106599 Al 10 / 2006 WO 2006 / 116002 11/ 2006 Application No. PCT/ US2014 / 036933 , dated Nov . 19 , 2015 . WO 2007 /050793 A2 3 / 2007 Taylor, R .G . et al ., 1985 . 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

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Growth Factor Dimer Growth factor monomer Non - reducing Reducing Colloidal blue stain US 9 , 758 ,577 B2 COMPOSITIONS AND METHODS FOR arm regions, fingers regions, N -terminal regions for extra GROWTH FACTOR MODULATION cellular associations, latency loops, alpha 1 helical regions, alpha 2 helical regions , RGD sequence regions, trigger loop CROSS REFERENCE TO RELATED regions and bowtie regions. In some embodiments , recom APPLICATIONS 5 binant proteins of the present invention may comprise one or more protein modules from a vertebrate species. In some This application is a divisional application of U . S . Utility embodiments , recombinant proteins of the present invention may comprise one or more protein modules comprising one application Ser . No. 14 /795 , 033 , now U . S . Pat . No. 9 ,399 , or more mutations . In some embodiments , recombinant 656 , filed Jul. 9, 2015 entitled Compositions and Methodsf 10 proteins of the present invention may comprise one or more for Growth Factor Modulation , which is a continuation of 10 mutations comprising one or more furin cleavage site International Application No. PCT/ US2014 / 036933 , filed regions . In some embodiments , such mutations may prevent May 6 , 2014 entitled Compositions and Methods for Growth enzymatic cleavage of recombinant proteins of the present Factor Modulation , which claims priority to U . S . Provi invention . In some embodiments , recombinant proteins of sional Patent Application No. 61/ 819 ,840 filed May 6 , 2013of , 15 the present invention may comprise one or more mutations entitled Compositions and Methods for Growth Factor comprising a mutation of the amino acid sequence RXXR to Modulation , U . S . Provisional Patent Application No . the amino acid sequence RXG . In some embodiments , 61 /823 ,552 filed May 15 , 2013 , entitled Compositions and recombinant proteins of the present invention may comprise Methods for Growth Factor Modulation and U .S . Provi one or more mutations comprising a mutation of the amino sional Patent Application No . 61/ 900 ,438 filed Nov . 6 , 2013etor , 20 acid sequence RXXR to the amino acid sequence AXXA . In entitled Compositions and Methods for Growth Factor 20 some embodiments , recombinant proteins of the present Modulation , the contents of each of which are herein incor invention may comprise one or more mutations comprising porated by reference in their entireties . N -terminal regions for extracellular associations. In some embodiments , recombinant proteins of the present invention SEQUENCE LISTING 25 may comprise one or more mutations comprising substitu The instant application contains a Sequence Listing which tion and / or deletion of at least one cysteine residue present has been submitted electronically in ASCII format and is within about the first 4 , 5 , 6 or 7 N - terminal amino acid hereby incorporated by reference in its entirety . Said ASCII residues. In some embodiments , recombinant proteins of the copy , created on Jun . 17 , 2016 , is named 2035 _ 1001USDI present invention may comprise one or more substitution of V _ SL .txt and is 705 ,464 bytes in size . 30 at least one cysteine residue with at least one serine residue. In some embodiments , recombinant proteins of the pres FIELD OF THE INVENTION ent invention may be complexed with a protein selected from the group consisting of LTBP1, LTBP1S , LTBP2 , Embodiments of the present invention may include LTBP3 , LTBP4 , fibrillin - 1 , fibrillin - 2 , fibrillin - 3 , fibrillin - 4 , recombinant proteins as well as antibodies directed to such 35 GARP , LRRC33 and a combination or fragment thereof. In proteins . In some embodiments , such proteins and antibod some embodiments , recombinant proteins of the present ies may be related to the field of TGF- B family member invention may comprise one or more detectable labels . Such biology. detectable labels may comprise biotin labels, polyhistidine tags and /or flag tags . BACKGROUND OF THE INVENTION 40 In some embodiments , the present invention provides chimeric proteins comprising one or more protein modules Cell signaling molecules stimulate a variety of cellular from at least two TGF- b - related proteins wherein said activities . Such signaling is often tightly regulated , often protein modules may be selected from the group consisting through interactions with other biomolecules , the extracel - of growth factor prodomain complexes (GPCs ) , latency lular and /or cellular matrix or within a particular cell envi- 45 associated peptides (LAPs ) , LAP - like domains, straight ronment or niche . Such interactions may be direct or indi- jacket regions , growth factor domains, fastener regions , rect. furin cleavage site regions , arm regions, fingers regions, Cell signaling cascades are involved in a number of N -terminal regions for extracellular associations , latency diverse biological pathways including , but not limited to loops, alpha 1 helical regions, RGD sequence regions , modulation of cell growth , modulation of tissue homeosta - 50 trigger loop regions , bowtie regions and any of those listed sis , extracellular matrix ( ECM ) dynamics , modulation of in Tables 2 , 3 and 11. In some embodiments , chimeric cell migration , invasion and immune modulation / suppres - proteins of the present invention may comprise one or more sion . In some cases, proteins involved in cell signaling are protein modules selected from one or more vertebrate spe synthesized and/ or are sequestered in latent form , requiring cies . In some embodiments, chimeric proteins of the present stimulus of some kind to participate in signaling events . 55 invention may comprise GPCs. In some embodiments , such There remains a need in the art for agents , tools and methods GPCs may comprise at least one LAP or LAP - like domain for modulating cell signaling and / or cellular activities . from a TGF - B family member and at least one growth factor domain from a TGF - B family member wherein the LAP or SUMMARY OF THE INVENTION LAP - like domain and the growth factor domain are from 60 different TGF - B family members. In some embodiments , In some embodiments , the present invention provides chimeric proteins of the present invention may comprise at recombinant proteins comprising one or more TGF - B - re - least one LAP or LAP - like domain and at least one growth lated proteins comprising one or more protein modules factor domain , each of which is selected from the group selected from the group consisting of growth factor prodo - consisting of TGF- B1 , TGF -B2 , TGF -B3 , GDF - 8 , GDF - 11 main complexes (GPCs ) , latency associated peptides 65 and inhibin beta A . In some embodiments , chimeric proteins (LAPs ) , LAP - like domains , straight jacket regions, growth of the present invention may comprise one or more GPC factor domains , fastener regions, furin cleavage site regions, wherein at least one N -terminal region is from a TGF - B US 9 , 758 ,577 B2 family member, at least one C - terminal region is from a (ELISA ) and /or a fluorescence -associated cell sorting TGF - B family member and wherein the N -terminal region (FACS ) -based assay. In some cases, recombinant proteins of and C - terminal region are from different TGF - B family such assays may be complexed with a protein selected from members . In some embodiments , chimeric proteins of the the group consisting of SEQ ID NOs: 153 - 161 and 286 - 292 present invention may comprise at least one N - terminal 5 or complexed with a protein selected from the group con region and at least one C -terminal region selected from sisting of LTBP1, LTBP1S , LTBP2 , LTBP3 , LTBP4, fibril TGF - B1 terminal regions, TGF - B2 terminal regions , TGF lin - 1 , fibrillin -2 , fibrillin - 3 , fibrillin - 4 , GARP , LRRC33 , B3 terminal regions , GDF - 8 terminal regions , GDF - 11 ter minal regions and inhibin beta A terminal regions . In some perlecan , decorin , elastin and collagen . In some cases , embodiments , chimeric proteins of the present invention 10 recombinant proteins may comprise a chimeric protein com may comprise a GPC from at least one TGF- B family prising an amino acid sequence selected from the group member comprising at least one arm region from a different consisting of SEQ ID NOs: 199 - 236 and 273 . TGF - B family member. In some embodiments , chimeric Other methods of selecting a desired antibody may com proteins of the present invention may comprise a GPC prisepri the steps of 1 ) providing a growth factor activity assay , comprising at least one TGF - B family member comprising at 15 2 ) contacting the growth factor activity assay with one or least one trigger loop region from a different TGF - B family more candidate antibodies, 3 ) obtaining growth factor activ member. In some embodiments , chimeric protein of the ity data and 4 ) selecting a desired antibody based on the present invention may comprise any of the protein module growth factor activity data . Growth factor activity assays combinations listed in Table 12 . according to such methods may comprise cell -based assays In some embodiments , chimeric protein of the present 20 selected from the group consisting of luciferase- based invention may be complexed with a protein selected from assays and proliferation assays . Such cell -based assays may the group consisting of LTBP1, LTBP1S , LTBP2 , LTBP3 , comprise one or more expression cells that express one or LTBP4 , fibrillin - 1, fibrillin - 2 , fibrillin - 3 , fibrillin - 4 , GARP more recombinant protein of the invention or a complex and LRRC33 and a combination or fragment thereof. In thereof . Such assays may further comprise one or more some embodiments , chimeric proteins of the present inven - 25 responsive cells that yield gene expression data and /or tion may comprise one or more detectable labels . In some embodiments , such detectable labels may comprise at least In some embodiments , the present invention provides one biotin label, polyhistidine tag and /or flag tag . pharmaceutical compositions comprising one or more of any In some embodiments , the present invention provides an of the recombinant proteins described herein , one or more of antibody directed to any of the recombinant proteins and /or 30 any of the chimeric proteins described herein and /or one or chimeric proteins disclosed herein . In some embodiments , more of any of the antibodies described herein and at least such antibodies comprise monoclonal antibodies . In some one pharmaceutically excipient. embodiments , antibodies of the present invention are sub - Some methods of the invention comprise treatment of a stantially isolated . In some embodiments , monoclonal anti - TGF- B -related indication in a subject comprising contacting bodies of the present invention are stabilizing antibodies . In 35 said subject with a composition of the invention . TGF - B some embodiments , stabilizing antibodies of the present related indications may include fibrotic indications ( e . g . lung invention reduce the level of free growth factor relative to fibrosis , kidney fibrosis , liver fibrosis , cardiovascular fibro the level of growth factor associated with one or more GPC . sis , skin fibrosis , and bone marrow fibrosis ) , myelofibrosis , In some embodiments , stabilizing antibodies may reduce cancer or cancer- related conditions ( e. g . colon cancer, renal growth factor- dependent cellular signaling . In some embodi- 40 cancer , breast cancer , malignant melanoma and glioblas ments , monoclonal antibodies of the present invention may toma ) and muscle disorders and /or injuries [ e . g . cachexia , comprise releasing antibodies . Such antibodies may increase muscular dystrophy, chronic obstructive pulmonary disease the level of free growth factor relative to the level of growth (COPD ) , motor neuron disease , trauma , neurodegenerative factor associated with one or more GPC . In some embodi- disease , infection , rheumatoid arthritis , immobilization , sar ments , releasing antibodies of the present invention may 45 copenia , inclusion body myositis and diabetes. ] increase growth factor- dependent cellular signaling . In some embodiments , the invention provides a kit com In some embodiments , the present invention provides prising a composition of the invention and instructions for compositions comprising one or more of any of the recom use thereof. binant proteins , one or more of any of the chimeric proteins and /or one or more of any of the antibodies described herein 50 BRIEF DESCRIPTION OF THE FIGURES combined with at least one excipient. In some embodiments , the present invention provides The foregoing and other objects , features and advantages methods of modulating the level of free growth factor in a will be apparent from the following description of particular subject or cell niche comprising the use of one or more embodiments of the invention , as illustrated in the accom compositions described herein . In some such methods , the 55 panying drawings . The drawings are not necessarily to scale , level of growth factor signaling is modulated . emphasis instead being placed upon illustrating the prin In some embodiments , the present invention provides ciples of various embodiments of the invention . methods for selecting a desired antibody comprising the use FIG . 1 is a diagram of the TGF- beta superfamily tree , of one or more assays, wherein such assays comprise one or where divergence is proportional to branch length . more recombinant protein of the invention . Some such 60 FIG . 2 is a schematic of one embodiment of a linear methods comprise the steps of 1 ) providing an antibody representation of a translated growth factor monomer . In binding assay , 2 ) contacting the binding assay with one or such embodiments , translated growth factors may comprise more candidate antibodies, 3 ) obtaining binding data related secretion signal peptides , prodomains and growth factor to candidate antibody affinity for the one or more recombi - domains . In embodiments according to embodiment nant protein and 4 ) selecting a desired antibody based on the 65 depicted here , translated growth factors may also comprise binding data . Binding assays according to such methods a cleavage site between prodomain and growth factor may include an enzyme- linked immunosorbent assay regions. US 9 , 758 ,577 B2 FIG . 3 is a schematic of one embodiment of a growth influence within biological systems , growth factor signaling factor- prodomain complex (GPC ) as well as an embodiment is tightly regulated , often through interactions with other of a free growth factor dimer and a free latency associated biomolecules, the extracellular and / or cellular matrix or peptide (LAP ) dimer . The arrow indicates the ability of within a particular cell environment or niche . These inter proteins according to this embodiment to alter between free 5 actions may be direct or indirect. and complexed forms. Growth factors of the transforming growth factor beta FIG . 4 is a schematic of one embodiment of a free LAP ( TGF - B ) family are involved in a variety of cellular pro dimer and a free growth factor dimer with labeled features cesses . Growth factor binding to type II receptors leads to and / or protein modules . type I receptor phosphorylation and activation (Denicourt , FIG . 5 is a schematic of an embodiment of a recombinant , GPC . C . et al. , Another twist in the transforming growth factor FIG . 6 is a schematic of embodiments of mutant recom B - induced cell -cycle arrest chronicle . PNAS. 2003. 100 ( 26 ) : binant GPCs. 15290 - 1 . ) Activated type I receptors may in turn phospho FIG . 7 depicts schematic representations of five recom rylate receptor- associated SMADs ( R - SMADs) promoting binant proteins alone or in complex with LTBP or GARP. co - SMAD ( e . g . SMAD4 ) dimer/ trimer formation and FIGS. 8A - 8G show structure -based alignment between 15 nuclear translocation . SMAD complexes collaborate with TGF - B family member proteins ( SEO ID NOS 1 , 117 , 116 , cofactors to modulate expression of TGF - ß family member 296 , 2 - 4 , 137 , 5 , 131, 125 , 6 , 14 , 21, 23 - 24 , 27 , 26 , 28 , and target genes. 10 , respectively , in order of appearance ) [adapted from Shi TGF - B family member signaling cascades are involved in et al ( Shi, M . et al. , Latent TGF - B structure and activation . a number of diverse biological pathways including , but not Nature . 2011 Jun . 15 ; 474 (7351 ) :343 - 9 , the contents of 20 limited to inhibition of cell growth , tissue homeostasis , which are herein incorporated by reference in their extracellular matrix ( ECM ) remodeling , endothelial to mes entirety. ) ] Cysteine residues required for interaction with enchymal transition (EMT ) in cell migration and invasion LTBPs and / or GARPs are boxed . Residues mutated in and immune modulation / suppression as well as in mesen Camurati - Engelmann syndrome are indicated with a star. chymal to epithelial transition . TGF - B signaling related to Protease cleavage sites are indicated with an up arrow . 25 growth inhibition and tissue homeostasis may affect epithe Protein modules and secondary structural elements are indi - lial , endothelial , hematopoietic and irimmune cells through cated with solid bars . Residues underlined at the N -terminus the activation of p21 and p15 /NK to mediate cell cycle arrest ofGDF - 8 correspond to alternatively predicted signal pep and repress myc . In relation to ECM remodeling , TGF - B tide processing sites. “ Chimeric module breakpoints” indi signaling may increase fibroblast populations and ECM cate regions where structural features are conserved and provide modules for chimeric protein construction (swap 30 deposition ( e . g . collagen ) . TGF- B signaling related to cell ping of modules between family members) in all family migration and invasion may affect epithelial and / or endothe members . N - terminal regions are shown in FIGS . 8A and lial cells , inducing stem cell - like phenotypes . This aspect of 8B , internal regions are shown in FIGS . 8C and 8D and signaling may play a role in smooth muscle cell proliferation C -terminal regions are shown in FIGS . 8E - 8G . following vascular surgery and / or stenting. In the immune FIGS . 9A - 9C present 3 tables showing the percent iden - 35 system , TGF - ß ligand is necessary for T regulatory cell tity between amino acid sequences found in the TGF - B function and maintenance of immune precursor cell growth family . FIG . 9A demonstrates percent identity among pro and homeostasis . Nearly all immune cells comprise recep proteins (prodomain and growth factor. ) Percent identity tors for TGF -B and TGF - B knockout mice die postnataly due among growth factor domains is presented in FIG . 9B while in part to inflammatory pathologies . Finally , TGF - B sup percent identity among prodomains is presented in FIG . 9C . 40 presses interferon gamma- induced activation of natural FIG . 10 presents an alignment conducted between GDF - 8 killer cells (Wi , J. et al. , 2011. Hepatology. 53 ( 4 ) : 1342 - 51 , (myostatin ) (SEQ ID NO : 5 ) , GDF - 11 (SEQ ID NO : 4 ) , the contents ofwhich are herein incorporated by reference in Inhibin A (SEQ ID NO : 6 ) and a GDF - 8 dimer ( SEQ ID NO : their entirety .) 297 ) . Arrows indicate cleavage sites . Regions involved in The recent solution of the crystal structure of the latent internal interactions are boxed . Solid rectangles appear 45 form of TGF - beta is a first for the entire TGF -beta family above residues predicted to be involved in steric clashes in chimeric constructs . Stars denote important break points in and offers deep insights into these complexes ( Shi, M . et al. , protein modules . Latent TGF - B structure and activation . Nature. 2011 Jun . FIG . 11 depicts the expression and purification of recom 15 ; 474 ( 7351) :343 - 9 ). Almost all signaling in the TGF -beta binant antigens and antigen complexes ( Coomassie Blue family goes through a common pathway whereby a dimeric stained SDS - PAGE ) . 50 ligand is recognized by a heterotetrameric receptor complex FIG . 12 presents results from analyses of cell lines stably containing two type 1 and two type II receptors . Each expressing TGF -B1 /GARP complexes. 300 . 19 cells stably receptor has a serine -threonine kinase domain . Type II transfected with empty vector control ( A ) , proTGF -B1 - receptors phosphorylate type I receptors, which in turn GARP ( B ) or TGF -B1 LAP -GARP ( C ) were fluorescently phosphorylate receptor- regulated Smads that translocate to labeled with antibodies directed to expressed proteins and 55 and accumulate in the nucleus and regulate transcription . examined for fluorescence intensity by flow cytometry . There are 33 differentmembers of the TGF -beta family in Luciferase assay data is presented in ( D ) showing TGF - B humans ( FIG . 1 ) . Members include the bone morphogenetic signaling activity resulting from co - culture of these cells proteins (BMP ) , inhibin , activin , growth and differentiation with cells expressing avßo integrin . factor (GDF ) , myostatin , nodal, anti -Mullerian hormone , FIG . 13 depicts recombinant histidine -tagged proGDF - 8 , 60 and lefty proteins. A review of TGF- B family members, separated by SDS - PAGE under reducing and non - reducing related signaling molecules as well as their relationships can conditions , as visualized by Coomassie staining. be found in Massague ., 2000 . Nature Reviews Molecular Cell Biology. 1: 169 - 78 , the contents of which are herein DETAILED DESCRIPTION incorporated by reference in their entirety . In some embodi 65 ments, mature growth factors are synthesized along with Growth factors are cell signaling molecules that stimulate their prodomains as single polypeptide chains (see FIG . 2 ). a variety of cellular activities . Due to their broad - reaching In some embodiments , such polypeptide chains may com US 9 , 758 ,577 B2 prise cleavage sites for separation of prodomains from invention may comprise antigens comprising one or more mature growth factors . In some embodiments , such cleavage components of one or more TGF- b -related proteins . Some sites are furin cleavage sites recognized and cleaved by tools may comprise antibodies directed toward antigens of the present invention . In additional embodiments , tools of proprotein convertases . v member 5 the present invention may comprise assays for the detection In general, homology among TGF- B family member 5 and / or characterization of TGF - B -related proteins , the detec growth factor domains is relatively high . Interestingly , pro tion and/ or characterization of antibodies directed toward domain homology is much lower . This lack of homology TGF - B - related proteins and / or the detection and / or charac may be an important factor in altered growth factor regula terization of cellular activities and / or their cellular signaling tion among family members. In some cases, prodomains related to TGF- b -related proteins. may guide proper folding and / or dimerization of growth Proteins of Interest factor domains . Prodomains have very recently been recog TGF - B - related proteins are involved in a number of nized , in some cases , to have important functions in direct cellular processes. In embryogenesis , the 33 members of the ing growth factors (after secretion ) to specific locations in TGF - B family of proteins are involved in regulating major the extracellular matrix ( ECM ) and/ or cellular matrix , until - developmental processes and the details of the formation of other signals are received that cause growth factor release many organs . Much of this regulation occurs before birth ; from latency . Release from latency may occur in highly however , the family continues to regulate many processes localized environments whereby growth factors may act after birth , including, but not limited to immune responses, over short distances ( e . g . from about 1 cell diameter to about wound healing , bone growth , endocrine functions and a few cell diameters, from about 2 cell diameters to about muscle mass. TGF - B -related proteins are listed and 100 cell diameters and /or from about 10 cell diameters to described in U .S . Provisional Patent Applications 61/ 722 , about 10 , 000 cell diameters ) and cleared once they reach the 919 , filed Nov. 6 , 2012 ; 61 /722 , 969, filed Nov. 6 , 2012 and circulation . Some growth factor- prodomain complexes are 61/ 823 , 552 , filed May 15 , 2013 the contents of each of secreted as homodimers . In some embodiments , prodomain which are herein incorporated by reference in their entire growth factor complexes may be secreted as heterodimers . -cties . As used herein , the term “ TGF - B -related protein ” refers to - A list of exemplary TGF- B family pro -proteins , i . e . the a TGF- B isoform , a TGF - B family member or a TGF- B protein after removal of the secretion signal sequence, is family member- related protein . TGF - B family members may shown in Table 1 . The pro - protein contains, and is the include , but are not limited to any of those shown in FIG . 1 precursor of, the prodomain and the growth factor. Shown in and / or listed in Table 1. These include , but are not limited to the Table are the names of the originating TGF - B family TGF- B proteins , BMPs, myostatin , GDFs and inhibins. In member and the pro - protein sequence . Also identified in some embodiments, the present invention provides tools “ bold ” and “ underlined ” are proprotein convertase cleavage and / or methods for isolating, characterizing and or modu sites. Upon cleavage , the resulting prodomain retains this lating TGF - B -related proteins . Aspects of the present inven site , whereas the mature growth factor begins following the tion provide tools and / or methods for characterizing and/ or ac cleavage site . It is noted that Leftyl and Lefty2 are not modulating cellular activities related to TGF - B -related pro cleaved by proprotein convertases just prior to the start of tein signaling . In other embodiments , tools of the present the mature growth factor. TABLE 1 Pro - proteins of the TGF - beta family

SEO TGF Member Prodomain and growth factor Sequence ID NO TGF - B1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLA 1 LYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKOSTH SIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEOHVELYOKYSNNSW RYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAHCSCDSRD NTLOVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAOHLOSSRHRRA LDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYI WSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEOL SNMIVRSCKCS TGF - B2 SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVIS 2 IYNSTRDLLOEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIP PTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELY QILKSKDLTSPTORYIDSKVVKTRAEGEWLSFDVTDAVHEWLEIHKDRNL GFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIK STRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCC LRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTOHSRVLSLYN TINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS TGF - B3 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVL 3 ALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNEL AVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEORIEL FQILRPDEHIAKORYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLG LEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQ KDEIHNPHLILMMIPPHRLDNPGOGGORKKRALDTNYCFRNLE LYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHS TVLGLYNTLN PEASASPCCVPODLEPLTILYYVGRTPKVEQLSNMVVKSCKCS US 9 , 758 ,577 B2

TABLE 1 - continued Pro -proteins of the TGF -beta family

SEO TGF Member Prodomain and growth factor Sequence ID NO GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSREL 4 RLESIKSOILSKLRLKEAPNISREVVKOLLPKAPPLOOILDLHDFOGDAL QPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTK VLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSL KIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSL GPGAEGLHPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGQCEYMFMOKYPHTHLVQQANPRGSAGPCCTP TKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF - 8 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSKLRLETAPNIS 5 (myostatin ) KDVIROLLPKAPPLRELIDQYDVORDDSSDGSLEDDDYHATTETIITMPT ESDFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIYLRPVETPTTVFVOI LRLIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLQNWLKQPESNL GIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCD EHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPH THLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQI IYGKIPAMVVDRCG CS Inhibin - beta ASPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEMVEAVKKHILNMLHLK KRPDVTOPVPKAALLNAIRKLHVGKVGENGYVEI EDDIGRRAEMNELMEO TSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRT KVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWH VFPVSSSIQRLLDOGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEG KKKGGGEGGAGADEEKEOSHRPFLMLOAROSEDHPHRRRRRGLECDGKVN ICCKKOFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFH STVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGONIIKKDIONMI VEECGCS Inhibin - beta BSPTPPPTPAAPPPPPPPGSPGGSODTCTSCGGFRRPEELGRVDGDFLEA VKRHILSRLOMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLD GHASPGADGOERVSEIISFAETDGLASSRVRLYFFISNEGNONLFVVOAS LWLYLKLLPYVLEKGSRRKVRVKVYFQEQGHGDRWNMVEKRVDLKRSGWH TFPLTEAIQALFERGERRLNLDVOCDSCOELAVVPVFVDPGEESHRPFVV VQARLGDSRHRIRKRGLECDGRTNLCCRQQFFIDFRLIGWNDWIIAPTGY YGNYCEGSCPAYLAGVPGSASSFHTAVVNOYRMRGLNPGTVNSCCIPTKL STMSMLYFDDEYNIVKRDVPNMIVEECGCA

Inhibin - beta CTPRAGGOCPACGGPTLELESORELLLDLAKRSILDKLHLTORPTLNRPV SRAALRTALOHLHGV POGALLEDNREOECEIISFAETGLSTINOTRLDFH FSSDRTAGDREVOOASLMFFVOLPSNTTWTLKVRVLVLGPHNTNLTLATO YLLEVDASGWHQLPLGPEAQAACSQGHLTLELVLEGQVAQSSVILGGAAH RPFVAARVRVGGKHQIHRRGIDCQGGSRMCCRQEFFVDFREIGWHDWIIQ PEGYAMNFCIGQCPLHIAGMPGIAASFHTAVLNLLKANTAAGTTGGGSCC VPTARRPLSLLYYDRDSNIVKTDIPDMVVEACGCS Inhibin - beta EOGTGSVCPSCGGSKLAPOAERALVLELAKOOILDGLHLTSRPRI THPPP 9 OAALTRALRRLOPGSVAPGNGEEVISFATVTDSTSAYSSLLTFHLSTPRS HEILYHARLWLHVLPTLPGTLCLRIFRWGPRRRROGSRTLLAEHHITNLG WHTLTLPSSGLRGEKSGVLKLQLDCRPLEGNSTVTGQPRRLLDTAGHQQP FLELKIRANEPGAGRARRRTPTCEPATPLCCRRDHYVDFOELGWRDWILO PEGYQLNYCSGQCPPHLAGSPGIAASFHSAVFSLLKANNPWPASTSCCVP TARRPLSLLYLDHNGNVVKTDVPDMVVEACGCS

Lefty1 LTGEOLLGSLLROLOLKEVPTLDRADMEELVIPTHVRAQYVALLORSHGD 10 RSRGKRFSOS FREVAGRFLALEASTHLLVFGMEORLPPNSELVOAVLRLF OEPVPKAALHRHGRLSPRSARARVTVEWLRVRDDGSNRTSLIDSRLVSVH ESGWKAFDVTEAVNFWOQLSRPROPLLLOVSVQREHLGPLASGAHKLVRF ASOGAPAGLGEPOLELHTLDLGDYGAOGDCDPEAPMTEGTRCCROEMYID LOGMKWAENWVLEPPGFLAYECVGTCROPPEALAFKWPFLGPROCIASET DSLPMIVSIKEGGRTRPQVVSLPNMRVQKCSCASDGALVPRRLQP Lefty2 LTEEQLLGSLLRQLQLSEVPVLDRADMEKLVIPAHVRAQYVVLLRRSHGD 11 RSRGKRFSOSFREVAGRFLASEASTHLLVFGMEORLPPNSELVOAVLRLF OEPVPKAALHRHGRLSPRSAQARVTVEWLRVRDDGSNRTSLIDSRLVSVH ESGWKAFDVTEAVNFWOOLSRPROPLLLOV SVOREHLGPLASGAHKLVRF ASQGAPAGLGEPQLELHTLDLRDYGAQGDCDPEAPMTEGTRCCRQEMYID LQGMKWAKNWVLEPPGFLAYECVGTCQQPPEALAFNWPFLGPROCIASET ASLPMIVSIKEGGRTRPQVVSLPNMRVQKCSCASDGALVPRRLQP GDF - 15 LSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTD 12 LVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSP TASRSWDVTRPLRROLSLARPOAPALHLRLSPPPSOSDOLLAESSSARPO LELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWV LSPREVOVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLICKTDTGVSLQTYDDLLAKDCHCI US 9 ,758 ,577 B2 11 TABLE 1 - continued Pro -proteins of the TGF -beta family

SEO TGF Member Prodomain and growth factor Sequence ID NO

Anti - Mullerian LLGTEALRAEEPAVGTSGLIFREDLDWPPGIPQEPLCLVALGGDSNGSSS 13 hormone PLRVVGALSAYEQAFLGAVQRARWGPRDLATFGVCNTGDRQAALPSLRRL GAWLRDPGGQRLVVLHLEEVTWEPTPSLRFQEPPPGGAGPPELALLVLYP GPGPEVTVTRAGLPGAQSLCPSRDTRYLVLAVDRPAGAWRGSGLALTLOP RGEDSRLSTARLOALLFGDDHRCFTRMTPALLLLPRSEPAPLPAHGOLDT VPFPPPRPSAELEESPPSADPFLETLTRLVRALRVPPARASAPRLALDPD ALAGFPOGLVNLSDPAALERLLDGEEPLLLLLRPTAATTGDPAPLHDPTS APWATALARRVAAELOAAAAELRSLPGLPPATAPLLARLLALCPGGPGGL GDPLRALLLLKALOGLRVEWRGRDPRGPGRAORSAGATAADGPCALRELS VDLRAERSVLIPETYQANNCQGVCGWPQSDRNPRYGNHVVLLLKMQVRGA ALARPPCCVPTAYAGKLLISLSEERISAHHVPNMVATECGCR

Inhibin - alpha COGLELARELVLAKVRALFLDALGPPAVTREGGDPGVRRLPRRHALGGFT 14 HRGSEPEEEEDVSOAILFPATDASCEDKSAARGLAQEAEEGLFRYMFRPS OHTRSROVTSAOLWFHTGLDROGTAASNSSEPLLGLLALSPGGPVAVPMS LGHAPPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAH TRTRPPSGGERARRSTPLMSWPWSPSALRLLORPPEEPAAHANCHRVALN ISFOELGWERWIVYPPSFIFHYCHGGCGLHIPPNLSLPVPGAPPTPAOPY SLLPGAOPCCAALPGTMRPLHVRTTSDGGYSFKYETVPNLLTOHCACI GDF - 1 PVPPGPAAALLQALGLRDEPQGAPRLRPVPPVMWRLFRRRDPQETRSGSR 15 RTSPGVTLOPCHVEELGVAGNIVRHIPDRGAP TRASEPASAAGHCPEWTV VFDLSAVEPAERPSRARLELRFAAAAAAAPEGGWELSVAOAGOGAGADPG PVLLROLVPALGPPVRAELLGAAWARNASWPRSLRLALALRPRAPAACAR LAEASLLLVTLDPRLCHPLARPRRDAEPVLGGGPGGACRARRLYVSFREV GWHRWVIAPRGFLANYCQGQCALPVALSGSGGPPALNHAVLRALMHAAAP GAADLPCCVPARLSPISVLFFDNSDNVVLROYEDMVVDECGCR GDF - 3 QEYVFLQFLGLDKAPSPQKFQPVPYILKKIFODREAAATTGVSRDLCYVK 16 FLPDOGFFLYPKKISQASSCLOKLLYFNLSAIKEREOLTL AQLGLDLGPNSYYNLGPELELALFLVQEPHVWGQTTPKPGKMFVLRSVPW PQGAVHFNLLDVAKDWNDNPRKNFGLFLEILVKEDRDSGVNFQPEDTCAR LRCSLHASLLVTLNPDOCHPSRKRRAAIPVPKLSCKNLCHRHOLFINFR DLGWHKWIIAPKGFMANYCHGECPFSLTISLNSSNYAFMQALMHAVDPEI PQAVCIPTKLSPISMLYQDNNDNVILRHYEDMVVDECGCG

GDF - 5 APDLGQRPQGTRPGLAKAEAKERPPLARNVFRPGGHSYGGGATNANARAK 17 GGTGQTGGLTQPKKDEPKKLPPRPGGPEPKPGHPPOTRQATARTVTPKGQ LPGGKAPPKAGSVPSSFLLKKAREPGPPREPKEPFRPPPITPHEYMLSLY RTLSDADRKGGNSSVKLEAGLANTITSFIDKGQDDRGPVVRKQRYVFDIS ALEKDGLLGAELRILRKKPSDTAKPAAPGGGRAAQLKLSSCPSGROPASL LDVRSVPGLDGSGWEVFDIWKLFRNFKNSAQLCLELEAWERGRAVDLRGL GFDRAAROVHEKALFLVFGRTKKRDLFFNEIKARSGODDKTVYEYLFSOR RKRRAPLATROGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAF HCEGLCEFPLRSHLEPTNHAVIOTLMNSMDPESTPPTCCVPTRLSPISIL FIDSANNVVYKQYEDMVVESCGCR GDF - 6 FOQASISSSSSSAELGSTKGMRSRKEGKMQRAPRDSDAGREGQEPQPRPO 18 DEPRAQQPRAQEPPGRGPRWPHEYMLSIYRTYSIAEKLGINASFFQSSK SANTITSFVDRGLDDLSHTPLRROKYLFDVSMLSDKEELVGAELRLFRQA PSAPWGPPAGPLHVOLFPCLSPLLLDARTLDPOGAPPAGWEVFDVWOGLR HQPWKQLCLELRAAWGELDAGEAEARARGPOOPPPPDLRSLGFGRRVRPP QERALLVVFTRSQRKNLFAEMREQLGSAEAAPGAGAEGSWPPPSGAPDA RPWLPSPGRRRRRTAFASRHGKRHGKKSRLRCSKKPLHVNFKELGWDDWI IAPLEYEAYHCEGVCDFPLRSHLEPTNHAIIQTLMNSMDPGSTPPSCCVP TKLTPISILYIDAGNNVVYKQYEDMVVESCGCR

GDF - 7 RDGLEAAAVLRAAGAGPVRSPGGGGGGGGGGRTLAQAAGAAAVPAAAVPR 19 ARAARRAAGSGFRNGSVVPHHFMMSLYRSLAGRAPAGAAAVSASGHGRAD TITGFTDQATQDESAAETGQSFLFDVSSLNDADEVVGAELRVLRRGSPES GPGSWTSPPLLLLSTCPGAARAPRLLYSRAAEPLVGORWEAFDVADAMRR HRREPRPPRAFCLLLRAVAGPVPSPLALRRLGFGWPGGGGSAAEERAVLV VSSRTORKESLFREIRAQARALGAALASEPLPDPGTGTASPRAVIGGRRR RRTALAGTRTAOGSGGGAGRGHGRRGRSRCSRKPLHVDFKELGWDDWIIA PLDYEAYHCEGLCDFPLRSHLEPTNHAIIQTLLNSMAPDAAPASCCVPAR LSPISILYIDAANNVVYKQYEDMVVEACGCR US 9 ,758 ,577 B2 13 14 TABLE 1 - continued Pro -proteins of the TGF -beta family

SEO TGF Member Prodomain and growth factor Sequence ID NO BMP - 10 SPIMNLEQSPLEEDMSLFGDVFSEQDGVDFNTLLQSMKDEFLKTLNLSDI 20 PTQDSAKVDPPEYMLELYNKFATDRTSMPSANIIRSFKNEDLFSQPVSFN GLRKYPLLFNVSIPHHEEVIMAELRLYTLVQRDRMIYDGVDRKITIFEVL ESKGDNEGERNMLVLVSGEIYGTNSEWETFDVTDAIRRWOKSGSSTHOLE VHIESKHDEAEDASSGRLEIDTSAONKHNPLLIVFSDDOSSDKERKEELN EMISHEQLPELDNLGLDSFSSGPGEEALLQMRSNIIYDSTARIRRNAKGN IDFKEIGWDSWIIAPPGYEAYECRGVCNYPLAEHLTPTKHAI IQALVHLKNSOKASKACCVPTKLEPISILYLDKGVVTYKFKYEGMAVSEC GCR BMP - 9 (GDF - 2 ) KPLQSWGRGSAGGNAHSPLGVPGGGLPEHTFNLKMFLENVKVDFLRSLNL 21 KTRVEPPOYMIDLYNRYTSDKSTTPASNIVRSFSMEDAISITA TEDFPFQKHILLFNISIPRHEQI TRAELRLYVSCONHVDPSHDLKGSVI YDVLDGTDAWDSATETKTFLVSODIQDEGWETLEVSSAVKRWVRSDSTKS KNKLEVTVESHRKGCDTLDISVPPGSRNLPFFVFSNDHSSGTKETRLEL REMI SHEQESVLKKLSKDGSTEAGESSHEEDTDGHVAAGSTLARRKRSAG AGSHCQKTSLRVNFEDIGWDSWIIAPKEYEAYECKGGCFFPLADDVTPTK HAIVOTLVHLKFPTKVGKACCVPTKLSPISVLYKDDMGVPTLKYHYEGMS VAECGCR Nodal TVATALLRTRGQPSSPSPLAYMLSLYRDPLPRADIIRSLQAEDVAVDGON 22 WTFAFDFSFLSQQEDLAWAELRLQLSSPVDLPTEGSLAIEIFHQPKPDTE QASDSCLERFQMDLFTVTLSQVTFSLGSMVLEVTRPLSKWLKRPGALEKO MSRVAGECWPRPPTPPATNVLLMLYSNLSOEOROLGGSTLLWEAESSWRA QEGOLSWEWGKRHRRHHLPDRSQLCRKVKFQVDFNLIGWGSWITYPKQYN AYRCEGECPNPVGEEFHPTNHAYIOSLLKRYOPHRVPSTCCAPVKTKPLS MLYVDNGRVLLDHEIKDMIVEECGCL BMP - 2 LVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLLSMFGLKORPTPSRDA 23 VVPPYMLDLYRRHSGOPGSPAPDHRLERAASRANTVRSFHHEESLEELPE TSGKTTRRFFFNLSSIPTEEFITSAELQVFREQMODALGNNSSFFIHRIN IYEIIKPATANSKFPVTRLLDTRLVNQNASRWESFDVTPAVMRWTAQGHA NHGFVVEVAHLEEKOGVSKRHVRISRSLHODEHSWSOIRPLLVTFGHDGK GHPLHKREKROAKHKORKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHA FYCHGECPFPLADHLNS TNHAIVOTLVNSVNSKIPKACCVPTELSAISML YLDENEKVVLKNYODMVVEGCGCR

BMP - 4 GASHASLIPETGKKKVAEIQGHAGGRRSGQSHELLRDFEATLLQMFGLRR 24 RPOPSKSAVIPDYMRDLYRLOSGEEEEEQIHSTGLEYPERPASRANTVRS FHHEEHLENIPGTSENSAFRFLFNLSSIPENEVISSAELRLFREQVDQGP DWERGFHRINIYEVMKPPAEVVPGHLITRLLDTRLVHHNVTRWETFDVSP AVLRWTREKQPNYGLAIEVTHLHQTRTHQGQHVRISRSLPQGSGNWAQLR PLLVTFGHDGRGHALTRRRRAKRSPKHHSQRARKKNKNCRRHSLYVDFSD VGWNDWIVAPPGYQAFYCHGDCPFPLADHLNS TNHAIVOTLVNSVNSSIP KACCVPTELSAISMLYLDEYDKVVLKNYQEMVVEGCGCR

BMP - 5 DNHVHSSFIYRRLRNHERREIOREILSILGLPHRPRPFSPGKOASSAPLF 25 MLDLYNAMTNEENPEESEYSVRASLAEETRGARKGYPASPNGYPRRIQLS RTTPLTTOSPPLASLHDTNFLNDADMVMSFVNLVERDKDFSHORRHYKEF RFDLTQIPHGEAVTAAEFRIYKDRSNNRFENETIKISIYQIIKEYTNRDA DLFLLDTRKAQALDVGWLVFDITVTSNHWVINPONNLGLOLCAETGDGRS INVKSAGLVGROGPOSKOPFMVAFFKASEVLLRSVRAANKRKNONRNKSS SHQDSSRMSSVGDYNTSEQKQACKKHELYVSFRDLGWODWIIAPEGYAAF YCDGECSFPLNAHMNATNHAIVOTLVHLMFPDHVPKPCCAPTKLNAISVL YFDDSSNVILKKYRNMVVRSCGCH

BMP - 6 CCGPPPLRPPLPAAAAAAAGGQLLGDGGSPGRTEQPPPSPQSSSGFLYRR 26 LKTQEKREMQKEILSVLGLPHRPRPLHGLQQPQPPALRQQEEQQQQQQLP RGEPPPGRLKSAPLFMLDLYNALSADNDEDGASEGERQQSWPHEAASSSO RROPPPGAAHPLNRKSLLAPGSGSGGASPLTSAODSAFLNDADMVMSFVN LVEYDKEFSPRORHHKEFKFNLSOIPEGEVVTAAEFRIYKDCVMGSFKNO TFLISIYQVLQEHQHRDSDLFLLDTRVVWASEEGWLEFDITATSNLWVVT PQHNMGLQLSVTRDGVHVHPRAAGLVGRDGPYDKOPFMVAFFKVSEVHV RTTRSASSRRROQSRNRSTQSQDVARVSSASDYNSSELKTACRKHELYVS FODLGWODWIIAPKGYAANYCDGECSFPLNAHMNATNHAIVOTLVHLMNP EYVPKPCCAPTKLNAISVLYFDDNSNVILKKYRNMVVRACGCH US 9 , 758 ,577 B2 15 TABLE 1 - continued Pro -proteins of the TGF -beta family

SEQ TGF Member Prodomain and growth factor Sequence ID NO BMP - 7 DFSLDNEVHSSFIHRRLRSQERREMOREILSILGLPHRPRPHLOGKHNSA 27 PMFMLDLYNAMAVEEGGGPGGOGFSYPYKAVFSTOGPPLASLODSHFLTD ADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKD YIRERFDNETFRISVYQVLQEHLGRESDLFLLDSRTLWASEEGWLVFDIT ATSNHWVVNPRHNLGLOLSVETLDGOSINPKLAGLIGRHGPONKOPFMVA FFKATEVHERSIRSTGSKORSQNRSKTPKNQEALRMANVAENSSSDQRQA CKKHELYVSFRDLGWODWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIV QTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACG CH BMP - 8A GGGPGLRPPPGCPQRRLGARERRDVOREILAVLGLPGRPRPRAPPAASRL 28 PASAPLFMLDLYHAMAGDDDEDGAPAEORLGRADLVMSFVNMVERDRALG HQEPHWKEFRFDLTQIPAGEAVTAAEFRIYKVPSIHLLNRTLHVSMFQVV QEQSNRESDLFFLDLOTLRAGDEGWLVLDVTAASDCWLLKRHKDLGLRLY VETEDGHSVDPGLAGLLGQRAPRSQQPFVVTFFRASPSPIRTPRAVRPLR RRQPKKSNELPQANRLPGIFDDVRGSHGROVCRRHELYVSFODLGWLDWV IAPOGYSAYYCEGECSFPLDSCMNATNHAILOSLVHLMKPNAVPKACCAP TKLSATSVLYYDSSNNVILRKHRNMVVKACGCH BMP - 8B GGGPGLRPPPGCPORRLGARERRDVOREILAVLGLPGRPRPRAPPAASRL 29 PASAPLFMLDLYHAMAGDDDEDGAPAERRLGRADLVMSFVNMVERDRALG HOEPHWKEFRFDLTOIPAGEAVTAAEFRIYKVPSIHLLNRTLHVSMFOVV OEOSNRESDLFFLDLOTLRAGDEGWLVLDVTAASDCWLLKRHKDLGLRLY VETEDGHSVDPGLAGLLGQRAPRSQQPFVVTFFRASPSPIRTPRAVRPLR RROPKKSNELPQANRLPGIFDDVHGSHGROVCRRHELYVSFODLGWLDWV IAPQGYSAYYCEGECSFPLDSCMNATNHAILOSLVHLMMPDAVPKACCAP TKLSATSVLYYDSSNNVILRKHRNMVVKACGCH BMP - 15 MEHRAQMAEGGOSSIALLAEAPTLPLIEELLEESPGEQPRKPRLLGHSLR 30 YMLELYRRSADSHGHPRENRTIGATMVRLVKPLTSVARPHRGTWHIQILG FPLRPNRGLYOLVRATVVYRHHLOL TRFNLSCHVEPWVOKNPTNHFPSSE GDSSKPSLMSNAWKEMDITOLVOQRFWNNKGHRILRLRFMCQQQKDSGGL ELWHGTSSLDIAFLLLYFNDTHKSIRKAKFLPRGMEEFMERESLLRRTRO ADGISAEVTASSSKHSGPENNOCSLHPFQISFRQLGWDHWIIAPPFYTPN YCKGTCLRVLRDGLNSPNHAI IQNLINQLVDQSVPRPSCVPYKYVPISVL MIEANGSILYKEYEGMIAESCTCR

GDF - 9 SOASGGEAQIAASAELESGAMPWSLLOHIDERDRAGLLPALFKVLSVGRG 31 GSPRLQPDSRALHYMKKLYKTYATKEGIPKSNRSHLYNTVRLFTPCTRHK QAPGDQVTGILPSVELLFNLDRITTVEHLLKSVLLYNINNSVSFSSAVKC VCNLMIKEPKSSSRTLGRAPYSFTFNSOFEFGKKHKWIQIDVTSLLOPLV ASNKRSIHMS INFTCMKDOLEHPSAONGLFNMTLVSPSLILYLNDTSAOA YHSWYSLHYKRRPSQGPDQERSLSAYPVGEEAAEDGRSSHHRHRRGQETV SSELKKPLGPASFNLSEYFROFLLPQNECELHDFRLSFSQLKWDNWIVAP HRYNPRYCKGDCPRAVGHRYGSPVHTMVONIIYEKLDSSVPRPSCVPAKY SPLSVLTIEPDGSIAYKEYEDMIATKCTCR BMP - 3 ERPKPPFPELRKAVPGDRTAGGGPDSELQPODKVSEHMLRLYDRYSTVQA 32 ARTPGSLEGGSQPWRPRLLREGNTVRSFRAAAAETLERKGLYIFNLTSLT KSENILSATLYFCIGELGNISLSCPVSGGCSHHAORKHIQIDLSAWTLKF SRNOSOLLGHLSVDMAKSHRDIMSWLSKDI TOLLRKAKENEEFLIGFNIT SKGROLPKRRLPFPEPYILVYANDAAISEPESVVSSLQGHRNFPTGTVPK WDSHIRAALSIERRKKRSTGVLLPLONNELPGAEYQYKKDEVWEERKPYK TLQAQAPEKSKNKKKORKGPHRKSOTLQFDEQTLKKARRKOWIEPRNCAR LKVDFADIGWSEWIISPKSFDAYYCSGACQFPMPKSLKPSNHATIOSI VRAVGVVPGIPEPCCVPEKMSSLSILFFDENKNVVLKVYPNMTVESCACR GDF - 10 SHRAPAWSALPAAADGLQGDRDLQRHPGDAAATLGPSAQDMVAVHMHRLY 33 EKYSROGARPGGGNTVRSFRARLEVVDOKAVYFFNLTSMODSEMILTATF WPRALEVLCKPRAKNASGRPLPLGPPTROHLLFRSLSONTAT OGLLRGAMALAPPPRGLWOAKDISPIVKAARRDG?LLLSAOLDSEERDPG VPRPSPYAPYILVYANDLAISEPNSVAVTLORYDPFPAGDPEPRAAPNNS ADPRVRRAAQATGPLODNELPGLDERPPRAHAQHFHKHQLWPSPFRALKP RPGRKDRRKKGQEVFMAASQVLDFDEKTMQKARRKQWDEPRVCSRRYLKV DFADIGWNEWIISPKSFDAYYCAGACEFPMPKIVRPSNHATIOSIVRAVG IIPGIPEPCCVPDKMNSLGVLFLDENRNVVLKVYPNMSVDTCACR GDNF FPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQFDDVMDFIQ 34 ATIKRLKRSPDKOMAVLPRRERNRQAAAANPENSRGKGRRGORGKNRGCV LTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRL VSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI US 9 , 758 ,577 B2 17 TABLE 1 - continued Pro -proteins of the TGF -beta family

SEO TGF Member Prodomain and growth factor Sequence ID NO NRTN IWMCREGLLLSHRLGPALVPLHRLPRTLDARIARLAQYRALLQGAPDAME 35 LRELTPWAGRPPGPRRRAGPRRRRARARLGARPCGLRELEVRVSELGLGY ASDETVLFRYCAGACEAAARVYDLGLRRLRORRRLRRERVRAQPCCRPTA YEDEVSFLDAHSRYHTVHELSARECACV PSPN WGPDARGVPVADGEFSSEQVAKAGGTWLGTHRPLARLRRALSGPCOLWSL 36 TLSVAELGLGYASEEKVIFRYCAGSCPRGARTQHGLALARLQGQGRAHGG PCCRPTRYTDVAFLDDRHRWORLPQLSAAACGCGG ARTN SLGSAPRSPAPREGPPPVLASPAGHLPGGRTARWCSGRARRPPPQPSRPA 37 PPPPAPPSALPRGGRAARAGGPGSRARAAGARGCRLRSQLVPVRALGLGH RSDELVRFRFCSGSCRRARSPHDLSLASLLGAGALRPPPGSRPVSOPCCR PTRYEAVSFMDVNSTWRTVDRLSATACGCLG

It is noted that some prodomains may be cleaved by . Depending on a variety of factors , growth factors may be proprotein convertase enzymes . As used herein , the term free or associated with one or more LAP or LAP - like " proprotein convertase ” refers to an enzyme that cleaves a domains. FIG . 3 is a schematic depicting an embodiment prodomain from a translated protein to facilitate protein wherein a growth factor dimer may associate with a LAP dimer. In some embodiments , GPCs comprise protein mod maturation . Some proprotein convertases of the present ules necessary for different aspects of growth factor signal invention include the subtilisin - like proprotein convertase 255 ing , secretion , latency and / or release from latent GPCs. As (SPC ) family member enzymes. The SPC family comprises used herein , the term " protein module ” refers to any com calcium -dependent serine endoproteases that include , but ponent, region and / or feature of a protein . Protein modules are not limited to furin / PACE , PC1/ 3 , PC2, PC4, PC5 / 6 , may vary in length , comprising one or more amino acids . PACE4 and PC7 ( Fuller et al. , 2009 . Invest Ophthalmol Vis Protein modules may be from about 2 amino acid residues Sci. 50 ( 12 ) :5759 -68 , the contents of which are herein incor - 30 in length to about 50 amino acid residues in length , from porated by reference in their entirety . ) GDF - 11 may in , in about 5 amino acid residues in length to about 75 amino acid some cases , be cleaved by PC5 /6 . In some cases, proprotein residues in length , from about 10 amino acid residues in convertases may cleave proproteins at additional sites , other length to about 100 amino acid residues in length , from about 25 amino acid residues in length to about 150 amino than those indicated in Table 1 . In some embodiments( the first , 35 acid residues in length , from about 125 amino acid residues pro - proteins may be cleaved at a first cleavage site (the first 3 ina length to about 250 amino acid residues in length , from site being the site closest to the N - terminus ). In other about 175 amino acid residues in length to about 400 amino embodiments , pro -proteins may be cleaved at a cleavage site acid residues in length , from about 200 amino acid residues other than a first cleavage site . In some cases, proprotein in length to about 500 amino acid residues in length and / or convertase cleavage may occur intracellularly . In some 40 at least 500 amino acid residues in length . cases, proprotein convertase cleavage may occur extracel In some embodiments, protein modules comprise one or lularly . more regions with known functional features ( e . g . protein Many TGF- ß family member proteins are synthesized in binding domain , nucleic acid binding domain , hydrophobic conjunction with prodomains . Some prodomains may pocket, etc . ) Protein modules may comprise functional pro remain associated with growth factors after cleavage . Such 45 tein domains necessary for different aspects of growth factor associationsmay form latent growth factor -prodomain com - signaling , secretion , latency and /or release from latent con plexes (GPCs ) that modulate the availability of growth formations . factors for cell signaling. Growth factors may be released In some embodiments , protein modules may be derived from latency in GPCs through associations with one or more from TGF - B - related proteins . Such protein modules may extracellular proteins . In some cases , growth factor release 50 include , but are not limited to latency - associated peptides may rely on force applied to GPCs through extracellular (LAPs ) , LAP - like domains, growth factor domains , fastener protein interactions . Such forces may pull from C - terminal regions , proprotein convertase cleavage sites ( e . g . furin and / or N -terminal regions ofGPCs resulting in the release of cleavage sites ) , B / TP cleavage sites , arm regions, finger associated growth factors . regions , residues ( such as cysteine residues for example ) for In some TGF -ß family members , the prodomain portion 55 extracellular protein [e . g . latent TGF -B binding protein of the GPC is responsible for growth factor retention and (LTBP ) , fibrillin and / or glycoprotein A repetitions predomi blocking the interaction of retained growth factors with their nant (GARP ) protein ] associations , latency loops (also receptors . Prodomain portions ofGPCs that function in this referred to herein as latency lassos ) alpha 1 helical regions, regard are referred to as latency associated peptides (LAPs ). alpha 2 helical regions, RGD sequences and bowtie regions . TGF -B1 , 2 and 3 are know to comprise LAPs. Some 60 FIG . 4 is a schematic diagram of an embodiment depicting prodomains may comprise LAP - like domains . As used LAP and growth factor dimers comprising protein modules . herein , the term “ LAP - like domain ” refers to prodomain In some embodiments, protein modules may be derived portions of GPCs and / or free prodomains that may be from one or more TGF - B isoform ( e. g . TGF- B1, TGF -B2 structurally similar or synthesized in a similar manner to and /or TGF - B3 ) . Such protein modules may comprise the LAPs, but that may not function to prevent growth factor/ 65 protein modules and / or amino acid sequences listed in Table receptor interactions . GDF - 8 and GDF - 11 prodomains com - 2 . Some protein modules of the present invention may prise LAP - like domains . comprise amino acid sequences similar to those in Table 2, US 9 ,758 ,577 B2 19 20 but comprise additional or fewer amino acids than those 6 more or fewer amino acids , about 7 more or fewer amino listed . Such amino acid sequences may comprise about 1 acids , about 8 more or fewer amino acids, about 9 more or more or fewer amino acids, about 2 more or fewer amino fewer amino acids, about 10 more or fewer amino acids or acids, about 3 more or fewer amino acids , about 4 more or greater than 10 more or fewer amino acids on N - terminal fewer amino acids, about 5 more or fewer amino acids, about and / or C - terminal ends . TABLE 2 TGF - B protein modules TGF - B Family SEO Member Protein Module Prodomain and growth factor Sequence ID NO TGF - Bi latency associated LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPPSO 38 peptide GEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEA DYYAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNT SELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQK YSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVROWLS RGGEIEGFRLSAHCSCDSRDNTLOVDINGFTTGRRG DLATIHGMNRPFLLLMATPLERAOHLOSSRHRR

TGF - B2 latency associated SLSTCSTLDMDOFMRKRIEAIRGOILSKLKLTSPPE 39 peptide DYPEPEEVPPEVISIYNSTRDLLOEKASRRAAACER ERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYF RIVRFDVSAMEKNASNLVKAEFRVFRLONPKARVPE ORIELYQILKSKDLTSPTORYIDSKVVKTRAEGEWL SFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSN NYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTR KKNSGKTPHLLLMLLPSYRLESQQTNRRKKR TGF - B3 latency associated SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 40 peptide PEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCT OENTESEYYAKEIHKFDMIOGLAEHNELAVCPKGIT SKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRN EORIELFQILRPDEHIAKORYIGGKNLPTRGTAEWL SFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNG DILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKOKD HHNPHLILMMIPPHRLDNPGQGGQRKKR TGF - B1 straight jacket LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQ 41 region GEVPPGPLP TGF - B2 straight jacket SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPE 42 region DYPEPEEVP TGF - B3 straight jacket SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 43 region PEPTVMTHVP TGF - B1 growth factor ALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP 44 domain KGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGA SAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRS CKCS TGF - B2 growth factor ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEP 45 domain KGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEA SASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKS CKCS

TGF - B3 growth factor ALDTNYCFRNLEENCCVRPLYIDFRODLGWKWVHEP 46 domain KGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEA SASPCCVPODLEPLTILYYVGRTPKVEQLSNMVVKS CKCS TGF - B1 fastener region residues 74 - 76 , YYA TGF - B2 fastener region residues 79 -81 , YYA TGF - B3 fastener region residues 80 - 82 , YYA TGF - ßi furin cleavage site RHRR region TGF - B2 furin cleavage site RKKR region i

TGF - B3 furin cleavage site RK theopt region US 9 , 758 ,577 B2 21 TABLE 2 - continued TGF - B protein modules TGF - B Family SEO Member Protein Module Prodomain and growth factor Sequence ID NO TGF - B1 arm region EAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTR 49 VLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPE PVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYL SNRLLAPSDSPEWLSFDVTGVVROWLSRGGEIEGFR LSAHCSCDSRDNTLOVDINGFTTGRRGD LATIHGMN RPFLLLMATPLERAQHLOSSRHRR

TGF - B2 arm region PEVISIYNSTRDLLOEKASRRAAACERERSDEEYYA 50 KEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSA MEKNASNLVKAEFRVFRLONPKARVPEQRIELYQIL KSKDLTSPTORYIDSKVVKTRAEGEWLSFDVTDAVH EWLHHKDRNLGFKISLHCPCCTFVPSNNYI IPNKSE ELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPH LLLMLLPSYRLESQQTNRRKKR TGF - B3 arm region YOVLALYNSTRELLEEMHGEREEGCTOENTESEYYA 51 KEIHKFDMIOGLAEHNELAVCPKGITSKVFRFNVSS VEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQIL RPDEHIAKORYIGGKNLPTRGTAEWLSFDVTDTVRE WLLRRESNLGLEISIHCPCHTFOPNGDILENIHEVM EIKFKGVDNEDDHGRGDLGRLKKOKDHHNPHLILMM IPPHRLDNPGQGGQRKKR TGF - B1 fingers region 1 CVROLYIDFRKDLGWKWIHEPKGYHANFC 52 TGF -B2 fingers region 1 CLRPLYIDFKRDLGWKWIHEPKGYNANFCA 53 TGF - B3 fingers region 1 CVRPLYIDFRQDLGWKWVHEPKGYYANFCS 54 TGF - B1 fingers region 2 CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS sú TGF - B2 fingers region 2 CVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS 56 TGF - B3 fingers region 2 CVPODLEPLTILYYVGRTPKVEQLSNMVVKSCKCS 57 a TGF - B1 residue for LTBP Cys 4 association l TGF - B2 residue for LTBP Cys 5 i association

TGF - B3 residue for LTBP Cys 7 I association

TGF - B1 residue for GARP Cys 4 i association

TGF - B2 residue for GARP Cys 5 I association

TGF - B3 residue for GARP Cys 7 i association TGF - B1 latency loop LASPPSQGEVPPGPL 58 TGF - B2 latency loop LTSPPEDYPEPEE 59 LTSPPEPTVMTHV 60 TGF -B3 latency loop ö TGF - B1 alpha 1 helical LSTCKTIDMELVKRKRIEAIRGQILSKLR 61 region n

TGF - B2 alpha 1 helical LSTCSTLDMDQFMRKRIEAIRGQILSKLK e62 region TGF - B3 alpha 1 helical LSLSTCTTLDFGHIKKKRVEAIRGOILSKLR 63 region TGF - B1 trigger loop region NGFTTGRRGDLATIHGMNRP 64 US 9 , 758 ,577 B2 23 24 TABLE 2 - continued TGF - B protein modules TGF - B Family SEO Member Protein Module Prodomain and growth factor Sequence ID NO TGF - B2 trigger loop region FAGIDGTSTYTSGDQKTIKSTRKKNSGKTP 65 ( long ) TGF - B3 trigger loop region GVDNEDDHGRGDLGRLKKOKDHHNP TGF - B1 RGD sequence residue 215 - 217 , RGD region TGF - B3 RGD sequence residue 241 - 243 , RGD region TGF - B1 bowtie region CSCDSRDNTLQVD TGF - B2 bowtie region CPCCTFVPSNNYIIPNKSEELEAR TGF - B3 bowtie region CPCHTFQPNGDILENIHEVMEIK

In some embodiments , LAPs or LAP - like domains com - 1 helical regions comprise N - terminal regions of LAPs or prise the prodomain portion of a TGF - B - related protein LAP- like domains . Alpha 1 helical regions may also com and /or GPC . Some LAPs or LAP - like domains may asso - 25 prise N - terminal regions for extracellular associations . Such ciate with growth factors in GPCs . Some LAPs may steri - extracellular associations may comprise extracellular matrix cally prevent growth factor association with one or more proteins and /or proteins associated with the extracellular cellular receptors . LAPs or LAP - like domains may comprise matrix . Some extracellular associations may comprise asso arm regions and / or straight jacket regions. Some LAP or ciations with proteins that may include , but are not limited LAP - like domains may comprise C - terminal regions 30 to LTBPs ( e . g . LTBP1, LTBP2 , LTBP3 and / or LTBP4 ) , referred to herein as "bowtie regions. ” In some LAP or fibrillins ( e . g . fibrillin - 1 , fibrillin - 2 , fibrillin - 3 and /or fibril LAP - like domain dimers , bowtie regions of each monomer lin - 4 ) perlecan , decorin and /or GARPs ( e . g . GARP and /or may associate and/ or interact. Such associations may com prise disulfide bond formation , as is found between mono LRRC33 ) . N -terminal extracellular associations may com mers of TGF - B isoform LAPs. prise disulfide bonds between cysteine residues . In some In some embodiments , arm regions may comprise trigger cases , extracellular matrix proteins and /or proteins associ loop regions . Trigger loops may comprise regions that ated with the extraceullar matrix may comprise bonds with associate with integrins . Such regions may comprise amino one or more regions of LAPs/ LAP - like domains other than acid sequences comprising RGD ( Arg -Gly - Asp ). Regions N - terminal regions. comprising RGD sequences are referred to herein as RGD 40 In some embodiments , growth factor domains comprise sequence regions. In some embodiments , LAPs or LAP - like one or more growth factor monomers . Some growth factor domains comprise latency loops ( also referred to herein as domains comprise growth factor dimers . Such growth factor latency lassos) . Some latency loops may maintain associa domains may comprise growth factor homodimers or het tions between LAPs or LAP -like domains and growth fac erodimers (comprising growth factor monomers from dif tors present within GPCs. LAPs or LAP - like domains may 45 ferent TGF - b -related proteins. ) Some growth factor domains also comprise fastener regions . Such fastener regions may m ay comprise fingers regions . Such fingers regions may maintain associations between LAPs or LAP - like domains comprise B -pleated sheets . Fingers regions may associate and growth factors present within GPCs. Some fastener with LAPs or LAP - like domains. Some fingers regions may regions may maintain LAP or LAP - like domain conforma - maintain association between growth factor domains and tions that promote growth factor retention . 50 LAPs or LAP - like domains. In some cases , GPCs may require enzymatic cleavage for In some embodiments , recombinant proteins of the pres dissociation of bound growth factors. Such cleavage may be ent invention may comprise protein modules from growth carried out in some instances by members of the BMP - 1 ) differentiation factor (GDF ) proteins . Such GDF protein Tolloid - like proteinase ( B / TP ) family (Muir et al. , 2011. J modules may comprise the protein modules and /or amino Biol Chem . 286 ( 49 ) :41905 - 11 , the contents of which are 55 acid sequences listed in Table 3 . In some embodiments , herein incorporated by reference in their entirety . ) These protein modules of the present invention may comprise metaloproteinases may include , but are not limited to BMP - amino acid sequences similar to those in Table 3 , but 1 , mammalian tolloid protein (mTLD ) mammalian tolloid - comprise additional or fewer amino acids than those listed . like 1 (mTLL1 ) and mammalian tolloid - like 2 (mTLL2 . ) Some such amino acid sequences may comprise about 1 Exemplary GPCs that may be cleaved by such metallopro - 60 more or fewer amino acids , about 2 more or fewer amino teinases may include , but are not limited to GDF - 8 and acids, about 3 more or fewer amino acids , about 4 more or GDF - 11 . In some cases, GDF - 8 may be cleaved by mTLL2 . fewer amino acids, about 5 more or fewer amino acids, about In some cases, tolloid cleavage may occur intracellularly . In 6 more or fewer amino acids , about 7 more or fewer amino some cases , tolloid cleavage may occur extracellularly . acids , about 8 more or fewer amino acids , about 9 more or Straight jacket regions may comprise alpha 1 helical 65 fewer amino acids , about 10 more or fewer amino acids or regions. In some embodiments , alpha 1 helical regions may greater than 10 more or fewer amino acids on N -terminal be positioned between growth factor monomers . Some alpha and /or C - terminal ends. US 9 ,758 , 577 B2 25 26 TABLE 3 GDF protein modules TGF - B Family SEO Member Protein Module Prodomain and growth factor Sequence ID NO GDF - 8 prodomain NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIO 70 ILSKLRLETAPNISKDVIROLLPKAPPLRELIDOYD VORDDSSDGSLEDDDYHATTETIITMPTESDFLMQV DGKPKCCFFKFSSKIOYNKVVKAOLWIYLRPVETPT TVFVOILRLIKPMKDGTRYTGIRSLKLDMNPGTGIW QSIDVKTVLQNWLKQPESNLGIEIKALD ENGHDLAV TFPGPGEDGLNPFLEVKVTDTPKRSRR

GDF - 11 prodomain AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 71 GCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNIS REVVKOLLPKAPPLQQILDLHD FOGDALOPEDFLEE DEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSP KVMFTKVLKAOLWVYLRPVPRPATVYLOILRLKPLT GEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDF KOVLHSWFROPOSNWGIEINAFDPSGTD LAVTSLGP GAEGLHPFMELRVLENTKRSRR GDF - 8 straight jacket NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIO 72 region ILSKLRLETAPNISKDVIROLLPKAPPL GDF - 11 straight jacket AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 73 region GCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNIS REVVKQLLPKAPPL GDF - 8 growth factor DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPK 74 domain RYKANYCSGECEFVFLOKYPHTHLVHOANPRGSAGP CCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGC

GDF - 11 growth factor NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPK 75 domain RYKANYCSGOCEYMFMOKYPHTHLVQQANPRGSAGP CCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGC

GDF - 8 fastener region residues 87 - 89 , DYH GDF - 11 fastener region residues 110 - 112 , EYH GDF - 8 furin cleavage site RSRR region GDF - 11 furin cleavage site RSRR region GDF - 8 BMP / Tolloid between residues R75 and 176 cleavage site GDF - 11 BMP / Tolloid between residues G97 and 198 cleavage site GDF - 8 arm region RELIDOYDVORDDSSDGSLEDDDYHATTETIITMPT 77 ESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIY LRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWOSIDVKTVLQNWLKQPESNLGIEIKALD ENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRR GDF - 11 arm region QQILDLHDFOGDALOPEDFLEEDEYHATTETVISMA 78 QETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWV YLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRH IRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSN WGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVL ENTKRSRR GDF - 8 fingers region 1 CRYPLTVDFEAFGWDWIIAPKRYKANYCS ? GDF - 11 fingers region 1 CRYPLTVDFEAFGWDWIIAPKRYKANYCS GDF - 8 fingers region 2 CTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS o GDF - 11 fingers region 2 CTPTKMSPINMLYFNDKQQI LYGKIPGMVVDRCGCS GDF - 8 latency loop RLETAPNISKDVIROLLPKAPPL corona GDF - 11 latency loop RLKEAPNISREVV KOLLPKAPP enuna US 9 , 758 ,577 B2 27 28 TABLE 3 - continued GDF protein modules TGF - B Family SEO Member Protein Module Prodomain and growth factor Sequence ID NO

GDF - 8 alpha i helical GLCNACTWRONTKSSRIEAIKIOIL SK 84 region GDF - 11 alpha 1 helical DGCPVCVWROHSRELRLESIKSOILSKL oop region

GDF - 8 bowtie r DENGHDLAVTFPGP k GDF - 11 bowtie region DPSGTDLAVTSLG og

Some recombinant proteins of the present invention may Elevated GDF - 15 in the blood is also associated with comprise GDF - 15 , GDF - 15 signaling pathway - related pro - ineffective and /or apoptotic erythropoiesis , such as in sub teins and /or modules and /or portions thereof. GDF - 15 is a 20 jects suffering from beta - thalassemia or dyserythropoietic anemias. TGF- B family protein that is highly expressed in liver. In some embodiments , recombinant proteins of the pres Expression of GDF - 15 is dramatically upregulated follow ent invention may comprise protein modules from activin ing liver injury (Hsiao et al . 2000 . Mol Cell Biol. 20 ( 10 ) : subunits . Such protein modules may comprise the protein 3742 -51 . ) Additionally , its expression in macrophages may 25 modules and / or amino acid sequences of the activin subunit serve a protective function in the context of atherosclerosis , 23 inhibin beta A , listed in Table 4 . In some embodiments , possibly through regulation of adhesion molecule expres protein modules of the present invention may comprise sion (Preusch et al. , 2013 . Eur J Med Res. 18 : 19 . ) While a amino acid sequences similar to those in Table 4 . but member of the TGF- B family , GDF - 15 comprises less than comprise additional or fewer amino acids than those listed . 30 % homology with other members , making it the most 20 Some such amino acid sequences may comprise about 1 divergent member of the family (Tanno et al ., 2010 . Curr ** more or fewer amino acids, about 2 more or fewer amino Opin Hematol. 17 ( 3 ): 184 - 90 , the contents of which are acids, about 3 more or fewer amino acids, about 4 more or incorporated herein by reference in their entirety . ) The fewer amino acids, about 5 more or fewer amino acids , about mature form is soluble and can be found in the blood stream . 6 more or fewer amino acids , about 7 more or fewer amino Interestingly , GDF - 15 levels in circulation have been found 35 acids , about 8 more or fewer amino acids , about 9 more or to negatively correlate with hepcidin levels , suggesting a fewer amino acids , about 10 more or fewer amino acids or role for GDF - 15 in iron load and /or metabolism (Finkenstedt greater than 10 more or fewer amino acids on N -terminal et al. , 2008 . British Journal of Haematology . 144 :789 -93 . ) and /or C - terminal ends . TABLE 4 Inhibin beta Aprotein modules

SEO Protein Module Prodomain and growth factor Sequence ID NO latency associated SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEMVEAVK 88 peptide ( LAP ) KHILNMLHLKKRPDVTOPVPKAALLNAIRKLHVGKVGEN GYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARKTLH FEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLF QQOKHPOGSLDTGEEAEEVGLKGERSELLLSEKVVDARK STWHVFPVSSSIORLLDOGKSSLDVRIACEOCOESGASL VLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEOSHRPFL MLQAROSEDHPHRRRRR straight jacket SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVK 89 region KHILNMLHLKKRPDVTQPVPKAALLN growth factor RGLECDGKVNICCKKOFFVSFKDIGWNDWIIAPSGYHAN domain YCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANL KSCCVPTKLRPMSMLYYDDGONIIKKDIQNMIVEECGCS fastener region residues 89 - 91 , RRA furin cleavage site RRRR region

arm region LNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEOTSE 92 IITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKV PKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGE RSELLLSEKVVDARKSTWHVFPVSSSIQRLLDOGKSSLD VRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGG AGADEEKEQSHRPFLMLQARQSEDHPHRRRRR US 9 ,758 ,577 B2 29 30 TABLE 4 - continued Inhibin beta Aprotein modules

SEO Protein Module Prodomain and growth factor Sequence ID NO fingers region 1 KKQFFVSFKDIGWNDWIIAPSGYHANYC ñ93 fingers region 2 CVPTKLRPMSMLYYDDGONIIKKDIQNMIVEECGCS 94 latency loop LKKRPDVTOPVPKAALL 95 alpha 1 helical ALAALPKDVPNSQPEMVEAVKKHILNML 96 region bowtie region QESGASLVLLGKKKKKEEEGEGKKKGGGEGGAG 97

Growth factor domains among TGF -B family members proteins while TGF -B isoforms (e .g . TGF- B1 , TGF -B2 and are more highly conserved while prodomains comprise a TGF- B3 ) signal through SMAD2 and SMAD3 . much lower percent identity among family members ( FIG . 20 Some BMP receptors and / or co - receptors are also distinct 9 .) Table 5 demonstrates this trend among TGF - B isoforms. from other TGF- ß family member proteins. Among these is the repulsive guidance molecule (RGM ) family of proteins . TABLE 5 RGM proteins act as co -receptors for BMP signaling. There are three RGM family members , RGMA , RGMB and Percent identity among TGF - B isoforms: LAP vs growth factor 25 RGMC [ also known as hemojuvelin (Hjv . ) ] Recombinant TGF- B1 TGF- B2 TGF- B3 proteins of the present invention comprising one or more TGF -B1 31. 2 % vs 71 . 2 % 31. 9 % vs 76 . 7 % BMP protein module may be useful for the development of TGF -B2 31. 2 % vs 71. 2 % 44 . 4 % vs 79 . 4 % antibodies and / or assays to study , enhance and / or perturb TGF -B3 31 . 9 % vs 76 . 7 % 44. 4 % vs 79 . 4 % BMP interactions with RGM proteins. 30 Another family of GDF /BMP interacting proteins is C - ter Prodomains may vary in length from about 50 to about minal cysteine knot- like (CTCK ) domain - containing pro 200 , from about 100 to about 400 or from about 300 to about teins . In some cases, CTCK domain -containing proteins may 500 amino acids residues. In some embodiments , prodo act antagonistically with regard to GDF /BMP signal trans mains range from about 169 to about 433 residues. Prodo - 35 duction . CTCK domain -containing proteins include , but are mains may be unrelated in sequence and / or low in homol- not limited to Cerberus , Connective tissue growth factor ogy . Some prodomains may have similar folds and / or three (CTGF ) , DAN domain family member 5 (DAND5 ) , Grem dimensional structures . Prodomains of TGF - B family mem - lin - 1 (GREM1 ) , Gremlin - 2 (GREM2 ) , Mucin - 19 (MUC19 ) , bers may comprise latency loops. Such loops may be pro - Mucin - 2 (MUC2 ) , Mucin - 5AC (MUC5AC ) , Mucin - 5B line - rich . Latency loop length may determine the ability of 40 (MUC5B ), Mucin - 6 (MUC6 ) , Neuroblastoma suppressor of such loops to encircle growth factor finger regions. tumorigenicity 1 (NBL1 ) , Norrin (NDP ), Otogelin (OTOG ), In some embodiments , protein modules from some Otogelin - like protein (OTOGL ) , Protein CYR61 (CYR61 ) , TGF- B family members comprise low sequence identity Protein NOV homolog (NOV ), Sclerostin (SOST ), Scle with protein modules from other TGF - B family members. rostin domain - containing protein 1 ( SOSTDC1) , SCO - spon Such low sequence identity may indicate specialized roles 45 din ( SSPO ), Slit homolog 1 protein ( SLIT1) , Slit homolog 2 for such family members with distinct protein modules . protein (SLIT2 ) , Slit homolog 3 protein ( SLIT3 ) , von Wille Association of GPCs with extracellular proteins may brand factor (VWF ) , WNT1- inducible- signaling pathway strengthen prodomain - growth factor interactions . In some protein 1 (WISP1 ) and WNT1 - inducible - signaling pathway embodiments , such extracellular proteins may include , but protein 3 (WISP3 ) . are not limited to LTBPs, fibrillins and /or GARP . In some 50 Recombinant Proteins cases , extracellular protein associations are required to keep In some embodiments , the present invention provides growth factors latent in GPCs. recombinantproteins . As used herein , the term “ recombinant GARP expression has been shown to be required for protein ” refers to a protein produced by an artificial gene surface expression of GPCs on the surface of cells of and /or process (e . g . genetic engineering ). Such recombinant hematopoietic origin ( Tran , D . Q . et al . ,GARP (LRRC32 ) is 55 proteins may comprise one or more protein modules from essential for the surface expression of latent TGF - B on one or more TGF - b - related proteins . Some recombinant platelets and activated FOXP3 + regulatory T cells . PNAS. proteins disclosed herein may be useful as recombinant 2009 , Jun . 2 . 106 ( 32 ) : 13445 -50 . ) GARP may act as a tether antigens . As used herein , the term “ recombinant antigen " to hold GPCs in place on the surface of these cells , includ - refers to a recombinant protein that may be used to immu ing , but not limited to regulatory T - cells and / or platelets . 60 nize one or more hosts for the production of antibodies In some embodiments , recombinant proteins of the pres - directed toward one or more epitopes present on such ent invention may comprise bone morphogenetic proteins recombinant antigens. Some recombinant antigens may be (BMPs ) , a family of TGF -B - related proteins. Protein mod cell- based antigens. As used herein , the term “ cell- based ules comprising sequences from BMPs may comprise antigen ” refers to recombinant antigens that are expressed in sequences from any of those BMP modules disclosed in 65 cells for presentation of such antigens on the cell surface . FIGS . 8A - 8G . While related to other TGF - B family member Such cells may be used to immunize hosts for the production proteins, BMPs generally signal through SMAD1, 5 and 8 of antibodies directed to such cell- based antigens. US 9 , 758 ,577 B2 31 32 In some embodiments , recombinant proteins disclosed residues ( SEQ ID NO : 295 ). Some polyhistidine tags may be herein may be used as therapeutics . Recombinant proteins present at the N - terminus of recombinant proteins disclosed disclosed herein may modulate growth factor ( e . g . growth herein . Some polyhistidine tags may be present at the factors comprising TGF - B - related proteins ) levels and / or C -terminus of recombinant proteins disclosed herein . Pro activity ( e . g . signaling ) upon administration and /or intro - 5 teins produced may comprise additional amino acids encod duction to one or more subjects and /or niches. ing one or more 3C protease cleavage site. Such sites allow In some embodiments , recombinant proteins disclosed herein may be used to assay growth factor ( e . g . growth for cleavage at the 3C protease cleavage site upon treatment factors comprising TGF - B -related proteins ) levels and / or with 3C protease , including, but not limited to rhinovirus 3C activity ( e . g . signaling ) . Some recombinant proteins dis - 10 protease . Some cleavage sites may be introduced to allow closed herein may be used in the isolation of antibodies for removal of detectable labels from recombinant proteins . directed to TGF - B - related proteins . Recombinant proteins of In some embodiments of the present invention , recombi the present invention may also be used as recombinant nant GPCs may comprise mutations in one or more amino antigens in the development of stabilizing [ reducing or acids as compared to wild type sequences . In some cases , preventing dissociation between two agents , ( e. g . growthwth - 15 one or more regions of proteolytic processing may be factor release from GPCs, GPC release from one or more mutated . Such regions may comprise proprotein convertase protein interactions ) and /or releasing ?enhancing the disso cleavage sites. Proprotein convertase ( e. g . furin ) cleavage ciation between two agents ( e . g . growth - factor release from site mutations prevent enzymatic cleavage at that site and /or GPCs , GPC release from one or more protein interactions ) ] prevent enzymatic cleavage of growth factors from their antibodies . Recombinant proteins of the present invention 20 prodomains (see FIG . 6 .) Some proprotein convertase cleav may include TGF - B family member proteins as well as age sites comprising RXXR sequences may be mutated to components and /or protein modules thereof. Some recom - RXG (wherein X indicates a site where amino acid residues binant proteins of the present invention may comprise may be variable ) . Such mutations are herein abbreviated as prodomains without associated growth factors , furin cleav - “ D2G ” mutations and may be resistant to enzymatic cleav age - deficient mutants, mutants deficient in extracellular pro - 25 age . In some embodiments , furin cleavage sites comprising tein associations and / or combinations thereof. RXXR sequences are mutated to AXXA . Such AXXA In some embodiments , recombinant proteins may com - sequences may also be resistant to enzymatic cleavage . prise detectable labels . Detectable labels may be used to In some embodiments, regions of proteolytic processing allow for detection and /or isolation of recombinant proteins . Some detectable labels may comprise biotin labelsroteins, poly -. 30 by tolloid and / or tolloid -like proteins may be mutated to histidine tags and / or flag tags. Such tags may be used to prevent such proteolytic processing. In some embodiments , isolate tagged proteins . Proteins produced may comprise tolloid processing regions on GDF - 8 and /or GDF - 11 may be additional amino acids encoding one or more 3C protease mutated . In some embodiments , mutation of aspartic acid cleavage site . Such sites allow for cleavage at the 3C residues to alanine residues within tolloid processing regions protease cleavage site upon treatment with 3C protease, 35s Pprevents tolloid processing . Mutation of aspartic acid resi including , but not limited to rhinovirus 3C protease . Such due 76 (D76 ) of the GDF - 8 (myostatin ) proprotein has been cleavage sites are introduced to allow for removal of detect shown to prevent proteolytic activation of latent GDF - 8 able labels from recombinant proteins. (Wolfman , N . M . et al. , PNAS . 2003 , Oct. 6 . 100 ( 26 ) : 15842 Recombinant GPCs 6 . ) In some embodiments , Asp 120 (D120 , residue number FIG . 5 is a schematic depicting an embodiment of a 40 counted from the translated protein , D98 from the proprotein recombinant GPC . Recombinant proteins according to FIG . of SEQ ID NO : 4 ) in GDF - 11 may be mutated to prevent 5 comprising TGF- B - family member proteins may comprise tolloid processing (Ge et al ., 2005. Mol Cell Biol. 25 (14 ) : features including , but not limited to C - terminal regions of 5846 - 58 , the contents of which are herein incorporated by the mature growth factor, N -terminal regions of the prodo - reference in their entirety . ) main and / or proprotein cleavage sites. The proprotein cleav - 45 In some embodiments , one or more amino acids may be age site of recombinant TGF - B GPCs may , for example , mutated in order to form recombinant GPCs with reduced comprise the furin consensus sequence RXXR wherein R is latency . Such mutations are referred to herein as “ activating arginine and X indicates amino acid residues that may vary mutations . " These mutations may introduce one or more among TGF - B family members. Furin cleavage site regions of steric clash between complex prodomains and sequences (although not limited to cleavage by furin alone 50 growth factor domains . As used herein , the term " steric and may include cleavage by other proprotein convertase clash , ” when referring to the interaction between two pro enzymes ) for each TGF - B family member are indicated in teins or between two domains and /or epitopes within the Table 1 . Recombinant GPCs according to the embodiment same protein , refers to a repulsive interaction between such depicted in FIG . 5 may also comprise one or more cysteine proteins , domains and /or epitopes due to overlapping posi residues within and /or near the N -terminal region of the 55 tion in three - dimensional space . Steric clash within GPCs prodomain . Such cysteine residues may be from about 1 to may reduce the affinity between prodomains and growth about 10 amino acids, from about 4 to about 15 amino acids, factor domains, resulting in elevated ratios of free growth from about 5 to about 20 amino acids and /or from about 7 factor to latent growth factor. In some embodiments , one or to about 50 amino acids from the N -terminus of the prodo - more amino acids may be mutated in order to form recom main . Recombinant GPCs may also comprise detectable 60 binant GPCs with increased latency . Such mutations are labels. Such detectable labels may be useful for detection referred to herein as “ stabilizing mutations .” These muta and / or isolation of recombinant GPCs. Detectable labels tions may increase the affinity between prodomains and may comprise 2 or more histidine (His ) residues. Such growth factor domains, resulting in decreased ratios of free detectable labels may also be referred to herein as polyhis - growth factor to latent growth factor. tidine tags. Polyhistidine tags may include hexa histidine 65 In some embodiments , recombinant proteins of the pres tags (SEQ ID NO : 295 ) or HIS - TAGTM (EMD Biosciences, ent invention may comprise any of the sequences listed in Darmstadt, Germany ) comprising a chain of six histidine Table 6 or fragments thereof . US 9 ,758 , 577 B2 33 34 TABLE 6 Recombinant proteins

SEO Protein Sequence ID NO iproTGF -B1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPPGP la EAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVE THNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRAELRLL RLKLKVEQHVELYOKYSNNSWRYLSNRLLAPSDSPEWLSFDVT GVVROWLSRGGEIEGFRLSAHCSCDSRDNTLOVDINGFTTGRR GDLATIHGMNRPFLLLMATPLERAOHLOSSRHRRALDTNYCFS STEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIW SLDTOYSKVLALYNOHNPGASAAPCCVPOALEPLPIVYYVGRK PKVEOLSNMIVRSCKCS proTGF - B1 C4S LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 98 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVE THNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRAELRLL RLKLKVEOHVELYOKY SNNSWRYLSNRLLAPSDSPEWLSFDVT GVVROWLSRGGEIEGFRLSAHCSCDSRDNTLOVDINGFTTGRR GDLATIHGMNRPFLLLMATPLERAQHLOSSRHRRALDTNYCFS STEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIW SLDTQYSKVLAL YNQHNPGASAAPCCVPQALEPLPIVYYVGRK PKVEQLSNMIVRSCKCS proTGF - B1 C4S LSTSKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPPGP 99 ( LAP ) LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVE THNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRAELRLL RLKLKVEOHVELYOKY SNNSWRYLSNRLLAPSDSPEWLSFDVT GVVROWLSRGGEIEGFRLSAHCSCDSRDNTLOVDINGFTTGRR GDLATIHGMNRPFLLLMATPLERAQHLOSSRHRR proTGF - B1 D2G LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPPGP 100 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVE THNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRAELRLL RLKLKVEOHVELYOKYSNNSWRYLSNRLLAPSDSPEWLSFDVT GVVROWLSRGGEIEGFRLSAHCSCDSRDNTLOVDINGFTTGRR GDLATIHGMNRPFLLLMATPLERAOHLOSSRHGALDTNYCFSS TEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWS LDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKP KVEQLSNMIVRSCKCS proTGF - B1 C4S D2G LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 101 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVE THNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRAELRLL RLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVT GVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRR GDLATIHGMNRPFLLLMATPLERAQHLOSSRHGALDTNYCFSS TEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWS LDTOYSKVLALYNOHNPGASAAPCCVPOALEPLPIVYYVGRKP KVEQLSNMIVRSCKCS proTGF - B1 LAP LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 38 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVE THNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRAELRLL RLKLKVEOHVELYOKY SNNSWRYLSNRLLAPSDSPEWLSFDVT GVVROWLSRGGEIEGFRLSAHCSCDSRDNTLOVDINGFTTGRR GDLATIHGMNRPFLLLMATPLERAQHLOSSRHRR proTGF - B2 SLSTCSTLDMDOFMRKRIEAIRGOILSKLKLTSPPEDYPEPEE 2 VPPEVISIYNSTRDLLOEKASRRAAACERERSDEEYYAKEVYK IDMPPFFPSENAIPPTFYRPYFRIVRFDV SAMEKNASNLVKAE FRVFRLONPKARVPEORIELYOILKSKDLTSPTORYIDSKVVK TRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPS NNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGK TPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLR PLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTOHSRV LSLYNTINPEASASPCCVSODLEPLTILYYIGKTPKIEOLSNM IVKSCKCS proTGF - B2 C5S SLSTSSTLDMDOFMRKRIEAIRGOILSKLKLTSPPEDYPEPEE 102 VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYK IDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAE FRVFRLONPKARVPEORIELYOILKSKDLTSPTORYIDSKVVK TRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPS NNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIKSTRKKNSGK TPHLLLMLLPSYRLESQOTNRRKKRALDAAYCFRNVODNCCLR PLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRV LSLYNTINPEASASPCCVSODLEPLTILYYIGKTPKIEQLSNM IVKSCKCS US 9 , 758 ,577 B2 35 36 TABLE 6 - continued Recombinant proteins SEQ Protein Sequence ID NO pro TGF - B2 LAP C5S SLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 103 VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYK IDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAE FRVFRLONPKARVPEORIELYOILKSKDLTSPTORYIDSKVVK TRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPS NNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIKSTRKKNSGK TPHLLLMLLPSYRLESQOTNRRKKR pro TGF - B2 C5S D2G SLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 104 VPPEVISIYNSTRDLLOEKASRRAAACERERSDEEYYAKEVYK IDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAE FRVFRLONPKARVPEORIELYOILKSKDLTSPTORYIDSKVK TRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPS NNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGK TPHLLLMLLPSYRLESQQTNRRKGALDAAYCFRNVQDNCCLRP LYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTOHSRVL SLYNTINPEASASPCCVSODLEPLTILYYIGKTPKIEOLSNMI VKSCKCS proTGF - B2 D2G SLSTCSTLDMDOFMRKRIEAIRGOILSKLKLTSPPEDYPEPEE 105 VPPEVISIYNSTRDLLOEKASRRAAACERERSDEEYYAKEVYK IDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAE FRVFRLONPKARVPEORIELYOILKSKDLTSPTORYIDSKVVK TRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPS NNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIKSTRKKNSGK TPHLLLMLLPSYRLESQQTNRRKGALDAAYCFRNVQDNCCLRP LYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVL SLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKI EQLSNMI VKSCKCS proTGF - B2 LAP SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 39 VPPEVISIYNSTRDLLOEKASRRAAACERERSDEEYYAKEVYK IDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAE FRVFRLONPKARVPEQRIELYQILKSKDLTSPTORYIDSKVVK TRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPS NNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIKSTRKKNSGK TPHLLLMLLPSYRLESQQTNRRKKR proTGF - B3 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIH KFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRA EFRVLRVPNPSSKRNEORIELFOILRPDEHIAKORYIGGKNLP TRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFOPN GDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKOKDHEINPH LILMMIPPHRLDNPGOGGORKKRALDTNYCFRNLEENCCVRPL YIDFRODLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLG LYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVV KSCKCS proTGF -B3 C7S SLSLSTSTTLDFGHIKKKRVEAIRGOILSKLRLTSPPEPTVMT 106 HVPYOVLALYNSTRELLEEMHGEREEGCTOENTESEYYAKEIH KFDMIOGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRA EFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKORYIGGKNLP TRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFOPN GDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKOKDHEINPH LILMMIPPHRLDNPGOGGORKKRALDTNYCFRNLEENCCVRPL YIDFRODLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLG LYNTLNPEASASPCCVPODLEPLTILYYVGRTPKVEOLSNMVV KSCKCS proTGF - B3 LAP C7S SLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT 107 HVPYOVLALYNSTRELLEEMHGEREEGCTOENTESEYYAKEIH KFDMIOGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRA EFRVLRVPNPSSKRNEORIELFOILRPDEHIAKORYIGGKNLP TRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPN GDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKOKDHEINPH LILMMIPPHRLDNPGQGGORKKR proTGF - B3 C7S D2G SLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT 108 HVPYOVLALYNSTRELLEEMHGEREEGCTOENTESEYYAKEIH KFDMIOGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRA EFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKORYIGGKNLP TRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFOPN GDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHEINPH US 9 , 758 ,577 B2 37 38 TABLE 6 - continued Recombinant proteins

SEO Protein Sequence ID NO LILMMIPPHRLDNPGOGGQRKGALDTNYCFRNLEENCCVRPLY IDFRODLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGL YNTLNPEASASPCCVPODLEPLTILYYVGRTPKVEOLSNMVVK SCKCS proTGF - B3 D2G SLSLSTCTTLDFGHIKKKRVEAIRGOILSKLRLTSPPEPTVMT 109 HVPYQVLALYNSTRELLE EMHGEREEGCTCENTESEYYAKEIH KFDMIOGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRA EFRVLRVPNPSSKRNEORIELFQILRPDEHIAKORYIGGKNLP TRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPN GDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKOKDHEINPH LILMMIPPHRLDNPGOGGQRKGALDTNYCFRNLEENCCVRPLY IDFRODLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGL YNTLNPEASASPCCVPODLEPLTILYYVGRTPKVEOLSNMVVK SCKCS proTGF - B3 LAP SLSLSTCTTLDFGHIKKKRVEAIRGOILSKLRLTSPPEPTVMT 40 HVPYOVLALYNSTRELLE EMHGEREEGCTOENTESEYYAKEIH KFDMIOGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRA EFRVLRVPNPSSKRNEORIELFOILRPDEHIAKORYIGGKNLP TRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFOPN GDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKOKDHEINPH LILMMIPPHRLDNPGOGGORKKR

In some embodiments , activating mutations may com dimerization . Mutations to R218 , H222 , C223 and /or C225 prise residues critical for LAP or LAP - like protein dimeriza may lead to weakened or disrupted disulfide bond formation tion . Some activating mutations may comprise TGF- B iso - 20 and LAP dimerization . In some embodiments , CED muta forms ( TGF -B1 , TGF -B2 and/ or TGF -B3 ) . Mutant GPCs with activating mutations may comprise mutations that tions lead to elevated release of TGF -B and /or increased correspond to mutations identified in Camurati - Engelmann TGF - B activity . In some embodiments , recombinant GPCs disease (CED ) . Subjects suffering from CED typically have comprising TGF- B1 with CED mutations comprise genetic defects in TGF -B1 . Mutations identified in such sequences listed in Table 7 . The amino acid substitutions subjects include , but are not limited to mutations in residues 35 indicated in these proteins reflect the residue number as Y81, R218 , H222 , C223 and C225 . Residues C223 and counted from the start of the translated protein (before C225 are necessary for disulfide bond formation in LAP removal of the secretion signal sequence ). TABLE 7 Recombinant GPCs with Camurati - Engelmann mutations

SEQ ID Protein Sequence NO proTGF - B1 781H LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPPGP 110 LPEAVLALHNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAOHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTOYSKVLALYNOHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF -B1 R218C LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 111 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEOHVELYOKY SNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFCLSAHCSCDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF -B1 H222D LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 112 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSADCSCDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAOHLOSS US 9 ,758 ,577 B2 39 TABLE 7 - continued Recombinant GPCs with Camurati - Engelmann mutations

SEO ID Protein Sequence NO RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTOYSKVLALYNOHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF -B1 C223R LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPPGP 113 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHRSCDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTQYSKVLALYNCHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF - B1 C225R LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 114 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYOKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHCSRDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF - B1 C223R LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPPGP 115 C225R LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHRSRDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAOHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTOYSKVLALYNOHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS 35 GPCs comprising CED mutations may find several uses in Furin cleavage of recombinant proteins of the invention the context of the present invention . In some embodiments , may in some cases occur intracellularly . In some cases furin such GPCs may be used to produce recombinant proteins cleavage of recombinant proteins of the invention may occur comprising LAPs or LAP - like domains complexed with GARP. Coexpression of the entire GPC with GARP may be 40o extracellularly . necessary in some embodiments , for proper association and In some embodiments , recombinant GPCs of the present folding . Through expression of GPCs comprising CED invention may comprise mutations in one or more N - termi mutations, growth factors may be able to dissociate leaving nal regions for extracellular associations . As used herein , the the desired GARP -LAP complex . Y81H mutations may be term “ N -terminal region for extracellular association ” refers useful in this regard . Y81H mutations lead to growth factor 45 to regions at or near protein N - termini thatmay be necessary release , but do not disrupt disulfide bonding between LAP for extracellular associations with one or more N - terminal monomers at residues C223 and C225 . Therefore , GARP - regions . Such regions may comprise at least the first N - ter LAP complexes formed through expression of Y81H GPC minal residue , at least the first 5 N - terminal residues, at least mutants may comprise intact LAP dimers wherein growth the first 10 N - terminal residues , at least the first 20 amino factors have become dissociated . In some embodiments , 50 acid residues and / or at least the first 50 amino acid residues . additional co - expression or addition of excess furin during Some mutations may comprise from about 1 amino acid the production process may enhance growth factor disso - residue to about 30 amino acid residues, from about 5 amino ciation as well . acid residues to about 40 amino acid residues and / or from GPCs comprising CED mutations may be expressed to about 10 amino acid residues to about 50 amino acid allow for the production and release ofmature growth factor. 55 residues at or near protein N - termini. Such regions may Some GPC - free growth factors expressed according to this comprise residues for LTBP, fibrillin and /or GARP associa method may be used to assess antibody reactivity , for tion . In some cases, one or more cysteine residues present example in enzyme- linked immunosorbent assays ( ELI- within and/ or near N - terminal regions for extracellular asso SAs. ) Some GPCs comprising CED mutations may be ciations may be necessary for such associations . In some expressed to allow for the production and release of GPC - 60 embodiments , cysteine residues present within and / or near bound growth factors. GPCs comprising CED mutations N - terminal regions for extracellular associations are present may be expressed to allow for the production and release of within about the first 2 N - terminal residues , about the first 3 chimeric proteins comprising the TGF- B1 LAP (or protein N - terminal residues , about the first 4 N - terminal residues , modules or fragments thereof ) expressed with one or more about the first 5 N - terminal residues , about the first 6 protein modules from other TGF - B family members . Such 65 N - terminal residues, about the first 7 N -terminal residues chimeric proteins may comprise TGF -B1 LAP and TGF -B2 and / or at least the first 30 N - terminal residues . Some muta or TGF -B3 growth factor domains . tions in one or more N - terminal regions for extracellular US 9 , 758 ,577 B2 42 associations comprise substitution and /or deletion of such mutated In recombinant GPCs comprising N - terminal pro cysteine residues. Such mutations may modulate the asso domain regions from TGF - B3, cysteine residues at position ciation of GPCs and /or prodomains with one or more 7 may be mutated . extracellular proteins, including , but not limited to LTBPs, In some cases, one or more cysteine in one or more other fibrillins and /or GARP. These mutations may also comprise 5 region of GPCs may be substituted or deleted . In some substitution of one or more cysteine with another amino embodiments , such GPC modifications may promote the acid . Cysteine residue substitutions are abbreviated herein as release ofmature growth factor from prodomains . In some “ C # X ” wherein # represents the residue number [counting cases , such cysteines may include those present in one or from the N -terminus of the pro -protein (without the signal more of mature growth factors , alpha 2 helices, fasteners , peptide ) ] of the original cysteine residue and X represents 10 latency lassos and / or bow - tie regions . the one letter amino acid code for the amino acid that is used In some embodiments , recombinant proteins of the pres for substitution . Any amino acid may be used for such ent invention may comprise protein modules derived from substitutions . In some cases , serine ( S ) residues are used to one or more species , including mammals , including , but not substitute cysteine residues . Nonlimiting examples of such limited to mice, rats , rabbits , pigs , monkeys and / or humans . mutations may include C4S , C5S and / or C7S . In recombi- 15 Recombinant proteins may comprise one or more amino nant GPCs comprising N - terminal prodomain regions from acids from one or more amino acid sequences derived from TGF- B1, cysteine residues residing at amino acid position one or more non -human protein sequences listed in Table 8 . number 4 may be mutated . In recombinant GPCs comprising In some cases , recombinant proteins of the present invention N -terminal prodomain regions from TGF -B2 , cysteine resi - may comprise such sequences with or without the native dues residing at amino acid position number 5 may be signal peptide . TABLE 8 Non - human proteins

SEO ID Protein Species Sequence NO proTGF - B1 Mouse LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 116 GPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEV TRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPP LLSRAELRLORLKSSVEOHVELYOKY SNNSWRYLGNRL LTPTDTPEWLSFDVTGVVROWLNQGDGIQGFRFSAHCS CDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMAT PLERAOHLHSSRHRRALDTNYCFSSTEKNCCVROLYIDF RKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTOYSKVL ALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCS proTGF - B1 Cyno LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPP 117 GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVT RVLMVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPV LLSRAELRLLRLKLKVEOHVELYOKY SNNSWRYLSNRL LAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAHCSC DSKDNTLOVDINGFTTGRRGDLATIHGMNRPFLLLMAT PLERAOHLOSSRHRRALDTNYCFSSTEKNCCVROLYIDF RKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVL ALYNOHNPGASAAPCCV POALEPLPIVYYVGRKPKVEO LSNMIVRSCKCS proTGF - B1 Mouse LSTSKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPP 118 C4S ( LAP ) GPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEV TRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPP LLSRAELRLORLKSSVEOHVELYOKY SNNSWRYLGNRL LTPTDTPEWLSFDVTGVVROWLNOGDGIOGFRFSAHCS CDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMAT PLERAOHLHSSRHRR proTGF - B1 Cyno LSTSKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPP 119 C4S ( LAP ) GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVT RVLMVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPV LLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRL LAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAHCSC DSKDNTLOVDINGFTTGRRGDLATIHGMNRPFLLLMAT PLERAQHLOSSRHRR proTGF - B1 Mouse LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 120 C4S D2G GPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEV TRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPP LLSRAELRLORLKSSVEOHVELYOKY SNNSWRYLGNRL LTPTDTPEWLSFDVTGVVROWLNQGDGIQGFRFSAHCS CDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMAT PLERAQHLHSSRHGALDTNYCFSSTEKNCCVROLYIDFR KDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLA LYNCHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLS NMIVRSCKCS US 9 , 758 ,577 B2 43 44 TABLE 8 - continued Non - human proteins SEO ID Protein Species Sequence NO proTGF - B1 Mouse LSTSKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPP 121 C4S GPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEV TRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPP LLSRAELRLORLKSSVEOHVELYOKYSNNSWRYLGNRL LTPTDTPEWLSFDVTGVVROWLNQGDGIQGFRFSAHCS CDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMAT PLERAOHLHSSRHRRALDTNYCFSSTEKNCCVROLYIDF RKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVL ALYNOHNPGASASPCCVPOALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCS proTGF - B1 Cyno LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPP 122 C4S GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVT RVLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPV LLSRAELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRL LAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAHCSC DSKDNTLOVDINGFTTGRRGDLATIHGMNRPFLLLMAT PLERAOHLOSSRHRRALDTNYCFSSTEKNCCVROLYIDF RKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTOYSKVL ALYNOHNPGASAAPCCVPOALEPLPIVYYVGRKPKVEO LSNMIVRSCKCS proTGF - B1 Cyno LSTSKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPP 123 CHS D2G GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVT RVLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPV LLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRL LAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAHCSC DSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMAT PLERAOHLOSSRHGALDTNYCFSSTEKNCCVROLYIDFR KDLGWKWIHEPKGYHANFCLGPCPYIWSLDTOYSKVLA LYNOHNPGASAAPCCVPOALEPLPIVYYVGRKPKVEOL SNMIVRSCKCS LRRC32 Cyno MSPQILLLLALLTLGLAAQHQDKVACKMVDKKVSCOG 124 LGLLQVPLVLPPDTETLDLSGNQLRSILASPLGFYTALRH LDLSTNEINFLQPGAFQALTHLEHLSLAHNRLAMATALS AGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSL AENSLTRLTRHTFRDMPALEOLDLHSNVLMDIEDGAFE GLPHL THLNLSRNSLTCISDFSLQQLRVLDLSCNSIEAFQ TASQPQAEFQLTWLDLRENKLLHFPDLAALPRLIYLNLS NNLIRLPTGPPODSKGIHAPSEGWSALPLSTPNGNVSARP LSOLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTF EARRSGSLPCLMLLDLSHNALETLELGARALGSLRTLLL OGNALRDLPPYTFANLASLORLNLOGNRVSPCGGPNEP GPASCVAFSGIASLRSLSLVDNEIELLRAGAFLHTPLTEL DLSSNPGLEVATGALTGLEASLEVLALOGNGLTVLOVD LPCFICLKRLNLAENRLSHLPAWTOAVSLEVLDLRNNSF SLLPGSAMGGLETSLRRLYLOGNPLSCCGNGWLAAOLH QGRVDVDATQDLICRFSSQEEVSLSHVRPEDCEKGGLK NINLIIILTFILVSAILLTTLATCCCVRRQKFNQQYKA proGDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIOILS 125 KLRLETAPNISKDAIROLLPRAPPLRELIDOYDVORDDSS DGSLEDDDYHATTETIITMPTESDFLMOADGKPKCCFFK FSSKIOYNKVVKAOLWIYLRPVKTPTTVFVOILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLONWLKO PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGW DWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPR GSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRC GCS proGDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIQILS 126 ???? KLRLETAPNISKDAIROLLPRAPPLRELIDQYDVORDDSS DGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFK FSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLQNWLKQ PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGW DWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPR GSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRC GCS US 9 , 758 ,577 B2 45 46 TABLE 8 - continued Non - human proteins SEO ID Protein Species Sequence NO proGDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIQILS 127 D76A KLRLETAPNISKDAIROLLPRAPPLRELIDOYDVORADSS DGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFK FSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLONWLKO PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGW DWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPR GSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRC GCS proGDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIQILS 128 AXXA D76A KLRLETAPNISKDAIROLLPRAPPLRELIDOYDVORADSS DGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFK FSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLONWLKO PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGW DWIIAPKRYKANYCSGECEFVFLOKYPHTHLVHOANPR GSAGPCCTPTKMSPINMLYFNGKEOIIYGKIPAMVDRC GCS

GDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIQILS 129 prodomain KLRLETAPNISKDAIROLLPRAPPLRELIDOYDVORDDSS DGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFK FSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLONWLKO PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKRSRR

GDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIQILS 130 prodomain KLRLETAPNISKDAIROLLPRAPPLRELIDQYDVORADSS D76A DGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFK FSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLONWLKO PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKRSRR proGDF - 8 Cyno NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 131 LRLETAPNISKDAIROLLPKAPPLRELIDQYDVORDDSSD GSLEDDDYHATTETII TMPTESDFLMOVDGKPKCCFFKF SSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWD WIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRG SAGPCCTPTKMSPINMLYFNGKEQI IYGKIPAMVVDRCG CS profDF - 8 Cyno NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 132 ???? LRLETAPNISKDAIROLLPKAPPLRELIDQYDVORDDSSD GSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKF SSKIOYNKVVKAOLWIYLRPVETPTTVFVOILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLONWLKOP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGWD WIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRG SAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS proGDF - 8 Cyno NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 133 D76A LRLETAPNISKDAIRQLLPKAPPLRELIDQYDVORADSSD GSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKF SSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLONWLKOP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWD WIIAPKRYKANYCSGECEFVFLOKYPHTHLVHOANPRG SAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS US 9 , 758 ,577 B2 47 48 TABLE 8 - continued Non - human proteins SEO ID Protein Species Sequence NO proGDF - 8 Cyno NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 134 AXXA D76A LRLETAPNISKDAIROLLPKAPPLRELIDOYDVORADSSD GSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKF SSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLONWLKOP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGWD WIIAPKRYKANYCSGECEFVFLOKYPHTHLVHOANPRG SAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS GDF - 8 Cyno NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 135 prodomain LRLETAPNISKDAIROLLPKAPPLRELIDOYDVORDDSSD GSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKF SSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKRSRR GDF - 8 Cyno NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 136 prodomain LRLETAPNISKDAIROLLPKAPPLRELIDOYDVORADSSD D76A GSLEDDDYHATTETII TMPTESDFLMOVDGKPKCCFFKF SSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLONWLKOP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKRSRR

proGDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 137 CVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKOLL PKAPPLQQILDLHDFOGDALOPEDFLEEDEYHATTETVIS MAQETDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOLW VYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRN LGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKA NYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPT KMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 138 ???? CVWROHSRELRLESIKSOILSKLRLKEAPNISREVV KOLL PKAPPLOOILDLHD FOGDALOPEDFLEEDEYHATTETVIS MAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLW VYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKASRA NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYK ANYCSGQCEYMFMOKYPHTHLVQQANPRGSAGPCCTP TKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 139 AXXA D96A CVWROHSRELRLESIKSQILSKLRLKEAPNISREVVKOLL PKAPPLQQILDLHD FOGAALOPEDFLEEDEYHATTETVIS MAQETDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOLW VYLRPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKASRA NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYK ANYCSGQCEYMFMOKYPHTHLVQQANPRGSAGPCCTP TKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS

proGDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 140 D96A CVWROHSRELRLESIKSOILSKLRLKEAPNISREVV KOLL PKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVIS MAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLW VYLRPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRN LGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKA NYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPT KMSPINMLYFNDKOQI LYGKIPGMVVDRCGCS US 9 , 758 ,577 B2 49 50 TABLE 8 - continued Non - human proteins SEO ID Protein Species Sequence NO GDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 141 prodomain CVWROHSRELRLESIKSOILSKLRLKEAPNISREV KOLL PKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVIS MAQETDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOLW VYLRPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRR GDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 142 prodomain CVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKOLL D96A PKAPPLOOILDLHD FOGAALOPEDFLEEDEYHATTETVIS MAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLW VYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRR LTBP3 CYNO MPGPRGAPGGLAPEMRGAGAAGLLALLLLLGLGGRVE 143 GGPAGERGAGGGGALARERFKVVFAPVICKRTCLKGOC RDSCOOGSNMTLIGENGHSTDTLTGSGFRVVVCPLPCM NGGOCSSRNOCLCPPDFTGRFCOVPAGGAGGGTGGSGP GLSRAGALSTGALPPLAPEGDSVAS KHAIYAVOVIADPP GPGEGPPAQHAAFLVPLGPGQISAEVQAPPPVVNVRVH HPPEASVOVHRIESSNAEGAAPSOHLLPHPKPSHPRPPTO KPLGRCFODTLPKQPCGSNPLPGLTKQEDCCGSIGTAWG QSKCHKCPQLQYTGVQKPGPVRGEVGADCPQGYKRLN STHCODINECAMPGVCRHGDCLNNPGSYRCVCPPGHSL GPSRTOCIADKPEEKSLCFRLVSPEHQCQHPLTTRLTROL CCCSVGKAWGARCQRCPADGTAAFKEICPAGKGYHILT SHQTLTIQGESDFSLFLHPDGPPKPQQLPESPSQAPPPEDT EEERGVTTDSPVSEERSVOOSHPTATTSPARPYPELISRPS PPTMRWFLPDLPPSRSAVEIAPTOVTETDECRLNONICG HGECVPGPPDYSCHCNPGYRSHPOHRYCVDVNECEAEP CGPGRGI CMNTGGSYNCHCNRGYRLHVGAGGRSCVDL NECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPP VCEDIDECRDPSSCPDGKCENKPGSFKCIACOPGYRSOG GGACRDVNECAEGSPCSPGWCENLPGSFRCTCAOGYAP APDGRSCVDVDECEAGDVCDNGICTNTPGSFQCQCLSG YHLSRDRSHCEDIDECDFPAACIGGDCINTNGSYRCLCP QGHRLVGGRKCODIDECTQDPGLCLPHGACKNLQGSYV CVCDEGFTPTQDQHGCEEVEQPHHKKECYLNFDDTVFC DSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEF HSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGAEICK EGKCVNTOPGYECYCKOGFYYDGNLLECVDVDECLDE SNCRNGVCENTRGGYRCACTPPAEYSPAOROCLSPEEM DVDECODPAACRPGRCVNLPGSYRCECRPPWVPGPSGR DCOLPESPAERAPERRDVCWSORGEDGMCAGPOAGPA LTFDDCCCROGRGWGAOCRPCPPRGAGSOCPTSOSESN SFWDTSPLLLGKPRRDEDSSEEDSDECRCVSGRCVPRPG GAVCECPGGFOLDASRARCVDIDECRELNORGLLCKSE RCVNTSGSFRCVCKAGFARSRPHGACVPORRR

LTBP3 Mouse MPGPRGAAHGLAPAMHOAGALGLLALLLLALLGPGGG 144 AEGGPAGERGTGGGGALARERFKVVFAPVICKRTCLKG QCRDSCQQGSNMTLIGENGHSTDTLTGSAFRVVVCPLPC MNGGOCSSRNOCLCPPDFTGRFCOV PAAGTGAGTGSSG PGLARTGAMSTGPLPPLAPEGESVASKHAIYAVQVIADP PGPGEGPPAOHAAFLVPLGPGOISAEVOAPPPVVNVRVH HPPEASVOVHRIEGPNAEGPASSOHLLPHPKPPHPRPPTO KPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWG QSKCHKCPQLQYTGVQKPVPVRGEVGADCPQGYKRLN STHCODINECAMPGNV CHGDCLNNPGSYRCVCPPGHSL GPLAAOCIADKPEEKSLCFRLVSTEHOCOHPLTTRLTRO LCCCSVGKAWGARCORCPADGTAAFKEICPGKGYHILT SHOTLTIOGESDFSLFLHPDGPPKPOOLPESPSRAPPLEDT EEERGVTMDPPVSEERSVOOSHPTTTTSPPRPYPELISRPS PPTFHRFLPDLPPSRSAVEIAPTOVTETDECRLNONICGH GOCVPGPSDYSCHCNAGYRSHPOHRYCVDVNECEAEPC GPGKGICMNTGGSYNCHCNRGYRLHVGAGGRSCVDLN ECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPPIC EDIDECRDPSTCPDGKCENKPGSFKCIACOPGYRSOGGG ACRDVNECSEGTPCSPGWCENLPGSYRCTCAQYEPAQD GLSCIDVDECEAGKVCODGICTNTPGSFQCQCLSGYHLS RDRSRCEDIDECDFPAACIGGDCINTNGSYRCLCPLGHR LVGGRKCKKDIDECSODPGLCLPHACENLOGSYVCVCD US 9 , 758 ,577 B2 51 52 TABLE 8 - continued Non - human proteins SEO ID Protein Species Sequence NO EGFTLTODOHGCEEVEOPHHKKECYLNFDDTVFCDSVL ATNVTOOECCCSLGAGWGDHCEIYPCPVYSSAEFHSLV PDGKRLHSGQQHCELCIPAHRDIDECILFGAEICKEGKCV NTOPGYECYCKOGFYYDGNLLECVDVDECLDESNCRN GVCENTRGGYRCACTPPAEYSPAQAQCLIPERWSTPOR DVKCAGASEERTACVWGPWAGPALTFDDCCCROPRLG TOCRPCPPRGTGSOCPTSOSESNSFWDTSPLLLGKSPRDE DSSEEDSDECRCVSGRCVPRPGGAVCECPGGFOLDASR ARCVDIDECRELNQRGLLCKSERCVNTSGSFRCVCKAGF TRSRPHGPACLSAAADDAAIAHTSVIDHRGYFH

LTBP1 Cyno MAGAWLRWGLLLWAGLLASSAHGRLRRITYVVHPGPG 145 LAAGALPLSGPPRSRTFNVALNARYSRSSAAAGAPSRAS PGVPSERTRRTSKPGGAALOGLRPPPPPPPEPARPAAPGG QLHPKPGGHPAAAPFAKQGRQVVRSKVPQETQSSGGSR LQVHQKQQLQGVNVCGGRCCHGWSKAPGSQRCTKRSC VPP CONGGMCLRPOLCVCKPGTKGKACETIAAODTSSP VFGGOSPGAASSWGPPEQAAKHTSSKKADTLPRVSPVA QMTLTLKPKPSVGLPQQIHSQVTPLSSQSVMIHHSQTQE YVLKPKYFPAOKGISGEOSTEGSFPLRYVODOVAAP FOL SNHTGRIKVVFTPSICKVTCTKGSCONSCEKGNTTTLISE NGHAADTLTATNFRWVLCHLPCMNGGQCSSRDKCOCPP NFTGKLCQIPVHGASVPKLYQHSQQPGKALGTHVIHSTH TLPLTVTSOOGVKVKFPPNIVNIHVKHPPEASVQIHOVSR IDGPTGQKTKEAQPGQSQVSYQGLPVQKTQTIHSTYSHQ QVIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSKQED CCGTVGTSWGFNKCOKCPKKPSYHGYNOMMECLPGYK RVNNTFCQDINECOLOGVCPNGECLNTMGSYRCTCKIG FGPDPTFSSCVPDPPVISEEKGPCYRLVSSGRQCMHPLSV HLTKOLCCCSVGKAWGPHCEKCPLPGTAAFKEICPGGM GYTVSGVHRRRPIHHHVGKGPVFVKPKNTOPVAKSTHP PPLPAKEEPVEALTFSREHGPGVAEPEVATAPPEKEIPSL DQEKTKLEPGQPQLSPGISTIHLHPQFPVVIEKTSPPVPVE VAPEASTSSASOVIAPTOVTEINECTVNPDICGAGHCINL PVRYTCICYEGYKFSEQORKCVDIDECTOVOHLCSOGRC ENTEGSFLCICPAGFMASEEGTNCIDVDECLRPDVCGEG HCVNTVGAFRCEYCDSGYRMTORGRCEDIDECLNPSTC PDEOCVNSPGSYOCVPCTEGFRGWNGOCLDVDECLEPN VCTNGDCSNLEGSYMCSCHKGYTRTPDHKHCKDIDECO QGNLCVNGQCKNTEGSFRCTCGQGYQLSAAKDQCEDID ECQHHHLCAHGQCRNTEGSFQCVCDQGYRASGLGDHC EDINECLEDKSVCORGDCINTAGSYDCTCPDGFOLDDN KTCODINECEHPGLCGPQGECLNTEGSFHCVCQQGFSIS ADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYOG FOAPODGOGCVDVNECELLSGVCGEAFCENVEGSFLCV CADENOEYSPMTGOCRSRTSTDLDVEOPKEEKKECYYN LNDASLCDNVLAPNVTKOECCCTSGAGWGDNCEIFPCP VLGTAEFTEMCPKGKGFVPAGESSSEAGGENYKDADEC LLFGOEICKNGFCLNTRPGYECYCKOGTYYDPVKLOCF DMDECODPSSCIDGQCVNTEGSYNCFCTHPMVLDASEK RCIRPAESNEQIEETDVYQDLCWEHLSDEYVCSRPLVGK OTTYTECCCLYGEAWGMOCALCPMKDSDDYAOLCNIP VTGRROPYGRDALVDFSEQYAPEADPYFIQDRFLNSFEE LQAEECGILNGCENGRCVRVQEGYTCDCFDGYHLDTAK MTCVDVNECDELNNRMSLCKNAKCINTEGSYKCLCLPG YVPSDKPNYCTPLNTALNLEKDSDLE

LTBP1S mouse NHTGRIKVVFTPSICKVTCT KGNCONSCOKGNTTTLISE 146 NGHAADTLTATNFRWVICHLPCMNGGQCSSRDKCQCPP NFTGKLCQIPVLGASMPKLYQHAQQQGKALGSHVIHST HTLPLTMTSQQGVKVKFPPNIVNIHVKHPPEASVQIHOV SRIDSPGGQKVKEAQPGQSQVSYQGLPVQKTQTVHSTY SHQQLIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSK QEDCCGTVGTSWGFNKCQKCPKKQSYHGYTQMMECL QGYKRVNNTFCQDINECQLQGVCPNGECLNTMGSYRCS CKMGFGPDPTFSSCVPDPPVISEEKGPCYRLVSPGRHCM HPLSVHLTKOICCCSVGKAWGPHCEKCPLPGTAAFKEIC PGGMGYTVSGVHRRRPIHOHIGKEAVYVKPKNTOPVAK STHPPPLPAKEEPVEALTSSWEHGPRGAEPEWTAPPEK EIPSLDQEKTRLEPGQPQLSPGVSTIHLHPQFPVWEKTSP PVPVEVAPEASTSSASOVIAPTOVTEINECTVNPDICGAG HCINLPVRYTCICYEGYKFSEQLRKCVDIDECAQVRHLC SOGRCENTEGSFLCVCPAGFMASEEGTNCIDVDECLRPD MCRDGRCINTAGAFRCEYCDSGYRMSRRGYCEDIDECL US 9 , 758 ,577 B2 53 54 TABLE 8 - continued Non - human proteins SEO ID Protein Species Sequence NO KPSTCPEEQCVNTPGSYQCVPCTEGFRGWNGOCLDVDE CLOPKVCTNGSCTNLEGSYMCSCHRGYSPTPDHRHCOD IDECOQGNLCMNGQCRNTDGSFRCTCGQGYQLSAAKD QCEDIDECEHHHLCSHGQCRNTEGSFQCVCNQGYRASV LGDHCEDINECLEDSSVCQGGDCINTAGSYDCTCPDGFQ LNDNKGCODINECAQPGLCGSHGECLNTQGSFHCVCEQ GFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCL CYOGFQAPODGOGCVDVNECELLSGVCGEAFCENVEGS FLCVCADENQEYSPMTGQCRSRVTEDSGVDRQPREEKK ECYYNLNDASLCDNVLAPNVTKOECCCTSGAGWGDNC EIFPCPVOGTAEFTEMCPRGKGLVPAGESSYDTGGENYK DADECLLFGEEICKNGYCLNTQPGYECYCKQGTYYDPV KLQCFDMDECODPNSCIDGQCVNTEGSYNCFCTHPMVL DASEKRCVOPTESNEOIEETDVYODLCWEHLSEEYVCSR PLVGKQTTYTECCCLYGEAWGMQCALCPMKDSDDYA QLCNIPVTGRRRPYGRDALVDFSEQYGPETDPYFIQDRF LNSFEELOAEECGILNGCENGRCVRVOEGYT CDCFDGY HLDMAKMTCVDVNECSELNNRMSLCKNAKCINTEGSY KCLCLPGYIPSDKPNYCTPLNSALNLDKESDLE

GARP mouse ISQRREQVPCRTVNKEALCHGLGLLQVPSVLSLDIQALY 147 LSGNQLQSILVSPLGFYTALRHLDLSDNQISFLQAGVFQA LPYLEHLNLAHNRLATGMALNSGGLGRLPLLVSLDLSG NSLHGNLVERLLGETPRLRTLSLAENSLTRLARHTFWG MPAVEOLDLHSNVLMDIEDGAFEALPHLTHLNLSRNSL TCISDFSLOOLOVLDLSCNSIEAFOTAPEPQAQFOLAWL DLRENKLLHFPDLAVFPRLIYLNVSNNLIQLPAGLPRGSE DLHAPSEGWSASPLSNPSRNASTHPLSQLLNLDLSYNEIE LVPASFLEHLTSLRFLNLSRNCLRSFEAROVDSLPCLVLL DLSHNVLEALELGTKVLGSLOTLLLODNALQELPPYTFA SLASLQRLNLQGNQVSPCGGPAEPGPPGCVDFSGIPTLH VLNMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATG ALVGLEASLEVLELOGNGLTVLRVDLPCFLRLKRLNLAE NOLSHLPAWTRAVSLEVLDLRNNSFSLLPGNAMGGLET SLRRLYLOGNPLSCCGNGWLAAOLHOGRVDVDATODL ICRFGSQEELSLSLVRPEDCEKGGLKNVNLILLLSFTLVS AIVLTTLATICFLRROKLSQQYKA

SGARP mouse ISORREQVPCRTVNKEALCHGLGLLQVPSVLSLDIQALY 148 LSGNQLQSILVSPLGFYTALRHLDLSDNQISFLQAGVFQA LPYLEHLNLAHNRLATGMALNSGGLGRLPLLVSLDLSG NSLHGNLVERLLGETPRLRTLSLAENSLTRLARHTFWG MPAVEOLDLHSNVLMDIEDGAFEALPHLTHLNLSRNSL TCISDFSLOOLOVLDLSCNSIEAFOTAPEPOAOFOLAWL DLRENKLLHFPDLAVFPRLIYLNVSNNLIOLPAGLPRGSE DLHAPSEGWSASPLSNPSRNASTHPLSQLLNLDLSYNEIE LVPASFLEHLTSLRFLNLSRNCLRSFEAROVDSLPCLVLL DLSHNVLEALELGTKVLGSLOTLLLODNALOELPPYTFA SLASLORLNLOGNOVSPCGGPAEPGPPGCVDFSGIPTLH VLNMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATG ALVGLEASLEVLELOGNGLTVLRVDLPCFLRLKRLNLAE NOLSHLPAWTRAVSLEVLDLRNNSFSLLPGNAMGGLET SLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDL ICRFGSQEELSLSLVRPEDCEKGGLKNVN

LRRC33 mouse WRSGPGTATAASQGGCKVVDGVADCRGLNLASVPSSLP 149 PHSRMLILDANPLKDLWNHSLOAYPRLENLSLHSCHLD RISHYAFREOGHLRNLVLADNRLSENYKESAAALHTLL GLRRLDLSGNSLTEDMAALMLONLSSLEVVSLARNTLM RLDDSIFEGLEHLVELDLQRNYIFEIEGGAFDGLTELRRL NLAYNNLPCIVDFSLTOLRFLNVSYNILEWFLAAREEVA FELEILDLSHNOLLFFPLLPOCGKLHTLLLODNNMGFYR ELYNTSSPOEMVAOFLLVDGNVTNITTVNLWEEFSSSDL SALRFLDMSONOFRHLPDGFLKKTPSLSHLNLNONCLK MLHIREHEPPGALTELDLSHNQLAELHLAPGLTGSLRNL RVFNLSSNOLLGVPTGLFDNASSITTIDMSHNOISLCPOM VPVDWEGPPSCVDFRNMGSLRSLSLDGCGLKALODCPF OGTSLTHLDLSSNWGVLNGSISPLWAVAPTLOVLSLRD VGLGSGAAEMDFSAFGNLRALDLSGNSLTSFPKFKGSLA LRTLDLRRNSLTAL PORVVSEOPLRGLOTIYLSONPYDC CGVEGWGALQQHFKTVADLSMVTCNLSSKIVRVVELPE GLPQGCKWEQVDTGLFYLVLILPSCLTLLVACTVVFLTF KKPLLQVIKSRCHWSSIY US 9 , 758 ,577 B2 55 56 TABLE 8 - continued Non -human proteins SEQ ID Protein Species Sequence NO

SLRRC33 mouse WRSGPGTATAASQGGCKVVDGVADCRGLNLASVPSSLP 150 PHSRMLILDANPLKDLWNHSLQAYPRLENLSLHSCHLD RISHYAFREQGHLRNLVLADNRLSENYKESAAALHTLL GLRRLDLSGNSLTEDMAALMLONLSSLEVVSLARNTLM RLDDSIFEGLEHLVELDLQRNYIFEIEGGAFDGLTELRRL NLAYNNLPCIVDFSLTQLRFLNVSYNILEWFLAAREEVA FELEILDLSHNOLLFFPLLPQCGKLHTLLLQDNNMGFYR ELYNTSSPQEMVAQFLLVDGNVTNITTVNLWEEFSSSDL SALRFLDMSONQFRHLPDGFLKKTPSLSHLNLNONCLK MLHIREHEPPGALTELDLSHNQLAELHLAPGLTGSLRNL RVFNLSSNQLLGVPTGLFDNASSITTIDMSHNQISLCPQM VPVDWEGPPSCVDFRNMGSLRSLSLDGCGLKALQDCPF QGTSLTHLDLSSNWGVLNGSISPLWAVAPTLQVLSLRD VGLGSGAAEMDFSAFGNLRALDLSGNSLTSFPKFKGSLA LRTLDLRRNSLTALPQRVVSEQPLRGLQTIYLSQNPYDC CGVEGWGALQQHFKTVADLSMVTCNLSSKIVRVVELPE GLPQGCKWEQVDTGL

LRRC33 Cyno WRDRSVTATAASQRGCKLVGGDTDCRGOSLASVPSSLP 151 PHARTLILDANPLKALWNHSLQPYPLLESLSLHSCHLERI GRGAFQEQGHLRSLVLGDNCLSENYKETAAALHTLPGL QTLDLSGNSLTEDMAALMLQNLSSLQSVSLARNTIMRL DDSVFEGLERLRELDLORNYIFEIEGGAFDGLTELRHLNL AYNNLPCIVDFGLTQLRSLNVSYNVLEWFLAAGGEAAF ELETLDLSHNOLLFFPLLPQYSKLHTLLLRDNNMGFYRD LYNTSSPREMVAQFLLVDGNVTNITTVNLWEEFSSSDLA DLRFLDMSONQFQYLPDGFLRKMPSLSHLNLNQNCLMT LHIREHEPPGALTELDLSHNOLSELHLTPGLASCLGSLRL FNLSSNQLLGVPPGLFANARNITTLDMSHNQISLCPLPAA SDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFOGT SLTSLDLSSNWGVLNGSLAPLRDVAPMLQVLSLRNMGL HSNFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALET LDLRRNSLTALPQKAVSEQLSRGLRTIYLSQNPYDCCGV DGWGALQQGQTVADWATVTCNLSSKIIRLAELPGGVPR DCKWERLDLGLLYLVLILPSCLTLLVACTLIVLTFKKPLL QVIKSRCHWSSVY

SLRRC33 Cyno WRDRSVTATAASORGCKLVGGDTDCRGOSLASVPSSLP 152 PHARTLILDANPLKALWNHSLQPYPLLESLSLHSCHLERI GRGAFQEQGHLRSLVLGDNCLSENYKETAAALHTLPGL QTLDLSGNSLTEDMAALMLQNLSSLQSVSLARNTIMRL DDSVFEGLERLRELDLQRNYIFEIEGGAFDGLTELRHLNL AYNNLPCIVDFGLTQLRSLNVSYNVLEWFLAAGGEAAF ELETLDLSHNOLLFFPLLPQYSKLHTLLLRDNNMGFYRD LYNTSSPREMVAQFLLVDGNVTNITTVNLWEEFSSSDLA DLRFLDMSQNQFQYLPDGFLRKMPSLSHLNLNQNCLMT LHIREHEPPGALTELDLSHNOLSELHLTPGLASCLGSLRL FNLSSNOLLGVPPGLFANARNITTLDMSHNQISLCPLPAA SDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQGT SLTSLDLSSNWGVLNGSLAPLRDVAPMLQVLSLRNMGL HSNFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALET LDLRRNSLTALPQKAVSEQLSRGLRTIYLSQNPYDCCGV DGWGALQQGQTVADWATVTCNLSSKIIRLAELPGGVPR DCKWERLDLGL US 9 , 758 ,577 B2 57 58 In some embodiments , recombinant proteins may be In some embodiments , complexed LTBPs may include, combined and /or complexed with one or more additional but are not limited to LTBP1, LTBP2 ,LTBP3 and /or LTBP4 . recombinant components . Such components may include Complexed LTBPs may comprise LTBP fragments and /or extracellular proteins known to associate with GPCs includ ing , but not limited to LTBPs, fibrillins, perlecan , GASP1/ 2 5 mutations. Some recombinant forms of LTBPs complexed proteins , follistatin , follistatin - related gene ( FLRG ), decorin 5 with recombinant GPCs may comprise alternatively spliced and / or GARP ( including , but not limited to recombinant variants of LTBPs. Some such variants of LTBP1 are short forms of such proteins) . Some recombinant GPCs of the ened at the N - terminus , referred to herein as LTBP1S . Some present invention must be co -expressed with one or more of recombinant proteins of the present invention may comprise such extracellular proteins for proper expression and / or LTBPs, fragments or mutants thereof comprising the amino folding acid sequences listed in Table 9 . TABLE 9 LTBP sequences

SEQ ID Protein Sequence NO LTBP1 1265 - 1443 NECELLSGVCGEAFCENVEGSFLCVCADENOEYSPMTGOC 153 RSRTSTDLDVDVDQPKEEKKECYYNLNDASLCDNVLAPNV TKQECCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGF VPAGESSSEAGGENYKDADECLLFGOEICKNGFCLNTRPGY ECYCKOGTYYDPVKLOCE

LTBP1 1265 - 1698 NECELLSGVCGEAFCENVEGSFLCVCADENOEYSPMTGOC 154 RSRTSTDLDVDVDQPKEEKKECYYNLNDASLCDNVLAPNV TKOECCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGF VPAGESSSEAGGENYKDADECLLFGOEICKNGFCLNTRPGY ECYCKQGTYYDPVKLQCFDMDECODPSSCIDGQCVNTEGS YNCFCTHPMVLDASEKRCIRPAESNEQIEETDVYQDLCWE HLSDEYVCSRPLVGKOTTYTECCCLYGEAWGMQCALCPL KDSDDYAQLCNIPVTGRROPYGRDALVDFSEQYTPEADPY FIQDRFLNSFEELQAEECGILNGCENGRCVRVQEGYTCDCF DGYHLDTAKMTCVDVNECDELNNRMSLCKNAKCINTDGS YKCLCLPGYVPSDKPNYCTPLNTALNLEKDSDLE LTBP1 809 - 1698 PSLDOEKTKLEPGOPOLSPGISTIHLHPOFPVVIEKTSPPVPV 155 EVAPEASTSSASOVIAPTOVTEINECTVNPDICGAGHCINLP VRYTCICYEGYRFSEQQRKCVDIDECTQVQHLCSQGRCEN TEGSFLCICPAGFMASEEGTNCIDVDECLRPDVCGEGHCVN TVGAFRCEYCDSGYRMTQRGRCEDIDECLNPSTCPDEQCV NSPGSYOCVPCTEGFRGWNGOCLDVDECLEPNVCANGDC SNLEGSYMCSCHKGYTRTPDHKHCRDIDECOOGNLCVNG QCKNTEGSFRCTCGQGYQLSAAKDQCEDIDECQHRHLCAH GQCRNTEGSFQCVCDQGYRASGLGDHCEDINECLEDKSVC QRGDCINTAGSYDCTCPDGFOLDDNKTCODINECEHPGLC GPQGECLNTEGSFHCVCQQGFSISADGRTCEDIDECVNNTV CDSHGFCDNTAGSFRCLCYOGFQAPQDGQGCVDVNECEL LSGVCGEAFCENVEGSFLCVCADENOEYSPMTGOCRSRTS TDLDVDVDOPKEEKKECYYNLNDASLCDNVLAPNVTKOE CCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGFVPAG ESSSEAGGENYKDADECLLFGOEICKNGFCLNTRPGYECYC KOGTYYDPVKLOCFDMDECODPSSCIDGOCVNTEGSYNCF CTHPMVLDASEKRCIRPAESNEQIEETDVYODLCWEHLSDE YVCSRPLVGKOTTYTECCCLYGEAWGMOCALCPLKDSDD YAQLCNIPVTGRROPYGRDALVDFSEQYTPEADPYFIQDRF LNSFEELQAEECGILNGCENGRCVRVQEGYTCDCFDGYHL DTAKMTCVDVNECDELNNRMSLCKNAKCINTDGSYKCLC LPGYVPSDKPNYCTPLNTALNLEKDSDLE

LTBP1S NHTGRIKVVFTPSICKVTCTKGSCONSCEKGNTTTLISENGH 156 AADTLTATNFRVVICHLPCMNGGOCSSRDKCOCPPNFTGK LCQIPVHGASVPKLYQHSQQPGKALGTHVIHSTHTLPLTVT SQQGVKVKFPPNIVNIHVKHPPEASVQIHOVSRIDGPTGOK TKEAQPGQSQVSYQGLPVOKTQTIHSTYSHOOVIPHVYPVA AKTQLGRCFQETIGSQCGKALPGLSKQEDCCGTVGTSWGF NKCOKCPKKPSYHGYNOMMECLPGYKRVNNTFCODINEC OLOGVCPNGECLNTMGSYRCTCKIGFGPDPTFSSCVPDPPV ISEEKGPCYRLVSSGRQCMHPLSVHLTKQLCCCSVGKAWG PHCEKCPLPGTAAFKEICPGGMGYTVSGVHRRRPIFIHHVG KGPVFVKPKNTOPVAKS THPPPLPAKEEPVEALTFSREHGP GVAEPEVATAPPEKEIPSLDOEKTKLEPGOPOLSPGISTIHLH POFPWIEKTSPPVPVEVAPEASTSSASQVIAPTOVTEINECT VNPDICGAGHCINLPVRYTCICYEGYRFSEQORKCVDIDEC TOVOHLCSOGRCENTEGSFLCICPAGFMASEEGTNCIDVDE CLRPDVCGEGHCVNTVGAFRCEYCDSGYRMTQRGRCEDI DECLNPSTCPDEOCVNSPGSYOCVPCTEGFRGWNGOCLDV DECLEPNVCANGDCSNLEGSYMCSCHKGYTRTPDHKHCR US 9 ,758 , 577 B2 59 60 TABLE 9 - continued LTBP sequences SEQ ID Protein Sequence NO DIDECOQGNLCVNGQCKNTEGSFRCTCGQGYQLSAAKDO CEDIDECQHRHLCAHGQCRNTEGSFQCVCDQGYRASGLGD HCEDINECLEDKSVCQRGDCINTAGSYDCTCPDGFOLDDN KTCODINECEHPGLCGPQGECLNTEGSFHCVCQQGFSISAD GRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYQGFQAP ODGOGCVDVNECELLSGVCGEAFCENVEGSFLCVCADEN QEYSPMTGOCRSRTSTDLDVDVDOPKEEKKECYYNLNDAS LCDNVLAPNVTKOECCCTSGVGWGDNCEIFPCPVLGTAEF TEMCPKGKGFVPAGESSSEAGGENYKDADECLLFGQEICK NGFCLNTRPGYECYCKOGTYYDPVKLQCFDMDECODPSS CIDGOCVNTEGSYNCFCTHPMVLDASEKRCIRPAESNEOIE ETDVYQDLCWEHLSDEYVCSRPLVGKOTTYTECCCLYGEA WGMQCALCPLKDSDDYAQLCNIPVTGRROPYGRDALVDF SEQYTPEADPYFIQDRFLNSFEELQAEECGILNGCENGRCVR VQEGYTCDCFDGYHLDTAKMTCVDVNECDELNNRMSLCK NAKCINTDGSYKCLCLPGYVPSDKPNYCTPLNTALNLEKDS DLE

LTBP3 GPAGERGAGGGGALARERFKVVFAPVICKRTCLKGQCRDS 157 COOGSNMTLIGENGHSTDTLTGSGFRVWVCPLPCMNGGOC SSRNOCLCPPDFTGRFCOVPAGGAGGGTGGSGPGLSRTGA LSTGALPPLAPEGDSVASKHAIYAVOVIADPPGPGEGPPAO HAAFLVPLGPGOISAEVOAPPPVVNVRVHHPPEASVOVHRI ESSNAESAAPSOHLLPHPKPSHPRPPTOKPLGRCFODTLPKO PCGSNPLPGLTKQEDCCGSIGTAWGQS KCHKCPQLQYTGV OKPGPVRGEVGADCPOGYKRLNSTHCODINE CAMPGVCR HGDCLNNPGSYRCVCPPGHSLGPSRTOCIADKPEEKSLCFR LVSPEHQCQHPLTTRL TRQLCCCSVGKAWGARCORCPTDG TAAFKEICPAGKGYHILTSHQTLTIGESDFSLFLHPDGPPK POOLPESPSOAPPPEDTEEERGVTTDSPVSEERSVOOSHPTA TTTPARPYPELISRPSPPTMRWFLPDLPPSRSAVEIAPTOVTE TDECRLNONICGHGECVPGPPDYSCHCNPGYRSHPOHRYC VDVNECEAEPCGPGRGICMNTGGSYNCHCNRGYRLHVGA GGRSCVDLNECAKPHLCGDGGFCINFPGHYKCNCYPGYRL KASRPPVCEDIDECRDPSSCPDGKCENKPGSFKCIACOPGY RSOGGGACRDVNECAEGSPCSPGWCENLPGSFRCTCAOGY APAPDGRSCLDVDECEAGDVCDNGICSNTPGSFQCQCLSG YHLSRDRSHCEDIDECDFPAACIGGDCINTNGSYRCLCPQG HRLVGGRKCODIDECSODPSLCLPHGACKNLOGSYVCVCD EGFTPTQDQHGCEEVEQPHHKKECYLNFDDTVFCDSVLAT NVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLCPDGK GYTODNNIVNYGIPAHRDIDECMLFGSEICKEGKCVNTOPG YECYCKOGFYYDGNLLECVDVDECLDESNCRNGVCENTR GGYRCACTPPAEYSPAOROCLSPEEMDVDECODPAACRPG RCVNLPGSYRCECRPPWVPGPSGRDCOLPESPAERAPERRD VCWSORGEDGMCAGPLAGPALTFDDCCCROGRGWGAOC RPCPPRGAGSHCPTSOSESNSFWDTSPLLLGKPPRDEDSSEE DSDECRCVSGRCVPRPGGAVCECPGGFOLDASRARCVDID ECRELNQRGLLCKSERCVNTSGSFRCVCKAGFARSRPHGA CVPQRRR

In some embodiments , LTBPs may comprise detectable 50 (LRRC33 ) or fragments and / or mutants thereof may be labels . Detectable labels may be used to allow for detection substituted for GARP [ also referred to herein as leucine rich and / or isolation of recombinant proteins comprising LTBPs. repeat containing 32 (LRRC32 . ) ] Such LRRC33 fragments Some detectable labels may comprise biotin labels , poly - and /or mutants may comprise one or more regions from the histidine tags and /or flag tags. Such tags may be used to LRRC33 sequence listed in Table 10 below . Recombinant isolate tagged proteins . Proteins produced may comprise 55 GARPs may also comprise mutants and / or GARP frag additional amino acids encoding one or more 3C protease ments . Some recombinant GARPs may be soluble (referred cleavage site . Such sites allow for cleavage at the 3C protease cleavage site upon treatment with 3C protease , to herein as SGARP ) . including , but not limited to rhinovirus 3C protease . Such In some embodiments , recombinant GARPs may com cleavage sites may be introduced to allow for removal of 60 prise one or more amino acid sequences listed in Table 10 . detectable labels from recombinant proteins. Some recombinant GARPs used herein may be expressed In some embodiments, GARPs, including , but not limited without the N - terminal residues AQ . Expressed GARPs may to recombinant forms of GARP , may be complexed with comprise detectable labels . Such detectable labels may be recombinant GPCs. Some recombinant GPCs of the present used to allow for detection and /or isolation . Some detectable invention may be co -expressed with GARPs to ensure 65 labels may comprise biotin labels , polyhistidine tags and /or proper folding and /or expression . In other embodiments , the flag tags. Such tags may be used to isolate tagged proteins . GARP homologue , leucine rich repeat containing 33 Proteins produced may comprise additional amino acids US 9 , 758 ,577 B2 62 encoding one or more 3C protease cleavage site . Such sites rhinovirus 3C protease . 3C protease cleavage sites may be allow for cleavage at the 3C protease cleavage site upon introduced to allow for removal of detectable labels from treatment with 3C protease, including, but not limited to recombinant proteins . TABLE 10 GARP sequences

SEQ ID Protein Sequence NO GARP AQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTETLDLS 158 GNOLRSILASPLGFYTALRHLDLSTNEISFLOPGAFOAL THL EHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSG LLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEOLDL HSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLOOLRV LDLSCNSIEAFOTASOPOAEFOLTWLDLRENKLLHFPDLAA LPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSALPLSAPS GNASGRPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRN CLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRT LLLOGNALRDLPPYTFANLASLORLNLOGNRVSPCGGPDEP GPSGCVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLS SNPGLEVATGALGGLEASLEVLALOGNGLMVLOVDLPCFI CLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSFSLLPGSA MGGLETSLRRLYLQGNPLSCCGNGWLAAQLHOGRVDVDA TODLICRFSSOEEVSLSHVRPEDCEKGGLKNINLIIILTFILVS AILLTTLAACCCVRRQKFNQQYKA SGARP AQHQDKVPCKMVDKKVSCOVLGLLQVPSVLPPDTETLDLS 159 GNOLRSILASPLGFYTALRHLDLSTNEISFLQPGAFQALTHL EHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSG LLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEOLDL HSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLOOLRV LDLSCNSIEAFOTASOPOAEFOLTWLDLRENKLLHFPDLAA LPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSALPLSAPS GNASGRPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRN CLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRT LLLQGNALRDLPPYTFANLASLQRLNLQGNRVSPCGGPDEP GPSGCVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLS SNPGLEVATGALGGLEASLEVLALOGNGLMVLOVDLPCFI CLKRLNLAENRLSHLPAWTOAVSLEVLDLRNNSFSLLPGSA MGGLETSLRRLYLOGNPLSCCGNGWLAAOLHOGRVDVDA TODLICRFSSQEEVSLSHVRPEDCEKGGLKNIN LRRC33 WRNRSGTATAASQGVCKLVGGAADCRGOSLASVPSSLPPH 160 ARMLTLDANPLKTLWNHSLOPYPLLESLSLHSCHLERISRG AFOEOGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDL SGNALTEDMAALMLONLSSLRSVSLAGNTIMRLDDSVFEG ERLRELDLORNYIFEIEGGAFDGLAELRHLNLAPNNLPCIV DFGLTRLRVLNVSYNVLEWFLATGGEAAFELETLDLSHNQ LLFFPLLPQYSKLRTLLLRDNNMGFYRDLYNTSSPREMVA OFLLVDGNVTNITTVSLWEEFSSSDLADLRFLDMSONOFOY LPDGFLRKMPSLSHLNLHONCLMTLHIREHEPPGALTELDL SHNOLSELHLAPGLASCLGSLRLFNLSSNOLLGVPPGLFAN ARNITTLDMSHNQISLCPLPAASDRVGPPSCVDFRNMASLR SLSLEGCGLGALPDCPFQGTSLTYLDLSSNWGVLNGSLAPL ODVAPMLOVLSLRNMGLHSSFMALDFSGFGNLRDLDLSG NCLTTFPRFGGSLALETLDLRRNSLTALPOKAVSEQLSRGL RTIYLSONPYDCCGVDGWGALQHGQTVADWAMVTCNLSS KIIRVTELPGGVPRDCKWERLDLGLLYLVLILPSCLTLLVAC TVIVLTFKKPLLQVIKSRCHWSSVY WRNRSGTATAASQGVCKLVGGAADCRGOSLASVPSSLPPH 161 ARMLTLDANPLKTLWNHSLOPYPLLESLSLHSCHLERISRG AFQEQGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDL SGNALTEDMAALMLONLSSLRSVSLAGNTIMRLDDSVFEG LERLRELDLORNYIFEIEGGAFDGLAELRHLNLAFNNLPCIV DFGLTRLRVLNVSYNVLEWFLATGGEAAFELETLDLSHNO LLFFPLLPOYSKLRTLLLRDNNMGFYRDLYNTSSPREMVA OFLLVDGNV TNITTVSLWEEFSSSDLADLRFLDMSONOFOY LPDGFLRKMPSLSHLNLHQNCLMTLHIREHEPPGALTELDL SHNOLSELHLAPGLASCLGSLRLFNLSSNQLLGVPPGLFAN ARNITTLDMSHNOISLCPLPAASDRVGPPSCVDFRNMASLR SLSLEGCGLGALPDCPFQGTSLTYLDLSSNWGVLNGSLAPL QDVAPMLQVLSLRNMGLHSSFMALDFSGFGNLRDLDLSG NCLTTFPRFGGSLALETLDLRRNSLTALPOKAVSEQLSRGL RTIYLSONPYDCCGVDGWGALQHGQTVADWAMVTCNLSS KIIRVTELPGGVPRDCKWERLDLGL US 9 ,758 , 577 B2 63 64 GPCs bound to LTBPs may adopt three dimensional shown to reduce fibrosis in various tissues . Decorin expres conformations that are distinct from conformations found sion has also been shown to increase the expression of with GPCs bound to GARP or other matrix proteins. This follistatin , a known inhibitor of free GDF - 8 . may be due, in some cases, to the presence of cysteines available on LTBP for disulfide bond formation with GPCs 5 In some embodiments , recombinant proteins of the pres that comprise a different distance from one another than ent invention may comprise those depicted in FIG . 7 . Some corresponding cysteines available for disulfide bond forma recombinant proteins of the present invention may comprise tion on GARP. Such differences in three dimensional con one or more features and /or combinations of protein mod formations may provide unique conformation - dependent ules from the embodiments depicted in FIG . 7 . epitopes on GPCs. In some embodiments , antibodies of the 10 Recombinant Growth Differentiation Factors (GDFs ) invention are directed to such conformation - dependent Activins and Inhibins epitopes. Such antibodies may function selectively to acti vate or inhibit growth factor activity depending on the Growth differentiation factors GDFs( ) , activins and inhib identity of bound protein ( e . g . LTBP or GARP. ) In some ins are TGF - B family member proteins involved in a number cases , different conformation -dependent epitopes may be 15 of cellular and /or developmental activities. In some embodi present on N -terminal alpha helices of proTGF - B when ments of the present invention , recombinant proteins may bound to LTBP or GARP . comprise one or more protein modules from one or more Recombinant proteins of the present invention may be GDFs , activins and / or inhibins . In further embodiments , coexpressed with GDF -associated serum protein (GASP ) 1 GDF protein modules may comprise GDF - 8 and / or GDF - 11 and / or GASP - 2 . Such recombinant proteinsmay include , but 20 protein modules . are not limited to GDF - 8 and /or GDF - 11 . GASPs are cir GDF - 8 and GDF - 11 , which are secreted as latent com culating proteins that bind and prevent activity of GDF - 8 plexes ( Sengle et al. , 2011. J Biol Chem . 286 ( 7 ) : 5087 - 99 ; and GDF -11 (Hill , J . J . et al. , 2003 . Mol Endocrinology . 17 (6 ): 1144 -54 and Hill , J . J . et al. , 2002 . JBC . 277 (43 ) : Ge et al ., 2005. Mol Cel Biol. 25 ( 14 ) : 5846 - 58 ) show con 40735 - 41 , the contents of each of which are herein incor - 25 servation of the fastener residues (Lys 27 and Tyr 75 of porated by reference in their entirety . ) Interestingly , GDF - 8 TGF- B1 ; see FIGS. 8A - 8G .) GDF - 8 ( also referred to herein and GDF - 11 growth factors are not found free in serum . as myostatin ) is involved in regulating muscle mass, and its About 70 % are in GPCs with the remaining 30 % associated deficiency increases muscle mass in multiple species, with GASPs as well as other proteins (e . g . follistatin , including humans (Rodino -Klapac , L . R . et al. , 2009 . follistatin - like related gene and decorin .) Studies using mice 30 Muscle Nerve . 39 ( 3 ) : 283 - 96 ) . GDF - 8 may be found in the lacking expression of GASP - 1 and /or GASP - 2 display phe - circulation in latent form , but may also be stored in the notypes indicative of myostatin and / or GDF - 11 overactivity (Lee et al. , 2013 . PNAS . 110 (39 ) : E3713 - 22 . ) GASP bound extracellular matrix , bound to LTBP3 (Anderson et al. , 2007 . GDF - 8 and /or GDF - 11 are unable to bind type II receptors J Biol Chem . 283 ( 11) : 7027 -35 ) or perlecan (Sengle et al. , and transmit related cellular signals . 35 2011. J Biol Chem . 286 ( 7 ) : 5087 - 99 . ) While complexed with Some recombinant proteins may be coexpressed with its prodomain , GDF - 8 is unable to participate in receptor perlecan . Such recombinant proteins may include , but are binding with the type Il receptor, ActRIIB ( Sengle et al. , not limited to GDF -8 . Studies by Sengle et al (Sengle et al ., 2008 . J Mol Biol . 381 (4 ): 1025 - 39 . ) While GDF -8 is 2011. J Biol Chem . 286 ( 7 ) :5087 - 99 , the contents of which expressed primarily in muscle , GDF - 11 expression is more are herein incorporated by reference in their entirety ) found 40 systemic and its activity is thought to be involved in multiple that the GDF - 8 prodomain associates with perlecan . Further processes (Lee et al. , 2013 . PNAS . 110 ( 39 ): E3713 - 22 . ). It is studies indicate that perlecan knockout leads to muscular hypertrophy, suggesting that the interaction between GDF - 8 believed to be involved in development of multiple tissues , and perlecan may contribute to GDF - 8 activity (Xu et al. including, but not limited to the retina , kidney, pancreas and 2010 . Matrix Biol. 29 ( 6 ) : 461 -70 . ) 45 olfactory system . It is also believed to be a circulating factor In some cases, recombinant proteins of the invention may in the blood (Sinha , M . et al. , 2014 . Science Express. be coexpressed with follistatin and / or FLRG . Such recom 10 . 1126 / science . 1251152 , p 2 - 6 and Katsimpardi, L . et al. , binant proteins may include , but are not limited to GDF - 8 . 2014 . Science Express . 10 . 1126 / science . 1251141 , the con Both follistatin and FLRG are known to antagonize some tents of each of which are herein incorporated by reference TGF - B family member proteins, including , but not limited to 50 in their entirety . ) GDF - 8 ( Lee , S - J . et al. , 2010 . Mol Endocrinol . 24 ( 10 ) : 1998 GDF -8 and GDF- 11 also share considerable homology . 2008, Takehara -Kasamatsu , Y . et al. , 2007 . J Med Invest. While the prodomains only share 48 % homology , GDF - 8 54 ( 3 - 4 ) :276 -88 , the contents of each of which are herein incorporated by reference in their entirety . ) Follistatin has and GDF - 11 growth factor domains share 90 % homology been shown to block GDF - 8 activity by binding to the free 55 (60 % homology when prodomains and growth factors are growth factor and preventing receptor binding . Both fol s taken together .) listatin and FLRG are implicated in modulating growth Release ofGDF - 8 and GDF - 11 from latentGPCs requires factor activity during development. cleavage of the prodomains at the BMP/ tolloid cleavage site In some embodiments , recombinant proteins of the inven ( located between Arg 75 and Asp 76 in GDF - 8 and between tion may be coexpressed with decorin . Such recombinant 60 Gly 97 and Asp 98 in GDF - 11 ) by BMP1 / tolloid metallo proteins may include, but are not limited to TGF- B and proteinases . This cleavage is between the a2 helix and the GDF - 8 . Decorin is a known antagonist of TGF - B activity fastener. Thus at least two different methods of unfastening ( Zhu , J. et al. , 2007 . J Biol Chem . 282 : 25852 -63 , the cotents the straitjacket, force and proteolysis , can release family of which are herein incorporated by reference in their members from latency . entirety ) and may also antagonize other TGF - B family 65 In some embodiments , recombinant proteins of the pres members , including , but not limited to GDF - 8 . Decorin - ent invention comprising GDFs may comprise sequences dependent inhibition of TGF - B and GDF - 8 activity has been listed in Table 11 or fragments thereof. US 9 ,758 , 577 B2 65 66 TABLE 11 Recombinant GDFs SEQ ID Protein Sequence NO ProGDF - 8 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSKLRLE 5 TAPNISKDVIROLLPKAPPLRELIDOYDVORDDSSDGS LEDDDY HATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKA OLWIYLRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKOPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRY PLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLOKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKI PAMW DRCGCS GDF - 8 prodomain NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIOILSKLRLE TAPNISKDVIROLLPKAPPLRELIDQYDVORDDSSDGSLEDDDY HATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKA QLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKQPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKRSRR GDF - 8 prodomain NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIOILSKLRLE 162 D76A TAPNISKDVIROLLPKAPPLRELIDOYDVORADSSDGSLEDDDY HATTETIITMPTESDFLMOVDGKPKCCFFKFSSKIOYNKVVKA QLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKQPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKRSRR proGDF - 8 AXXA NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSKLRLE 163 TAPNISKDVIROLLPKAPPLRELIDOYDVORDDSSDGSLEDDDY HATTETIITMPTESDFLMOVDGKPKCCFFKFSSKIQYNKVVKA QLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLQNWLKQPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKASRADFGLDCDEHSTESRCCRY PLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLOKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMW DRCGCS proGDF - 8 D76A NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIOILSKLRLE 164 TAPNISKDVIROLLPKAPPLRELIDOYDVORADSSDGSLEDDDY HATTETII TMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKA OLWIYLRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKOPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRY PLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMW DRCGCS proGDF - 8 AXXA NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIQILSKLRLE 165 D76A TAPNISKDVIRQLLPKAPPLRELIDQYDVQRADSSDGSLEDDDY HATTETII TMPTESDFLMOVDGKPKCCFFKFSSKIOYNKVVKA OLWIYLRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKOPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKASRADFGLDCDEHSTESRCCRY PLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLOKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKI PAMW DRCGCS proGDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC VWROHSRELRLESIKSQILSKLRLKEAPNISREVVKOLLPKAPP LOQILDLHDFOGDALOPEDFLEEDEYHATTETVISMAQETDPA VOTDGSPLCCHFHFSPKVMFTKVLKAOLWVYLRPVPRPATVY LQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSI DFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGOCEYMFMOKYPHTHLVQQANPR GSAGPCCTPTKMSPINMLYFNDKOOLIYGKIPGMVVDRCGCS proGDF - 11 D98A AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 166 VWROHSRELRLESIKSQILSKLRLKEAPNISREVVKOLLPKAPP LQQILDLHDFQGAALQPEDFLEEDEYHATTETVISMAQETDPA VOTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVY LQILRLKPLTGEGTAGGGGGGRRHIRIRSLKI ELHSRSGHWOSI DFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPR GSAGPCCTPTKMSPINMLYFNDKOQIIYGKIPGMVVDRCGCS US 9 ,758 ,577 B2 67 68 TABLE 11 - continued Recombinant GDFs SEQ ID Protein Sequence NO proGDF - 11 D2G AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 167 VWROHSRELRLESIKSOILSKLRLKEAPNISREVVKOLLPKAPP LQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPA VOTDGSPLCCHFHFSPKVMFTKVLKAOLWVYLRPVPRPATVY LOILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWOSI DFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKRSGNLGLDCDEHSSESRCCRYPLTVDFEAF GWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRG SAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF - 11 Axxa AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 168 VWROHSRELRLESIKSOILSKLRLKEAPNISREVVKOLLPKAPP LOQILDLHDFOGDALOPEDFLEEDEYHATTETVISMAQETDPA VOTDGSPLCCHFHFSPKVMFTKVLKAOLWVYLRPVPRPATVY LQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSI DFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKASRANLGLDCDEHSSESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGOCEYMFMOKYPHTHLVOOANPR GSAGPCCTPTKMSPINMLYFNDKOOIIYGKIPGMVVDRCGCS proGDF - 11 AXXA AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 169 D98A VWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPP LQQILDLHDFQGAALQPEDFLEEDEYHATTETVISMAQETDPA VOTDGSPLCCHFHFSPKVMFTKVLKAOLWVYLRPVPRPATVY LQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSI DFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKASRANLGLDCDEHSSESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPR GSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 170 prodomain D9 8A VWROHSRELRLESIKSOILSKLRLKEAPNISREVVKOLLPKAPP LOOILDLHDFOGAALOPEDFLEEDEYHATTETVISMAQETDPA VOTDGSPLCCHFHFSPKVMFTKVLKAOLWVYLRPVPRPATVY LQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWOSI DFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKRSRR GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 71 prodomain VWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPP LQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPA VQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVY LOILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWOSI DFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKRSRR

45 Activins and inhibins are TGF - B family member proteins , In some embodiments , recombinant proteins of the pres the activity of each of which often results in opposing ent invention may comprise integrins. Integrins are cell functions (Bilezikjian et al 2012 . ) Like other family mem - surface heterodimers formed by alpha and beta subunits , bers, these proteins occur physiologically as dimers . each of which has a transmembrane domain and in the Activins and inhibins are constructed in part from the same 50 N - terminal portion of the extracellular domain come B -subunits , that may include inhibin -beta A , inhibin -beta B , together to form the ligand binding site . Recombinant pro inhibin - beta C and inhibin - beta E (referred to herein as teins of the present invention may comprise integrins and / or B -subunit A , B , C and E , respectively .) The difference integrin subunits . Such integrins and /or integrin subunits between activins and inhibins , structurally , is that activins may comprise any of those disclosed in U . S . Provisional are B -subunit dimers while inhibins are heterodimers , » Patent Application No . 61/ 722 , 919 filed Nov . 6 , 2012 , the wherein the second subunit is inhibin - a . Activins are named contents of which are herein incorporated by reference in for their subunit pairs , such that activin A comprises a their entirety . homodimer of two A subunits , activin AB comprises a dimer Recombinant proteins of the invention may include inter of A and B subunits , B comprises a dimer of B subunits , etc . so cellular adhesion molecule 1 ( ICAM - 1 ) . In some cases, (Muenster et al 2011. ) Activins are involved in a variety of ICAM -1 proteins of the present invention may be used as functions that may include , but are not limited to cell control proteins during antibody development and /or anti growth , differentiation , programmed cell death , endocrine body testing . In some cases, ICAM -1 may be used as a functions, cellular metabolism , bone growth , etc . They are control during selection of binding molecules using phage especially recognized for their control of reproductive hor - 65 display technologies . In some cases , ICAM - 1 proteins of the mone cycles. Activin and inhibin signaling often functions invention comprise one or more detectable label . Detectable antagonistically in this regard . labels may include , for example , histidine tags . US 9 , 758 ,577 B2 69 70 Chimeric Proteins pro -protein sequences listed in Table 1 .) Some chimeric In some embodiments , recombinant proteins of the pres - proteins of the present invention may comprise protein ent invention may comprise chimeric proteins . As used modules comprising amino acid sequences similar to those herein , the term " chimeric protein ” refers to a protein comprising one or more protein modules from at least two 5 inthan Table 12 , but comprising additional or fewer amino acids different proteins [ formed from the same gene ( e . g . variantsNo 5 than those listed . Such modules may comprise about 1 more arising from alternative splicing ) or from different genes ] . or fewer amino acids, about 2 more or fewer amino acids, Chimeric proteins may comprise protein modules from two about 3 more or fewer amino acids , about 4 more or fewer or more TGF- B family member proteins. Such chimeric amino acids , about 5 more or fewer amino acids, about 6 proteins may comprise protein modules from TGF -B1 , TGF - 101 more or fewer amino acids, about 7 more or fewer amino B2 and / or TGF -B3 . Some chimeric proteins of the present acids, about 8 more or fewer amino acids , about 9 more or invention may comprise protein modules including , but not fewer amino acids, about 10 more or fewer amino acids or limited to the protein modules and /or amino acid sequences greater than 10 more or fewer amino acids on N - terminal listed in Table 12 ( residue numbers correspond to the and /or C - terminal ends . TABLE 12 Protein modules

SEO ID Protein Residues Sequence NO TGF - B1 1 - 74 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEV 171 PPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADY TGF - B1 1 - 207 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEV 172 PPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYA KEVTRVLMVETHNEIYDKF KOSTHSIYMFFNTSELRE AVPEPVLLSRAELRLLRLKLKVEOHVELYOKYSNNS WRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGEIE GFRLSAHCSCDSRDNTLQVDI TGF - B1 46 - end EAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTR 173 VLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEP VLLSRAELRLLRLKLKVEOHVELYOKYSNNSWRYLS NRLLAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLS AHCSCDSRDNTLOVDINGFTTGRRGDLATIHGMNRPF LLLMATPLERAQHLOSSRHRRALDTNYCFSSTEKNCC VROLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIW SLDTOYSKVLALYNOHNPGASAAPCCVPOALEPLPIV YYVGRKPKVEQLSNMIVRSCKCS TGF - B1 47 - end AVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRV 174 LMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVL LSRAELRLLRLKLKVEOHVELYOKYSNNSWRYLSNR LLAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAH CSCDSRDNTLOVDINGFTTGRRGDLATIHGMNRPFLL LMATPLERAOHLOSSRHRRALDTNYCFSSTEKNCCVR OLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSL DTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYY VGRKPKVEQLSNMIVRSCKCS TGF - B1 74 - 249 YYAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTSE 175 LREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSN NSWRYLSNRLLAPSDSPEWLSFDVTGWROWLSRGG EIEGFRLSAHCSCDSRDNTLOVDINGFTTGRRGDLATI HGMNRPFLLLMATPLERAQHLOSSRHRR TGF - B1 74 - end YYAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTSE 176 LREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSN NSWRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGG EIEGFRLSAHCSCDSRDNTLOVDINGFTTGRRGDLATI HGMNRPFLLLMATPLERAQHLOSSRHRRALDTNYCF SSTEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFC LGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVP QALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS TGF - B1 75 - 249 YAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTSEL 177 REAVPEPVLLSRAELRLLRLKLKVEOHVELYOKY SNN SWRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGEI EGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIH GMNRPFLLLMATPLERAQHLOSSRHRR TGF - B1 75 - end YAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTSEL 178 REAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNN SWRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGEI EGFRLSAHCSCDSRDNTLOVDINGFTTGRRGDLATIH GMNRPFLLLMATPLERAQHLOSSRHRRALDTNYCFSS US 9 ,758 ,577 B2 71 TABLE 12 - continued Protein modules SEQ ID Protein Residues Sequence NO TEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFCLG PCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQA LEPLPIVYYVGRKPKVEQLSNMIVRSCKCS TGF - B1 228 - 361 FLLLMATPLERAOHLOSSRHRRALDTNYCFSSTEKNC 179 CVROLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYI WSLDTOYSKVLALYNOHNPGASAAPCCVPOALEPLPI VYYVGRKPKVEOLSNMIVRSCKCS TGF - B1 250 - 361 ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEP 44 KGYHANFCLGPCPYIWSLDTOYSKVLALYNCHNPGA SAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSC KCS TGF - B2 232 - 260 FAGIDGTSTYTSGDQKTIKSTRKKNSGKTP 65 TGF - B2 236 - 254 GTSTYTSGDQKTIKSTRKK 180 TGF - B3 1 - 46 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEP 43 TVMTHVP TGF - B3 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEP 181 TVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQEN TESE TGF - B3 80 - 280 YYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNV 182 SSVEKNRTNLFRAEFRVLRVPNPSSKRNEORIELFQIL RPDEHIAKORYIGGKNLPTRGTAEWLSFDVTDTVRE WLLRRESNLGLEISIHCPCHTFOPNGDILENIHEVMEIK FKGVDNEDDHGRGDLGRLKKOKDEIHNPHLILMMIPP HRLDNPGQGGQRKKR TGF - B3 281 - 392 ALDTNYCFRNLEENCCVRPLYIDFRODLGWKWVHEP 46 KGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEAS ASPCCVPODLEPLTILYYVGRTPKVEQLSNMVVKSCK CS

GDF - 8 1 - 75 NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIOIL 183 SKLRLETAPNISKDVIROLLPKAPPLRELIDQYDVOR GDF - 8 1 - 64 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQIL 72 SKLRLETAPNISKDVIRQLLPKAPPL GDF - 8 75 - end RDDSSDGSLEDDDYHATTETIITMPTESDFLMOVDGK 184 PKCCFFKFSSKIOYNKVVKAOLWIYLRPVETPTTVFV QILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDVK TVLONWLKOPESNLGIEIKALDENGHDLAVTFPGPGE DGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCR YPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLOK YPHTHLVHOANPRGSAGPCCTPTKMSPINMLYFNGK EOIIYGKIPAMVVDRCGCS

GDFS 65 - end RELIDOYDVORDDSSDGSLEDDDYHATTETII TMPTES 185 DFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIYLR PVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLDMNPG TGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHD LAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCD EHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCS GECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSP INMLYFNGKEQIIYGKIPAMVVDRCGCS GDF8 65 - 243 RELIDQYDVORDDSSDGSLEDDDYHATTETIITMPTES 77 DFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLR PVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPG TGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHD LAVTFPGPGEDGLNPFLEVKVTDTPKRSRR

GDF - 8 76 - 243 DDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKP 186 KCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQI LRLIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDVKT VLQNWLKOPESNLGIEIKALDENGHDLAVTFPGPGED GLNPFLEVKVTDTPKRSRR US 9 ,758 , 577 B2 73 74 TABLE 12 - continued Protein modules SEQ ID Protein Residues Sequence NO GDF - 8 244 - 352 DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKR 74 YKANYCSGECEFVFLOKYPHTHLVHOANPRGSAGPC CTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS GDF - 11 1 - 86 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 73 GCPVCVWROHSRELRLESIKSQILSKLRLKEAPNISRE VVKQLLPKAPPL GDF - 11 1 - 96 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 187 GCPVCVWROHSRELRLESIKSOILSKLRLKEAPNISRE VVKOLLPKAPPLOOILDLHDFO GDF - 11 1 - 108 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 188 GCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISRE VVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEE GDF - 11 97 - 274 GDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDG 189 SPLCCHFHFSPKVMFTKVLKAOLWVYLRPVPRPATV YLOILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSR SGHWOSIDFKOVLHSWFROPOSNWGIEINAFDPSGTD LAVTSLGPGAEGLHPFMELRVLENTKRSRR GDF - 11 87 - 274 QQILDLHDFOGDALOPEDFLEEDEYHATTETVISMAQ 78 ETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVY 78 LRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRI RSLKIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEI NAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRS RR

GDF - 11 275 - 383 NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKR 75 YKANYCSGOCEYMFMOKYPHTHLVOOANPRGSAGP CCTPTKMSPINMLYFNDKQQI IYGKIPGMVVDRCGCS Inhibin 1 - 64 SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEA 190 Beta A VKKHILNMLHLKKRPDVTQPVPKAALL Inhibin 1 - 76 SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEA 191 Beta A VKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVG KVG Inhibin 65 - 288 NAIRKLHVGKVGENGYVEI EDDIGRRAEMNELMEQT 192 Beta A SEIITFAESGTARKTLHFEISKEGSDLSVERAEVWLFL KVPKANRTRTKVTIRLFOOOKHPOGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIORLLDO GKSSLDVRIACEOCOESGASLVLLGKKKKKEEEGEGK KKGGGEGGAGADEEKEQSHRPFLMLQAROSEDHPHR RR

Inhibin 65 - 289 NAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEOT 193 Beta A SEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFL KVPKANRTRTKVTIRLFOOOKHPOGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDO GKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGK KKGGGEGGAGADEEKEQSHRPFLMLQARQS EDHPHR niiililililililili RRR Inhibin 65 - 290 NAIRKLHVGKVGENGYVEI EDDIGRRAEMNELMEQT 194 Beta A SEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFL KVPKANRTRTKVTIRLFOOOKHPOGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDO GKSSLDVRIACEOCOESGASLVLLGKKKKKEEEGEGK KKGGGEGGAGADEEKEQSHRPFLMLQARQS EDHPHR RRRR Inhibin 77 - 289 ENGYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARK 195 Beta A TLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKV TIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEK VVDARKSTWHVFPVSSSIQRLLDOGKSSLDVRIACEQ CCESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGA DEEKEQSHRPFLMLQARQS EDHPHRRRR Inhibin 77 - 290 ENGYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARK 196 Beta A TLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKV TIRLFOOOKHPOGSLDTGEEAEEVGLKGERSELLLSEK US 9 ,758 , 577 B2 75 TABLE 12 - continued Protein modules SEQ ID Protein Residues Sequence NO VVDARKSTWHVFPVSSSIQRLLDOGKSSLDVRIACEQ COESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGA DEEKEQSHRPFLMLQAROSEDHPHRRRRR Inhibin 77 - end ENGYVEIEDDIGRRAEMNELMEOTSEIITFAESGTARK 197 Beta A TLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKV TIRLFOOOKHPOGSLDTGEEAEEVGLKGERSELLLSEK VVDARKSTWHVFPVSSSIQRLLDOGKSSLDVRIACEO CQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGA DEEKEOSHRPFLMLQAROSEDHPHRRRRRGLECDGK VNICCKKOFFVSFKDIGWNDWIIAPSGYHANYCEGEC PSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCV PTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS Inhibin 291 - 406 GLECDGKVNICCKKOFFVSFKDIGWNDWIIAPSGYHA 198 Beta A NYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFA NLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEE CGCS

In some embodiments , chimeric proteins of the present proteins contains the a2 helix . Furthermore , F95 may be an invention may comprise combinations of any of the protein 25 important residue in conferring latency for GDF11. This modules listed in Table 12 . Some chimeric proteins com - residue is in a similar position as a Camurati - Engelmann prising GPCsmay comprise protein modules that have been mutation found in TGF - B1 , Y81H ( see FIGS. 8A - 8G ) , thus , substituted with any of the protein modules listed in Table mutation of this residue to a smaller amino acid , such as an 12 . Alanine , may be carried out to promote dissociation of the In some embodiments , chimeric proteins may comprises mature GDF11 growth factor from the GPC . Such mutants protein modules from GDFs and / or inhibins . Such GDFs may be useful as positive control molecules in designing may include GDF - 11 and /or GDF - 8 . Some such chimeric assays to screen for GDF11 activating antibodies . proteins may comprise a prodomain from GDF - 11 and a In some embodiments , chimeric proteins of the present growth factor from GDF - 8 . In such embodiments, chimeric invention may comprise protein module combinations proteins may comprise substituted N - terminal regions » including, but not limited to the combinations of protein between GDF - 11 and GDF -8 . In other embodiments , chi modules and /or amino acid sequences listed in Table 13 . meric proteins may comprise a prodomain from GDF - 8 and Some chimeric proteins of the present invention may com a growth factor from GDF - 11 . Such chimeric proteins may prise protein modules comprising amino acid sequences comprise amino acid residues 1 - 108 from GDF - 11 and similar to those in Table 13 , but comprising additional or amino acid residues 90 - the end of the protein from GDF - 8 . 40 fewer amino acids than those listed . Such amino acid Some chimeric proteins may comprise an arm region from sequences may comprise about 1 more or fewer amino acids, GDF - 11 . about 2 more or fewer amino acids , about 3 more or fewer Some chimerics of the present invention may comprise amino acids, about 4 more or fewer amino acids, about 5 GDF - 8 comprising an arm region ofGDF - 11 . Such chime- more or fewer amino acids, about 6 more or fewer amino rics may be unstable due to steric clash between residue F95 43 acids, about 7 more or fewer amino acids , about 8 more or from the GDF - 11 arm and the a2 helix of the chimeric GPC . fewer amino acids, about 9 more or fewer amino acids, about Therefore , in some cases, GDF8 /GDF11 / Activin chimeras 10 more or fewer amino acids or greater than 10 more or may be designed so that the ARM region of such chimeric fewer amino acids on N -terminal and / or C - terminal ends . TABLE 13 Protein module combinations Protein Protein Protein SEQ module module module ID Chimeric Sequence NO TGF - B2 TGF - B1 NA SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPE 199 LAP growth DYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACE factor RERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPY FRIVRFDVSAMEKNASNLVKAEFRVFRLONPKAR VPEORIELYOILKSKDLTSPTORYIDSKVVKTRAE GEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPC CTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDO KTIKSTRKKNSGKTPHLLLMLLPSYRLESOOTNRR KKRALDTNYCFSSTEKNCCVROLYIDFRKDLGWK WIHEPKGYHANFCLGPCPYIWSLDTOYSKVLALY NOHNPGASAAPCCVPOALEPLPIVYYVGRKPKVE OLSNMIVRSCKCS US 9 , 758 ,577 B2 77 TABLE 13 - continued Protein module combinations Protein Protein Protein SEO module module module ID Chimeric Sequence NO TGF - B3 TGF - B1 N / A SLSLSTCTTLDFGHI KKKRVEAIRGOILSKLRLTSP 200 LAP growth PEPTVMTHVPYOVLALYNSTRELLEEMHGEREEG factor CTCENTESEYYAKEIHKFDMI OGLAEHNELAVCP KGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNP SSKRNEORIELFQILRPDEHIAKORYIGGKNLPTRG TAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCH TFOPNGDILENIHEVMEIKFKGVDNEDDHGRGDL GRLKKOKDHHNPHLILMMIPPHRLDNPGOGGOR KKRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWK WIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALY NOHNPGASAAPCCVPQALEPLPIVYYVGRKPKVE QLSNMIVRSCKCS TGF - B3 TGF -B1 N / A SLSLSTCTTLDFGHI KKKRVEAIRGOILSKLRLTSP 201 ( 1 - 46 ) ( 47 - end ) PEPTVMTHVPAVLALYNSTRDRVAGESAEPEPEP EADYYAKEVTRVLMVETHNEIYDKFKOSTHSIYM FFNTSELREAVPEPVLLSRAELRLLRLKLKVEOHV ELYOKYSNNSWRYLSNRLLAPSDSPEWLSFDVTG VVROWLSRGGEIEGFRLSAHCSCDSRDNTLOVDI NGFTTGRRGDLATIHGMNRPFLLLMATPLERAQH LOSSRHRRALDTNYCFSSTEKNCCVROLYIDFRK DLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYS KVLALYNOHNPGASAAPCCVPOALEPLPIVYYVG RKPKVEOLSNMIVRSCKCS TGF - B3 TGF - B1 N / A SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 202 ( 1 - 79 ) ( 75 - end ) PEPTVMTHVPYOVLALYNSTRELLEEMHGEREEG CTOENTESEYAKEVTRVLMVETHNEIYDKFKOST HSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLK VEOHVELYOKYSNNSWRYLSNRLLAPSDSPEWLS FDVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNT LOVDINGFTTGRRGDLATIHGMNRPFLLLMATPL ERAQHLOSSRHRRALDTNYCFSSTEKNCCVROLY IDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLD TQYSKVLALYNQHNPGASAAPCCVPQALEPLPIV YYVGRKPKVEQLSNMIVRSCKCS TGF - B1 TGF - B3 TGF - B1 LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPPSO 203 ( 1 - 74 ) ( 80 - 280 ) ( 250 GEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPE 361 ) ADYYYAKEIHKFDMIQGLAEHNELAVCPKGITSK VFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNE ORIELFOILRPDEHIAKORYIGGKNLPTRGTAEWL SFDVTDTVREWLLRRESNLGLEISIHCP CHTFOPN GDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKK OKDHHNPHLILMMIPPHRLDNPGOGGORKKRAL DTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTOYSKVLALYNQHNP GASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNM IVRSCKCS TGF - B3 TGF - B1 TGF - B3 SLSLSTCTTLDFGHI KKKRVEAIRGOILSKLRLTSP 204 ( 1 - 79 ) ( 75 - 249 ) ( 281 PEPTVMTHVPYOVLALYNSTRELLEEMHGEREEG 392 ) CTCENTESEYAKEVTRVLMVETHNEIYDKFKOST HSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLK VEQHVELYOKYSNNSWRYLSNRLLAPSDSPEWLS FDVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNT LOVDINGFTTGRRGDLATIHGMNRPFLLLMATPL ERAQHLOSSRHRRALDTNYCFRNLEENCCVRPLY IDFRODLGWKWVHEPKGYYANFCSGPCPYLRSA DTTHSTVLGLYNTLNPEASASPCCVPODLEPLTIL YYVGRTPKVEQLSNMVVKSCKCS TGF - B1 TGF - B2 TGF - B1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQ 205 ( 1 - 207 ) trigger ( 228 GEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPE loop 361 ) ADYYAKEVTRVLMVETHNEIYDKFKOSTHSIYMF Short FNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVE ( 236 LYOKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGV 254 ) VROWLSRGGEIEGFRLSAHCSCDSRDNTLOVDIG TSTYTSGDOKTIKSTRKKFLLLMATPLERAQHLOS SRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLG WKWIHEPKGYHANFCLGPCPYIWSLDTOYSKVL . ALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKP KVEQLSNMIVRSCKCS US 9 , 758 ,577 B2 79 80 TABLE 13 - continued Protein module combinations Protein Protein Protein SEO module module module ID 1 Chimeric Sequence NO TGF - B1 TGF - B2 TGF - B1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSO 206 ( 1 - 207 ) trigger ( 228 GEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPE loop 361 ) ADYYAKEVTRVLMVETHNEIYDKFKOSTHSIYMF Long FNTSELREAVPEPVLLSRAELRLLRLKLKVEOHVE ( 232 LYOKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGV 260 ) VROWLSRGGEIEGFRLSAHCSCDSRDNTLOVDIA GIDGTSTYTSGDOKTIKSTRKKNSGKTPFLLLMAT PLERAOHLOSSRHRRALDTNYCFSSTEKNCCVRO LYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWS LDTOYSKVLALYNOHNPGASAAPCCV POALEPLP IVYYVGRKPKVEOLSNMIVRSCKCS GDF - 11 GDF - 8 GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 207 ( 1 - 96 ) ( 76 - 243 ) ( 275 EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA 383 ) PNISREVVKQLLPKAPPLQQILDLHDFODDSSDGS LEDDDYHATTETIITMPTESDFLMQVDGKPKCCFF KFSSKIOYNKVVKAOLWIYLRPVETPTTVFVOILR LIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDVKT VLONWLKOPESNLGIEIKALDENGHDLAVTFPGP GEDGLNPFLEVKVTDTPKRSRRNLGLDCDEHSSE SRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQ CEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKM SPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF - 11 GDF - 8 GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 208 ( 1 - 86 ) (65 - 243 ) ( 275 EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA 383 ) PNISREVVKOLLPKAPPLRELIDOYDVORDDSSDG SLEDDDYHATTETIITMPTESDFLMQVDGKPKCCF FKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQIL RLIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDV KTVLONWLKOPESNLGIEIKALDENGHDLAVTFP GPGEDGLNPFLEVKVTDTPKRSRRNLGLDCDEHS SESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCS GOCEYMFMOKYPHTHLVOOANPRGSAGPCCTPT KMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF - 11 GDF - 8 N / A AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 209 ( 1 - 96 ) ( 76 - 243 ) EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA PNISREVVKOLLPKAPPLQQILDLHDFODDSSDGS LEDDDYHATTETIITMPTESDFLMQVDGKPKCCFF KFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILR LIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDVKT VLONWLKQPESNLGIEIKALDENGHDLAVTFPGP GEDGLNPFLEVKVTDTPKRSRR GDF - 11 GDF - 8 NA AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 210 ( 1 - 86 ) ( 65 - 243 ) EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA PNISREVV KOLLPKAPPLRELIDOYDVORDDSSDG SLEDDDYHATTETII TMPTESDFLMOVDGKPKCCF FKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQIL RLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDV KTVLONWLKOPESNLGIEIKALDENGHDLAVTFP GPGEDGLNPFLEVKVTDTPKRSRR GDF - 11 Inhibin GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 211 ( 1 - 96 ) Beta A ( 275 EPDGCPVCVWROHSRELRLESIKSQILSKLRLKEA ( 77 - 290 ) 383 ) PNISREVV KOLLPKAPPLOOILDLHDFOENGYVEI EDDIGRRAEMNELMEOTSEIITFAESGTARKTLHF EISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTI RLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLS EKVVDARKSTWHVFPVSSSIQRLLDOGKSSLDVRI ACEOCOESGASLVLLGKKKKKEEEGEGKKKGGG EGGAGADEEKEOSHRPFLMLQAROSEDHPHRRR RRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWI IAPKRYKANYCSGQCEYMFMQKYPHTHLVQQAN PRGSAGPCCTPTKMSPINMLYFND KOOIIYGKIPG MVVDRCGCS GDF - 11 Inhibin GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 212 ( 1 - 86 ) Beta A ( 275 EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA ( 65 - 290 ) 383 ) PNISREVV KOLLPKAPPLNAIRKLHVGKVGENGY VEIEDDIGRRAEMNELMEQTSEIITFAESGTARKTL HFEISKEGSDLSVVERAEVWLFLKVPKANRTRTK VTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELL US 9 , 758 ,577 B2 81 TABLE 13 - continued Protein module combinations Protein Protein Protein SEO module module module ID Chimeric Sequence NO LSEKVVDARKS TWHVFPVSSSIORLLDOGKSSLD VRIACEOCOESGASLVLLGKKKKKEEEGEGKKKG GGEGGAGADEEKEOSHRPFLMLOAROSEDHPHR RRRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWD WIIAPKRYKANYCSGOCEYMFMOKYPHTHLVOO ANPRGSAGPCCTPTKMSPINMLYFNDKOOLIYGKI PGMVVDRCGCS GDF - 11 Inhibin N / A AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 213 ( 1 - 96 ) Beta A EPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEA ( 77 - 290 ) PNISREVV KOLLPKAPPLOOILDLHD FOENGYVEI EDDIGRRAEMNELMEQTSEIITFAESGTARKTLHF EISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTI RLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLS EKVVDARKSTWHVFPVSSSIQRLLDOGKSSLDVRI ACEQCQESGASLVLLGKKKKKEEEGEGKKKGGG EGGAGADEEKEOSHRPFLMLOAROSEDHPHRRR RR GDF - 11 Inhibin NA AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 214 ( 1 - 86 ) Beta A EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA ( 65 - 290 ) PNISREVV KOLLPKAPPLNAIRKLHVGKVGENGY VEIEDDIGRRAEMNELMEOTSEIITFAESGTARKTL HFEISKEGSDLSVVERAEVWLFLKVPKANRTRTK VTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELL LSEKVVDARKS TWHVFPVSSSIORLLDOGKSSLD VRIACEQCQESGASLVLLGKKKKKEEEGEGKKKG GGEGGAGADEEKEQSHRPFLMLQARQSEDHPHR RRRR GDF - 8 GDF - 11 GDF - 8 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKI 215 ( 1 - 75 ) ( 97 - 274 ) ( 244 QILSKLRLETAPNISKDVIROLLPKAPPLRELIDOY 352 ) DVORGDALOPEDFLEEDEYHATTETVISMAQETD PAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVY LRPVPRPATVYLOILRLKPLTGEGTAGGGGGGRR HIRIRSLKIELHSRSGHWOSIDFKOVLHSWFROPOS NWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELR VLENTKRSRRDFGLDCDEHSTESRCCRYPLTVDFE AFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTH LVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQII YGKIPAMVVDRCGCS GDF - 8 GDF - 11 GDF - 8 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKI 216 ( 1 - 64 ) ( 87 - 274 ) ( 244 OILSKLRLETAPNISKDVIROLLPKAPPLOQILDLH 352 ) DFOGDALOPEDFLEEDEYHATTETVISMAQETDP AVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYL RPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHI RIRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSN WGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRV LENTKRSRRDFGLDCDEHSTESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGECEFVFLOKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIY GKIPAMVVDRCGCS GDF - 8 GDF - 11 N / A NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKI 217 ( 1 - 75 ) ( 97 - 274 ) OILSKLRLETAPNISKDVIROLLPKAPPLRELIDOY DVORGDALOPEDFLEEDEYHATTETVISMAQETD PAVOTDGSPLCCHFHFSPKVMFTKVLKAOLWVY LRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRR HIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQS NWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELR VLENTKRSRR

GDF - 8 GDF - 11 GDF - 8 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKI 218 ( 1 - 64 ) ( 87 - 274 ) ( 244 QILSKLRLETAPNISKDVIROLLPKAPPLQQILDLH 352 ) DFOGDALQPEDFLEEDEYHATTETVISMAQETDP AVOTDGSPLCCHFHFSPKVMFTKVLKAOLWVYL RPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHI RIRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSN WGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRV LENTKRSRRDFGLDCDEHSTESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIY GKIPAMVVDRCGCS US 9 , 758 ,577 B2 83 84 TABLE 13 - continued Protein module combinations Protein Protein Protein SEO module module module ID 1 Chimeric Sequence NO

GDF - 8 Inhibin GDF - 8 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKI 219 ( 1 - 75 ) Beta A ( 244 QILSKLRLETAPNISKDVIROLLPKAPPLRELIDOY ( 77 - 289 ) 352 ) DVORENGYVEI EDDIGRRAEMNELMEOTSEIITFA ESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVP KANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIORLL DOGKSSLDVRIACEOCOESGASLVLLGKKKKKEE EGEGKKKGGGEGGAGADEEKEQSHRPFLMLQAR OSEDHPHRRRRDFGLDCDEHSTESRCCRYPLTVD FEAFGWDWIIAPKRYKANYCSGECEFVFLOKYPH THLVHQANPRGSAGPCCTPTKMSPINMLYFNGKE OLIYGKIPAMVVDRCGCS GDF - 8 Inhibin GDF - 8 NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKI 220 ( 1 - 64 ) Beta A ( 244 OILSKLRLETAPNISKDVIROLLPKAPPLNAIRKLH ( 65 - 290 ) 352 ) VGKVGENGYVEIEDDIGRRAEMNELMEOTSEIITF AESGTARKTLHFEISKEGSDLSWERAEVWLFLK VPKANRTRTKVTIRLFOOOKHPOGSLDTGEEAEE VGLKGERSELLLSEKVVDARKSTWHVFPVSSSIOR LLDOGKSSLDVRIACEOCOESGASLVLLGKKKKK EEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQ AROSEDHPHRRRRRDFGLDCDEHSTESRCCRYPL TVDFEAFGWDWIIAPKRYKANYCSGECEFVFLOK YPHTHLVHOANPRGSAGPCCTPTKMSPINMLYFN GKEQIIYGKIPAMVVDRCGCS GDF - 8 Inhibin N / A NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKI 221 ( 1 - 75 ) Beta A OILSKLRLETAPNISKDVIROLLPKAPPLRELIDOY ( 77 - 290 ) DVORENGYVEIEDDIGRRAEMNELMEOTSEIITFA ESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVP KANRTRTKVTIRLFOOOKHPOGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLL DOGKSSLDVRIACEOCOESGASLVLLGKKKKKEE EGEGKKKGGGEGGAGADEEKEQSHRPFLMLQAR OSEDHPHRRRRR GDF - 8 Inhibin NA NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKI 222 ( 1 - 64 ) Beta A QILSKLRLETAPNISKDVIROLLPKAPPLNAIRKLH (65 - 290 ) VGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITF AESGTARKTLHFEISKEGSDLSVVERAEVWLFLK VPKANRTRTKVTIRLFOOOKHPOGSLDTGEEAEE VGLKGERSELLLSEKVVDARKSTWHVFPVSSSIOR LLDOGKSSLDVRIACEOCOESGASLVLLGKKKKK EEEGEGKKKGGGEGGAGADEEKEOSHRPFLMLO ARQSEDHPHRRRRR Inhibin GDF - 8 Inhibin SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 223 Beta A ( 76 - 243 ) Beta A VEAVKKHILNMLHLKKRPDVTOPVPKAALLNAIR ( 1 - 76 ) ( 291 KLHVGKVGDDSSDGSLEDDDYHATTETIITMPTES 406 ) DFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIY LRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWOSIDVKTVLONWLKOPESNLGIEIKA LDENGHDLAVT FPGPGEDGLNPFLEVKVTDTPKR SRRGLECDGKVNICCKKOFFVSFKDIGWNDWIIAP SGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYR MRGHSPFANLKSCCVPTKLRPMSMLYYDDGONII KKDIONMIVEECGCS

Inhibin GDF - 8 Inhibin SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 224 Beta A ( 65 - 243 ) Beta A VEAVKKHILNMLHLKKRPDVTOPVPKAALLRELI ( 1 - 64 ) ( 291 DOYDVORDDSSDGSLEDDDYHATTETIITMPTES 406 ) DFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIY LRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKA LDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKR SRRGLECDGKVNICCKKOFFVSFKDIGWNDWIIAP SGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYR MRGHSPFANLKSCCVPTKLRPMSMLYYDDGONII KKDIQNMIVEECGCS Inhibin GDF - 8 N / A SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 225 Beta A ( 76 - 243 ) VEAVKKHILNMLHLKKRPDVTOPVPKAALLNAIR ( 1 - 76 ) KLHVGKVGDDSSDGSLEDDDYHATTETIITMPTES US 9 , 758 ,577 B2 85 86 TABLE 13 - continued Protein module combinations Protein Protein Protein SEO module module module ID 1 2 Chimeric Sequence NO DFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIY LRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWOSIDVKTVLONWLKOPESNLGIEIKA LDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKR SRR

Inhibin GDF - 8 ?? SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 226 Beta A ( 65 - 243 ) VEAVKKHILNMLHLKKRPDVTOPVPKAALLRELI ( 1 - 64 ) DQYDVORDDSSDGSLEDDDYHATTETIITMPTES DFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIY LRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWOSIDVKTVLONWLKOPESNLGIEIKA LDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKR SRR Inhibin GDF - 11 Inhibin SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 227 Beta A ( 97 - 274 ) Beta A VEAVKKHILNMLHLKKRPDVTQPVPKAALLNAIR ( 1 - 76 ) ( 291 KLHVGKVGGDALOPEDFLEEDEYHATTETVISMA 406 ) OETDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOL WVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGG GRRHIRIRSLKIELHSRSGHWOSIDFKOVLHSWFR QPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPF MELRVLENTKRSRRGLECDGKVNICCKKOFFVSF KDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSS LSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMS MLYYDDGONIIKKDIQNMIVEECGCS Inhibin GDF - 11 Inhibin SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 228 Beta A ( 87 - 274 ) Beta A VEAVKKHI LNMLHLKKRPDVTOPVPKAALLQOIL ( 1 - 64 ) ( 291 DLHDFQGDALOPEDFLEEDEYHATTETVISMAQE 406 ) TDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOLW VYLRPVPRPATVYLOILRLKPLTGEGTAGGGGGG RRHIRIRSLKI ELHSRSGHWOSIDFKOVLHSWFRO POSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFM ELRVLENTKRSRRGLECDGKVNICCKKOFFVSFK DIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSL SFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMS MLYYDDGONIIKKDIQNMIVEECGCS Inhibin GDF - 11 N / A SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 229 Beta A ( 97 - 274 ) VEAVKKHILNMLHLKKRPDVTOPVPKAALLNAIR ( 1 - 76 ) KLHVGKVGGDALOPEDFLEEDEYHATTETVISMA OETDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOL WVYLRPVPRPATVYLOILRLKPLTGEGTAGGGGG GRRHIRIRSLKIELHSRSGHWOSIDFKOVLHSWFR QPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPF MELRVLENTKRSRR Inhibin GDF - 11 NA SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 230 Beta A ( 87 - 274 ) VEAVKKHILNMLHLKKRPDVTOPVPKAALLOOIL ( 1 - 64 ) DLHDFQGDALQPEDFLEEDEYHATTETVISMAQE TDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLW VYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGG RRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRO POSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFM ELRVLENTKRSRR

Chimeric proteinsmay be used to characterize and / or map 55 TABLE 14 epitopes associated with GPCs. As used herein , the terms " map ” or “ mapping ” refer to the identification , character Chimeric proteins ization and /or determination of one or more functional regions of one or more proteins . Such characterizations may SEQ be necessary for determining interactions between one or 60 ID more protein modules and another agent ( e . g . another pro Protein Sequence NO tein and / or protein module . ) Some chimeric proteins may be proTGF - LSTSKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVP231 used to characterize functions associated with one or more Blarm3 PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE proteins and /or protein modules . C4S IHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKN In some embodiments , chimeric proteins of the present 65 RTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIA invention may comprise the sequences listed in Table 14 or KORYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESN fragments thereof US 9 , 758 ,577 B2 87 88 TABLE 14 - continued phils (Palmer , E . L . et al. , Sequence and tissue distribution of the integrin a , subunit, a novel partner of B , that is widely Chimeric proteins distributed in epithelia and muscle . Journal of Cell Biology . 1993 . 123 ( 5 ) : 1289 - 97 . ) Expression of d , is not detected SEQ Searearlier than E12 . 5 , suggesting that it does not play a major ID role in the earliest tissue morphogenesis (Wang , A . et al. , Protein Sequence NO Expression of the integrin subunit a , in the murine embryo . LGLEISIHCPCHTFOPNGDILENIHEVMEIKFKGVDNED Developmental Dynamics. 1995 . 204 :421 - 31 . ) In vivo func DHGRGDLGRLKKOKDHHNPHLILMMI PPHRLDNPGOG tions of a , are unclear. Phenotypes observed in knockout GORKKRALDTNYCFSSTEKNCCVROLYIDFRKDLGWK 10 mice suggest a role in lymphatic valve development ( Ba WIHEPKGYHANFCLGPCPYIWSLDTOYSKVLALYNOH zigou , E . et al. , Integrin - d , is required for fibronectin matrix NPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIV assembly during lymphatic valve morphogenesis . Dev Cell . RSCKCS 2009 Aug . 17 ( 2 ) : 175 - 86 . ) Reported interaction partners of proTGF LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP232 integrin aß, include VCAM -1 , the third FnlIl domain on B1 PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE tenascin C , osteopontin , polydom / SVEP1, VEGF - A and Trigger VTRVLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVP 15 NGF ( Yokasaki, Y . et al. , Identification of the ligand binding Loop EPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYL site for the integrin aoß , in the third fibronectin type III ( short ) SNRLLAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLS B2 AHCSCDSRDNTLQVDINGFTGTSTYTSGDQKTIKSTRK repeat of tenascin C . The Journal of Biological Chemistry. C4S KHGMNRPFLLLMATPLERAQHLOSSRHRRALDTNYCF 1998 . 273 ( 19 ) : 11423 - 8 ; Marcinkiewicz , C . et al. , Inhibitory al SSTEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFCL effects of MLDG - containing heterodimeric disintegrins GPCPYIWSLDTOYSKVLALYNOHNPGASAAPCCVPOA 20 reveal distinct structural requirements for interaction of the LEPLPIVYYVGRKPKVEQLSNMIVRSCKCS integrin a , ß , with VCAM - 1 , tenascin - C , and osteopontin . JBC . 2000 . 275 (41 ) :31930 - 7 ; Oommen , S . et al. , Vacular proTGF - SLSLSTSTTLDFGHI KKKRVEAIRGOILSKLRLTSPPEPT233 endothelial growth factor A (VEGF - A ) induces endothelial B3 armi VMTHVPYOVLALYNSTRELLEEMHGEREEGCTOENTE C7S SEYYAKEVTRVLMVETHNEIYDKF KOSTHSIYMFFNTS and cancer cell migration through direct binding to integrin ELREAVPEPVLLSRAELRLLRLKLKVEOHVELYOKYSN Coß . JBC . 2011 . 286 ( 2 ) : 1083 - 92 ; Sato -Nishiuchi , R . et al. , NSWRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGE Polydom /SVEP1 is a ligand for integrin ayß . JBC . 2012 . IEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHG 287 ( 30 ) : 25615 - 30 ; Staniszewska , I . et al. , Integrin a , ß , is a MNRPFLLLMATPLERAOHLOSSRHRRALDTNYCFRNL receptor for nerve growth factor and other neurotrophins . EENCCVRPLYIDFRODLGWKWVHEPKGYYANFCSGPC Journal of Cell Science . 2007 . 121 (Pt 4 ): 504 - 13 ; Yokosaki, PYLRSADTTHSTVLGLYNTLNPEASASPCCVPODLEPL Y . et al ., The integrin a , ß , binds to a novel recognition TILYYVGRTPKVEQLSNMVVKSCKCS sequence (SVVYGLR ; SEQ ID NO : 238 ) in the thrombin TGF LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP234 cleaved amino - terminal fragment of osteopontin . JBC . 1999 . Blarm3 PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE 274 ( 51 ) :36328 - 34 . ) C4S IHKFDMIOGLAEHNELAVCPKGITSKVFRFNVSSVEKN Binding sites on proteins that interact with aoß , have been ( LAP ) RTNLFRAEFRVLRVPNPSSKRNEORIELFQILRPDEHIA mapped using linear peptides. These sites include binding KORYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESN LGLEISIHCPCHTFOPNGDILENIHEVMEIKFKGVDNED 35 sites on tenascin C ( AEIDGIEL ; SEQ ID NO : 237 ) , osteo DHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQG pontin ( SVVYGLR ; SEQ ID NO : 238 ) , polydom / SVEP1 GORKKR (EDDMMEVPY ; SEQ ID NO : 239 ) and VEGF- A ( EYP ) . Unlike auß , and asß?, A , B , does not require a canonical TGF SLSLSTSTTLDFGHI KKKRVEAIRGOILSKLRLTSPPEPT235 RGD sequence motif . Some, but not all reported targets have B3c75 armi VMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTS 40 an acidic residue/ hydrophobic residue /proline motif. Some ( LAP ) ELREAVPEPVLLSRAELRLLRLKLKVEOHVELYOKYSN also comprise a tyrosine residue . NSWRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGE The trigger loop of TGF -B1 and TGF- B3 carries an RGD IEGFRLSAHCSCDSRDNTLOVDINGFTTGRRGDLATIHG sequence where a ,B6 and / or a , ß , bind to enable growth MNRPFLLLMATPLERAQHLOSSRHRR factor release. The TGF - B2 trigger loop region is different TGF - B1 LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP236 45 from those of TGF - B1 and TGF -B3 , comprising the Trcigger PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE ?? oop VTRVLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVP sequence FAGIDGTSTYTSGDQKTIKSTRKKNSGKTP ( short ) EPVLLSRAELRLLRLKLKVEOHVELYOKYSNNSWRYL ( SEQ ID NO : 65 ) , without an RGD trimer . Of this region , B2 SNRLLAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLS residues AGIDGTST (SEQ ID NO : 240 ) align with the C4S AHCSCDSRDNTLQVDINGFTGTSTYTSGDQKTIKSTRKAuces peptide on the third FnlII domain of tenascin - C that has been ( LAP ) KHGMNRPFLLLMATPLERAQHLOSSRHRR 50 mapped as an agß , binding site . Also , the tyrosine following this region may play a role in potential aoß , binding . In some embodiments, chimeric proteins may comprise Therefore, aoß , binding to TGF -B2 could be physiologically one or more protein modules from TGF- B2 . Although the relevant. In some embodiments , chimeric proteins of the crystal structure for the TGF - B2 growth factor has been present invention may comprise trigger loop sequences elucidated ( Daopin , S . et al. , Crystal structure of transform - 55 comprising any of the sequences listed in Table 15 . ing growth factor- B2 : an unusual fold for the superfamily . Science . 1992 . 257 (5068 ) : 369 -73 ) activation mechanisms TABLE 15 remain to be fully understood . Activation may be dependent upon one or more interactions between the TGF -B2 trigger Trigger loop sequences loop and agß , integrin . The TGF -B2 trigger loop may 60 SEO comprise similar structural and /or functional features asso Source ID ciated with RGD sequences . TGF- B2 trigger loops may bind protein Trigger loop sequence NO integrins, including , but not limited to aoß , integrins. According to mouse tissue staining , integrin subunit a , is TGF - B2 FAGIDGTSTYTSGDQKTIKSTRKKNSGKTP 65 widely expressed in skeletal and cardiac muscle , visceral 65 TGF - B2 AGIDGTST 240 smooth muscle , hepatocytes, airway epithelium , squamous epithelium , choroid plexus epithelium and also on neutro US 9 , 758 ,577 B2 89 90 isolation of chimeric proteins . Such detectable labels may TABLE 15 - continued comprise biotin labels , polyhistidine tags and / or flag tags . Trigger loop sequences Tags may be used to identify and / or isolate tagged proteins . SEO Proteins produced may comprise additional amino acids Source ID encoding one or more 3C protease cleavage site . Such sites protein Trigger loop sequence NO allow for cleavage at the 3C protease cleavage site upon TGF - B2 GTSTYTSGDQKTIKSTRKK 180 treatment with 3C protease , including , but not limited to ( short ) rhinovirus 3C protease . 3C protease cleavage sites may be introduced to allow for removal of detectable labels from TGF - B1 INGFTTGRRGDLATIHGMNRP 241 10 chimeric proteins. Protein Expression TGF - B1 SGRRGDLATI 242 In some embodiments , synthesis of recombinant proteins TGF - B1 TGRRGDLATI 243 of the present invention may be carried out according to any method known in the art. Some protein synthesis may be TGF - B3 FKGVDNEDDHGRGDLGRLKKOKDHHNP 244 carried out in vitro . Some protein synthesis may be carried GDF - 8 PGEDGLNP 245 out using cells . Such cells may be bacterial and / or eukary otic . In some embodiments , eukaryotic cells may be used for GDF - 11 PGAEGLHP 246 protein synthesis . Some such cells may be mammalian . Some mammalian cells used for protein expression may Inhibin A RPEATP 247 20 include , but are not limited to mouse cells , rabbit cells , rat BMP - 9 SHRKGCDTLDISVPPGSRNLP 248 cells , monkey cells , hamster cells and human cells . Such cells may be derived from a cell line . In other embodiments , BMP - 2 RHVRISRSLHODEHSWSQIRP 249 human cells may be used . In further embodiments , cell lines QHVRISRSLPQGSGNWAQLRP 250 may include , but are not limited to HEK293 cells , CHO BMP - 4 25 cells, Hela cells , Sw -480 cells , EL4 T lymphoma cells , BMP - 7 IGRHGPONKQP 251 TMLC cells , 293T/ 17 cells , Hs68 cells , CCD1112sk cells , HFF - 1 cells , Keloid fibroblasts , A204 cells , L17 RIB cells BMP - 6 VGRDGPYDKQP 252 N and C2C12 cells. BMP - 8 LGQRAPRSQQP 253W In some embodiments , 293 cells are used for synthesis of 0 30 recombinant proteins of the present invention . These cells Lefty 1 RFASQGAPAGLGEP 254 0 are human cells that post - translationally modify proteins osteopontin SVVYGLR 238 with human - like structures ( e . g . glycans ) . Such cells are W easily transfectable and scalable and are able to grow to high tenascinc AEIDGIEL 237 densities in suspension culture . 293 cells may include 293E W 35 cells . 293E cells are HEK293 cells stably expressing polydom / SVEP1 EDDMMEVPY 239 EBNA1 (Epstein -Barr virus nuclear antigen - 1 ) . In some VEGF - A EYP I cases, 293E cells may be grown in serum - free medium to simplify down - stream purification . In some cases , 293 -6E In some embodiments , chimeric proteins of the present cells (NRC Canada , Ottawa , CA ) may be used . Such cells invention may comprise one or more TGF - B2 trigger loops . 4040 express truncated EBNA1 ( EBNA1t ) and may comprise Such chimeric proteins may exhibit activation ( e . g . growth enhanced production of recombinant proteins and may be factor release ) regulated in a manner similar to that of optimized for growth and /or protein expression in serum TGF -B2 . Some chimeric proteins of the present invention free medium to simplify down - stream purification . In some may comprise TGF - B -related proteins wherein one or more cases , insect cells may be used to express recombinant protein modules are substituted with one or more protein 45 proteins of the invention . In some cases , insect cell expres modules comprising one or more TGF - B2 trigger loops . sion may be carried out using Spodoptera frugiperda cells Some chimeric proteins comprise TGF - B - related proteins including , but not limited to Sf9 and / or Sf -21 cells . In some wherein one or more protein modules comprising at least cases , insect cell cultures may comprise Trichoplusia ni one RGD sequence are substituted with one or more protein cells , including , but not limited to Tn -368 and /or HIGH modules comprising one or more TGF -B2 trigger loops. In 50 FIVETM BTI- TN -5B1 - 4 cells . A further list of exemplary other embodiments, chimeric proteins may comprise TGF- insect cell lines can be found in U . S . Pat. No . 5 , 024 , 947 , the B1 and / or TGF- b3 proteins wherein one or more protein contents of which are herein incorporated by reference in modules comprising at least one RGD sequence are substi - their entirety . tuted with one or more protein modules comprising one or In some embodiments , recombinant proteins of the inven more TGF -B2 trigger loops . Such chimeric proteins may 55 tion may comprise an antibody Fc domain to create an Fc exhibit TGF- B1 activity . fusion protein . The formation of an Fc fusion protein with In some embodiments , chimeric proteins of the present any of the recombinant proteins described herein may be invention may comprise one or more protein modules from carried out according to any method known in the art , BMPs. Protein modules comprising sequences from BMPs including as described in Czajkowsky , D . M . et al. , 2012 . may comprise sequences from any of those BMP modules 60 EMBO Mol Med . 4 (10 ): 1015 - 28 and U . S . Pat . Nos . 5 , 116 , disclosed in FIGS. 8A - 8G . Chimeric proteins of the present 964 , 5 ,541 ,087 and 8 ,637 ,637 , the contents of each of which invention comprising one or more BMP protein module may are herein incorporated by reference in their entirety. Fc be useful for the development of antibodies and / or assays to fusion proteins of the invention may be linked to the hinge study , enhance and / or perturb BMP interactions with other region of an IgG Fc via cysteine residues in the Fc hinge proteins , including , but not limited to RGM proteins . 65 region . Resulting Fc fusion proteins may comprise an anti Chimeric proteins may comprise detectable labels . body - like structure , but without Cy domains or light chains . Detectable labels may be used to allow for detection and / or In some cases, Fc fusion proteins may comprise pharma US 9 , 758 ,577 B2 91 92 cokinetic profiles comparable to native antibodies . In some GACTGGTATTCTTAACTATGTT GCTCCTTTTACGC cases , Fc fusion proteins of the invention may comprise an TATGTGGATACGCTGCTTTAATGCCTTTGTATCAT extended half - life in circulation and / or altered biological GCTATTGCTT activity . In some cases, Fc fusion proteins of the invention CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATC may be prepared using any of the TGF - B family proteins or 5 CTGGTTGCTGTCTCTTTATGAG GAGTTGTGGC TGF - B - related proteins described herein . In some cases , Fc CCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGT fusion proteins may comprise TGF- B , GDF - 8 and /or GDF GTTTGCTGACGCA 11 . ACCCCCACTGGTTGGGGCATTGCCACCACCTGTCA Sequences encoding recombinant proteins of the present GCTCCTTTCCGGGACTTTCGCTT TCCCCCTCCCTAT invention may be inserted into any number of DNA vectors 10 TGCCACGGCGGAACTCATCGCCGCCTGCCTTGC known in the art for expression . Such vectors may include CCGCTGCTGGA plasmids . In some embodiments , sequences encoding CAGGGGCTCGGCTGTTGGGCACTGACAATTC recombinant proteins of the present invention are cloned into CGTGGTGTTGTCGGGGAAGCTGACGT CCTTTC pTT5 vectors (NRC Biotechnology Research Institute , Mon CATGGCTGCTCGCCTGTGTTGCCACCTGGATTCT treal, Québec . ) In other embodiments pTT22 , PTT28 , PYD5 , 15 GCGCGGGACGTCCTTCTG pYD7, pYD11 (NRC Biotechnology Institute , Montréal , CTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTC Québec ) and /or PMA vectors (Life Technologies , Carlsbad , CTTCCCGCGGCCTGCTGCCGGCT CTGCGGCCTCT Calif .) may be used . Vectors may comprise promoter TCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCG sequences to modulate expression of sequences encoding GATCTCCCTTTGGG CCGCCTCCCCGCCTG (SEQ ID recombinant proteins of the present invention . Such promot - 20 NO : 256 ). WPREs may enhance translation of nucleic acids ers may be constitutively active and/ or may be regulated by comprising WPREs. Such enhanced translation may be due extrinsic and / or intrinsic factors. Some extrinsic factors may to increased cytoplasmic export of newly transcribed be used to enhance or suppress expression of sequences mRNA . encoding recombinant proteins of the present invention . In some embodiments , recombinant proteins may com Some vectors may encode nuclear localization signals that 25 prise one or more secretion signal sequences . As used may be incorporated into recombinant proteins of the pres - herein , the term “ secretion signal sequence ” refers to a chain ent invention upon translation . Some vectors may produce of amino acids (or nucleotides that encode them at the mRNA transcripts that comprise nuclear export signals . nucleic acid level ) that when part of a protein , modulate RNA transcribed from a modified pTT5 vector ( pTT5 - secretion of such proteins from cells. Some secretion signal WPRE ) contains an element that facilitates nuclear export of 30 sequences may be located at protein termini . In other the transcripts . Some vectors may be modified by insertion embodiments , secretion signal sequences may be N -terminal of one ormore ligation - independent cloning (LIC ) cassettes amino acid sequences. Other secretions signal sequences to provide for simpler cloning . may comprise the secretion signal of the Ig kappa chains . Vectors encoding recombinant proteins of the present Such Ig kappa chains may be human Ig kappa chains. In invention may be delivered to cells according to any method 35 some embodiments , secretion signal sequences may com known in the art , including, but not limited to transfection , prise the amino acid sequence MDMRVPAQLLGLLLLWF electroporation and /or transduction . In some embodiments , SGVLG (SEQ ID NO : 257 ). vectors may comprise one or more elements to enhance In some embodiments , recombinant proteins of the pres vector replication in host cells . In some embodiments , ent invention may require coexpression with one or more vectors may comprise oriP sites for episomal replication in 40 other proteins for proper expression , folding , secretion , cells that express EBNA - 1 . activity and / or function . Some recombinant GPCs of the In some cases , cells are stably transfected to produce present invention may be coexpressed with LTBPs, fibrillins recombinant proteins of the present invention . Stably trans - and /or GARP. fected cells pass transfected genes to daughter cells during I n some embodiments , recombinant proteins of the pres cell division , thus eliminating the need for repeated trans - 45 ent invention may be biotinylated . As used herein , the term fection . In some cases , the transfected genes are stably “ biotinylating ” refers to the attaching of one or more biotin inserted into the genome of the transfected cells . Transfected labels . Such biotin labels may facilitate interactions of genes may comprise genes for cell selection , such as genes biotinylated recombinant proteins with avidin and /or that confer resistance to one or more toxic or repressive streptavidin coated surfaces and / or proteins . As used herein , compounds . Such genes may be used to support the growth 50 a “ biotin label” refers to a detectable label comprising one of only cells with stable incorporation of the transfected or more biotin molecules . The term “ biotinylated ” refers to genes when grown in the presence of such one or more toxic a molecule or protein that comprises one or more biotin or repressive compounds ( e . g . puromycin , kenomycin , etc . ) labels. Biotin molecules bind with high affinity to avidin and Cell selection may also comprise selecting cells based on streptavidin molecules . This property may be used to capture overall recombinant protein expression levels . Determina - 55 biotinylated proteins using avidin and / or stretavidin coated tion of such levels may be carried out, for example , by materials . Some recombinant GPCs of the present invention Western Blot and / or ELISA . may be biotinylated near the N - terminus . Such recombinant In some embodiments , nucleotide sequences encoding GPCs may be introduced to avidin / streptavidin coated cell recombinant proteins of the present invention may comprise culture surfaces, allowing biotinylated recombinant GPCs to one or more woodchuck hepatitis virus posttranscriptional 60 adhere to the surface in a manner such that the orientation regulatory element (WPRE ) . RNA nucleic acids comprising and bonding of such bound GPCsmimics the orientation and such elements may comprise the sequence GCCACGGCG - bonding of GPCs to LTBPs, fibrillins and /or GARPs . GAACUCAUCGCCGCCUGCCUUGCCCGCUGCUG - In some embodiments, recombinant proteins produced GACAGGGGCUCGGC UGUUGGGCACUGACAAUUC may be analyzed for quality control purposes to assess both CGUGGU (SEQ ID NO : 255 ) . RNA comprising WPRES 65 biophysical properties as well as bioactive properties. Bio may be transcribed from DNA comprising the sequence physical characterization may include assessing protein AATCAACCTCTGGATTACAAAATTTGTGAAAGATT - migration patterns after reducing and / or non -reducing SDS US 9 ,758 ,577 B2 93 94 PAGE . Biophysical characterization may also comprise gel associated growth factors, furin cleavage -deficient mutants , filtration , mass spectrometric analysis and /or analysis of mutants deficient in extracellular protein associations and /or association/ dissociation between LAPs or LAP - like combinations thereof. domains and growth factor domains. Bioactive properties In some embodiments , antigenic proteins may comprise may be analyzed by assessing reactivity with antibodies 5 TGF - ß - related proteins and / or modules thereof. Such anti and / or signaling activity of dissociated growth factors and / or genic proteins may comprise epitopes from regions where growth factors associate with or comprise stereological latent GPCs . proximity with prodomain regions . Antibodies ofthe present Some proteins produced may comprise additional amino invention directed to such epitopes may bind overlapping acids encoding one or more detectable labels for purification 10 regions between growth factors and prodomains . Such anti [ e . g . polyhistidine tag, flag tag , etc .] In some embodiments , bodies may stereologically inhibit the dissociation of growth proteins are N -terminally labeled . In some embodiments , factors from GPCs . proteins are C - terminally labeled . In some embodiments , In some embodiments , antigenic proteins comprise only proteins are biotinylated . In some embodiments , recombi the prodomain or only the growth factor from a particular nant proteins of the present invention are N -terminally terminally 15 GPCGP . Epitopes present on such antigenic proteins may be biotinylated . shielded or unexposed in intact GPCs. Some antibodies of Proteins produced may comprise additional amino acids the present invention may be directed to such epitopes. Such encoding one or more 3C protease cleavage site . Such sites antibodies may be releasing antibodies , promoting growth allow for cleavage between residues Q and G of the 3C factor dissociation from GPCs. Further antibodies may protease cleavage site upon treatment with 3C protease , 20 compete with free growth factor for prodomain binding, including, but not limited to rhinovirus 3C protease . In some thereby promoting growth factor dissociation from GPCs. embodiments , such cleavage sites are introduced to allow In some embodiments , antigenic proteins may comprise for removal of detectable labels from recombinant proteins . proprotein convertase (e . g . furin ) cleavage site mutations . In some embodiments , modification of expressed growth Such mutations may prevent enzymatic cleavage of growth factor proproteins may be carried out by enzymatic cleav - 25 factors from their prodomains . Some antibodies of the age . In some cases , proprotein convertases may be used . present invention may be directed to epitopes present on Such proprotein convertases may include , but are not limited such mutant proteins . Such antibodies may stabilize the to furin / PACE3 , PC1/ 3 , PC2, PC4 , PC5/ 6 , PACE4 and PC7. association between prodomains and growth factors. In Proprotein convertase cleavage may be caried out in solution some embodiments , furin cleavage site mutants comprise or in tissue culture . In some cases , proprotein convertases 30 D2G mutants as described herein . are expressed in cells expressing proproteins to be cleaved . In some embodiments , antigenic proteins comprising pro In some cases, proprotein convertases are added to tissue domains may comprise N - terminal mutations that lead to cultures of cells expressing proproteins to be cleaved . decreased prodomain association with LTBPs and / or GARP Antibodies and therefore may present epitopes in the N - terminal region In some embodiments, compounds and /or compositions 35 that may otherwise be shielded by those associations . Some of the present invention may comprise antibodies or frag - antibodies of the present invention may be directed to such ments thereof. As used herein , the term " antibody ” is eniepitopes . Some antigenic proteins comprising TGF- B1 pro referred to in the broadest sense and specifically covers domains may comprise C4S mutations . Such mutations may various embodiments including, but not limited to mono - prevent association of antigenic proteins with LTBPs and /or clonal antibodies, polyclonal antibodies , multispecific anti - 40 GARP, making these proteins useful for presenting N - ter bodies ( e . g . bispecific antibodies formed from at least two minal epitopes . Antibodies directed to C4S mutants may intact antibodies ) , and antibody fragments such as diabodies prevent GPC association with LTBPs and /or GARP. Some so long as they exhibit a desired biological activity . Anti antibodies directed to C4S mutants may reduce growth bodies are primarily amino -acid based molecules but may factor signaling in a particular niche . Some such antibodies also comprise one or more modifications ( including , but not 45 may reduce or prevent the release of growth factor by limited to the addition of sugar moieties , fluorescent moi- blocking the ability of the GPCs to associate securely with eties , chemical tags, etc . ) the extracellular matrix . Recombinant and Chimeric Protein Use in Antibody Gen - In some embodiments , antigenic proteins may comprise eration one or more recombinant LTBP . Such recombinant LTBPs In some embodiments, recombinant and /or chimeric pro - 50 may comprise LTBP1, LTBP2 , LTBP3 , LTBP4 , alternatively teins described herein may be used as antigens (referred to spliced variants and /or fragments thereof. Recombinant herein as antigenic proteins ) to generate antibodies. Such LTBPs may also be modified to comprise one or more antigenic proteins may comprise epitopes that may be less detectable labels . Such detectable labels may include , but accessible for antibody generation in similar wild type are not limited to biotin labels , polyhistidine tags, myc tags, proteins . Some antibodies directed to antigenic proteins of 55 HA tags and / or fluorescent tags . the present invention may modulate the release of one or In some embodiments , antigenic proteins may comprise more growth factors from one or more GPCs .) Some such one or more recombinant protein and /or chimeric protein antibodies may be stabilizing [reducing or preventing dis complexed with one or more recombinant LTBP . Some sociation between two agents, ( e . g . growth - factor release antigenic proteins may comprise proprotein convertase from GPCs, GPC release from one or more protein interac - 60 cleavage site mutants ( e . g . D2G mutants , AXXA mutants ) tions ) ] and / or releasing ?enhancing the dissociation between complexed with one or more recombinant LTBP . Some such two agents (e .g . growth - factor release from GPCs, GPC recombinant LTBPs may comprise LTBP1S . Some recom release from one or more protein interactions ) ] antibodies . binant LTBPs may comprise one or more detectable labels, Antigenic proteins of the present invention may comprise including, but not limited to biotin labels , polyhistidine tags TGF - B - related proteins as well as components and / or pro - 65 and /or flag tags . tein modules thereof. In some cases, antigenic proteins of In some embodiments , antigenic proteins may comprise the present invention may comprise prodomains without GARP (or homologues thereof, including , but not limited to US 9 ,758 ,577 B2 95 96 LRRC33 ) . Such GARP may be recombinant, referred to may be characterized as agonists . As used herein , the term herein as recombinant GARP . Some recombinant GARPS " natural depot” refers to a location within a cell , tissue or may comprise one or more modifications , truncations and /or organ where increased levels of a biomolecule or ion are mutations as compared to wild type GARP. Recombinant stored . For example , the extracellular matrix may act as a GARPs may be modified to be soluble . In other embodi- 5 natural depot for one or more growth factors . ments , recombinant GARPs are modified to comprise one or The contact necessary for growth - factor release may be more detectable labels . In further embodiments , such detect- defined as direct or indirect contact of antibody with a GPC able labels may include , but are not limited to biotin labels , or a component thereof or with a cellular structure such as polyhistidine tags , flag tags , myc tags , HA tags and / or an extracellular and /or cellular matrix protein and /or protein fluorescent tags . In some embodiments , antigenic proteins 10 associated with the extracellular and / or cellular matrix ?e . g . may comprise one or more recombinant protein and /or LTBPs ( e . g . LTBP1 , LTBP2, LTBP3 and /or LTBP4) , chimeric protein complexed with one or more recombinant fibrillins ( e . g . fibrillin - 1 , fibrillin - 2 , fibrillin - 3 and /or fibril GARP. In some embodiments , antigenic proteins comprise lin - 4 ) perlecan , decorin , elastin , collagen and /or GARPs LAPs ( e . g . TGF - B LAPs ) and / or LAP - like domains com - ( e . g . GARP and / or LRRC33 ) ] for release of growth factor. plexed with recombinant GARP . In some embodiments , 15 Release of at least 5 % , 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , antigenic proteins comprise D2G mutants ( e . g . TGF - B D2G 70 % , 80 % , 90 % or more of growth factor is sufficient to mutants ) complexed with recombinant GARP. In some characterize antibodies of the present invention as releasing embodiments , complexed recombinant GARPs may be antibodies . It is understood that growth factor release after soluble forms of GARP ( SGARP ) . In some embodiments, antibody administration may be local and may occur over a SGARPs comprises one or more biotin labels , polyhistidine 20 sustained period of time and may include peaks or spikes of tags and /or flag tags . release . Antibodies of the present invention may act to In some embodiments , GARPs complexed with LAP release one or more growth factor over minutes, hours , days and/ or LAP - like domains are desired as antigens, in assays or longer. and / or for antibody development. In such embodiments, Release profiles may have an initial peak or burst within LAPs and /or LAP - like domains may comprise CED muta - 25 from about 4 hours to about 7 days of contacting in vivo or tions . Such LAPs and /or LAP -like domains may be shorter periods in vitro . For example, initial peak or burst expressed as GPCs to facilitate proper protein folding , may occur from about 4 hours to about 5 hours , or from conformation and /or expression , but the CED mutations about 4 hours to about 6 hours , or from about 4 hours to present may enhance growth factor release , leaving the about 7 hours , or from about 4 hours to about 8 hours , or desired GARP -LAP (or LAP - like domain ) complex behind . 30 from about 4 hours to about 9 hours, or from about 4 hours GARP -LAP (or LAP - like domain ) complexes may be useful to about 10 hours, or from about 4 hours to about 11 hours , as antigens in the production of releasing antibodies that or from about 4 hours to about 12 hours , or from about 4 specifically target GARP - associated GPCs . hours to about 24 hours , or from about 4 hours to about 36 In some embodiments , GPCs comprising CED mutations hours , or from about 4 hours to about 48 hours , or from may act to stabilize a natively populated conformation of 35 about 1 day to about 7 days , or from about 1 day to about LAP (or LAP -like domain ) characterized by reduced growth 2 days, or from about 1 day to about 3 days , or from about factor association (both as a free LAP or LAP - like domains 1 day to about 4 days , or from about 4 days to about 5 days, and / or as a GARP and / or LTBP/ LAP complex ) , thereby or from about 4 days to about 6 days , or from about 4 days exposing epitopes that may be less exposed in wild - type to about 7 days . Compounds and / or compositions of the proteins . Such mutations may shift the conformational equi - 40 present invention may stimulate the release of 5 to 100 % of librium of LAP or LAP - like domains to facilitate the pro - the growth factor present. For example, the percent of duction of activating antibodies. growth factor release may be from about 5 % to about 10 % , In some embodiments , antigenic proteins of the present or from about 5 % to about 15 % , or from about 5 % to about invention may comprise one or more protein modules from 20 % , or from about 5 % to about 25 % , or from about 10 % to GDFs ( e . g . GDF - 11 and / or GDF - 8 ) . In some embodiments , 45 about 30 % , or from about 10 % to about 40 % , or from about antibodies of the present invention may be directed toward 10 % to about 50 % , or from about 10 % to about 60 % , or from antigenic proteins comprising GDF - 8 protein modules . In about 20 % to about 70 % , or from about 20 % to about 80 % , some embodiments , such antibodies may modulate GDF - 8 or from about 40 % to about 90 % , or from about 40 % to levels and / or activity in one or more niches . In some about 100 % . embodiments , antibodies of the present invention may pre - 50 Releasing antibodies generated according to methods vent the release of GDF - 8 growth factors from GPCs. In described herein may be generated to release growth factors some embodiments , antibodies of the present invention may from GPCs comprising any of the pro - proteins listed in be used to repair and / or enhance muscle tissues . Table 1 . In some cases , releasing antibodies are directed to In some embodiments , recombinant proteins ( including , GPCs comprising TGF - B isoforms and / or one or more but not limited to chimeric proteins) described herein may 55 modules of such isoforms. In some cases , releasing antibod be used in studies to identify and map epitopes that may be ies are directed to GPCs comprising GDFs and / or one or important targets for antibody development. Such studies more modules from GDFs. may be used to identify epitopes that may promote growth Stabilizing Antibodies factor release or stabilization of GPCs upon antibody bind As used herein , the term " stabilizing antibody ” refers to ing. 60 an antibody that decreases the ratio of active and / or free Releasing Antibodies growth factor relative to inactive and / or prodomain - associ As used herein , the term “ releasing antibody ” refers to an ated growth factor upon the introduction of the antibody to antibody that increases the ratio of active and / or free growth one or more GPC , cell , niche , natural depot and / or any other factor relative to inactive and/ or prodomain - associated site of growth factor sequestration . In this context, antibod growth factor upon the introduction of the antibody to a 65 ies may be characterized as antagonists . As used herein , an GPC , cell , niche , natural depot or any other site of growth “ antagonist ” is one which interferes with or inhibits the factor sequestration . In this context, releasing antibodies physiological action of another . Antagonist action may even US 9 , 758 ,577 B2 97 98 result in stimulation or activation of signaling downstream growth factors from GPCs comprising any of the pro and hence may act agonistically relative to another pathway, proteins listed in Table 1 . Such antibodies may physically separate from the one being antagonized . Pathways are interact with GPC protease cleavage sites and / or block the interrelated , so , in one nonlimiting example , a TGF - B interaction of proteolytic enzymes that may target such antagonist could act as a BMP agonist and vice versa . In the 5 cleavage sites . In some cases, stabilizing antibodies are context of cellular events , as used herein , the term “ down - directed to GPCs comprising TGF - B isoforms and /or one or stream ” refers to any signaling or cellular event that happens more modules of such isoforms. In some cases, stabilizing after the action , binding or targeting by compounds and / or antibodies are directed to GPCs comprising GDFs and / or compositions of the present invention . one or more modules from GDFs . Contact necessary for inhibition or stabilization may be 10 Stabilizing antibodies directed to GPCs comprising direct or indirect contact between antibody and GPC or GDF - 8 may block metalloproteinase cleavage of such com components thereof or with cellular structures such as an plexes . Such agents may bind to GPCs comprising GDF - 8 in extracellular and /or cellular matrix protein and / or protein such a way as to physically prevent interactions between associated with the extracellular and /or cellular matrix [e . g . such GPCs and metalloproteinases targeting such GPCs . LTBPs ( e . g . LTBP1, LTBP2, LTBP3 and / or LTBP4 ) , 15 Agents that actually target metalloproteinases themselves fibrillins ( e . g . fibrillin - 1 , fibrillin - 2 , fibrillin - 3 and / or fibril- have been described previously ( see U . S . Pat. No . 7 ,572 , lin - 4 ) perlecan , decorin , elastin , collagen and /or GARPs 599 , the contents of which are herein incorporated by ( e . g . GARP and/ or LRRC33 ) ] whereby release of growth reference in their entirety .) factor is inhibited . Inhibition of release of at least 5 % , 10 % , Antibody Selection 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % or more of 20 A desired antibody may be selected from a larger pool of growth factors may be sufficient, in some cases , to charac - two or more candidate antibodies based on the desired terize antibodies of the present invention as inhibitory or antibody ' s ability to associate with desired antigens and/ or stabilizing . Inhibitory antibodies may stabilize GPCs and epitopes. Such antigens and /or epitopes may include, but are trap them as heterodimers . not limited to any of those described herein , including , but It is understood that inhibition of growth factor release 25 not limited to recombinant proteins, chimeric proteins, after contact with one or more antibodies of the present GPCs, prodomains ( e . g . LAPs or LAP - like domains) , invention may be local and may occur over a sustained growth factors , protein modules , LTBPs, fibrillins, GARP, period of time and may include peaks , troughs or spikes . TGF - B -related proteins and /or mutants and / or variants and / Inhibitory antibodies which may also function to stabilize or complexes and / or combinations thereof. Selection of GPCs may be defined by their release kinetics . Release of 30 desired antibodies may be carried out using an antibody growth factor and corresponding release kinetics , even binding assay , such as a surface Plasmon resonance - based locally , may be directly measured or inferred by downstream assay , an enzyme- linked immunosorbent assay (ELISA ) or signaling events. In some embodiments , changes in protein fluorescence - associated cell sorting ( FACS ) - based assay . or nucleic acid concentrations or phenotypic responses may Such assays may utilize a desired antigen to bind a desired be indicative of the effects of compounds and / or composi- 35 antibody and then use one or more detection methods to tions of the present invention . detect binding . Antibodies of the present invention may act to inhibit In some embodiments, antibodies of the present invention release of a growth factor over minutes , hours or days . may be selected from a larger pool of two or more candidate Inhibition and / or stabilization profiles may have an initial antibodies based on their ability to associate with desired trough within from about 4 hours to about 7 days of 40 antigens and/ or epitopes from multiple species (referred to introduction in vivo or shorter periods in vitro . For example , herein as “ positive selection . ” ) initial trough of inhibition or stabilization may occur from In some embodiments , such species may comprise verte about 4 hours to about 5 hours , or from about 4 hours to brate species. In some embodiments , such species may about 6 hours, or from about 4 hours to about 7 hours , or comprise mammalian species . In some embodiments , such from about 4 hours to about 8 hours , or from about 4 hours 45 species may include , but are not limited to mice , rats , to about 9 hours , or from about 4 hours to about 10 hours , rabbits , goats , sheep , pigs, horses, cows and/ or humans. or from about 4 hours to about 11 hours , or from about 4 In some embodiments , negative selection is used to hours to about 12 hours , or from about 4 hours to about 24 remove antibodies from a larger pool of two or more hours, or from about 4 hours to about 36 hours , or from candidate antibodies . As used herein the term " negative about 4 hours to about 48 hours , or from about 1 day to about 50 selection ” refers to the elimination of one or more factors 7 days , or from about 1 day to about 2 days, or from about from a group based on their ability to bind to one or more 1 day to about 3 days , or from about 1 day to about 4 days , undesired antigens and /or epitopes . In some embodiments , or from about 4 days to about 5 days , or from about 4 days undesired antigens and / or epitopes may include , but are not to about 6 days, or from about 4 days to about 7 days . limited to any of those described herein , including , but not Introduction of compounds and /or compositions of the pres - 55 limited to recombinant proteins , chimeric proteins , GPCs , ent invention may lead to inhibition and/ or stabilization of prodomains ( e . g . LAPs or LAP - like domains ) , growth fac 5 % to 100 % of growth factor present. For example , the tors, protein modules, LTBPs , fibrillins , GARPs , TGF - B percent of growth factor inhibition or stabilization may be related proteins and / or mutants and / or variants and /or com from about 5 % to about 10 % , from about 5 % to about 15 % , binations and / or complexes thereof. from about 5 % to about 20 % , from about 5 % to about 25 % , 60 In some embodiments , antibodies of the present invention from about 10 % to about 30 % , from about 10 % to about may be directed to prodomains ( e . g . the prodomain portion 40 % , from about 10 % to about 50 % , from about 10 % to of a GPC and /or free LAP or LAP - like domains ) that about 60 % , from about 20 % to about 70 % , from about 20 % decrease growth factor signaling and / or levels ( e . g . TGF - B to about 80 % , from about 40 % to about 90 % or from about growth factor signaling and /or levels ) in a given niche . In 40 % to about 100 % . 65 some embodiments , antibodies of the present invention may Stabilizing antibodies generated according to methods directed to LAPs or LAP - like domains that increase growth described herein may be generated to block the release of factor signaling and/ or levels in a given niche . In some US 9 , 758 ,577 B2 99 100 embodiments , antibodies of the present invention may be growth - factor releasing , GPC stabilizing , activating and / or directed to prodomains ( e .g . LAPs or LAP - like domains ) inhibitory ) and /or by the cell niche in which they function . and / or GPCs only when complexed with LTBPs, fibrillins As used herein the term , “ antibody fragment” refers to and / or GARP. any portion of an intact antibody . In some embodiments , In some embodiments , antibodies of the present invention 5 antibody fragments comprise antigen binding regions from may be selected from a larger pool of two or more candidate intact antibodies. Examples of antibody fragments may antibodies based on their ability to modulate growth factor include , but are not limited to Fab , Fab ' , F (ab ') 2 , and Fv levels and / or activity . In some cases , growth factor activity fragments ; diabodies; linear antibodies; single - chain anti assays may be used to test the ability of candidate antibodies body molecules ; and multispecific antibodies formed from to modulate growth factor activity . Growth factor activity antibody fragments . Papain digestion of antibodies produces assays may include, cell - based assays as described herein - two identical antigen - binding fragments , called “ Fab ” frag below . Additional assays that may be used to determine the ments , each with a single antigen -binding site . Also pro effect of candidate antibodies on growth factor activity may duced is a residual “ Fc " fragment, whose name reflects its include , but are not limited to enzyme- linked immunosor - 16 ability to crystallize readily . Pepsin treatment yields an bent assay ( ELISA ) , Western blotting , reporter assays ( e . g . F (ab ') 2 fragment that has two antigen -binding sites and is luciferase -based reporter assays or other enzyme- based still capable of cross- linking antigen . Compounds and /or reporter assays ), PCR analysis , RT -PCR analysis and / or compositions of the present invention may comprise one or othermethods known in the art including any of the methods more of these fragments . For the purposes herein , an “ anti described in U . S . Provisional Patent Applications 61/ 722 , 20 body ” may comprise a heavy and light variable domain as 919, filed Nov. 6 , 2012 and 61 /722 , 969, filed Nov . 6 , 2012 , well as an Fc region . the contents of each of which are herein incorporated by As used herein , the term “ native antibody ” refers to a reference in their entireties . usually heterotetrameric glycoprotein of about 150 ,000 dal In some embodiments , one or more recombinant proteins tons , composed of two identical light ( L ) chains and two or antibodies disclosed herein may be used in assays to test, 25 identical heavy ( H ) chains . Each light chain is linked to a develop and /or select antibodies . Recombinant GPCs may heavy chain by one covalent disulfide bond , while the be expressed to test releasing and / or stabilizing abilities of number of disulfide linkages varies among the heavy chains one or more antibodies being assayed . In some embodi- of different immunoglobulin isotypes . Each heavy and light ments, recombinant proteins may be expressed as positive or chain also has regularly spaced intrachain disulfide bridges . negative control components of assays. In some embodi - 30 Each heavy chain has at one end a variable domain (VH ) ments , multiple recombinant proteins may be expressed at followed by a number of constant domains . Each light chain once to modulate growth factor release and / or activity , has a variable domain at one end (V2 ) and a constant domain wherein such recombinant proteins may act synergistically at its other end ; the constant domain of the light chain is or antagonistically in such modulation . aligned with the first constant domain of the heavy chain , In some embodiments GPCs comprising CED mutations 35 and the light chain variable domain is aligned with the may provide a baseline level of growth factor activity in variable domain of the heavy chain . assays designed to test releasing antibodies, as these mutant As used herein , the term “ variable domain ” refers to proteins are sufficient for producing a biological effect in specific antibody domains that differ extensively in sequence humans . In some embodiments , GPCs comprising CED among antibodies and are used in the binding and specificity mutations may be used as positive controls in activity assays 40 of each particular antibody for its particular antigen . geared toward screening for releasing antibodies . In some As used herein , the term “ Fv ” refers to antibody frag embodiments , GPCs comprising CED mutations may be ments comprising complete antigen -recognition and anti used for screening for stabilizing antibody activity , as they gen - binding sites . These regions consist of a dimer of one can be presumably activated in the absence of integrins . In heavy chain and one light chain variable domain in tight, such assays , GPCs comprising CED mutations may be 45 non - covalent association . expressed in cell lines ( e . g . 293 cells or others ) and growth As used herein , the term “ light chain ” refers to a com factor activity and / or release may be assessed in the pres - ponent of an antibody from any vertebrate species assigned ence or absence of antibodies being tested . In some embodi- to one of two clearly distinct types, called kappa and lambda ments, co -expression of GPCs comprising CED mutation based on amino acid sequences of constant domains . with wild type GPCs ( including , but not limited to TGF -B1 , 50 Depending on the amino acid sequence of the constant TGF -B2 , or TGF - B3 ) could also be used to regulate free domain of their heavy chains , antibodies can be assigned to growth factor levels . In such embodiments , modulation of different classes . There are five major classes of intact free growth factor levels may accomplished by co - transfec - antibodies : IgA , IgD , IgE , IgG , and IgM , and several of tion of different ratios of wild type and mutant GPCs ( e . g . these may be further divided into subclasses ( isotypes ), e . g . , 1 :1 , 1: 2 , 1: 3 , 1: 4 , 1: 5 , 1: 10 ) . In some embodiments , further 55 IgG1, IgG2, IgG3, IgG4, IgA , and IgA2 . As used herein , the co - expression of LTBPs, fibrillins or GARPs may be carried term “ Single - chain Fv ” or “ scFv ” refers to a fusion protein out to add one ormore additional levels of free growth factor of Vy and V , antibody domains, wherein these domains are modulation . linked together into a single polypeptide chain . In some Antibody Development embodiments, the Fv polypeptide linker enables the scFv to In some embodiments, compounds and /or compositions 60 form the desired structure for antigen binding . of the present invention comprising antibodies , antibody As used herein , the term “ bispecific antibody” refers to an fragments , their variants or derivatives as described above antibody capable of binding two different antigens. Such are specifically immunoreactive with antigenic proteins as antibodies typically comprise regions from at least two described herein . different antibodies . Bispecific antibodies may include any Antibodies of the present invention may be characterized 65 of those described in Riethmuller, G . 2012 . Cancer Immu by their target molecule (s ), by the antigens used to generate nity . 12 : 12 - 18, Marvin , J. S . et al. , 2005 . Acta Pharmaco them , by their function (whether as agonists , antagonists , logica Sinica . 26 ( 6 ) : 649 - 58 and Schaefer , W . et al ., 2011 . US 9 , 758 ,577 B2 101 102 PNAS. 108 ( 27 ) : 11187 - 92 , the contents of each ofwhich are 6 ,673 , 901 ; U . S . Pat . No. 6 ,348 ,584 ) . In some embodiments , herein incorporated by reference in their entirety . antibody mimetics may be those known in the art including , As used herein , the term “ diabody ” refers to a small but are not limited to affibody molecules , affilins, affitins , antibody fragment with two antigen - binding sites. Diabodies anticalins, avimers , Centyrins, DARPINSTM , Fynomers and comprise a heavy chain variable domain Vh connected to a 5 Kunitz and domain peptides. In other embodiments , anti light chain variable domain V . in the same polypeptide body mimetics may include one or more non -peptide region . chain . By using a linker that is too short to allow pairing between the two domains on the same chain , the domains are As used herein , the term “ antibody variant” refers to a forced to pair with the complementary domains of another biomolecule resembling an antibody in structure and /or chain and create two antigen - binding sites . Diabodies are 10 function comprising some differences in their amino acid described more fully in , for example , EP 404 , 097 ; WO sequence , composition or structure as compared to a native 93 / 11161; and Hollinger et al. (Hollinger , P. et al. , “ Diabod antibody . ies” : Small bivalent and bispecific antibody fragments . The preparation of antibodies, whether monoclonal or PNAS . 1993 . 90 :6444 - 8 ) the contents of each of which are polyclonal, is known in the art. Techniques for the produc incorporated herein by reference in their entirety . tion of antibodies are well known in the art and described , As used herein , the term “ monoclonal antibody ” refers to e .g . in Harlow and Lane “ Antibodies , A Laboratory an antibody obtained from a population of substantially Manual” , Cold Spring Harbor Laboratory Press , 1988 ; Har homogeneous cells (or clones) , i. e ., the individual antibodies low and Lane “ Using Antibodies: A Laboratory Manual” comprising the population are identical and /or bind the same Cold Spring Harbor Laboratory Press, 1999 and “ Therapeu epitope , except for possible variants that may arise during 20 tic Antibody Engineering : Current and Future Advances production of the monoclonal antibodies, such variants Driving the Strongest Growth Area in the Pharmaceutical generally being present in minor amounts . In contrast to Industry ” Woodhead Publishing , 2012 . polyclonal antibody preparations that typically include dif - Standard Monoclonal Antibody Generation ferent antibodies directed against different determinants In some embodiments , antibodies are generated in knock ( epitopes ) , each monoclonal antibody is directed against a 25 out mice , lacking the gene that encodes for desired target single determinant on the antigen antigens . Such mice may not be tolerized to target antigens The modifier “ monoclonal” indicates the character of the and therefore may be better suited for generating antibodies antibody as being obtained from a substantially homoge - against such antigens that may cross react with human and neous population of antibodies , and is not to be construed as mouse forms of the antigen . For the production of mono requiring production of the antibody by any particular 30 clonal antibodies , host mice may be immunized with recom method . The monoclonal antibodies herein include " chime- binant proteins to elicit lymphocytes that specifically bind ric ” antibodies ( immunoglobulins ) in which a portion of the such proteins . Resulting lymphocytes may be collected and heavy and / or light chain is identical with or homologous to fused with immortalized cell lines . Resulting hybridoma corresponding sequences in antibodies derived from a par cells may be cultured in suitable culture medium with ticular species or belonging to a particular antibody class or 35 selection agents to support the growth of only fused cells . subclass , while the remainder of the chain ( s ) is identical Desired hybridoma cell lines may be identified through with or homologous to corresponding sequences in antibod binding specificity analysis of secreted antibodies for target ies derived from another species or belonging to another peptides and clones of such cells may be subcloned through antibody class or subclass , as well as fragments of such limiting dilution procedures and grown by standard meth antibodies . 40 ods . Antibodies produced by subcloned hybridoma cells As used herein , the term “ humanized antibody ” refers to may be isolated and purified from culture medium by a chimeric antibody comprising a minimal portion from one standard immunoglobulin purification procedures or more non - human ( e . g . , murine ) antibody source with the Recombinant Antibodies remainder derived from one or more human immunoglobu - Recombinant antibodies of the present invention may be lin sources. For the most part , humanized antibodies are 45 generated according to any of the methods disclosed in U . S . human immunoglobulins (recipient antibody ) in which resi - Provisional Patent Applications 61 /722 ,919 , filed Nov . 6 , dues from the hypervariable region from an antibody of the 2012 and 61/ 722 , 969 , filed Nov . 6 , 2012 , the contents of recipient are replaced by residues from the hypervariable each of which are herein incorporated by reference in their region from an antibody of a non - human species (donor entireties. In some embodiments , recombinant antibodies antibody ) such as mouse , rat, rabbit or nonhuman primate 50 may be produced using hybridoma cells produced according having the desired specificity , affinity , and /or capacity to methods described herein . Heavy and light chain variable As used herein , the term “ hypervariable region ” refers to region cDNA sequences of antibodies may be determined regions within the antigen binding domain of an antibody using standard biochemical techniques . Total RNA may be comprising amino acid residues responsible for antigen extracted from antibody -producing hybridoma cells and binding . The amino acids present within the hypervariable 55 converted to cDNA by reverse transcriptase (RT ) poly regions determine the structure of the complementarity m erase chain reaction ( PCR ) . PCR amplification may be determining region (CDR ). As used herein , the term “ CDR ” carried out on resulting cDNA to amplify variable region refers to regions of antibodies comprising a structure that is genes . Such amplification may comprise the use of primers complimentary to its target antigen or epitope . specific for amplification of heavy and light chain In some embodiments, compounds and /or compositions 60 sequences . Resulting PCR products may then be subcloned of the present invention may be antibody mimetics . As used into plasmids for sequence analysis. Once sequenced , anti herein , the term “ antibody mimetic ” refers to any molecule body coding sequences may be placed into expression which mimics the function or effect of an antibody and vectors. For humanization , coding sequences for human which binds specifically and with high affinity to their heavy and light chain constant domains may be used to molecular targets . In some embodiments , antibody mimetics 65 substitute for homologous murine sequences . The resulting may be monobodies , designed to incorporate the fibronectin constructs may then be transfected into mammalian cells for type III domain (Fn3 ) as a protein scaffold ( U . S . Pat. No. large scale translation . US 9 ,758 ,577 B2 103 104 Development of Cytotoxic Antibodies enrichment, target antigens are bound to a substrate ( e . g . by In some embodiments , antibodies of the present invention passive adsorption ) and contacted with one or more solu may be capable of inducing antibody -dependent cell -medi tions comprising phage particles . Phage particles with affin ated cytotoxicity (ADCC ) , complement- dependent cytotox - ity for such target antigens are precipitated out of solution . icity (CDC ) and / or antibody -dependent cell phagocytosis 5 In some cases , phage enrichment comprises solution - phase ( ADCP. ) ADCC is an immune mechanism whereby cells are phage enrichment where target antigens are present in a lysed as a result of immune cell attack . Such immune cells solution that is combined with phage solutions . According to may include CD56 + cells, CD3 -natural killer ( NK ) cells , such methods, target antigens may comprise detectable monocytes and neutrophils ( Strohl, W . R . Therapeutic Anti- labels ( e . g . biotin labels ) to facilitate retrieval from solution body Engineering . Woodhead Publishing , Philadelphia Pa . 10 and recovery of bound phage . 2012 . Ch . 8 , p 186 , the contents of which are herein After selection , cDNA encoding CDRs of precipitated incorporated by reference in their entirety .) library members may be sequenced from the bound phage . In some cases, antibodies of the present invention may be Such sequences may be directly incorporated into antibody engineered to comprise a given isotype depending on sequences for recombinant antibody production , or mutated whether or not ADCC or ADCP is desired upon antibody 15 and utilized for further optimization through in vitro affinity binding . Such antibodies , for example , may be engineered maturation . according to any of the methods disclosed by Alderson , K . In some cases phage display screening may be used to L . et al ., J Biomed Biotechnol . 2011 . 2011 :379123 . ) In the generate broadly diverse panels of antibodies. Such diversity case ofmouse antibodies , different isotypes of antibodies are may be measured by diversity of antibody sequences and / or more effective at promoting ADCC . IgG2a , for example , is 20 diversity of epitopes targeted . more effective at inducing ADCC than is IgG2b . Some Affinity Maturation Techniques antibodies of the present invention , comprising mouse Affinity maturation techniques of the present invention IgG2b antibodies may be reengineered to comprise IgG2a may comprise any of those disclosed in U . S . Provisional antibodies . Such reengineered antibodies may be more Patent Applications 61 /722 , 919 , filed Nov. 6 , 2012 and effective at inducing ADCC upon binding cell- associated 25 61/ 722 , 969 , filed Nov . 6 , 2012 , the contents of each of which antigens. are herein incorporated by reference in their entireties. After In some embodiments , genes encoding variable regions of antibody fragments capable of binding target antigens are antibodies developed according to methods of the present identified (e . g. through the use of phage display libraries as invention may be cloned into mammalian expression vectors described above ) high affinity mutants may be derived from encoding human Fc regions. Such Fc regionsmay comprise 30 these through the process of affinity maturation . Affinity Fc regions from human IgGlk . IgGlk Fc regions may maturation technology is used to identify sequences encod comprise amino acid mutations known to enhance Fc ing CDRs that have the highest affinity for target antigens. receptor binding and antibody -dependent cell -mediated Using such technologies , select CDR sequences ( e . g . ones cytotoxicity ADCC . that have been isolated or produced according to processes In some cases , antibodies may be engineered to reduce 35 described herein ) may be mutated randomly as a whole or at ADCC . Antibodies that do not activate ADCC or that are specific residues to create millions to billions of variants . associated with reduced levels of ADCC may be desireable Such variants may be subjected to repeated rounds of affinity for antibody embodiments of the present invention , in some screening ( e . g . display library screening ) for their ability to cases due to no or limited immune -mediated clearance , bind target antigens . Such repeated rounds of selection , allowing longer half- lives in circulation . 40 mutation and expression may be carried out to identify Antibody Fragment Display Library Screening Techniques antibody fragment sequences with the highest affinity for In some embodiments , antibodies of the present invention target antigens . Such sequences may be directly incorpo may be produced and / or optimized using high throughput rated into antibody sequences for recombinant antibody methods of discovery. Such methods may include any of the production . display techniques ( e . g . display library screening tech - 45 Antibody Characterization niques ) disclosed in U . S . Provisional Patent Applications Compounds and /or compositions of the present invention 61/ 722 , 919 , filed Nov . 6 , 2012 and 61 / 722 , 969, filed Nov . 6 , comprising antibodies may act to decrease local concentra 2012 , the contents of each of which are herein incorporated tion of one or more GPC through removal by phagocytosis , by reference in their entireties . In some embodiments , pinocytosis , or inhibiting assembly in the extracellular synthetic antibodies may be designed , selected or optimized 50 matrix and /or cellular matrix . Introduction of compounds by screening target antigens using display technologies ( e . g . and /or compositions of the present invention may lead to the phage display technologies. ) Phage display libraries may removal of 5 % to 100 % of the growth factor present in a comprise millions to billions of phage particles , each given area . For example , the percent of growth factor expressing unique antibody fragments on their viral coats . In removal may be from about 5 % to about 10 % , from about some cases, cDNA encoding each fragmentmay contain the 55 5 % to about 15 % , from about 5 % to about 20 % , from about same sequence with the exception of unique sequences 5 % to about 25 % , from about 10 % to about 30 % , from about encoding variable loops of the complementarity determining 10 % to about 40 % , from about 10 % to about 50 % , from regions (CDRs ) . Vy chains of CDRsmay be expressed as a about 10 % to about 60 % , from about 20 % to about 70 % , fusion protein , linked to viral coat proteins ( e . g . the N - ter - from about 20 % to about 80 % , from about 40 % to about minus of the viral pIII coat protein . ) V chains may be 60 90 % or from about 40 % to about 100 % . expressed separately for assembly with Vy chains in the Measures of release, inhibition or removal of one or more periplasm prior to complex incorporation into viral coats . growth factors may be made relative to a standard or to the For selection , target antigens may be incubated , in vitro , natural release or activity of growth factor under normal with phage display library particles for precipitation of physiologic conditions , in vitro or in vivo . Measurements positive binding partners . This process is referred to herein 65 may also be made relative to the presence or absence of as “ phage enrichment. " In some cases , phage enrichment antibodies . Such methods ofmeasuring growth factor levels , comprises solid -phase phage enrichment. According to such release , inhibition or removal include standard measurement US 9 , 758 ,577 B2 105 106 in tissue and /or fluids ( e . g . serum or blood ) such as Western factor not typically associated with a particular GPC tar blot, enzyme- linked immunosorbent assay (ELISA ) , activity geted by the antibody , would be a neomorphic antibody and assays , reporter assays , luciferase assays , polymerase chain the release would be a neomorphic change . reaction (PCR ) arrays, gene arrays , Real Time reverse In some embodiments , compounds and /or compositions transcriptase (RT ) PCR and the like . 5 of the present invention may act to alter and / or control Antibodies of the present invention may bind or interact proteolytic events . In some embodiments , such proteolytic with any number of epitopes on or along GPCs or their events may be intracellular or extracellular . In some embodi associated structures to either enhance or inhibit growth ments , such proteolytic events may include the alteration of factor signaling. Such epitopes may include any and all furin cleavage and / or other proteolytic processing events . In possible sites for altering , enhancing or inhibiting GPC 10 some embodiments , such proteolytic events may comprise function . In some embodiments, such epitopes include , but proteolytic processing of growth factor signaling molecules are not limited to epitopes on or within growth factors , or downstream cascades initiated by growth factor signaling regulatory elements , GPCs, GPC modulatory factors , molecules . growth factor receiving cells or receptors , LAPs or LAP - like In some embodiments , compounds and /or compositions domains , fastener regions , furin cleavage sites , arm regions , 15 of the present invention may induce or inhibit dimerization fingers regions, LTBP binding domains , fibrillin binding or multimerization of growth factors (ligands ) or their domains, glycoprotein A repetitions predominant (GARP ) receptors. In some embodiments , such actions may be binding domains , latency lassos , alpha 1 regions , RGD through stabilization of monomeric , dimeric or multimeric sequences, bowtie regions, extracellular matrix and / or cel- forms or through the disruption of dimeric or multimeric lular matrix components and /or epitopes formed by com - 20 complexes . bining regions or portions of any of the foregoing . In some embodiments , compounds and /or compositions Compounds and / or compositions of the present invention of the present invention may act on homo and /or heterodi exert their effects via binding ( reversibly or irreversibly ) to mers of the monomeric units comprising either receptor one or more epitopes and / or regions of antibody recognition . groups or GPCs or other signaling molecule pairs . While not wishing to be bound by theory , such binding sites 25 Antibodies of the present invention may be internalized for antibodies , are most often formed by proteins , protein into cells prior to binding target antigens . Upon internaliza domains or regions . Binding sites may ; however, include tion , such antibodies may act to increase or decrease one or biomolecules such as sugars , lipids, nucleic acid molecules more signaling events , release or stabilize one or more or any other form of binding epitope. GPCs, block or facilitate growth factor release and /or alter In some embodiments , antagonist antibodies of the pres - 30 one or more cell niche . ent invention may bind to TGF - B prodomains, stabilizing In some embodiments , compounds and /or compositions and preventing integrin -mediated release , for example , by of the present invention may also alter the residence time of blocking the RGD site or by stabilizing the structure . Such one or more growth factor in one or more GPC and / or alter antibodies would be useful in the treatment of Camurati - the residence time of one or more GPC in the extracellular Engelmann disease , in which mutations in the prodomain 35 matrix and / or cellular matrix . Such alterations may result in cause excessive TGF- B activation . Such antibodies would irreversible localization and / or transient localization . also be useful in Marfan ' s syndrome , in which mutations in Antibodies of the present invention may be designed , fibrillins or LTBPs alter TGF - B and BMP activation . manufactured and /or selected using any methods known to In some embodiments, antibodies of the present invention one of skill in the art. In some embodiments, antibodies selectively inhibit the release of TGF - B from GPCs associ- 40 and / or antibody producing cells of the present invention are ated with LTBPs but not those associated with GARP . Such produced according to any of the methods listed in U . S . antibodies function as anti - fibrotic therapeutics but exhibit Provisional Patent Applications 61 /722 , 919 , filed Nov. 6 , minimal inflammatory effects . In some embodiments , GPC 2012 and 61/ 722 , 969 , filed Nov. 6 , 2012 , the contents of LTBP complex - binding antibodies do not bind GPC -GARP each of which are herein incorporated by reference in their complexes . In some embodiments , such antibodies , may not 45 entireties . be specific to a particular LTBP or GPC , but may bind to Antibody Generation in Knockout Mice GPCs close to or overlapping with GARP binding sites, such In some embodiments , antibodies of the current invention that binding is impeded by GARP, but not by LTBPs. In may be generated in knockout mice that lack a gene encod some embodiments , antibodies are provided that selectively ing one or more desired antigens . Such mice would not be bind one or more combinatorial epitopes between GARP and 50 tolerized to such antigens and therefore may be able to proTGF -B . In some embodiments of the present invention , generate antibodies against them that could cross react with compounds and / or compositions are provided which induce human and mouse forms of the antigen . For the production release of TGF- B from GARP - proTGF- B complexes . Such of monoclonal antibodies , hostmice are immunized with the antibodies may be selected for their ability to bind to GARP target peptide to elicit lymphocytes that specifically bind prodomain binary complexes but not GARP -proTGF - B ter - 55 that peptide . Lymphocytes are collected and fused with an nary complexes , GARPs alone, or prodomains alone . immortalized cell line . The resulting hybridoma cells are Alternatively or additionally , antibodies of the present cultured in a suitable culture medium with a selection agent invention may function as ligand mimetics which would to support the growth of only the fused cells . induce internalization of GPCs . Such antibodies may act as In some embodiments , knocking out one or more growth nontraditional payload carriers , acting to deliver and / or ferry 60 factor gene may be lethal and / or produce a fetus or neonate bound or conjugated drug payloads to specific GPC and / or that is non - viable . In some embodiments , neonatal animals GPC - related sites . may only survive for a matter of weeks ( e . g . 1 , 2 , 3 , 4 or 5 Changes elicited by antibodies of the present invention weeks ). In such embodiments , immunizations may be car may result in neomorphic changes in the cell . As used ried out in neonatal animals shortly after birth . Oida et al herein , the term “ neomorphic change ” refers to a change or 65 (Oida , T . et al . , TGF - B induces surface LAP expression on alteration that is new or different. For example , an antibody Murine CD4 T cells independent of FoxP3 induction . PLOS that elicits the release or stabilization of one or more growth One . 2010 . 5 ( 11 ) :e15523 ) demonstrate immunization of US 9 , 758 ,577 B2 107 108 neonatal TGF- B knockoutmice through the use of galectin - 1 dissociation of free growth factors from GPCs. Such disso injections to prolong survival (typically 3 - 4 weeks after birth ciation may be induced upon antibody binding to a GPC or in these mice ) . Mice were immunized with cells expressing dissociation may be promoted by preventing the reassocia murine TGF - B every other day for 10 days beginning on the tion of free growth factor with LAP or LAP - like protein . In 8th day after birth and spleen cells were harvested on day 22 5 some cases , anti- TGF - B LAP antibodies are provided . Anti after birth . Harvested spleen cells were fused with myeloma TGF - B LAP antibodies may comprise TGF - B - activating cells and of the resulting hybridoma cells , many were found antibodies . Such antibodies may increase TGF - B activity , in to successfully produce anti- LAP antibodies . In some some cases through by releasing TGF - B free growth factor embodiments of the present invention , these methods may from latent GPCs and /or preventing the reassociation of free be used to generate antibodies. In some embodiments , such 10 TGF - ß growth factor with LAP. In some cases , anti - TGF - B methods may comprise the use of human antigens . In some LAP antibodies may increase TGF - B activity more favorably embodiments , cells used for immunization may express when proTGF -B is associated with LTBP . In some cases , TGF - B and GARP. In such embodiments , GARPs may be anti- TGF - B LAP antibodies may increase TGF - B activity expressed with native transmembrane domains to allow for more favorably when proTGF - B is associated with GARP. In GARP - TGF - B complexes to remain tethered to the cell 15 some cases, anti - TGF - B LAP antibodies may function syn surface of the transfected cells used from immunization . ergistically with other TGF - B activators ( e . g . avßo and / or Some antigens may comprise proTGF - B1 tethered to LTBP a , Bs ) to increase TGF - B activity . ( e . g . LTBP1S .) In some cases , recombinant proteins related Variations to other TGF - B family members may be used as antigens . Compounds and / or compositions of the present invention Methods of the present invention may also comprise one 20 may exist as a whole polypeptide, a plurality of polypeptides or more steps of the immunization methods described by or fragments of polypeptides , which independently may be Oida et al combined with one or more additional and / or encoded by one or more nucleic acids, a plurality of nucleic modified steps. Modified steps may include, but are not acids , fragments of nucleic acids or variants of any of the limited to the use of alternate cell types for fusions , the aforementioned . As used herein , the term “ polypeptide ” pooling of varying number of spleen cells when performing 25 refers to a polymer of amino acid residues ( natural or fusions , altering the injection regimen , altering the date of unnatural) linked together most often by peptide bonds . The spleen cell harvest , altering immunogen and / or altering term , as used herein , refers to proteins, polypeptides, and immunogen dose . Additional stepsmay include the harvest - peptides of any size , structure , or function . In some instances ing of other tissues ( e . g . lymph nodes ) from immunized the polypeptide encoded is smaller than about 50 amino mice . 30 acids and the polypeptide is then termed a peptide . If the Activating and Inhibiting Antibodies polypeptide is a peptide, it will be at least about 2 , 3 , 4 , or Antibodies of the present invention may comprise acti at least 5 amino acid residues long. Thus , polypeptides vating or inhibiting antibodies . As used herein , the term include gene products , naturally occurring polypeptides , " activating antibody ” refers to an antibody that promotes synthetic polypeptides , homologs, orthologs, paralogs, frag growth factor activity . Activating antibodies include anti- 35 ments and other equivalents , variants , and analogs of the bodies targeting any epitope that promotes growth factor foregoing . A polypeptide may be a single molecule or may activity . Such epitopes may lie on prodomains ( e . g . LAPS be a multi- molecular complex such as a dimer , trimer or and LAP - like domains ) growth factors or other epitopes that tetramer . They may also comprise single chain or multichain when bound by antibody, lead to growth factor activity . polypeptides and may be associated or linked . The term Activating antibodies of the present invention may include , 40 polypeptide may also apply to amino acid polymers in which but are not limited to TGF - ß - activating antibodies, GDF - 8 one or more amino acid residues are an artificial chemical activating antibodies , GDF - 11 -activating antibodies and analogue of a corresponding naturally occurring amino acid . BMP- activating antibodies . As used herein , the term “ polypeptide variant” refers to As used herein , the term “ inhibiting antibody ” refers to an molecules which differ in their amino acid sequence from a antibody that reduces growth factor activity . Inhibiting anti- 45 native or reference sequence . The amino acid sequence bodies include antibodies targeting any epitope that reduces variants may possess substitutions , deletions, and /or inser growth factor activity when associated with such antibodies. tions at certain positions within the amino acid sequence , as Such epitopes may lie on prodomains ( e . g . LAPs and compared to a native or reference sequence . Ordinarily , LAP - like domains ) growth factors or other epitopes that lead variants will possess at least about 50 % identity (homology ) to reduced growth factor activity when bound by antibody. 50 to a native or reference sequence , and preferably , they will Inhibiting antibodies of the present invention may include , be at least about 80 % , more preferably at least about 90 % but are not limited to TGF - b - inhibiting antibodies , GDF - 8 - identical ( homologous) to a native or reference sequence . inhibiting antibodies , GDF - 11- inhibiting antibodies and In some embodiments " variantmimics ” are provided . As BMP -inhibiting antibodies. used herein , the term “ variant mimic ” refers to a variant Embodiments of the present invention include methods of 55 which contains one ormore amino acids which would mimic using activating and /or inhibiting antibodies in solution , in an activated sequence . For example , glutamate may serve as cell culture and /or in subjects to modify growth factor a mimic for phospho - threonine and /or phospho -serine . signaling . Alternatively , variant mimics may result in deactivation or in Anti -LAP and Anti -LAP -Like Domain Antibodies an inactivated product containing the mimic , e . g . , phenyl In some embodiments, compounds and /or compositions 60 alanine may act as an inactivating substitution for tyrosine ; of the present invention may comprise one or more antibody or alanine may act as an inactivating substitution for serine . targeting a prodomain , including LAP and /or LAP - like The amino acid sequences of the compounds and /or com domains . Such antibodies may reduce or elevate growth positions of the invention may comprise naturally occurring factor signaling depending on the specific LAP or LAP -like amino acids and as such may be considered to be proteins , domain that is bound and / or depending on the specific 65 peptides , polypeptides , or fragments thereof. Alternatively , epitope targeted by such antibodies . Anti -LAP and /or anti - the compounds and/ or compositions may comprise both LAP -like protein antibodies of the invention may promote naturally and non -naturally occurring amino acids. US 9 , 758 ,577 B2 109 110 As used herein , the term “ amino acid sequence variant” has been substituted , or they may be multiple , where two or refers to molecules with some differences in their amino acid more amino acids have been substituted in the same mol sequences as compared to a native or starting sequence . The ecule . amino acid sequence variants may possess substitutions, As used herein , the term " conservative amino acid sub deletions, and /or insertions at certain positions within the 5 stitution ” refers to the substitution of an amino acid that is amino acid sequence . As used herein , the terms “ native” or normally present in the sequence with a different amino acid “ starting ” when referring to sequences are relative terms of similar size , charge , or polarity . Examples of conservative substitutions include the substitution of a non - polar (hydro referring to an original molecule against which a comparison phobic ) residue such as isoleucine , valine and leucine for may be made . Native or starting sequences should not be another non -polar residue . Likewise , examples of conserva confused with wild type sequences. Native sequences or tive substitutions include the substitution of one polar (hy molecules may represent the wild - type (that sequence found drophilic ) residue for another such as between arginine and in nature ) but do not have to be identical to the wild - type lysine , between glutamine and asparagine , and between sequence . glycine and serine . Additionally , the substitution of a basic Ordinarily , variants will possess at least about 70 % 15 residue such as lysine , arginine or histidine for another, or homology to a native sequence, and preferably , they will be the substitution of one acidic residue such as aspartic acid or at least about 80 % , more preferably at least about 90 % glutamic acid for another acidic residue are additional homologous to a native sequence . examples of conservative substitutions . Examples of non As used herein , the term “ homology ” as it applies to conservative substitutions include the substitution of a non amino acid sequences is defined as the percentage of resi- 20 polar (hydrophobic ) amino acid residue such as isoleucine , dues in the candidate amino acid sequence that are identical valine , leucine , alanine , methionine for a polar (hydrophilic ) with the residues in the amino acid sequence of a second residue such as cysteine , glutamine, glutamic acid or lysine sequence after aligning the sequences and introducing gaps , and /or a polar residue for a non -polar residue . if necessary , to achieve the maximum percent homology . As used herein , the term “ insertional variants " when Methods and computer programs for the alignment are well 25 referring to proteins are those with one or more amino acids known in the art . It is understood that homology depends on inserted immediately adjacent to an amino acid at a particu a calculation ofpercent identity but may differ in value due lar position in a native or starting sequence . As used herein , to gaps and penalties introduced in the calculation . the term “ immediately adjacent” refers to an adjacent amino As used herein , the term “ homolog ” as it applies to amino acid that is connected to either the alpha- carboxy or alpha acid sequences is meant the corresponding sequence of other 30 amino functional group of a starting or reference amino acid . species having substantial identity to a second sequence of As used herein , the term “ deletional variants ” when a second species . referring to proteins, are those with one or more amino acids As used herein , the term “ analog ” is meant to include in the native or starting amino acid sequence removed . polypeptide variants which differ by one or more amino acid Ordinarily , deletional variants will have one or more amino alterations , e . g ., substitutions, additions or deletions of 35 acids deleted in a particular region of the molecule . amino acid residues that still maintain the properties of the As used herein , the term " derivatives , " as referred to parent polypeptide . herein includes variants of a native or starting protein As used herein , the term " derivative ” is used synony - comprising one or more modifications with organic pro mously with the term “ variant” and refers to a molecule that teinaceous or non - proteinaceous derivatizing agents , and has been modified or changed in any way relative to a 40 post - translational modifications . Covalent modifications are reference molecule or starting molecule . traditionally introduced by reacting targeted amino acid The present invention contemplates several types of com - residues of the protein with an organic derivatizing agent pounds and / or compositions which are amino acid based that is capable of reacting with selected side - chains or including variants and derivatives . These include substitu terminal residues , or by harnessing mechanisms of post tional, insertional, deletional and covalent variants and 45 translational modifications that function in selected recom derivatives . As such , included within the scope of this binant host cells . The resultant covalent derivatives are invention are compounds and / or compositions comprising useful in programs directed at identifying residues important substitutions , insertions , additions, deletions and / or covalent for biological activity , for immunoassays , or for the prepa modifications . For example , sequence tags or amino acids, ration of anti -protein antibodies for immunoaffinity purifi such as one or more lysines , can be added to peptide 50 cation of the recombinant glycoprotein . Such modifications sequences of the invention ( e . g ., at the N - terminal or C - ter- are within the ordinary skill in the art and are performed minal ends ) . Sequence tags can be used for peptide purifi - without undue experimentation . cation or localization . Lysines can be used to increase Certain post - translational modifications are the result of peptide solubility or to allow for biotinylation . Alternatively , the action of recombinant host cells on the expressed poly amino acid residues located at the carboxy and amino 55 peptide . Glutaminyl and asparaginyl residues are frequently terminal regions of the amino acid sequence of a peptide or post- translationally deamidated to the corresponding gluta protein may optionally be deleted providing for truncated myl and aspartyl residues . Alternatively , these residues are sequences . Certain amino acids ( e . g . , C - terminal or N - ter- deamidated under mildly acidic conditions. Either form of minal residues ) may alternatively be deleted depending on these residues may be present in the proteins used in the use of the sequence , as for example , expression of the 60 accordance with the present invention . sequence as part of a larger sequence which is soluble , or Other post- translational modifications include hydroxy linked to a solid support . lation of proline and lysine, phosphorylation of hydroxyl “ Substitutional variants" when referring to proteins are groups of seryl or threonyl residues, methylation of the those that have at least one amino acid residue in a native or alpha -amino groups of lysine , arginine , and histidine side starting sequence removed and a different amino acid 65 chains ( T . E . Creighton , Proteins : Structure and Molecular inserted in its place at the same position . The substitutions Properties , W . H . Freeman & Co . , San Francisco , pp . 79 - 86 may be single , where only one amino acid in the molecule (1983 ) ) . US 9 , 758 ,577 B2 111 112 Covalent derivatives specifically include fusion mol As used herein , the term " half- domain ,” when referring to ecules in which proteins of the invention are covalently proteins, refers to a portion of an identified domain having bonded to a non -proteinaceous polymer. The non - proteina - at least half the number of amino acid resides as the domain ceous polymer ordinarily is a hydrophilic synthetic polymer, from which it is derived . It is understood that domains may i . e . a polymer not otherwise found in nature . However , 5 not always contain an even number of amino acid residues . polymers which exist in nature and are produced by recom - Therefore , in those cases where a domain contains or is binant or in vitro methods are useful, as are polymers which identified to comprise an odd number of amino acids, a are isolated from nature . Hydrophilic polyvinyl polymers half - domain of the odd - numbered domain will comprise the fall within the scope of this invention , e . g . polyvinylalcohol whole number portion or next whole number portion of the and polyvinylpyrrolidone . Particularly useful are polyvinyl- 10 domain number of amino acids of the domain / 2 + / - 0 . 5 alkylene ethers such a polyethylene glycol, polypropylene amino acids ) . For example , a domain identified as a 7 amino glycol . The proteins may be linked to various non -proteina acid domain could produce half -domains of 3 amino acids or ceous polymers, such as polyethylene glycol, polypropylene 4 amino acids ( 7 / 2 = 3 . 5 + / - 0 . 5 being 3 or 4 ) . It is also glycol or polyoxyalkylenes, in the manner set forth in U . S . understood that sub -domains may be identified within Pat. Nos. 4 ,640 , 835 ; 4 ,496 ,689 ; 4, 301, 144 ; 4 ,670 ,417 ; 15 domains or half -domains , these subdomains possessing less 4 ,791 , 192 or 4 ,179 , 337 . than all of the structural or functional properties identified in As used herein , the term " features ” when referring to the domains or half domains from which they were derived . proteins are defined as distinct amino acid sequence -based It is also understood that the amino acids that comprise any components of a molecule . Features of the proteins of the of the domain types herein need not be contiguous along the present invention include surface manifestations, local con - 20 backbone of the polypeptide ( i . e . , nonadjacent amino acids formational shape, folds , loops, half -loops , domains , half - may fold structurally to produce a domain , half - domain or domains , sites, termini or any combination thereof. subdomain ) . As used herein , the term " surface manifestation ” when As used herein , the terms “ site ,” as it pertains to amino referring to proteins refers to a polypeptide based compo - acid based embodiments is used synonymously with “ amino nent of a protein appearing on an outermost surface . 25 acid residue " and " amino acid side chain " . A site represents As used herein , the term “ local conformational shape” a position within a peptide or polypeptide that may be when referring to proteins refers to a polypeptide based modified , manipulated , altered , derivatized or varied within structural manifestation of a protein which is located within the polypeptide based molecules of the present invention . a definable space of the protein . As used herein , the terms " termini” or “ terminus ,” when As used herein , the term " fold ” , when referring to pro - 30 referring to proteins refers to an extremity of a peptide or teins , refers to the resultant conformation of an amino acid polypeptide. Such extremity is not limited only to the first or sequence upon energy minimization . A fold may occur at the final site of the peptide or polypeptide but may include secondary or tertiary level of the folding process . Examples additional amino acids in the terminal regions . The poly of secondary level folds include beta sheets and alpha peptide based molecules of the present invention may be helices . Examples of tertiary folds include domains and 35 characterized as having both an N - terminus ( terminated by regions formed due to aggregation or separation of energetic an amino acid with a free amino group (NH2 ) ) and a forces . Regions formed in this way include hydrophobic and C - terminus ( terminated by an amino acid with a free car hydrophilic pockets , and the like . boxyl group (COOH ) ) . Proteins of the invention are in some As used herein , the term “ turn ” as it relates to protein cases made up of multiple polypeptide chains brought conformation , refers to a bend which alters the direction of 40 together by disulfide bonds or by non -covalent forces (mul the backbone of a peptide or polypeptide and may involve timers , oligomers ) . These sorts of proteins will have mul one, two , three or moreraamino amino acid residuesresidues . tiple N - and C - termini. Alternatively, the termini of the As used herein , the term “ loop , ” when referring to pro - polypeptides may be modified such that they begin or end , teins , refers to a structural feature of a peptide or polypeptide as the case may be , with a non -polypeptide based moiety which reverses the direction of the backbone of a peptide or 45 such as an organic conjugate . polypeptide and comprises four or more amino acid resi - Once any of the features have been identified or defined dues. Oliva et al. have identified at least 5 classes of protein as a component of a molecule of the invention , any of loops (Oliva , B . et al. , An automated classification of the several manipulations and /or modifications of these features structure of protein loops . J Mol Biol. 1997 . 266 ( 4 ): 814 - 30 .) may be performed by moving, swapping , inverting , deleting , As used herein , the term " half - loop , " when referring to 50 randomizing or duplicating . Furthermore , it is understood proteins , refers to a portion of an identified loop having at that manipulation of features may result in the same out least half the number of amino acid resides as the loop from come as a modification to the molecules of the invention . which it is derived . It is understood that loops may not For example , a manipulation which involved deleting a always contain an even number of amino acid residues. domain would result in the alteration of the length of a Therefore , in those cases where a loop contains or is 55 molecule just as modification of a nucleic acid to encode less identified to comprise an odd number of amino acids , a than a full length molecule would . half -loop of the odd -numbered loop will comprise the whole Modifications and manipulations can be accomplished by number portion or next whole number portion of the loop methods known in the art such as site directed mutagenesis . (number of amino acids of the loop / 2 + / - 0 . 5 amino acids) . The resulting modified molecules may then be tested for For example , a loop identified as a 7 amino acid loop could 60 activity using in vitro or in vivo assays such as those produce half - loops of 3 amino acids or 4 amino acids described herein or any other suitable screening assay ( 7 / 2 = 3 . 5 + / - 0 . 5 being 3 or 4 ) . known in the art. As used herein , the term " domain ,” when referring to In some embodiments , compounds and/ or compositions proteins , refers to a motif of a polypeptide having one or of the present invention may comprise one or more atoms more identifiable structural or functional characteristics or 65 that are isotopes. As used herein , the term “ isotope ” refers to properties ( e . g . , binding capacity , serving as a site for a chemical element that has one or more additional neutrons . protein - protein interactions .) In some embodiments , compounds of the present invention US 9 ,758 ,577 B2 113 114 may be deuterated . As used herein , the term " deuterate ” In some embodiments , targeting groups may be proteins, refers to the process of replacing one or more hydrogen e . g ., glycoproteins, or peptides , e . g ., molecules having a atoms in a substance with deuterium isotopes . Deuterium specific affinity for a co -ligand , or antibodies e . g . , an anti isotopes are isotopes of hydrogen . The nucleus of hydrogen body , that binds to a specified cell type such as a cancer cell, contains one proton while deuterium nuclei contain both a 5 endothelial cell , or bone cell. Targeting groups may also proton and a neutron . The compounds and /or compositions comprise hormones and /or hormone receptors. of the present invention may be deuterated in order to In some embodiments , targeting groups may be any change one or more physical property , such as stability , or to ligand capable of targeting specific receptors . Examples allow compounds and /or compositions to be used in diag - include, without limitation , folate , GalNAc , galactose, man nostic and / or experimental applications. nose , mannose -6 - phosphate , apatamers , integrin receptor Conjugates and Combinations ligands , chemokine receptor ligands, transferrin , biotin , It is contemplated by the present invention that the serotonin receptor ligands, PSMA, endothelin , GCPII, compounds and /or compositions of the present invention somatostatin , LDL , and HDL ligands . In some embodi may be complexed , conjugated or combined with one or 15 ments , targeting groups are aptamers . Such aptamers may be more homologous or heterologous molecules. As used unmodified or comprise any combination of modifications herein , the term “ homologous molecule ” refers to a mol disclosed herein . ecule which is similar in at least one of structure or function In still other embodiments , compounds and / or composi relative to a starting molecule while a " heterologous mol- tions of the present invention may be covalently conjugated ecule ” is one that differs in at least one of structure or 20 to cell penetrating polypeptides. In some embodiments , function relative to a starting molecule . Structural homologs cell -penetrating peptides may also include signal sequences. are therefore molecules which may be substantially struc- In some embodiments , conjugates of the invention may be turally similar. In some embodiments , such homologs may designed to have increased stability , increased cell transfec be identical. Functional homologs are molecules which may tion and /or altered biodistribution ( e . g . , targeted to specific be substantially functionally similar. In some embodiments , 25 tissues or cell types . ) such homologs may be identical . In some embodiments , conjugating moieties may be Compounds and /or compositions of the present invention added to compounds and / or compositions of the present may comprise conjugates . Such conjugates of the invention may include naturally occurring substances or ligands, such invention such that they allow the attachment of detectable as proteins (e . g. , human serum albumin (HSA ), low -density 30 labels to targets for clearance . Such detectable labels lipoprotein (LDL ), high - density lipoprotein (HDL ) , or include , but are not limited to biotin labels , ubiquitins, globulin ); carbohydrates (e .g ., a dextran , pullulan , chitin , fluorescent molecules , human influenza hemaglutinin (HA ) , chitosan , inulin , cyclodextrin or hyaluronic acid ) ; or lipids . c -myc , histidine (His ), flag , glutathione S - transferase (GST ) , Conjugates may also be recombinant or synthetic molecules, V5 (a paramyxovirus of simian virus 5 epitope ), biotin , such as synthetic polymers , e. g. , synthetic polyamino acidsde , 35 aavidin , streptavidin , horse radish peroxidase (HRP ) and an oligonucleotide ( e . g . an aptamer ). Examples of digoxigenin . polyamino acids may include polylysine (PLL ), poly L - as In some embodiments, compounds of the invention may partic acid , poly L - glutamic acid , styrene -maleic acid anhy - be conjugated with an antibody Fc domain to create an Fc dride copolymer , poly ( L - lactide -co - glycolied ) copolymer, fusion protein . The formation of an Fc fusion protein with divinyl ether- maleic anhydride copolymer , N - ( 2 -hydroxy - 40 any of the compounds described herein may be carried out propyl) methacrylamide copolymer (HIVIPA ) , polyethylene according to any method known in the art , including as glycol ( PEG ), polyvinyl alcohol (PVA ) , polyurethane , poly described in U . S. Pat. Nos . 5 ,116 , 964 , 5, 541 , 087 and 8, 637 , ( 2 - ethylacryllic acid ) , N - isopropylacrylamide polymers , or 637 , the contents of each of which are herein incorporated polyphosphazine . Example of polyamines include : polyeth - by reference in their entirety . Fc fusion proteins of the ylenimine , polylysine (PLL ) , spermine, spermidine, 45 invention may comprise a compound of the invention linked polyamine , pseudopeptide- polyamine , peptidomimetic to the hinge region of an IgG Fc via cysteine residues in the polyamine , dendrimer polyamine , arginine , amidine , Fc hinge region . Resulting Fc fusion proteins may comprise protamine, cationic lipid , cationic porphyrin , quaternary salt an antibody - like structure , but without Chi domains or light of a polyamine , or an alpha helical peptide . chains . In some cases , Fc fusion proteins may comprise In some embodiments , conjugates may also include tar - 50 pharmacokinetic profiles comparable to native antibodies . In geting groups . As used herein , the term " targeting group ” some cases, Fc fusion proteins of the invention may com refers to a functional group or moiety attached to an agent prise extended half- life in circulation and /or altered biologi that facilitates localization of the agent to a desired region , cal activity . tissue , cell and /or protein . Such targeting groups may In some embodiments , compounds and /or compositions include , but are not limited to cell or tissue targeting agents 55 of the present invention may be combined with one another or groups ( e . g . lectins, glycoproteins, lipids , proteins , an or other molecules in the treatment of diseases and /or antibody that binds to a specified cell type such as a kidney conditions. cell or other cell type ) . In some embodiments , targeting Nucleic Acids groups may comprise thyrotropins, melanotropins, lectins , In some embodiments , compounds and /or compositions glycoproteins , surfactant protein A , mucin carbohydrates, 60 of the present invention may be encoded by nucleic acid multivalent lactose, multivalent galactose , N -acetyl - galac - molecules . Such nucleic acid molecules include , without tosamine , N - acetyl- gulucosamine , multivalent mannose , limitation , DNA molecules , RNA molecules , polynucle multivalent fucose , glycosylated polyaminoacids, multiva - otides , oligonucleotides, mRNA molecules, vectors , plas lent galactose, transferrin , bisphosphonate , polyglutamate , mids and the like . In some embodiments , the present inven polyaspartate , lipids , cholesterol, steroids, bile acids, folates , 65 tion may comprise cells programmed or generated to express vitamin B12 , biotin , an RGD peptide , an RGD peptide nucleic acid molecules encoding compounds and / or com mimetic or an aptamer . positions of the present invention . US 9 ,758 , 577 B2 115 116 Methods of Use 0122007 , U .S . Pat. No . 8 ,415 , 459 or International Pub . No. Methods of the present invention include methods of WO 2011 / 151432 , the contents of each of which are herein modifying growth factor activity in one or more biological incorporated by reference in their entirety . system . Such methods may include contacting one or more Efficacy of treatment or amelioration of disease can be biological system with a compound and / or composition of 5 assessed , for example by measuring disease progression , the invention . In some cases, these methods include modi- disease remission , symptom severity, reduction in pain , fying the level of free growth factor in a biological system quality of life, dose of a medication required to sustain a ( e . g . in a cell niche or subject. ) Compounds and /or compos treatment effect, level of a disease marker or any other tions according to such methods may include , but are not measurable parameter appropriate for a given disease being limited to biomolecules , including, but not limited to recom - 10 treated or targeted for prevention . It is well within the ability binant proteins , protein complexes and /or antibodies of one skilled in the art to monitor efficacy of treatment or described herein . prevention by measuring any one of such parameters , or any In some embodiments , methods of the present invention combination of parameters . In connection with the admin may be used to initiate or increase growth factor activity , istration of compositions of the present invention , “ effective termed " activating methods ” herein . Some such methods 15 against” for example a cancer, indicates that administration may comprise growth factor release from a GPC and /or in a clinically appropriate manner results in a beneficial inhibition of growth factor reassociation into a latent GPC . effect for at least a statistically significant fraction of In some cases , activating methods may comprise the use of patients , such as an improvement of symptoms, a cure , a an antibody, a recombinant protein and / or a protein com - reduction in disease load , reduction in tumor mass or cell plex . According to some activating methods , one or more 20 numbers , extension of life , improvement in quality of life , or activating antibody is provided . In such methods , one or other effect generally recognized as positive by medical more growth factormay be released or prevented from being doctors familiar with treating the particular type of cancer. drawn back into a GPC . In one , non - limiting example , an treatment or preventive effect is evident when there is anti -LAP antibody may be provided that enhances dissocia - a statistically significant improvement in one or more tion between a growth factor and a GPC and / or prevents 25 parameters of disease status, or by a failure to worsen or to reformation of a GPC . develop symptoms where they would otherwise be antici Embodiments of the present invention include methods of pated . As an example , a favorable change of at least 10 % in using anti -LAP and /or anti- LAP - like domain antibodies to a measurable parameter of disease , and preferably at least modify growth factor activity . In some cases , such methods 20 % , 30 % , 40 % , 50 % or more can be indicative of effective may include the use of anti - TGF - B -LAP antibodies as TGF - 30 treatment. Efficacy for a given composition or formulation B -activating antibodies. In some cases , methods of using of the present invention can also be judged using an experi and/ or testing such antibodies may include any of the mental animal model for the given disease as known in the methods taught in Tsang , M . et al. 1995 . Cytokine 7 ( 5 ) : 389 - art . When using an experimental animal model , efficacy of 97 , the contents of which are herein incorporated by refer treatment is evidenced when a statistically significant ence in their entirety . 35 change is observed . In some embodiments , methods of the present invention Therapeutics for Fibrosis may be used to reduce or eliminate growth factor activity , In some embodiments , compounds and / or compositions termed “ inhibiting methods ” herein . Some such methods of the present invention may be useful for altering fibrosis . may comprise growth factor retention in a GPC and /or In some embodiments , such compounds and /or composi promotion of reassociation of growth factor into a latent 40 tions are antagonists of TGF - B . TGF- B is recognized as the GPC . In some cases , inhibiting methods may comprise the central orchestrator of the fibrotic response . Antibodies ususe of an antibody targeting TGF -B decrease fibrosis in numerous preclinical Therapeutics models . Such antibodies and / or antibody -based compounds In some embodiments , compositions and methods of the include LY2382770 (Eli Lilly , Indianapolis , Ind . ) . Also invention may be used to treat a wide variety of diseases , 45 included are those described in U .S . Pat. No . 6 ,492 ,497 , U . S . disorders and /or conditions . In some cases, such diseases, Pat. No . 7, 151 , 169 and U .S . Pat. No. 7 ,723 , 486 and U . S . disorders and / or conditions may be TGF - B - related indica - publication US2011 /0008364 , the contents of each of which tions. As used herein , the term “ TGF - B -related indication ” are herein incorporated by reference in their entirety . refers to any disease , disorder and / or condition related to Fibrosis is a common sequela of many types of tissue expression , activity and / or metabolism of a TGF - B family 50 destructive diseases . When new space is created by the member protein or any disease , disorder and /or condition disruption of differentiated cells , progenitors or stem cells that may benefit from modulation of the activity and / or that normally occupy a niche in the tissue , the default levels of one or more TGF - B family member protein . pathway appears to be the proliferation of connective tissue TGF- B - related indications may include, but are not limited cells , e . g . fibroblasts , to fill in the empty space. This is to , fibrosis , anemia of the aging, cancer ( including, but not 55 accompanied by the production of extracellular matrix con limited to colon , renal, breast , malignant melanoma and stituents including collagens that result in scarring and glioblastoma ) facilitation of rapid hematopoiesis following permanent effacement of the tissue . chemotherapy , bone healing , endothelial proliferation syn A difficult aspect of fibrosis is its chronicity, which may dromes , asthma and allergy , gastrointestinal disorders, aortic require continued therapy until the underlying destruction of aneurysm , orphan indications ( such as Marfan ' s syndrome 60 parenchymal cells is terminated or the cells are replaced by and Camurati -Engelmann disease ) obesity , diabetes , arthri- stem cell pools , or by transplantation . Fibrosis is thought to tis , multiple sclerosis , muscular dystrophy , amyotrophic be much easier to arrest than to reverse. The TGF -beta lateral sclerosis (ALS ) Parkinson 's disease, osteoporosis , family is of central importance in regulating the growth of osteoarthritis , osteopenia , metabolic syndromes , nutritional fibroblastic cells and the production of extracellular matrix disorders , organ atrophy, chronic obstructive pulmonary 65 constituents including collagen . Integrins aß and aß disease (COPD ) and anorexia . Additional indications may (and possibly a b ) may participate in activation of TGF include any of those disclosed in US Pub . No . 2013 / betal and 3 . The integrin VLA -1 is a receptor for collagen US 9 , 758 ,577 B2 117 118 and is expressed on lymphocytes only late after their acti - compositions of the present invention may be used in vation and is strongly implicated in the development of combination with pirfenidone . fibrotic disease . In some cases, compounds and /or composition of the In some embodiments , compounds and /or compositions invention may be used in the treatment of lung fibrosis . Lung of the present invention are designed to block integrin außo , 5 fibrosis models may be used in the development and / or aß , and aß, activation of TGF - beta for inhibiting fibrosis . testing of compounds and /or compositions of the invention . In some embodiments , compounds and / or compositions of Lung fibrosis models may include the bleomycin induced the present invention are designed to target interaction sites lung injury models and / or chronic bleomycin induced lung between GPCs and LTBPs while leaving interaction sites injury models . Bleomycin induced lung injury models may between GPCs and GARP unaffected . Such compounds 10 be carried out as described by Schaefer et al, and also by and/ or compositions of the present invention may act as Horan et al. (Horan G . S . et al. , 2008 . Am J Respir Crit Care inhibitory antibodies, preventing growth factor signaling Med , 177 ( 1 ) : 56 -65 . Epub 2007 Oct . 4 , the contents of each and inhibiting fibrosis . In some embodiments , compounds of which are herein incorporated by reference in their and / or compositions of the present invention are designed to entirety . ) According to the Horan study, SV129 mice are target one or more of TGF -B1 , 2 and 3 or chimeric antigens 15 tracheally exposed to bleomycin which results in the devel thereof. opment of lung fibrosis . With this model, potential thera Fibrotic indications for which compounds and / or compo - peutics are administered through intraperitoneal injections sitions of the present invention may be used therapeutically while postmortem lung tissue or bronchoalveolar lavage include , but are not limited to lung indications ( e . g . Idio - collections can be assayed for levels of hydroxyproline as an pathic Pulmonary Fibrosis ( IPF ), Chronic Obstructive Pul- 20 indicator of fibrotic activity . Using the same technique ,mice monary Disorder (COPD ), Allergic Asthma, Acute Lung carrying a luciferase reporter gene , driven by the collagen injury, Eosinophilic esophagitis, Pulmonary arterial hyper - la2 gene promoter may be used in themodel so that fibrotic tension and Chemical gas -injury , ] kidney indications [e . g . activity may be determined by luciferase activity assay as a Diabetic glomerulosclerosis , Focal segmental glomerulocle - function of collagen gene induction . Additional bleomycin rosis ( FSGS ) , Chronic kidney disease , Fibrosis associated 25 induced lung models may be carried out according to those with kidney transplantation and chronic rejection , IgA neph described by Thrall et al ( Thrall , R . S . et al ., 1979 . Am J ropathy and Hemolytic uremic syndrome, ] liver fibrosis [ e . g . Pathol. 95 : 117 - 30 , the contents of which are herein incor Non -alcoholic steatohepatitis (NASH ), Chronic viral hepa porated by reference in their entirety .) Additional lung titis , Parasitemia , Inborn errors ofmetabolism , Toxin -medi modelsmodal may include the mouse asthma models . Airway ated fibrosis , such as alcohol fibrosis , Non -alcoholic steato - 30 remodeling ( lung fibrosis ) may be a serious problem in hepatitis -hepatocellular carcinoma (NASH -HCC ) , Primary subjects with chronic asthma. Asthma models may include biliary cirrhosis and Sclerosing cholangitis , ] cardiovascular any of those described by Nials et al (Nials , A . T . et al. , 2008 . fibrosis (e . g . cardiomyopathy, hypertrophic cardiomyopathy , Disease Models and Mechanisms. 1 : 213 - 20 , the contents of atherosclerosis and restenosis ) systemic sclerosis , skin fibro wich are herein incorporated by reference in their entirety . ) sis ( e . g . Skin fibrosis in systemic sclerosis , Diffuse cutane - 35 Models of chronic obstructive pulmonary disease ( COPD ) ous systemic sclerosis , Scleroderma , Pathological skin scar- may be used . Such models may include any of those ring , Keloid , Post surgical scarring , Scar revision surgery , described by Vlahos et al (Vlahos , R . et al. , 2014 . Clin Sci . Radiation - induced scarring and Chronic wounds) and can - 126 :253 -65 , the contents of which are herein incorporated cers or secondary fibrosis ( e . g . Myelofibrosis , Head and by reference in their entirety .) Models of cigarette smoking Neck Cancer, M7 acute Megakaryoblastic Leukemia and 40 emphysema may be used . Such models may be carried out Mucositis . ) Other diseases, disorders or conditions related to as described in Ma et al . 2005 . J Clin Invest . 115 : 3460 - 72 , fibrosis that may be treated using compounds and / or com - the contents of which are herein incorporated by reference in positions of the present invention , include , but are not their entirety . Models of chronic pulmonary fibrosis may be limited to Marfan ' s Syndrome, Stiff Skin Syndrome, Sclero - used . Such models in rodents may be carried out according derma, Rheumatoid arthritis , bone marrow fibrosis , Crohn ' s 45 to the intratracheal fluorescein isothiocyanate ( FITC ) instil disease , Ulcerative colitis, Systemic lupus erythematosus, lation model described in Roberts , S . N . et al. 1995 . J Pathol. Muscular Dystrophy , Dupuytren ' s contracture , Camurati - 176 ( 3 ) :309 - 18 , the contents of which are herein incorpo Engelmann Disease , Neural scarring , Proliferative vitreo - rated by reference in their entirety. Models of asbestos and retinopathy , corneal injury , complications after glaucoma silica induced lung injury may also be used . Such models drainage surgery and Multiple Sclerosis . 50 may be carried out as described in Coin , P. G . et al ., 1996 . Assays useful in determining the efficacy of the com - Am J Respir Crit Care Med . 154 (5 ) : 1511 - 9 , the contents of pounds and / or compositions of the present invention for the which are herein incorporated by reference in their entirety . alteration of fibrosis include , but are not limited to , histo - In some cases, models of lung irradiation may be used . Such logical assays for counting fibroblasts and basic immuno - models may be carried out as described in Pauluhn , J . et al . histochemical analyses known in the art. 55 2001. Toxicology . 161: 153 -63 , the contents of which are Animal models are also available for analysis of the herein incorporated by reference in their entirety . In some efficacy of compounds and /or compositions of the present cases, phorbol myristate acetate ( PMA ) - induced lung injury invention in altering fibrosis . Examples of animal fibrosis models may be used . Such models may be carried out as models useful for such analysis may include , for example , described in Taylor , R . G . et al. , 1985 . Lab Invest. 52 ( 1 ) : any of those taught by Schaefer , D . W . et al. , 2011. Eur 60 61 - 70 , the contents of which are herein incorporated by Respir Rev. 20 : 120 , 85 - 97 , the contents of which are herein reference in their entirety . incorporated by reference in their entirety . Such models may Renal fibrosis models may be utilized to develop and /or include , but are not limited to those described in Table 1 of test compounds and /or compositions of the present inven that publication , including lung models , renal models , liver tion . In some embodiments , a well established model of models , cardiovascular models and / or collagen - induced 65 renal fibrosis , unilateral ureteral obstruction (UUO ) model , models . Schaefer et al also teach the use of pirfenidone in the may be used . In this model , mice are subjected to proximal treatment of fibrosis . In some cases, compounds and /or ureteral ligation . After a period of hours to days , fibrosis is US 9 ,758 ,577 B2 119 120 examined in the regions blocked by ligation (Ma , L . J . et al ., and transgenic mouse models , including , but not limited to 2003 . American Journal of Pathology . 163 ( 4 ): 1261- 73 , the the model described in Vannucchi, A . M . et al. , 2002 . Blood . contents of which are herein incorporated by reference in 100 ( 4 ) :1123 -32 , the contents of each of which are herein their entirety . ) In one example , this method was utilized by incorporated by reference in their entirety . Further models Meng, X . M . et al. (Meng , X . M . et al. , Smad2 Protects 5 may include models of thrombopoietin - induced myelofibro against TGF- beta /Smad3 -Mediated Renal Fibrosis . J Am sis . Such models may be carried out as described in Cha Soc Nephrol. 2010 September ; 21 ( 9 ) : 1477 - 87 . Epub 2010 graoui, H . et al . , 2002. Blood . 100 ( 10 ) :3495 -503 , the con Jul. 1 ) to examine the role of SMAD - 2 in renal fibrosis . tents of which are herein incorporated by reference in their SMAD - 2 is an intracellular member of the TGF -beta cell entirety . signaling pathway . In some cases, cyclosporine A - induced 10 In some embodiments , compounds and / or compositions nephropathy models may be used . Such models may be of the invention may be used to treat diseases , disorders carried out as described in Ling, H . et al . , 2003 . J Am Soc and /or conditions related to muscular dystrophy (MD ) Nephrol. 14 :377 - 88 , the contents of which are herein incor including , but not limited to Duchenne MD and Becker MD . porated by reference in their entirety . In some cases , renal In some cases MD models may be used to develop and / or models of Alport Syndrome may be used . Transgenic mice 15 test such compounds and / or compositions . Such models may with collagen III knockout may be used in Alport syndrome include those described in Ceco , E . et al. , 2013 . FEBS J . studies . These mice develop progressive fibrosis in their 280 ( 17 ) :4198 - 209, the contents of which are herein incor kidneys . Alport syndrome models may be carried out as porated by reference in their entirety . described in Koepke, M . L . et al. , 2007 . Nephrol Dial Compounds and /or compositions of the invention may, in Transplant. 22 ( 4 ) : 1062- 9 and /or Hahm , K . et al. , 2007 . Am 20 some cases, be combined with one or more other therapeu J Pathol. 170 ( 1 ) : 110 - 5 , the contents of each of which are tics for the treatment of one or more fibrotic indication . herein incorporated by reference in their entirety . Examples of such other therapeutics may include , but are not In some cases, models of cardiovascular fibrosis may be limited to LPA1 receptor antagonists , lysyl oxidase 2 inhibi used to develop and / or test compounds and / or compositions tors, hedgehog inhibitors, IL - 3 / IL -4 inhibitors , CTGF of the invention for treatment of cardiovascular fibrotic 25 inhibitors , anti - a po antibodies and anti - IL - 13 antibodies . indications . In some cases , vascular injury models may be In some cases , compounds and /or compositions of the used . Such models may include balloon injury models . In present invention are designed to increase TGF - B growth some cases, these may be carried out as described in Smith factor activity to promote fibrosis to treat diseases , disorders et al. , 1999 . Circ Res . 84 ( 10 ) : 1212 - 22 , the contents of which and /or conditions where fibrosis may be advantageous . Such are herein incorporated by reference in their entirety . Block - 30 compounds may include activating antibodies . ing TGF - B in this model was shown to block neointima Therapeutics for Myelofibrosis formation . Accordingly , TGF - B inhibiting antibodies of the Myelofibrosis is a chronic blood cancer caused by muta present invention may be used to reduce and / or block tions in bonemarrow stem cells . Disease is characterized by neointima formation . an impaired ability to make normal blood cells . Patients In some embodiments , models of liver fibrosis may be 35 develop splenomegaly and hepatomegaly and excessive used to develop and /or test compounds and / or compositions fibrosis occurs in the bone marrow . Myeloproliferative neo of the invention for treatment of liver fibrotic indications. plasms (MPNs ) are the collective name for three related Liver models may include any of those described in Iredale , types of myelofibrosis with different clinical features: pri J . P . 2007 . J Clin Invest. 117 ( 3 ): 539 -48 , the contents of mary myelofibrosis (PMF ) , essential thrombocythemia and which are herein incorporated by reference in their entirety. 40 polycythemia vera . All three have overactive signaling of These include , but are not limited to , any of the models listed the JAK -STAT cell signaling pathway (Klampfi , et al. , 2013 . in Tables 1 and / or 2 . In some cases , liver models may NEJM 369 :2379 - 90 , the contents of which are herein incor include carbon tetrachloride induced liver fibrosis models . porated by reference in their entirety .) Primary myelofibrosis Such models may be carried out according to the methods (PMF ) is characterized by increased angiogenesis , reticulin described in Fujii , T . et al. , 2010 . BMC Gastroenterology . 45 and collagen fibrosis. As the disease advances , the number 10 :79 , the contents of which are herein incorporated by of osteoclasts increase and bone marrow becomes unaspi reference in their entirety . rable . Some fibrosis of PMF may be reversed by stem cell In some embodiments , models of wound healing may be transplantation (SCT . ) 98 % of individuals with poly used to develop and /or test compounds and / or compositions cythemia vera have mutated JAK2 leading to overactive of the invention for treatment of fibrotic wound indications. 50 JAK -STAT signaling . Wound models may include chronic wound models . Current therapeutics for MPNs include allogeneic In some cases, models ofGI injury -related fibrosis may be hematopoietic cell transplantation (HCT ) and Janus kinase used to develop and / or test compounds and / or compositions ( JAK ) inhibition . Allogeneic HCT is associated with up to of the invention for treatment of GI- related fibrosis . Such 10 % mortality as well as graft failure and significant side injury models may include , but are not limited to 2 ,4 ,6 - 55 effects and toxicity . JAK inhibition therapy comprises the trinitrobenzenesulfonic acid ( TNBS ) induced colitis models . use of Ruxolitinib (Rux ) a smallmolecule inhibitor of JAK2 Such models may be carried out as described in Scheiffele , that was approved in 2011 to treat MPNs. Rux is marketed F . et al ., 2002 . Curr Protoc Immunol. Chapter 15 :Unit 15 . 19, under the names JAKAFI? and JAKAVI® by Incyte phar the contents of which are herein incorporated by reference in maceuticals (Wilmington , Del . ) and Novartis (Basel , Swit their entirety. 60 zerland ) . Although able to improve splenomegaly and In some embodiments , compounds and/ or compositions hepatomegaly , Rux is not curative and some studies do not of the invention may be used to treat diseases , disorders show much benefit (Odenike , O ., 2013 . Hematology. 2013 and / or conditions related to bone marrow fibrosis . In some ( 1 ) :545 - 52 , the contents of which are herein incorporated by cases , bone marrow fibrosis models may be used to develop reference in their entirety . ) and / or test such compounds and /or compositions. Models 65 In some cases, compounds and /or compositions of the may include the marrow cell adoptive transfer model invention may be used to treat myeloproliferative disorders , described in Lacout , C . et al ., 2006 . Blood . 108 ( 5 ) : 1652 -60 including, but not limited to primary myelofibrosis , second US 9 ,758 , 577 B2 121 122 ary myelofibrosis , essential thrombocythemia , polycythemia may be useful in the case of iron deficiency due to high vera , idiopathic myelofibrosis and chronic myeloid leuke hepcidin levels. In some embodiments , hepcidin antagonists mia . In some cases, treatments may be carried out in of the present invention may comprise antibodies that dis combination with one or more known therapies for myelo rupt BMP - 6 signaling through hemojuvelin . fibrosis , including, but not limited to allogeneic HCT, JAK 5 Anemias are conditions and / or diseases associated with inhibition , fresolimumab (GC1008 ; Genzyme, Cambridge , decreased numbers of red blood cells and / or hemoglobin . Mass. ) treatment to block TGF - B1 , 2 and 3 (Mascarenhas , J. Compounds and /or compositions of the present invention et al. , 2014 . Leukemia and Lymphoma . 55 :450 - 2 , the con - may be useful in treating anemias. Such anemias may tents of which are herein incorporated by reference in their include anemia of chronic disease ( ACD ), which is also entirety ) simtuzumab (Gilead Biosciences , Foster City , 10 referred to as anemia of inflammation (AI ) . Subjects with Calif . ) treatment to block lysyl oxidase activity and collagen ACD , may suffer from chronic renal failure or acute inflam cross - linking and Pentraxin - 2 (Promedior , Lexington , mation due to rheumatoid arthritis , cancer, infection , etc . Mass. ) treatment to stimulate regulatory macrophages and Subjects suffering from ACD typically comprise elevated inhibit myelofibroblasts . In some cases , models of myelo - levels of hepcidin and impaired erythropoiesis . In a study by proliferative disorders may be used to develop and / or test 15 Sasu et al (Sasu et al. , 2010 . Blood . 115 ( 17 ) :3616 - 24 ) an such compounds and /or compositions of the invention antibody with high affinity for hepcidin was effective in intended for the treatment of myelofibrosis . Models may treating murine anemia in a mouse model of inflammation . include the marrow cell adoptive transfer model described in The studies found that the most effective treatments Lacout, C . et al. , 2006 . Blood . 108 ( 5 ) : 1652 -60 and trans - involved combining the antibody with an erythropoiesis genic mouse models , including, but not limited to the model 20 stimulating agent (ESA . ) Accordingly , some compounds described in Vannucchi, A . M . et al . , 2002 . Blood . 100 ( 4 ) : and /or compositions of the present invention may be used in 1123 -32 , the contents of each of which are herein incorpo - combination with ESAs to increase efficacy . Current anti rarated by reference in their entirety . Myelofibrosis models hepcidin antibodies being tested for treatment of ACD may include thrombopoietin - induced myelofibrosis . Such include Ab12B9 (Amgen , Thousand Oaks, Calif .) and models may be carried out as described in Chagraoui, H . et 25 LY2787106 ( Eli Lilly , Indianapolis , Ind . ) FG4592 ( Fibro al. , 2002 . Blood . 100 ( 10 ) : 3495 - 503 , the contents of which Gen , San Francisco , Calif. ) is a small molecule inhibitor of are herein incorporated by reference in their entirety . TGF - hypoxia - inducible factor (HIF ) that is also currently used to B1 has been shown to be the primary agonist of fibrosis treat anemia . according to this model . Further myelofibrosis models may In some cases , compounds and / or compositions of the be carried out as described in Mullally , A . et al. , 2010 . 30 present invention may be used to treat subjects with iron Cancer Cell. 17 : 584 - 96 , the contents of which are herein deficiency anemia (IDA ) associated with gastric bypass incorporated by reference in their entirety . surgery and / or inflammatory bowel disease ( IBD . ) Gastric Therapeutics for Scarring and Wound Healing bypass surgery leaves subjects with a reduced ability to In some embodiments , compounds and / or compositions metabolize iron due to bypass of the proximal gastric pouch of the present invention may be useful in altering wound 35 and duodenum (Warsh et al ., 2013 , the contents of which are healing and / or scar formation . In some cases, compounds herein incorporated by reference in their entirety . ) IBD and/ or compositions of the invention may ensure proper patients often suffer from iron deficiency due to intestinal wound healing ( including, but not limited to chronic blood loss and decreased absorption due to inflammation . wounds. ) In some cases, compounds and /or compositions of Some compounds and / or compositions of the present the invention may be used for reducing, treating and or 40 invention may be used to treat subjects suffering from preventing scar formation . Such compounds and / or compo iron -refractory iron deficiency anemia ( IRIDA . ) IRIDA is a sitions may comprise anti - TGF - B antibodies. In some cases, genetic disease caused by a defect in the enzyme TGF -B -activating antibodies may be used to promote heal Matriptase - 2 (De Falco , L . et al. , 2013 , the contents of which ing in wounds. are herein incorporated by reference in their entirety . ) Therapeutics for Disorders of Iron Metabolism 45 Matriptase -2 , a transmembrane serine protease , is an impor In some embodiments , methods , compounds and /or com - tant hepcidin regulator. Matriptase - 2 is capable of enzymatic positions of the present invention may be used to treat cleavage of hemojuvelin . Subjects with defective disorders of iron metabolism . Such disorders may include Matriptase - 2 activity have elevated levels of hemojuvelin , disorders comprising reduced iron levels ( e . g . anemias ) or due to lack of degradation , and therefore hepcidin expres disorders comprising elevated iron levels ( e . g . hemochro - 50 sion remains high and iron levels are reduced . Characteris matosis. ) BMP- 6 and hemojuvelin interact to modulate tics of the disease include, but are not limited to microcytic hepcidin expression . Some methods , compounds and / or hypochromic anemia , low saturation of transferrin and nor compositions of disclosed herein may be used to alter m al to high levels of hepcidin . Some subjects with IRIDA hepcidin levels , thereby regulating bodily iron levels . are diagnosed soon after birth , but many are not diagnosed Some embodiments of the present invention may com - 55 until adulthood . Treatments described herein may be used to prise hepcidin agonists or hepcidin antagonists . Hepcidin modulate irregular hepcidin levels associated with IRIDA . agonists may activate or promote the expression and / or Iron overloading anemias can occur as a result of blood physiological action of hepcidin . Such agonists may be transfusion . Excess iron associated with transfused blood useful in the treatment or prevention of iron overload due to cannot be secreted naturally and requires additional treat low hepcidin levels and / or activity . In some cases , agonists 60 ments for removal, such as chelation therapy . Such therapy may not reverse established iron overload , but may diminish is generally not well tolerated and may comprise many side iron damage to tissues. Some hepcidin agonists of the effects . Thus, there is a clinical need for new , better tolerated present invention may elevate production of hepcidin therapies . Additional therapies include EXJADE® , for the through activating and/ or enhancing BMP -6 /hemojuvelin treatment of patients , age 10 and older , with non - transfu signaling . 65 sion - dependent thalassemia (NTDT ) syndromes . Also Hepcidin antagonists may block or reduce the expression included is ACE -536 , a ligand trap that blocks TGF - B and /or physiological action of hepcidin . Such antagonists superfamily members. Both EXJADE and ACE - 536 are US 9 , 758 ,577 B2 123 124 known to elevate erythropoiesis . In some embodiments , unfortunate side effect is the loss of red blood cells , platelets compounds and / or compositions of the present invention and white blood cells which depend on active cell division may be used to control iron overloading . Some such embodi- of bone marrow cells . In some embodiments , compounds ments may function to redistribute iron from parenchyma to and /or compositions of the present invention may be macrophages where iron is better tolerated . In some cases 5 designed to treat patients suffering from anemia ( the loss of this may be carried out through elevation of hepcidin levels . red blood cells ) , thrombocytopenia ( a decrease in the num In studies by Gardenghi et al (Gardenghi et al ., 2010 , JCI. ber of platelets ) and /or neutropenia (a decrease in the 120 ( 12 ) : 4466 - 77 ) overexpression of murine hepcidin was number of neutrophils ) . able to increase hemoglobin levels and decrease iron over - Therapeutics for Cancer load in mouse model of B - thalassemia and a mouse model of 10 Various cancers may be treated with compounds and /or hemochromatosis ( Viatte et al. , 2006 , Blood . 107: 2952 . ) compositions of the present invention . As used herein , the GDF -15 levels in circulation have been found to nega- term “ cancer ” refers to any of various malignant neoplasms tively correlate with hepcidin levels, suggesting a role for characterized by the proliferation of anaplastic cells that GDF - 15 in iron loading and / or metabolism (Finkenstedt et tend to invade surrounding tissue and metastasize to new al. , 2008 . British Journal of Haematology. 144 : 789- 93 , the 15 body sites and also refers to the pathological condition contents of which are herein incorporated by reference in characterized by such malignant neoplastic growths. Can their entirety. ) Transcription of the gene encoding GDF - 15 cers may be tumors or hematological malignancies , and may be upregulated under stress and / or hypoxic conditions . include but are not limited to , all types of lymphomas / In some cases, compounds and/ or compositions of the leukemias, carcinomas and sarcomas, such as those cancers present invention may be used to treat subjects suffering 20 or tumors found in the anus, bladder , bile duct , bone, brain , from iron disorders and / or anemias by altering GDF - 15 breast, cervix , colon /rectum , endometrium , esophagus , eye , signaling activity . Such compounds and/ or compositions gallbladder , head and neck , liver, kidney, larynx , lung , may comprise antibodies capable of stabilizing or destabi - mediastinum ( chest ) , mouth , ovaries, pancreas , penis , pros lizing the GDF - 15 GPC or through modulation of one or tate , skin , small intestine , stomach , spinal marrow , tailbone , more interaction between GDF - 15 and one or more co - 25 testicles, thyroid and uterus . factor. In cancer, TGF - B may be either growth promoting or Hemochromatosis is a disease characterized by iron over - growth inhibitory . As an example , in pancreatic cancers , load due to hyperabsorption of dietary iron . In hereditary SMAD4 wild type tumors may experience inhibited growth hemochromatosis (HH ) this overload is caused by inheri - in response to TGF - B , but as the disease progresses , con tance of a common autosomal recessive copy of the HFE 30 stitutively activated type II receptor is typically present. gene from both parents . In such cases , iron may be over - Additionally , there are SMAD4 - null pancreatic cancers . In loaded in plasma as well as in organs and tissues , including , some embodiments , compounds and/ or compositions of the but not limited to the pancreas, liver and skin , leading to present invention are designed to selectively target compo damage caused by iron deposits ( Tussing -Humphreys et al, nents of TGF - B signaling pathways that function uniquely in 2013 . ) Current therapies for HH may include phlebotomy, 35 one or more forms of cancer . Leukemias , or cancers of the multiple times per year. In some embodiments, compounds blood or bone marrow that are characterized by an abnormal and / or compositions of the present invention may be used to proliferation of white blood cells i . e . , leukocytes , can be treat HH by modulating subject iron levels . divided into four major classifications including Acute lym Mutations in the hepcidin (HAMP ) and / or hemojuvelin phoblastic leukemia ( ALL ) , Chronic lymphocytic leukemia (HFE2 ) genes are responsible for a severe form of 40 (CLL ) , Acute myelogenous leukemia or acute myeloid leu hemochromatosis known as juvenile hemochromatosiskemia ( AML ) ( AML with translocations between chromo (Roetto et al . , 2003 ; Papanikolauou et al. , 2004 . ) Some some 10 and 11 [ t ( 10 , 11 ) ] , chromosome 8 and 21 [ t ( 8 ; 21 ) ] , mutations of hemojuvelin associated with juvenile chromosome 15 and 17 [ t( 15 ; 17 ) ], and inversions in chro hemochromatosis lead to protein misfolding and reduce mosome 16 [inv ( 16 )] ; AML with multilineage dysplasia , hemojuvelin secretion from the cell , thus decreasing overall 45 which includes patients who have had a prior myelodys hemojuvelin signaling activity . Other mutations affect plastic syndrome (MDS ) or myeloproliferative disease that hemojuvelin interactions with other signaling molecules. transforms into AML ; AML and myelodysplastic syndrome Hemojuvelin comprising the mutation G99R , for example , (MDS ) , therapy - related , which category includes patients is unable to bind BMP - 2 . Hemojuvelin comprising the who have had prior chemotherapy and /or radiation and mutation L101P is unable to associate with either BMP - 2 or 50 subsequently develop AML or MDS ; d ) AML not otherwise neogenin . Some therapeutic embodiments of the present categorized , which includes subtypes of AML that do not fall invention may comprise the modulation of hemojuvelin into the above categories; and e ) Acute leukemias of signaling . ambiguous lineage, which occur when the leukemic cells During chemotherapy , cell division is temporarily halted cannot be classified as either myeloid or lymphoid cells , or to prevent the growth and spread of cancerous cells . An 55 where both types of cells are present ) ; and Chronic myel unfortunate side effect is the loss of red blood cells which ogenous leukemia (CML ) . depend on active cell division of bonemarrow cells . In some The types of carcinomas include , but are not limited to , embodiments , compounds and/ or compositions of the pres - papilloma / carcinoma, choriocarcinoma, endodermal sinus ent invention may be used to treat anemia associated che tumor , teratoma, adenoma/ adenocarcinoma , melanoma , motherapy. 60 fibroma, lipoma, leiomyoma , rhabdomyoma, mesothelioma , In some cases , compounds and / or compositions of the angioma, osteoma , chondroma , glioma , lymphoma/ leuke present invention may be combined with any of the thera - mia , squamous cell carcinoma , small cell carcinoma , large peutics described herein to increase efficacy . cell undifferentiated carcinomas, basal cell carcinoma and Therapeutics for Anemia , Thrombocytopenia and Neutrope - sinonasal undifferentiated carcinoma. nia 65 The types of sarcomas include , but are not limited to , soft During chemotherapy, cell division is temporarily halted tissue sarcoma such as alveolar soft part sarcoma, angiosar to prevent the growth and spread of cancerous cells . An coma, dermatofibrosarcoma, desmoid tumor, desmoplastic US 9 , 758 ,577 B2 125 126 small round cell tumor, extraskeletal chondrosarcoma, anti - cancer treatments , such as biological, chemotherapy extraskeletal osteosarcoma, fibrosarcoma , hemangiopericy and radiotherapy . Accordingly, a treatment can include , for toma, hemangiosarcoma, Kaposi' s sarcoma, leiomyosar example , imatinib (Gleevac ) , all -trans - retinoic acid , a mono coma, liposarcoma, lymphangiosarcoma, lymphosarcoma, clonal antibody treatment ( gemtuzumab , ozogamicin ) , che malignant fibrous histiocytoma , neurofibrosarcoma, rhab - 5 motherapy ( for example , chlorambucil, prednisone , predni domyosarcoma, synovial sarcoma , and Askin ' s tumor, solone, vincristine , cytarabine, clofarabine, farnesyl Ewing 's sarcoma (primitive neuroectodermal tumor) , malig transferase inhibitors , decitabine , inhibitors of MDR1) , nant hemangioendothelioma, malignant schwannoma, rituximab , interferon - a , anthracycline drugs ( such as dauno osteosarcoma, and chondrosarcoma. rubicin or idarubicin ), L - asparaginase , doxorubicin , cyclo In some embodiments , compositions and methods of the 10 phosphamide , doxorubicin , bleomycin , fludarabine , etopo invention may be used to treat one or more types of cancer side , pentostatin , or cladribine ) , bone marrow transplant, or cancer - related conditions that may include , but are not stem cell transplant, radiation therapy , anti -metabolite drugs limited to colon cancer , renal cancer , breast cancer, malig - (methotrexate and 6 -mercaptopurine ), or any combination nant melanoma and glioblastomas (Schlingensiepen et al. , thereof . 2008 ; Ouhtit et al ., 2013. ) 15 Radiation therapy (also called radiotherapy , X -ray High - grade gliomas ( e . g . anaplastic astrocytomas and therapy , or irradiation ) is the use of ionizing radiation to kill glioblastomas ) make up around 60 % of malignant brain cancer cells and shrink tumors . Radiation therapy can be tumors . TGF- B2 has been found to be overexpressed in over administered externally via external beam radiotherapy 90 % of such gliomas and expression levels correlate with (EBRT ) or internally via brachytherapy . The effects of tumor progression . Further , studies using TGF - 02 reduction 20 radiation therapy are localized and confined to the region at the mRNA level in cancer patients showed significant being treated . Radiation therapy may be used to treat almost improvement in tumor outcome (Bogdahn et al. , 2010 . ) In every type of solid tumor , including cancers of the brain , light of these studies , some compositions of the present breast, cervix , larynx , lung , pancreas , prostate , skin , stom invention may be used therapeutically to treat individuals ach , uterus , or soft tissue sarcomas . Radiation is also used to with high - grade gliomas . Such compositions may act to 25 treat leukemia and lymphoma . lower the levels of free TGF - B2 and /or the levels of TGF - B2 Chemotherapy is the treatment of cancer with drugs that activity . can destroy cancer cells . In current usage , the term “ che In some cases, TGF- B2 activity may contribute to tumor motherapy ” usually refers to cytotoxic drugs which affect development through modulation of metastasis , angiogen - rapidly dividing cells in general, in contrast with targeted esis , proliferation and /or immunosuppressive functions that 30 therapy . Chemotherapy drugs interfere with cell division in impair immunological tumor surveillance (Schlingensiepen various possible ways, e . g . with the duplication of DNA or et al. , 2008 . ) A study by Reed et al (Reed et al. , 1994 ) the separation of newly formed chromosomes . Most forms demonstrated TGF - B2 mRNA expression in a large percent of chemotherapy target all rapidly dividing cells and are not age ofmelanocytic lesions including primary invasive mela specific to cancer cells , although some degree of specificity nomas and metastatic melanomas . Some compounds and /or 35 may come from the inability of many cancer cells to repair compositions of the present invention may be used to DNA damage , while normal cells generally can . modulate TGF -B2 activity and / or levels in such lesions and Most chemotherapy regimens are given in combination . or prevent lesion formation . Melanoma cell growth in the Exemplary chemotherapeutic agents include , but are not brain parenchyma has also been shown to be influenced by limited to , 5 - FU Enhancer, 9 - AC , AG2037 , AG3340 , Aggre TGF -B2 activity (Zhang et al. , 2009. ) Some compounds 40 canase Inhibitor , Aminoglutethimide , Amsacrine and / or compositions of the present invention may be used to ( m - AMSA ) , Asparaginase , Azacitidine , Batimastat (BB94 ) , prevent or control such cell growth through modulation of BAY 12 - 9566 , BCH - 4556 , Bis -Naphtalimide , Busulfan , TGF - B2 activity and/ or levels . Capecitabine , Carboplatin , Carmustaine + Polifepr Osan , Among females worldwide , breast cancer is the most cdk4 /cdk2 inhibitors, Chlorombucil , CI- 994 , Cisplatin , prevalent form of cancer . Breast cancer metastasis is medi- 45 Cladribine , CS -682 , Cytarabine HC1, D2163 , Dactinomycin , ated in part through interactions between cancer cells and Daunorubicin HCI, DepoCyt, Dexifosamide, Docetaxel, extracellular matrix components , such as hyaluronic acid Dolastain , Doxifluridine , Doxorubicin , DX8951f, E 7070 , (HA .) CD44 has been shown to be the major receptor for HA EGFR , Epirubicin , Erythropoietin , Estramustine phosphate on cancer cells (Ouhtit et al. , 2013 .) The interaction between sodium , Etoposide (VP16 -213 ), Farnesyl Transferase Inhibi CD44 and HA leads to modulation of cell motility , survival 50 tor, FK 317 , Flavopiridol, Floxuridine , Fludarabine , Fluo adhesion and proliferation . TGF - B2 transcription is also rouracil ( 5 - FU ) , Flutamide , Fragyline , Gemcitabine , Hex upregulated by CD44 signaling activity and is believe to amethylmelamine (HMM ) , Hydroxyurea contribute to resulting changes in cell motility . Unfortu (hydroxycarbamide ) , Ifosfamide, Interferon Alfa - 2a , Inter nately , current therapies have limited efficacy and many feron Alfa - 2b , Interleukin - 2 , Irinotecan , ISI 641, Krestin , carry adverse effects due to a lack of specificity . In some 55 Lemonal DP 2202 , Leuprolide acetate ( LHRH - releasing cases, compounds and /or compositions of the present inven - factor analogue ) , Levamisole, LiGLA ( lithium - gamma lino tion may be used to alter cellular activities induced by lenate ), Lodine Seeds, Lometexol , Lomustine (CCNU ) , TGF - B2 upregulation . Marimistat, Mechlorethamine HCl (nitrogen mustard ), The invention further relates to the use of compounds Megestrol acetate , Meglamine GLA , Mercaptopurine , and/ or compositions of the present invention for treating one 60 Mesna , Mitoguazone (methyl - GAG ; methyl glyoxal bis or more forms of cancer, in combination with other phar - guanylhydrazone ; MGBG ) , Mitotane ( 0 . p '- DDD ) , Mitoxan maceuticals and/ or other therapeutic methods, e . g . , with trone , Mitoxantrone HC1, MMI 270 , MMP, MTA /LY known pharmaceuticals and / or known therapeutic methods, 231514 , Octreotide, ODN 698 , OK -432 , Oral Platinum , Oral such as , for example , those which are currently employed Taxoid , Paclitaxel ( TAXOL® ), PARP Inhibitors, PD for treating these disorders . For example , the compounds 65 183805 , Pentostatin ( 2 ' deoxycoformycin ), PKC 412 , Pli and / or compositions of the present invention can also be camycin , Procarbazine HCI, PSC 833 , Ralitrexed , RAS administered in conjunction with one or more additional Farnesyl Transferase Inhibitor, RAS Oncogene Inhibitor, US 9 ,758 , 577 B2 127 128 Semustine (methyl - CCNU ) , Streptozocin , Suramin , Tamox matrix , imparting resistance to fracture . The role of TGF ifen citrate , Taxane Analog , Temozolomide, Teniposide beta family members in bone remodeling makes them attrac ( VM - 26 ) , Thioguanine, Thiotepa , Topotecan , Tyrosine tive targets for potential therapeutics to treat bone disorder Kinase , UFT ( Tegafur /Uracil ) , Valrubicin , Vinblastine sul- and disease . fate, Vindesine sulfate , VX - 710 , VX -853 , YM 116 , ZD 5 Numerous diseases and /or disorders affect bones and 0101 , ZD 0473 / Anormed , ZD 1839 , ZD 9331. joints . Such diseases and / or disorders may be congenital , Biological therapies use the body ' s immune system , genetic and /or acquired . Such diseases and / or disorders either directly or indirectly , to fight cancer or to lessen the include , but are not limited to , bone cysts , infectious arthri side effects that may be caused by some cancer treatments. tis , Paget ' s disease of the bone , Osgood - Schlatter disease , In some embodiments , compounds and / or compositions of 10 Kohler ' s bone disease , bone spurs (osteophytes ), bone the present invention may be considered biological therapies tumors , craniosynostosis , fibrodysplasia ossificans progres in that they may stimulate immune system action against one sive, fibrous dysplasia , giant cell tumor of bone, hypophos or more tumor , for example . However , this approach may phatasia , Klippel- Feil syndrome, metabolic bone disease , also be considered with other such biological approaches , osteoarthritis , osteitis deformans, osteitis fibrosa cystica , e . g . , immune response modifying therapies such as the 15 osteitis pubis , condensing osteitis , osteitis condensans osteo administration of interferons, interleukins, colony - stimulat- chondritis dissecans, osteochondroma, osteogenesis imper ing factors , other monoclonal antibodies, vaccines, gene fecta , osteomalacia , osteomyelitis , osteopenia , osteopetro therapy , and nonspecific immunomodulating agents are also sis , osteoporosis, osteosarcoma, porotic hyperostosis , envisioned as anti -cancer therapies to be combined with the primary hyperparathyroidism , renal osteodystrophy and compounds and / or compositions of the present invention . 20 water on the knee . Small molecule targeted therapy drugs are generally Mouse models for evaluating the effectiveness of thera inhibitors of enzymatic domains on mutated , overexpressed , peutics on bone development and repair are well known in or otherwise critical proteins within the cancer cell, such as the art. In one such model demonstrated by Mohammad , et tyrosine kinase inhibitors imatinib (Gleevec /Glivec ) and al. (Mohammad , K . S . et al. , Pharmacologic inhibition of the gefitinib (Iressa ) . Examples of monoclonal antibody thera - 25 TGF - beta type I receptor kinase has anabolic and anti pies that can be used with compounds and /or compositions catabolic effects on bone . PLoS One . 2009 ; 4 (4 ) :e5275 . of the present invention include , but are not limited to , the Epub 2008 Apr. 16 ), inhibition of the TGF -beta type I anti -HER2 / neu antibody trastuzumab (Herceptin ) used in receptor was carried out in C57B1/ 6 mice through twice breast cancer , and the anti -CD20 antibody rituximab , used in daily administration of a potent inhibitor , SD - 208 , by gav a variety of B - cell malignancies . The growth of some 30 age . Subsequently , bone mineral density (BMD ) was ana cancers can be inhibited by providing or blocking certain l yzed using a PIXImus mouse densitometer (GE Lunar II , hormones . Common examples of hormone - sensitive tumors Faxitron Corp . , Wheeling , Ill . ) . Changes in BMD are include certain types of breast and prostate cancers. Remov - expressed as a percentage change in the area scanned . The ing or blocking estrogen or testosterone is often an important study found that after 6 weeks of treatment, male mice additional treatment . In certain cancers , administration of 35 exhibited a 4 .12 % increase in bone accrual while female hormone agonists, such as progestogens may be therapeu - mice exhibited a 5 . 2 % increase . tically beneficial . Compounds and /or compositions of the present invention Cancer immunotherapy refers to a diverse set of thera - may be useful as therapies for simple or complex bone peutic strategies designed to induce the patient' s own fractures and/ or bone repair . In such treatments, compounds immune system to fight the tumor, and include , but are not 40 and / or compositions of the present invention may be intro limited to , intravesical BCG immunotherapy for superficial duced to the site of injury directly or through the incorpo bladder cancer , vaccines to generate specific immune r ation into implantation devices and coated biomatrices . responses , such as for malignant melanoma and renal cell Additionally , treatments are contemplated in which com carcinoma, and the use of Sipuleucel - T for prostate cancer, pounds and/ or compositions of the present invention are in which dendritic cells from the patient are loaded with 45 supplied together with one or more GPC in a treatment area , prostatic acid phosphatase peptides to induce a specific facilitating the slow release of one or more growth factors immune response against prostate - derived cells . from such GPCs. In some embodiments, compounds and /or compositions Therapeutics for Angiogenic and Endothelial Proliferation of the present invention are designed to prevent T cell Conditions inhibition . Such compounds and / or compositions may pre - 50 The compounds and /or compositions of the present inven vent the dissociation of growth factors from the prodomain tion may be used to treat angiogenic and endothelial prolif of the GPC or from extracellular matrix and / or cellular eration syndromes, diseases or disorders . The term “ angio matrix components including , but not limited to GARPs, genesis ” , as used herein refers to the formation and /or fibrillins or LTBPs . reorganization of new blood vessels. Angiogenic disease Therapeutics for Bone Healing 55 involves the loss of control over angiogenesis in the body . In Compounds and / or compositions of the present invention such cases , blood vessel growth , formation or reorganization may be used to treat bone disorders and / or improve bone may be overactive ( including during tumor growth and healing or repair. Cellular remodeling of bone is a lifelong cancer where uncontrolled cell growth requires increased process that helps to maintain skeletal integrity . This process blood supply ) or insufficient to sustain healthy tissues. Such involves cycles of osteoclastic bone resorption and new 60 conditions may include , but are not limited to angiomas , bone formation that function to repair defects and areas of angiosarcomas , telangiectasia , lymphangioma, congenital weakness in bone . TGF -beta family members , preferably vascular anomalies, tumor angiogenesis and vascular struc BMPs, are thought to be important factors in coupling the tures after surgery. Excessive angiogenesis is noted in can processes of resorption and formation by osteoclasts. TGF - cer , macular degeneration , diabetic blindness , rheumatoid beta family members are prevalent in the bone matrix and 65 arthritis, psoriasis as well as many other conditions . Exces upregulated by bone injury . TGF -beta family members are sive angiogenesis is often promoted by excessive angiogenic also believed to impart strength to the fully formed bone growth factor expression . Compounds and /or compositions US 9 , 758 ,577 B2 129 130 of the present invention may act to block growth factors seminated Encephalomyelitis ( ADEM ) , Acute necrotizing involved in excessive angiogenesis . Alternatively , com hemorrhagic leukoencephalitis , Addison 's disease , Agam pounds and /or compositions of the present invention may be maglobulinemia , Alopecia areata , Amyloidosis , Ankylosing utilized to promote growth factor signaling to enhance spondylitis, Anti -GBM / Anti- TBM nephritis , Antiphospho angiogenesis in conditions where angiogenesis is inhibited . 5 lipid syndrome (APS ) , Autoimmune angioedema, Autoim Such conditions include , but are not limited to coronary mune aplastic anemia , Autoimmune dysautonomia , Autoim artery disease , stroke , diabetes and chronic wounds . mune hepatitis , Autoimmune hyperlipidemia, Autoimmune Therapeutics for Orphan Indications and Diseases immunodeficiency , Autoimmune inner ear disease (AIED ) , The compounds and /or compositions of the present inven - Autoimmune myocarditis , Autoimmune pancreatitis , Auto tion may be used to treat orphan indications and /or diseases . 10 immune retinopathy , Autoimmune thrombocytopenic pur Such diseases include Marfan ' s syndrome. This syndrome is pura (ATP ) , Autoimmune thyroid disease , Autoimmune urti a connective tissue disorder, effecting bodily growth and caria , Axonal & neuronal neuropathies , Balo disease , development. Tissues and organs that are most severely Behcet' s disease , Bullous pemphigoid , Cardiomyopathy, compromised include the heart, blood vessels , bones , eyes , Castleman disease , Celiac disease , Chagas disease , Chronic lungs and connective tissue surrounding the spinal cord . 15 fatigue syndrome, Chronic inflammatory demyelinating Unfortunately , the effects can be life threatening . Marfan ' s polyneuropathy ( CIDP ) , Chronic recurrent multifocal osto syndrome is caused by a genetic mutation in the gene that myelitis (CRMO ) , Churg - Strauss syndrome, Cicatricial produces fibrillin , a major component of bodily connective pemphigoid /benign mucosal pemphigoid , Crohn ' s disease , tissue . Latent TGF - B binding protein (LTBP ) is an important Cogans syndrome, Cold agglutinin disease , Congenital heart regulator of TGF - B signaling that exhibits close identity to 20 block , Coxsackie myocarditis , CREST disease , Essential fibrillin protein family members . Functional LTBP is mixed cryoglobulinemia , Demyelinating neuropathies , Der required for controlling the release of active TGF - B (Oklu , matitis herpetiformis , Dermatomyositis , Devic ' s disease R . et al ., The latent transforming growth factor beta binding (neuromyelitis optica ), Diabetes Type I , Discoid lupus, protein (LTBP ) family . Biochem J . 2000 Dec . 15 ; 352 Pt Dressler' s syndrome, Endometriosis , Eosinophilic esoph 3 :601 - 10 ) . In some embodiments , compounds and /or com - 25 agitis , Eosinophilic fasciitis , Erythema nodosum , Experi positions of the present invention are designed to alter the mental allergic encephalomyelitis , Evans syndrome, Fibro release profile of TGF - B . In such embodiments , compounds myalgia , Fibrosing alveolitis , Giant cell arteritis ( temporal and / or compositions may comprise antibodies characterized arteritis ) , Glomerulonephritis , Goodpasture ' s syndrome, as inhibitory antibodies . Granulomatosis with Polyangiitis (GPA ) see Wegener ' s , In some embodiments , compounds and /or compositions 30 Graves ' disease , Guillain - Barre syndrome, Hashimoto ' s of the present invention may be useful in the treatment of encephalitis, Hashimoto ' s thyroiditis , Hemolytic anemia , Camurati- Engelmann disease (CED ). This disease primarily Henoch - Schonlein purpura , Herpes gestationis , Hypogam affects the bones, resulting in increased bone density . Espe - maglobulinemia , Idiopathic thrombocytopenic purpura cially affected are the long bones of the legs and arms; (ITP ) , IgA nephropathy, IgG4 - related sclerosing disease , however, the bones of the skill and hips can also be affected . 35 Immunoregulatory lipoproteins, Inclusion body myositis , The disease results in leg and arm pain as well as a variety Insulin -dependent diabetes ( typel ) , Interstitial cystitis , Juve of other symptoms . CED is very rare , reported in approxi nile arthritis , Juvenile diabetes , Kawasaki syndrome, Lam mately 200 individuals worldwide and is caused by a bert- Eaton syndrome, Large vessel vasculopathy, Leukocy mutation in the TGF - B gene . TGF - B produced in the bodies toclastic vasculitis , Lichen planus, Lichen sclerosus, of these individuals has a defective prodomain , leading to 40 Ligneous conjunctivitis , Linear IgA disease (LAD ), Lupus overactive TGF -B signaling (Janssens , K . et al. , Transform - (SLE ) , Lyme disease , chronic , Meniere ' s disease , Micro ing growth factor -beta 1 mutations in Camurati- Engelmann scopic polyangiitis , Mixed connective tissue disease disease lead to increased signaling by altering either acti- (MCTD ), Mooren ’s ulcer, Mucha - Habermann disease ,Mul vation or secretion of the mutant protein . J Biol Chem . 2003 tiple endocrine neoplasia syndromes, Multiple sclerosis , Feb . 28 ; 278 ( 9 ) : 7718 - 24 . Epub 2002 Dec . 18 ). As described 45 Myositis , Myasthenia gravis , Narcolepsy , Neuromyelitis by Shi et al. , ( Shi, M . et al. , Latent TGF - beta structure and optica (Devic ' s ) , Neutropenia , Ocular cicatricial pemphig activation . Nature . 2011 Jun . 15 ; 474 (7351 ): 343 - 9 ) among oid , Optic neuritis , Palindromic rheumatism , PANDAS (Pe CED mutations, Y81H disrupts an a2 -helix residue that diatric Autoimmune Neuropsychiatric Disorders Associated cradles the TGF - B fingers . The charge -reversal E169K and with Streptococcus ) , Paraneoplastic cerebellar degeneration , H222D mutations disrupt a pH - regulated salt bridge 50 Paroxysmal nocturnal hemoglobinuria (PNH ) , Parry Rom between Glu 169 and His 222 in the dimerization interface berg syndrome, Parsonnage - Turner syndrome, Pars planitis of the prodomain . Residue Arg 218 is substantially buried : (peripheral uveitis ), Pemphigus, Peripheral neuropathy , it forms a cation - it bond with Tyr 171 and salt bridges across Perivenous encephalomyelitis , Pernicious anemia , POEMS the dimer interface with residue Asp 226 of the ' bowtie syndrome, Polyarteritis nodosa , Type I, II , & III autoimmune region of the growth factor prodomain complex (GPC ) . 55 polyglandular syndromes , Polyendocrinopathies , Polymyal Moreover , CED mutations in Cys 223 and Cys 225 demon - gia rheumatica , Polymyositis , Postmyocardial infarction strate the importance of disulphide bonds in the bowtie syndrome, Postpericardiotomy syndrome, Progesterone der region for holding TGF - B in inactive form . In this embodi matitis , Primary biliary cirrhosis , Primary sclerosing cho ment, compounds and /or compositions of the present inven - langitis , Psoriasis , Psoriatic arthritis , Idiopathic Pulmonary tion comprising one or more inhibitory antibodies would 60 fibrosis, Pyoderma gangrenosum , Pure red cell aplasia , serve to alleviate symptoms. In some embodiments , admin - Raynauds phenomenon , Reactive arthritis , Reflex sympa istration would be to the neonate subject. thetic dystrophy , Reiter ' s syndrome, Relapsing polychon Therapeutics for Immune and Autoimmune Diseases and dritis , Restless legs syndrome, Retroperitoneal fibrosis , Disorders Rheumatic fever, Rheumatoid arthritis , Sarcoidosis , Compounds and / or compositions of the present invention 65 Schmidt syndrome, Scleritis , Scleroderma, Sjogren ' s syn may be used to treat immune and autoimmune disorders . drome, Small vessel vasculopathy , Sperm & testicular auto Such disorders include , but are not limited to Acute Dis - immunity , Stiff person syndrome, Subacute bacterial endo US 9 , 758 ,577 B2 131 132 carditis (SBE ) , Susac ' s syndrome, Sympathetic ophthalmia , organisms or agents present in their body. Subjects at risk of Takayasu ' s arteritis , Temporal arteritis /Giant cell arteritis , having one or more infection are subjects that are predis Thrombocytopenic purpura ( TTP ) , Tolosa -Hunt syndrome, posed to developing one or more infection . Such subjects Transverse myelitis , Tubular autoimmune disorder , Ulcer- may include, for example , subjects with known or suspected ative colitis , Undifferentiated connective tissue disease 5 exposure to one or more infectious organisms or agents . In (UCTD ) , Uveitis , Vesiculobullous dermatosis , Vasculitis , some embodiments , subjects at risk of having infections Vitiligo and Wegener ’ s granulomatosis ( also known as may also include subjects with conditions associated with Granulomatosis with Polyangiitis (GPA ) ) . impaired abilities to mount immune responses to infectious TGF - B plays an active role in leukocyte differentiation , organisms and/ or agents , e . g ., subjects with congenital and / proliferation and activation making it an important factor in 10 or acquired immunodeficiency , subjects undergoing radia immune and autoimmune diseases . Additionally , TGF - B tion therapy and/ or chemotherapy , subjects with burn inju promotes chemotaxis of leukocytes and influences adhesion ries , subjects with traumatic injuries and subjects molecule -mediated localization . A role for TGF - B in cardiac , undergoing surgery or other invasive medical or dental pulmonary and gastric inflammation has been demonstrated procedures. Furthermore, SMAD3 - deficient mice are prone to chronic 15 Infections are broadly classified as bacterial, viral, fungal , mucosal infections as a result of T - cell activation impair - and / or parasitic based on the category of infectious organ ment and reduced mucosal immunity ( Blobe , G . C . et al. , isms and /or agents involved . Other less common types of Role of transforming growth factor beta in human disease . infection are also known in the art , including , e . g ., infections N Engl J Med . 2000 May 4 ; 342 ( 18 ): 1350 - 8 ). As an immu involving rickettsiae , mycoplasmas, and agents causing nosuppressant, TGF - B has been shown to both inhibit the 20 scrapie , bovine spongiform encephalopathy (BSE ) , and function of inflammatory cells as well as enhance the prion diseases ( e . g . , kuru and Creutzfeldt- Jacob disease ) . function of regulatory T cells. Recent studies have shown Examples of bacteria , viruses , fungi, and parasites which that the latent TGF - B growth factor prodomain complex cause infection are well known in the art. An infection can (GPC ) binds to regulatory T cells through an interaction with be acute , subacute , chronic , or latent, and it can be localized the Glycoprotein - A repetitions anonymous protein (GARP ) . 25 or systemic . As used herein , the term “ chronic infection ” In fact, GARP is necessary for TGF - B association with T refers to those infections that are not cleared by the normal cells ( Tran , D . Q . et al ., GARP (LRRC32 ) is essential for the actions of the innate or adaptive immune responses and surface expression of latent TGF - B on platelets and activated persist in the subject for a long duration of time, on the order FOXP3 + regulatory T cells . PNAS . 2009 . 106 ( 32 ) : 13445 - of weeks, months , and years. A chronic infection may reflect 50 ) . This interaction provides the platform necessary to 30 latency of the infectious agent, and may include periods in release active TGF - B from the GPC in an integrin -dependent which no infectious symptoms are present, i .e ., asymptom manner (Wang , R . et al ., GARP regulates the bioavailability atic periods. Examples of chronic infections include , but are and activation of TGF- B . Mol Biol Cell. 2012 March ; not limited to , HIV infection and herpesvirus infections. 23 (6 ) : 1129 - 39 . Epub 2012 Jan . 25 ) . In some embodiments , Furthermore , an infection can be predominantly intracellular compounds and / or compositions of the present invention 35 or extracellular during at least one phase of the infectious modulate the interaction between GARP and TGF - B . Such organism ' s or agent ' s life cycle in the host. modulation may selectively modulate T cell activity for Compounds and / or compositions of the present invention treatment of disease ( e . g . autoimmune disease and /or can - and additional therapeutic agents may be administered in cer . ) In some embodiments , compounds and / or composi combination in the same composition (e .g . , parenterally ) , as tions of the present invention may be used for the treatment 40 part of a separate composition or by another method of immune and /or autoimmune disorders . In some embodi- described herein . ments , compounds and/ or compositions of the present Therapeutics for Eye Related Diseases , Disorders and/ or invention may specifically target GARP -bound GPC , GARP Conditions or the interaction site between GARP and the GPC . In some In some embodiments , compounds and /or compositions embodiments , compounds and/ or compositions of the pres - 45 of the present invention may be useful in the treatment of ent invention comprising antibodies are designed to promote diseases , disorders and / or conditions related to eyes . These release of growth factors ( including , but not limited to may include, but are not limited to glaucoma, dry eye and /or TGF - B ) from GARP - bound GPCs while not affecting corneal wound healing . In some embodiments , compounds growth factor release from LTBP -bound GPCs. Treatment of and /or compositions may be useful in the treatment of immune and autoimmune disorders with compounds and / or 50 glaucoma. Evidence suggests that TGF - B2 is upregulated in compositions of the present invention may be in combina - glaucoma ( Picht, G . et al ., Transforming growth factor beta tion with standard of care ( SOC ) or synergistic combinations 2 levels in the aqueous humor in different types of glaucoma or with companion diagnostics . and the relation to filtering bleb development. Graefes Arch Therapeutics for Infectious Agents Clin Exp Ophthalmol. 2001 March 239 ( 3 ) : 199 - 207 ; Trip In some embodiments, compounds and /or compositions 55 athi, R . C . et al. , Aqueous humor in glaucomatous eyes of the present invention may be useful for treatment of contains an increased level of TGF - B2 . Exp Eye Res . 1994 infectious diseases and /or disorders, for example , in subjects December 59 (6 ): 723 -7 .) This includes primary open -angle with one or more infections . In some embodiments , subjects glaucoma and juvenile glaucoma. There is also evidence that have one or more infection or are at risk of developing one TGF- B2 may induce senescence - like effects in human tra or more infection . As used herein , the term “ infection ” refers 60 becular meshwork cells , which control intraocular pressure to a disease or condition in a host attributable to the presence ( often dysfunctional in glaucoma ) ( Yu , A . L . et al. , TGF - B2 of one or more foreign organism or agent capable of induces senescence -associated changes in human trabecular reproduction within the host. Infections typically comprise meshwork cells . Invest Ophthalmol Vis Sci. 2010 November breaching of one or more normal mucosal or other tissue 51 ( 11 ) : 5718 - 23 . ) In some embodiments , compounds and /or barriers by one or more infectious organisms or agents . 65 compositions of the present invention may be used to Subjects having one or more infection are subjects that decrease the ratio of free TGF -B2 to GPC - bound (inactive ) comprise one or more objectively measurable infectious TGF- B2 in or around eye tissues affected by or related to US 9 ,758 ,577 B2 133 134 glaucoma. TGF- B -related proteins may also impact on cor- In some embodiments , animal models may be used to neal wound healing ( e . g . after surgical repair and/ or LASIK develop and test compounds and /or compositions of the treatment) ( Huh , M . I. et al. , Distribution of TGF - B isoforms present invention for use in the treatment of cardiovascular and signaling intermediates in corneal fibrotic wound repair . diseases , disorders and / or conditions. In some cases , vascu J Cell Biochem . 2009 Oct. 1 . 108 ( 2 ): 476 - 88 ; Sumioka , T. et 5 lar injury models may be used . Such models may include al. , Inhibitory effect of blocking TGF - beta /Smad signal on balloon injury models . In some cases , these may be carried injury - induced fibrosis of corneal endothelium . Mol Vis . out as described in Smith et al. , 1999 . Circ Res . 84 ( 10 ) : 2008 ; 14 :2272 -81 . Epub 2008 Dec . 11; Carrington , L . M . et 1212 - 22, the contents of which are herein incorporated by al. , Differential regulation of key stages in early corneal reference in their entirety . wound healing by TGF- beta isoforms and their inhibitors . 10 Therapeutics Related to Muscle Disorders and /or Injuries Invest Ophthalmol Vis Sci. 2006 May ; 47 ( 5 ) : 1886 - 94 . ) In some embodiments , compounds and / or compositions Compounds and / or compositions of the present invention of the present invention may be used to treat one or more may be used to modulate TGF - B - related proteins in the muscle disorders and /or injuries . In some cases , such com cornea to enable and / or enhance wound healing . Such com pounds and/ or composition may include, but are not limited pounds and /or compositions would be welcomed in the field 15 to antibodies that modulate GDF - 8 , GDF - 11 and /or activin where previous attempts have been unsuccessful. Mead et al activity . Muscle comprises about 40 - 50 % of total body (Mead , A . L . et al ., Evaluation of anti - TGF -beta2 antibody weight, making it the largest organ in the body. Muscle as a new postoperative anti - scarring agent in glaucoma disorders may include cachexia ( e . g . muscle wasting . ) surgery . Invest Ophthalmol Vis Sci. 2003 August ; 44( 8 ) : Muscle wasting may be associated with a variety of diseases 3394 -401 ) developed anti- TGF -B2 antibodies to prevent 20 and catabolic disorders ( e . g . HIV /AIDS , cancer, cancer scarring in eye tissues; however, results of clinical trials cachexia , renal failure , congestive heart failure , muscular were inconclusive . In some embodiments , compounds and dystrophy , disuse atrophy , chronic obstructive pulmonary or compositions of the present invention may be used to disease , motor neuron disease , trauma, neurodegenerative modulate TGF -B2 levels ( free versus GPC - bound ) thereby disease , infection , rheumatoid arthritis , immobilization , dia providing an alternate method of approaching anti - scarring 25 betes, etc . ) In such disorders , GDF - 8 and /or activin signal therapy . ing activity may contribute to muscle catabolism (Han et al. , Therapeutics for Cardiovascular Indications 2013 . Int J Biochem Cell Biol. 45 ( 10 ) : 2333 - 47 ; Lee . , 2010 . In some embodiments, compounds and /or compositions Immunol Endocr Metab Agents Med Chem . 10 : 183 - 94 , the of the present invention may be used to treat one or more contents of each of which are herein incorporated by refer cardiovascular indications, including, but not limited to 30 ence in their entirety .) Other muscle disorders may comprise cardiac hypertrophy . Cardiac hypertrophy comprises sarcopenia . Sarcopenia is the progressive loss of muscle and enlargement of the heart due , typically due to increased cell function associated with aging . In the elderly , sarcopenia can volume of cardiac cells (Aurigemma 2006 . N Engl J Med . cause frailty , weakness , fatigue and loss ofmobility (Morely . 355 ( 3 ) : 308 - 10 . ) Age - related cardiac hypertrophy may be 2012 . Family Practice. 29 : 144 - 148 . ) With the aged popula due , in part, to reduced circulating levels ofGDF - 11. A study 35 tion increasing in numbers, sarcopenia is progressively by Loffredo et al (Loffredo et al. , 2013 . Cell . 153 : 828 -39 ) becoming a more serious public health concern . A study by found that fusion of the circulatory system between young Hamrick et al (Hamrick et al. , 2010 . 69( 3 ): 579 - 83 ) demon and old mice had a protective effect with regard to cardiac strated that GDF - 8 inhibition could repair muscle in a mouse hypertrophy. The study identified GDF - 11 as a circulating model of fibula osteotomoy comprising lateral compartment factor that decreased with age in mice and was able to show 40 muscle damage . Administration of GDF - 8 propeptides was that its administration could also reduce cardiac hypertro - sufficient to increase muscle mass by nearly 20 % as well as phy. Some compounds and / or compositions of the present improve fracture healing. Some compounds and / or compo invention may be used to treat and / or prevent cardiac sitions of the present invention may be used to treat muscle atrophy. Such compounds and /or compositions may com diseases, disorders and/ or injuries by modulating GDF - 8 prise GDF - 11 agonists that elevate levels of circulating 45 activity . In some cases , compounds of the present invention GDF - 11, in some cases through enhancing the dissociation may beGDF - 8 signaling antagonists , preventing or reducing of GDF - 11 growth factor from latent GPCs. GDF- 8 signaling activity . In some embodiments , compositions and methods of the Inclusion body myositis ( IBM ) is a disease characterized invention may be used to treat one or more types of arterial by progressive muscle loss, typically occurring in mid - to disorders. Such disorders may include , but are not limited to 50 late - life . The disease is thought to occur due to an autoim the development of aortic aneurysms. Aortic aneurysmsmay m une response to autoantigens in the muscle causing T -cell arise from a variety of causes , but most result ultimately in invasion of the muscle fiber and resulting in myofiber the overexpression of TGF -B2 . A study by Boileau et al destruction (Greenberg 2012 . Curr Opin Neurol. 25 ( 5 ) :630 ( Boileau et al. , Nature Genetics Letters . 2012 . 44 ( 8 ) : 916 - 23 , 9 . ) Therapeutic compounds are being investigated , including the contents of which are herein incorporated by reference in 55 Bimagrumab (BYM338 ; Novartis , Basel, Switzerland ) an their entirety ) uncovered causative mutations in TGF -B2 that antibody that targets type II activin receptors , preventing were associated with some inherited forms of susceptibility GDF - 8 and /or activin signal transduction , thereby stimulat to thoracic aortic disease . Interestingly , although the muta ing muscle production and strengthening ( see clinical trial tions were predicted to cause haploinsufficiency for TGF - B2 , number NCT01925209 entitled Efficacy and Safety of the aortic tissues of individuals with such mutations com - 60 Bimagrumab /BYM338 at 52 Weeks on Physical Function , prised increased levels of TGF - B2 , as determined by immu- Muscle Strength , Mobility in sIBM Patients (RESILIENT ) ] nostaining . Similar findings were found in aortic tissues Some compounds and /or compositions of the present inven from individuals suffering from Marfans syndrome (Nataat - tion may be used to treat subjects with IBM . In some cases , madja et al. , 2006 . ) In some cases , compounds and / or such compounds and / or compositions may block GDF - 8 compositions of the present invention may be used to reduce 65 activity ( e . g . through stabilization of GDF - 8 GPCs. ) In or prevent elevated TGF - B2 signaling in such instances addition to IBM , BYM338 is being investigated for treat thereby limiting aneurysm development and /or progression . ment of chronic obstructive pulmonary disease (COPD .) In US 9 ,758 , 577 B2 135 136 some cases , compounds and / or compositions of the present human vertebrate ” refers to any vertebrate with the excep invention utilized for IBM treatment, may be used to treat tion of humans (i . e . Homo sapiens ). Exemplary non -human COPD as well . In some cases , compounds and /or composi - vertebrates include wild and domesticated species such as tions of the present invention may be administered in companion animals and livestock . Livestock include domes combination and /or coordination with BYM338 . 5 ticated animals raised in an agricultural setting to produce Therapeutics for Diabetes materials such as food , labor, and derived products such as Skeletal muscle uses and stores glucose for fuel . Due to fiber and chemicals. Generally, livestock includes all mam this , skeletalmuscle is an important regulator of circulating mals , avians and fish having potential agricultural signifi glucose levels . Uptake of glucose by muscle can be stimu - cance . In particular , four -legged slaughter animals include lated by either contraction or by insulin stimulation 10 steers, heifers , cows, calves , bulls , cattle , swine and sheep . (McPherron et al. , 2013 . Adipocyte . 2 ( 2 ) : 92 - 8 , herein incor - Bioprocessing porated by reference in its entirety ). A recent study by Guo In some embodiments , the present invention provides et al (Guo , et al ., 2012 . Diabetes 61 ( 10 ) :2414 -23 ) found that methods for producing one or more biological products in when GDF - 8 receptor -deficient mice were crossed with host cells by contacting such cells with compounds and /or A - ZIP /F1 mice ( a lipodistrophic mouse strain , used as a 15 compositions of the present invention capable ofmodulating diabetic model) hybrid off - spring showed reduced levels of expression of target genes, or altering the level of growth blood glucose and improved sensitivity to insulin . Hyper - factor signaling molecules wherein such modulation or phagia ( excessive eating ) was also reduced in these mice . In alteration enhances production of biological products . some embodiments , compound and /or compositions of the According to the present invention , bioprocessing methods present invention may be used to treat diabetes and/ or 20 may be improved by using one or more compounds and/ or hyperphagia . Some such treatments may be used to reduce compositions of the present invention . They may also be blood glucose and / or improve insulin sensitivity . In some improved by supplementing , replacing or adding one or cases , such treatments may comprise GDF - 8 signaling more compounds and / or compositions . antagosists, such as one or more antibodies that prevent Pharmaceutical Compositions dissociation ofGDF -8 from its prodomain . 25 The pharmaceutical compositions described herein may Therapeutics for Gastro - Intestinal Diseases , Disorders and be characterized by one or more of bioavailability , thera or Conditions peutic window and / or volume of distribution . In some embodiments , compositions and methods of the Bioavailability invention may be used to treat one or more types of In some embodiments, pharmaceutical compositions gastro - intestinal (GI ) disorders . Such disorders may include, 30 comprise complexes of compounds and / or compositions of but are not limited to inflammatory bowel disease (IBD ) the present invention with GPCs. In such embodiments , ( e . g . Crohn ' s disease and ulcerative colitis . ) complexes may be implanted at desired therapeutic sites TGF - B2 may play a role in gut homeostasis and may have where steady dissociation of growth factors from complexes an anti - inflammatory role , protecting against GI- related dis may occur over a desired period of time. In some embodi orders such as mucositis and certain forms of colitis . In one 35 ments , implantation complexes may be carried out in asso study, TGF - B2 was shown to suppress macrophage inflam - ciation with sponge and /or bone- like matrices . Such implan matory responses in the developing intestine and protect tations may include , but are not limited to dental implant against inflammatory mucosal injury (Maheshwari et al. , sites and / or sites of bone repair . 2011. ) Interestingly , levels of TGF - B2 are high in breast In some embodiments , compounds and/ or compositions milk , suggesting that TGF - B2 may function , in some cases, 40 of the present invention are made in furin -deficient cells . topically . Indeed , TGF -B2 in breast milk may attenuate GPCs produced in such cells may be useful for treatment in inflammatory responses (Rautava et al ., 2011. ) Some com areas where release is slowed due to the fact that furin pounds, compositions and /or methods of the present inven - cleavage in vivo is rate -limiting during GPC processing . In tion may be used to modulate GI TGF - B2 levels and / or some embodiments , one or more tolloid and/ or furin sites in activity in the maintenance of homeostasis and / or in the 45 GPCs are mutated , slowing the action of endogenous tolloid management of GI- related disorders . and / or furin proteases . In such embodiments , growth factor In some cases, models of Gl- related diseases , disorders release may be slowed ( e .g . at sites of implantation . ) and / or conditions may be used to develop and / or test com - Antibodies of the present invention , when formulated into pounds and /or compositions of the invention for treatment of compositions with delivery / formulation agents or vehicles GI- related diseases , disorders and / or conditions . In some 50 as described herein , may exhibit increased bioavailability as cases , GI injury models may be used . Such injury models compared to compositions lacking delivery agents as may include, but are not limited to 2 ,4 , 6 - trinitrobenzenesul described herein . As used herein , the term “ bioavailability " fonic acid ( TNBS ) induced colitis models . Such models may refers to the systemic availability of a given amount of a be carried out as described in Scheiffele, F. et al ., 2002 . Curr particular agent administered to a subject. Bioavailability Protoc Immunol. Chapter 15 :Unit 15 . 19 , the contents of 55 may be assessed by measuring the area under the curve which are herein incorporated by reference in their entirety . (AUC ) or the maximum serum or plasma concentration Veterinary Applications (Cmor ) of the unchanged form of a compound following In some embodiments , it is contemplated that composi - administration of the compound to a mammal . AUC is a tions and methods of the invention will find utility in the area determination of the area under the curve plotting the serum of veterinary care including the care and treatment of 60 or plasma concentration of a compound along the ordinate non -human vertebrates . As described herein , the term “ ver - ( Y -axis ) against time along the abscissa ( X -axis ) . Generally , tebrate ” includes all vertebrates including , but not limited to the AUC for a particular compound may be calculated using fish , amphibians, birds, reptiles and mammals ( including methods known to those of ordinary skill in the art and as but not limited to alpaca , banteng, bison , camel , cat , cattle , described in G . S . Banker , Modern Pharmaceutics, Drugs deer, dog , donkey, gayal, goat, guinea pig , horse , llama, 65 and the Pharmaceutical Sciences, v . 72 , Marcel Dekker, New mice , monkeys, mule , pig , rabbit , rats , reindeer, sheep water York , Inc . , 1996 , the contents of which are herein incorpo buffalo , yak and humans .) As used herein the term “ non - rated by reference in their entirety. US 9 , 758 ,577 B2 137 138 Cmax values are maximum concentrations of compounds decrease at least about 2 % , at least about 5 % , at least about achieved in serum or plasma of a subject following admin - 10 % , at least about 15 % , at least about 20 % , at least about istration of compounds to the subject . Cmax values of par 25 % , at least about 30 % , at least about 35 % , at least about ticular compounds may be measured using methods known 40 % , at least about 45 % , at least about 50 % , at least about to those of ordinary skill in the art. As used herein , the 5 55 % , at least about 60 % , at least about 65 % , at least about phrases “ increasing bioavailability ” or “ improving the phar- 70 % . macokinetics, " refer to actions that may increase the sys - Formulation , Administration , Delivery and Dosing temic availability of a compounds and/ or compositions of In some embodiments , compounds and / or compositions the present invention (as measured by AUC , Cor, or Cmin ) of the present invention are pharmaceutical compositions . in a subject. In some embodiments , such actions maymin 10 As used herein , the term “ pharmaceutical composition ” comprise co -administration with one or more delivery refers to a compound and /or composition of the present agents as described herein . In some embodiments, the bio - invention that has been formulated with one or more phar availability of compounds and/ or compositions may maceutically acceptable excipients . In some embodiments , increase by at least about 2 % , at least about 5 % , at least pharmaceutical compositions may optionally comprise one about 10 % , at least about 15 % , at least about 20 % , at least 15 or more additional active substances , e. g . therapeutically about 25 % , at least about 30 % , at least about 35 % , at least and /or prophylactically active substances. General consid about 40 % , at least about 45 % , at least about 50 % , at least erations in the formulation and / or manufacture of pharma about 55 % , at least about 60 % , at least about 65 % , at least ceutical agents may be found, for example , in Remington : about 70 % , at least about 75 % , at least about 80 % , at least The Science and Practice of Pharmacy 21st ed ., Lippincott about 85 % , at least about 90 % , at least about 95 % or about 20 Williams & Wilkins, 2005 (incorporated herein by refer 100 % . ence ) . Therapeutic Window In some embodiments , compositions may be administered Compounds and /or compositions of the present invention , to humans, human patients or subjects . For the purposes of when formulated with one or more delivery agents as the present disclosure , the phrase " active ingredient” gen described herein , may exhibit increases in the therapeutic 25 erally refers to compounds and / or compositions of the window of compound and /or composition administration as present invention to be delivered as described herein . compared to the therapeutic window of compounds and/ or Although the descriptions of pharmaceutical composi compositions administered without one or more delivery tions provided herein are principally directed to pharmaceu agents as described herein . As used herein , the term " thera - tical compositions which are suitable for administration to peutic window ” refers to the range of plasma concentrations , 30 humans , it will be understood by the skilled artisan that such or the range of levels of therapeutically active substance at compositions are generally suitable for administration to the site of action , with a high probability of eliciting a other subjects , e . g . , to non -human animals, e . g . non -human therapeutic effect. In some embodiments , therapeutic win - mammals . Modification of pharmaceutical compositions dows of compounds and /or compositions when co - admin suitable for administration to humans in order to render the istered with one or more delivery agent as described herein 35 compositions suitable for administration to various animals may increase by at least about 2 % , at least about 5 % , at least is well understood , and the ordinarily skilled veterinary about 10 % , at least about 15 % , at least about 20 % , at least pharmacologist can design and / or perform such modifica about 25 % , at least about 30 % , at least about 35 % , at least tion with merely ordinary , if any , experimentation . Subjects about 40 % , at least about 45 % , at least about 50 % , at least to which administration of pharmaceutical compositions is about 55 % , at least about 60 % , at least about 65 % , at least 40 contemplated include , but are not limited to , humans and / or about 70 % , at least about 75 % , at least about 80 % , at least other primates ; mammals, including commercially relevant about 85 % , at least about 90 % , at least about 95 % or about mammals such as cattle , pigs , horses , sheep , cats , dogs , 100 % . mice , and /or rats ; and / or birds , including commercially Volume of Distribution relevant birds such as poultry , chickens, ducks, geese , and / or Compounds and/ or compositions of the present invention , 45 turkeys . when formulated with one or more delivery agents as In some embodiments , formulations of the pharmaceuti described herein , may exhibit an improved volume of dis - cal compositions described herein may be prepared by any tribution ( V dist) , e . g ., reduced or targeted , relative to formu - method known or hereafter developed in the art of pharma lations lacking one or more delivery agents as described cology . In general, such preparatory methods include the herein . V dist relates the amount of an agent in the body to the 50 step of bringing active ingredients into association with concentration of the same agent in the blood or plasma. As excipients and /or one or more other accessory ingredients , used herein , the term “ volume of distribution ” refers to the and then , if necessary and /or desirable , dividing, shaping fluid volume that would be required to contain the total and /or packaging products into desired single - or multi - dose amount of an agent in the body at the same concentration as units . in the blood or plasma: V dis equals the amount of an agent 55 In some embodiments , pharmaceutical compositions of in the body / concentration of the agent in blood or plasma. the present invention may be prepared , packaged , and /or For example , for a 10 mg dose of a given agent and a plasma sold in bulk , as single unit doses, and /or as a plurality of concentration of 10 mg/ L , the volume of distribution would single unit doses. As used herein , the term “ unit dose ” refers be 1 liter. The volume of distribution reflects the extent to to a discrete amount of the pharmaceutical composition which an agent is present in the extravascular tissue . Large 60 comprising a predetermined amount of active ingredient . volumes of distribution reflect the tendency of agents to bind Amounts of active ingredient are generally equal to the to the tissue components as compared with plasma proteins . dosage of active ingredients which would be administered to In clinical settings , Vlis may be used to determine loading subjects and / or convenient fractions of such a dosages such doses to achieve steady state concentrations. In some as, for example , one -half or one -third of such a dosages . embodiments , volumes of distribution of compounds and / or 65 In some embodiments , relative amounts of active ingre compositions of the present invention when co - administered dients , pharmaceutically acceptable excipients , and /or any with one or more delivery agents as described herein may additional ingredients in pharmaceutical compositions of the US 9 , 758 ,577 B2 139 140 present invention may vary , depending upon identity , size , In some embodiments , pharmaceutically acceptable and / or condition of subjects to be treated and further excipients of the present invention may include , but are not depending upon routes by which compositions are to be limited to , inert diluents , dispersing and / or granulating administered . By way of example , compositions may com agents , surface active agents and /or emulsifiers , disintegrat prise between about 0 . 1 % and 100 % , e . g . , from about 0 . 5 % 5 ing agents , binding agents , preservatives , buffering agents , to about 50 % , from about 1 % to about 30 % , from about 5 % lubricating agents , and / or oils . Such excipients may option to about 80 % or at least 80 % ( w /w ) active ingredient. In ally be included in pharmaceutical compositions . some embodiments , active ingredients are antibodies Exemplary diluents include , but are not limited to , cal directed toward regulatory elements and /or GPCs. cium carbonate , sodium carbonate , calcium phosphate , Formulations 10 dicalcium phosphate , calcium sulfate , calcium hydrogen Compounds and /or compositions of the present invention phosphate , sodium phosphate lactose , sucrose , cellulose , may be formulated using one or more excipients to : ( 1 ) microcrystalline cellulose , kaolin , mannitol , sorbitol, inosi increase stability ; ( 2 ) increase cell permeability ; ( 3 ) permit tol, sodium chloride , dry starch , cornstarch , powdered sugar, the sustained or delayed release ( e . g ., of compounds and/ or etc . , and /or combinations thereof. growth factors from such formulations ) ; and /or ( 4 ) alter the 15 Exemplary granulating and / or dispersing agents include , biodistribution ( e . g . , target compounds to specific tissues or but are not limited to , potato starch , corn starch , tapioca cell types) . In addition to traditional excipients such as any starch , sodium starch glycolate , clays , alginic acid , guar and all solvents , dispersion media , diluents , liquid vehicles , gum , citrus pulp , agar, bentonite , cellulose and wood prod dispersion aids, suspension aids, surface active agents, iso - ucts , natural sponge , cation - exchange resins, calcium car tonic agents , thickening agents , emulsifying agents and 20 bonate , silicates, sodium carbonate , cross - linked poly (vinyl preservatives, formulations of the present invention may pyrrolidone ) ( crospovidone ), sodium carboxymethyl starch comprise, without limitation , liposomes , lipid nanoparticles, (sodium starch glycolate ), carboxymethyl cellulose, cross polymers, lipoplexes, core - shell nanoparticles, peptides , linked sodium carboxymethyl cellulose (croscarmellose ) , proteins , cells transfected with the compounds and/ or com - methylcellulose , pregelatinized starch ( starch 1500 ) , micro positions of the present invention ( e . g . , for transplantation 25 crystalline starch , water insoluble starch , calcium car into subjects ) and combinations thereof. boxymethyl cellulose , magnesium aluminum silicate Excipients (VEEGUM® ), sodium lauryl sulfate , quaternary ammonium Various excipients for formulating pharmaceutical com - compounds, etc ., and /or combinations thereof. positions and techniques for preparing the composition are Exemplary surface active agents and /or emulsifiers known in the art ( see Remington : The Science and Practice 30 include , but are not limited to , natural emulsifiers ( e . g . of Pharmacy, 21st Edition , A . R . Gennaro , Lippincott, Wil acacia , agar, alginic acid , sodium alginate , tragacanth , chon liams & Wilkins, Baltimore , Md. , 2006 ; incorporated herein drux , cholesterol , xanthan , pectin , gelatin , egg yolk , casein , by reference ). wool fat, cholesterol, wax , and lecithin ) , colloidal clays ( e . g . In some embodiments , the use of conventional excipient bentonite [aluminum silicate ) and VEEGUM® [magnesium media are contemplated within the scope of the present 35 aluminum silicate ]) , long chain amino acid derivatives , high disclosure, except insofar as any conventional excipient molecular weight alcohols ( e . g . stearyl alcohol, cetyl alco media may be incompatible with substances and /or their hol, oleyl alcohol, triacetin monostearate , ethylene glycol derivatives , such as by producing any undesirable biological distearate , glyceryl monostearate , and propylene glycol effects or otherwise interacting in deleterious manners with monostearate , polyvinyl alcohol) , carbomers (e .g . carboxy any other component( s) of pharmaceutical compositions. 40 polymethylene, polyacrylic acid , acrylic acid polymer , and Formulations of pharmaceutical compositions described carboxyvinyl polymer ) , carrageenan , cellulosic derivatives herein may be prepared by any method known or hereafter ( e . g . carboxymethylcellulose sodium , powdered cellulose , developed in the art of pharmacology . In general, such hydroxymethyl cellulose , hydroxypropyl cellulose , preparatory methods include steps of associating active hydroxypropyl methylcellulose , methylcellulose ) , sorbitan ingredients with excipients and / or other accessory ingredi- 45 fatty acid esters ( e . g . polyoxyethylene sorbitan monolaurate ents . [ TWEEN®20 ] , polyoxyethylene sorbitan [ TWEENn®60 ] , Pharmaceutical compositions, in accordance with the polyoxyethylene sorbitan monooleate [ TWEEN®80 ) , sorbi present disclosure , may be prepared , packaged , and /or sold tan monopalmitate [SPAN®40 ] , sorbitan monostearate in bulk , as single unit doses , and /or as a plurality of single [Span®60 ) , sorbitan tristearate [ Span®65 ] , glyceryl unit doses . 50 monooleate , sorbitan monooleate [ SPAN®80 ] ) , polyoxyeth Relative amounts of active ingredients , pharmaceutically ylene esters (e . g . polyoxyethylene monostearate acceptable excipients , and /or additional ingredients in phar- [MYRJ®45 ], polyoxyethylene hydrogenated castor oil, maceutical compositions of the present disclosure may vary, polyethoxylated castor oil , polyoxymethylene stearate , and depending upon identity , size , and /or condition of subjects SOLUTOL® ), sucrose fatty acid esters, polyethylene glycol being treated and further depending upon routes by which 55 fatty acid esters ( e . g . CREMOPHOR® ) , polyoxyethylene pharmaceutical compositions may be administered . ethers , ( e. g . polyoxyethylene lauryl ether [BRIJ®30 ]) , poly In some embodiments, pharmaceutically acceptable (vinyl - pyrrolidone ) , diethylene glycol monolaurate, trietha excipient are at least 95 % , at least 96 % , at least 97 % , at least nolamine oleate , sodium oleate, potassium oleate , ethyl 98 % , at least 99 % or 100 % pure . In some embodiments , oleate , oleic acid , ethyl laurate , sodium lauryl sulfate , excipients are approved for use in humans and /or for vet- 60 PLUORINC®F 68, POLOXAMER®188 , cetrimonium bro erinary use . In some embodiments, excipients are approved mide, cetylpyridinium chloride, benzalkonium chloride , by the United States Food and Drug Administration . In some docusate sodium , etc . and / or combinations thereof . embodiments, excipients are pharmaceutical grade . In some Exemplary binding agents include , but are not limited to , embodiments , excipients meet the standards of the United starch ( e . g . cornstarch and starch paste ) ; gelatin ; sugars ( e . g . States Pharmacopoeia (USP ) , the European Pharmacopoeia 65 sucrose , glucose, dextrose, dextrin , molasses, lactose , lacti (EP ) , the British Pharmacopoeia , and / or the International tol, mannitol) ; natural and synthetic gums ( e . g . acacia , Pharmacopoeia . sodium alginate , extract of Irish moss , panwar gum , ghatti US 9 , 758 ,577 B2 141 142 gum , mucilage of isapol husks , carboxymethylcellulose , pyrogen - free water , isotonic saline , Ringer ' s solution , ethyl methylcellulose , ethylcellulose, hydroxyethylcellulose , alcohol, etc ., and / or combinations thereof. hydroxypropyl cellulose , hydroxypropyl methylcellulose , Exemplary lubricating agents include, but are not limited microcrystalline cellulose , cellulose acetate , poly (vinyl - pyr - to , magnesium stearate , calcium stearate , stearic acid , silica , rolidone ), magnesium aluminum silicate (Veegum® ) , and 5 talc , malt , glyceryl behanate , hydrogenated vegetable oils , larch arabogalactan ); alginates; polyethylene oxide ; poly - polyethylene glycol, sodium benzoate , sodium acetate , ethylene glycol; inorganic calcium salts; silicic acid ; sodium chloride , leucine , magnesium lauryl sulfate , sodium polymethacrylates ; waxes ; water ; alcohol, etc . , and combi - lauryl sulfate , etc ., and combinations thereof. nations thereof. Exemplary oils include, but are not limited to , almond , Exemplary preservatives may include , but are not limited " apricot kernel, avocado , babassu , bergamot, black current to , antioxidants, chelating agents, antimicrobial preserva seed , borage, cade, camomile , canola , caraway , carnauba , tives, antifungal preservatives , alcohol preservatives, acidic castor, cinnamon , cocoa butter, coconut , cod liver , coffee , preservatives, and / or other preservatives . Exemplary anti - corn , cotton seed , emu, eucalyptus , evening primrose , fish , oxidants include , but are not limited to , alpha tocopherol, 16 flaxseed , geraniol , gourd , grape seed , hazel nut, hyssop , ascorbic acid , acorbyl palmitate , butylated hydroxyanisole , isopropyl myristate , jojoba, kukui nut, lavandin , lavender , butylated hydroxytoluene, monothioglycerol, potassium lemon , litsea cubeba , macademia nut, mallow , mango seed , metabisulfite , propionic acid , propyl gallate , sodium ascor - meadowfoam seed , mink , nutmeg , olive, orange , orange bate , sodium bisulfite , sodium metabisulfite , and / or sodium roughy, palm , palm kernel, peach kernel , peanut, poppy sulfite . Exemplary chelating agents include ethylenedi- 20 seed , pumpkin seed , rapeseed , rice bran , rosemary, saf aminetetraacetic acid ( EDTA ) , citric acid monohydrate , flower, sandalwood , sasquana, savoury , sea buckthorn , disodium edetate , dipotassium edetate , edetic acid , fumaric sesame, shea butter, silicone , soybean , sunflower, tea tree , acid , malic acid , phosphoric acid , sodium edetate , tartaric thistle , tsubaki, vetiver , walnut, and wheat germ oils . Exem acid , and /or trisodium edetate . Exemplary antimicrobial plary oils include , but are not limited to , butyl stearate , preservatives include , but are not limited to , benzalkonium 25 caprylic triglyceride , capric triglyceride , cyclomethicone , chloride, benzethonium chloride , benzyl alcohol, bronopol, diethyl sebacate , dimethicone 360 , isopropylmyristate , min cetrimide , cetylpyridinium chloride , chlorhexidine, chlo eral oil, octyldodecanol, oleyl alcohol, silicone oil, and /or robutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin , hexetidine , imidurea , phenol , phenoxyethanol, combinations thereof. phenylethyl alcohol, phenylmercuric nitrate , propylene gly - 30 coloringExcipients ager such as cocoa butter and suppository waxes, col , and /or thimerosal. Exemplary antifungal preservatives coloring agents , coating agents , sweetening, flavoring, and / include , but are not limited to , butyl paraben , methyl para or perfuming agents can be present in the composition , ben , ethyl paraben , propyl paraben , benzoic acid , hydroxy according to the judgment of the formulator. benzoic acid , potassium benzoate , potassium sorbate , Formulation Vehicles : Liposomes, Lipoplexes , and Lipid sodium benzoate , sodium propionatehate , andand //or or sorbic acid . 3535 NanoparticlesNa Exemplary alcohol preservatives include , but are not limited Compounds and / or compositions of the present invention to , ethanol , polyethylene glycol, phenol, phenolic com - may be formulated using one or more liposomes, lipoplexes pounds, bisphenol, chlorobutanol, hydroxybenzoate , and /or and /or lipid nanoparticles. In some embodiments , pharma phenylethyl alcohol. Exemplary acidic preservatives ceutical compositions comprise liposomes . Liposomes are include , but are not limited to , vitamin A , vitamin C , vitamin 40 artificially -prepared vesicles which may primarily be com E , beta -carotene , citric acid , acetic acid , dehydroacetic acid , posed of a lipid bilayer and may be used as delivery vehicles ascorbic acid , sorbic acid , and /or phytic acid . Other preser for the administration of nutrients and pharmaceutical for vatives include, but are not limited to , tocopherol, tocoph mulations . Liposomes may be of different sizes such as , but erol acetate, deteroxime mesylate , cetrimide, butylated not limited to , multilamellar vesicles (MLVs ) which may be hydroxyanisol (BHA ), butylated hydroxytoluened (BHT ) , 45 hundreds of nanometers in diameter and may contain a ethylenediamine , sodium lauryl sulfate ( SLS ) , sodium lauryl series of concentric bilayers separated by narrow aqueous ether sulfate (SLES ) , sodium bisulfite , sodium metabisulfite , compartments , small unicellular vesicle ( SUVs) which may potassium sulfite , potassium metabisulfite , GLYDANT be smaller than 50 nm in diameter and large unilamellar PLUS® , PHENONIP® , methylparaben , GERMALL®115 , vesicle (LUVs ) which may be between 50 and 500 nm in GERMABEN?II, NEOLONETM KATHONTM , and / or 50 diameter. Liposome components may include , but are not EUXYL® . limited to , opsonins or ligands in order to improve the Exemplary buffering agents include , but are not limited attachment of liposomes to unhealthy tissue or to activate to , citrate buffer solutions , acetate buffer solutions , phos - events such as, but not limited to , endocytosis . Liposomes phate buffer solutions, ammonium chloride, calcium carbon - may comprise low or high pH . In some embodiments , ate , calcium chloride , calcium citrate , calcium glubionate , 55 liposome pH may be varied in order to improve delivery of calcium gluceptate , calcium gluconate , D - gluconic acid , pharmaceutical formulations. calcium glycerophosphate , calcium lactate , propanoic acid , In some embodiments , liposome formation may depend calcium levulinate , pentanoic acid , dibasic calcium phos - on physicochemical characteristics such as, but not limited phate, phosphoric acid , tribasic calcium phosphate , calcium to , the pharmaceutical formulation entrapped , liposomal hydroxide phosphate , potassium acetate , potassium chlo - 60 ingredients , the nature of themedium in which lipid vesicles ride, potassium gluconate , potassium mixtures , dibasic are dispersed , the effective concentration of entrapped sub potassium phosphate , monobasic potassium phosphate , stances , potential toxicity of entrapped substances, addi potassium phosphate mixtures , sodium acetate , sodium t ional processes involved during the application and / or bicarbonate , sodium chloride , sodium citrate , sodium lac delivery of vesicles , optimization size , polydispersity , shelf tate , dibasic sodium phosphate , monobasic sodium phos - 65 life of vesicles for the intended application , batch - to -batch phate , sodium phosphate mixtures , tromethamine , magne - reproducibility and possibility of large -scale production of sium hydroxide , aluminum hydroxide, alginic acid , safe and efficient liposomal products . US 9 ,758 ,577 B2 143 144 In some embodiments , formulationsmay be assembled or in vivo delivery of siRNA to hepatocytes. Proc Natl Acad compositions altered such that they are passively or actively Sci USA . 2007 104 : 12982 - 12887; Davis , M . E . et al. , The directed to different cell types in vivo . first targeted delivery of siRNA in humans via a self In some embodiments , formulations may be selectively assembling , cyclodextrin polymer- based nanoparticle : from targeted through expression of different ligands on formu - 5 concept to clinic . Mol Pharm . 2009 6 :659 -668 ; Davis , M . E . lation surfaces as exemplified by , but not limited by, folate , et al. , Evidence of RNAi in humans from systemically transferrin , N - acetylgalactosamine (GalNAc ), and antibody administered siRNA via targeted nanoparticles. Nature . targeted approaches . 2010 . 464 : 1067 -70 ; the contents of each of which are herein In some embodiments, pharmaceutical compositions of incorporated by reference in their entirety . ) the present invention may be formulated with liposomes , 10 Compounds and / or compositions of the present invention lipoplexes and / or lipid nanoparticles to improve efficacy of may be formulated as nanoparticles using combinations of function . Such formulations may be able to increase cell polymers , lipids , and /or other biodegradable agents , such as , transfection by pharmaceutical compositions . In some but not limited to , calcium phosphates. In some embodi embodiments , liposomes, lipoplexes , or lipid nanoparticles ments , components may be combined in core - shells , may be used to increase pharmaceutical composition stabil - 15 hybrids, and/ or layer- by - layer architectures, to allow for ity . fine - tuning of nanoparticle structure , so delivery may be In some embodiments , liposomes are specifically formu enhanced . For antibodies of the present invention , systems lated for pharmaceutical compositions comprising one or based on poly ( 2 - (methacryloyloxy ) ethyl phosphorylcho more antibodies . Such liposomesmay be prepared according line ) -block - ( 2 - ( diisopropylamino ) ethyl m ethacrylate ) , to techniques known in the art, such as those described by 20 (PMPC - PDPA ) , a pH sensitive diblock copolymer that self Eppstein et al . ( Eppstein , D . A . et al. , Biological activity of assembles to form nanometer -sized vesicles , also known as liposome- encapsulated murine interferon gamma is medi- polymersomes , at physiological pH may be used . These ated by a cell membrane receptor. Proc Natl Acad Sci USA . polymersomes have been shown to successfully deliver 1985 June; 82 ( 11 ) : 3688 - 92 ) ; Hwang et al. (Hwang , K . J . et relatively high antibody payloads within live cells. (Mas al ., Hepatic uptake and degradation of unilamellar sphingo - 25 signani, M . et al ., Cellular delivery of antibodies : effective myelin / cholesterol liposomes: a kinetic study . Proc Natl targeted subcellular imaging and new therapeutic tool. Acad Sci USA . 1980 July ; 77 ( 7 ) :4030 - 4 ) ; U . S . Pat. No. Nature Proceedings. 2010 . p 1 - 17 . ) 4 ,485 ,045 and U . S . Pat. No . 4 ,544 , 545 . Production of lipo - In some embodiments , PEG - charge - conversional poly somes with sustained circulation time are also described in mers (Pitella , F . et al. , Enhanced endosomal escape of U . S . Pat . No. 5 ,013 ,556 . 30 siRNA - incorporating hybrid nanoparticles from calcium In some embodiments, liposomes of the present invention phosphate and PEG - block charge - conversional polymer for comprising antibodies may be generated using reverse phase efficient gene knockdown with negligible cytotoxicity . Bio evaporation utilizing lipids such as phosphatidylcholine , materials. 2011 32 :3106 - 14 ) may be used to form nanopar cholesterol as well as phosphatidylethanolamine that have ticles for delivery of compounds and / or compositions of the been polyethylene glycol- derivatized . Filters with defined 35 present invention . In some embodiments, PEG - charge - con pore size are used to extrude liposomes of the desired versional polymers may improve upon PEG - polyanion diameter. In another embodiment, compounds and / or com - block copolymers by being cleaved into polycations at positions of the present invention may be conjugated to acidic pH , thus enhancing endosomal escape . external surfaces of liposomes by disulfide interchange In some embodiments , complexation , delivery and /or reactions as is described by Martin et al. (Martin , F . J . et al. , 40 internalization of polymeric nanoparticles may be precisely Irreversible coupling of immunoglobulin fragments to pre controlled by altering chemical compositions in both core formed vesicles . An improved method for liposome target and shell nanoparticle components (Siegwart , D . J . et al. , ing . J Biol Chem . 1982 Jan . 10 ; 257 (1 ): 286 - 8 ) . Combinatorial synthesis of chemically diverse core- shell Formulation Vehicles : Polymers and Nanoparticles nanoparticles for intracellular delivery . Proc Natl Acad Sci Compounds and / or compositions of the present invention 45 USA . 2011 108 : 12996 - 3001 ) . may be formulated using natural and / or synthetic polymers . In someembodiments , matrices of poly ( ethylene - co - vinyl Non - limiting examples of polymers which may be used for acetate ) , are used to deliver compounds and / or compositions delivery include, but are not limited to DMRI/ DOPE , polox of the invention . Such matrices have bee described by others amer, chitosan , cyclodextrin , and poly ( lactic - co - glycolic (Sherwood , J . K . et al. , Controlled antibody delivery sys acid ) ( PLGA ) polymers . In some embodiments , polymers 50 tems. Nature Biotechnology . 1992 . 10 : 1446 - 9 . ) may be biodegradable . AllyAntibody Formulations In some embodiments , polymer formulation may permit Antibodies of the present invention may be formulated for sustained and / or delayed release of compounds and / or com - intravenous administration or extravascular administration positions ( e . g ., following intramuscular and / or subcutaneous (Daugherty , et al. , Formulation and delivery issues for injection ). Altered release profile for compounds and /or 55 monoclonal antibody therapeutics . Adv Drug Deliv Rev . compositions of the present invention may result in , for 2006 Aug . 7 ; 58 (5 - 6 ) :686 -706 and US patent application example , compound release over an extended period of time. publication number US2011/ 0135570 , the contents of each Polymer formulations may also be used to increase the of which are herein incorporated by reference in their stability of compounds and / or compositions of the present entirety ) . Extravascular administration routes may include , invention . 60 but are not limited to subcutaneous administration , intrap In some embodiments , polymer formulations may be eritoneal administration , intracerebral administration , selectively targeted through expression of different ligands intraocular administration , intralesional administration , as exemplified by, but not limited by, folate , transferrin , and topical administration and intramuscular administration . N - acetylgalactosamine (GalNAc ) (Benoit , D . S . et al ., Syn - In some embodiments , antibody structures may be modi thesis of folate -functionalized RAFT polymers for targeted 65 fied to improve effectiveness as therapeutics. Improvements siRNA delivery . Biomacromolecules . 2011 12 : 2708 - 14 ; may include , but are not limited to improved thermody Rozema, D . B . et al. , Dynamic polyconjugates for targeted namic stability , reduced Fc receptor binding properties and / US 9 ,758 ,577 B2 145 146 or imporved folding efficiency. Modifications may include , of functional proteins into mammalian cells in vitro and in but are not limited to amino acid substitutions , glycosy - vivo using a supercharged protein . ACS Chem Biol . 2010 . lation , palmitoylation and / or protein conjugation . 5 :747 -52 ; McNaughton , B . R . et al ., Mammalian cell pen In some embodiments, antibodies of the present invention etration , siRNA transfection , and DNA transfection by may be formulated with antioxidants to reduce antibody 5 supercharged proteins . Proc Natl Acad Sci, USA . 2009 . oxidation . Antibodies of the present invention may also be 106 :6111 -6 ; Verdine , G . L . et al. , Stapled peptides for formulated with additives to reduce protein aggregation . intracellular drug targets. Methods Enzymol. 2012 . 503 : 3 Such additives may include , but are not limited to albumin , 33 ; the contents of each of which are herein incorporated by amino acids, sugars , urea , guanidinium chloride , polyalcho - reference in their entirety ) . hols , polymers ( such as polyethylene glycol and dextrans ) , 10 In some embodiments , the cell- penetrating polypeptides surfactants ( including , but not limited to polysorbate 20 and may comprise first and second domains . First domains may polysorbate 80 ) or even other antibodies . comprise supercharged polypeptides . Second domains may In some embodiments , antibodies of the present invention comprise protein -binding partner. As used herein , protein may be formulated to reduce the impact ofwater on antibody binding partners may include , but are not limited to , anti structure and function . Antibody preparartions in such for - 15 bodies and functional fragments thereof, scaffold proteins mulations may be may be lyophilized . Formulations subject and / or peptides . Cell - penetrating polypeptides may further to lyophilization may include carbohydrates or polyol com comprise intracellular binding partners for protein - binding pounds to protect and / or stabilize antibody structure . Such partners . In some embodiments , cell - penetrating polypep compounds may include , but are not limited to sucrose , tides may be capable of being secreted from cells where trehalose and mannitol . 20 compounds and /or compositions of the present invention In some embodiments , antibodies of the present invention may be introduced . may be formulated with polymers . In some embodiments , Compositions of the present invention comprising pep polymer formulations may comprise hydrophobic polymers . tides and / or proteins may be used to increase cell transfec Such polymers may be microspheres formulated with poly - tion and / or alter compound / composition biodistribution lactide - co - glycolide through solid - in - oil - in -water encapsu - 25 ( e . g . , by targeting specific tissues or cell types ) . lation methods . In some embodiments , microspheres com - Formulation Vehicles : Cells prising ethylene - vinyl acetate copolymer may also be used Cell- based formulations of compounds and / or composi for antibody delivery and /or to extend the time course of tions of the present invention may be used to ensure cell antibody release at sites of delivery . In some embodiments , transfection ( e . g . , in cellular carriers ) or to alter biodistri polymers may be aqueous gels . Such gels may , for example , 30 bution ( e . g ., by targeting cell carriers to specific tissues or comprise carboxymethylcellulose. In some embodiments , cell types. ) aqueous gels may also comprise hyaluronic acid hydrogels . Cell Transfer Methods In some embodiments , antibodies may be covalently linked A variety of methods are known in the art and suitable for to such gels through hydrazone linkages that allow for introduction of nucleic acids or proteins into cells , including sustained delivery in tissues , including but not limited to 35 viral and non - viral mediated techniques . Examples of typi tissues of the central nervous system . cal non -viral mediated techniques include , but are not lim Formulation Vehicles : Peptides and Proteins ited to , electroporation , calcium phosphate mediated trans Compounds and / or compositions of the present invention fer , nucleofection , sonoporation , heat shock , may be formulated with peptides and / or proteins . In some magnetofection , liposomemediated transfer, microinjection , embodiments, peptides such as, but not limited to , cell 40 microproj ectile mediated transfer (nanoparticles ) , cationic penetrating peptides and / or proteins/ peptides that enable polymer mediated transfer (DEAE - dextran , polyethylen intracellular delivery may be used to deliver pharmaceutical imine , polyethylene glycol (PEG ) and the like ) or cell formulations. Non - limiting examples of a cell penetrating fusion . peptides which may be used with pharmaceutical formula - The technique of sonoporation , or cellular sonication , is tions of the present invention include cell - penetrating pep - 45 the use of sound ( e . g . , ultrasonic frequencies ) for modifying tide sequences attached to polycations that facilitates deliv - the permeability of cell plasma membranes . Sonoporation ery to the intracellular space, e . g ., HIV -derived TAT peptide , methods are known to those in the art and are used to deliver penetratins , transportans, or hCT derived cell -penetrating nucleic acids in vivo ( Yoon , C . S . et al. , Ultrasound peptides ( see , e . g . Caron , N . J . et al. , Intracellular delivery of mediated gene delivery . Expert Opin Drug Deliv . 2010 a Tat -eGFP fusion protein into muscle cells . Mol Ther. 2001 . 50 7 : 321 - 30 ; Postema, M . et al ., Ultrasound - directed drug 3 ( 3 ) :310 - 8 ; Langel , U . , Cell - Penetrating Peptides: Processes delivery . Curr Pharm Biotechnol. 2007 8 : 355 -61 ; Newman , and Applications, CRC Press, Boca Raton Fla ., 2002; El C . M . et al ., Gene therapy progress and prospects : ultra Andaloussi, S . et al ., Cell- penetrating peptides :mechanisms sound for gene transfer. Gene Ther. 2007 . 14 (6 ): 465 -75 ; the and applications . Curr Pharm Des . 2003. 11 ( 28 ): 3597 -611 ; contents of each of which are herein incorporated by refer and Deshayes, S . et al ., Cell -penetrating peptides : tools for 55 ence in their entirety ). Sonoporation methods are known in intracellular delivery of therapeutics . Cell Mol Life Sci. the art and are also taught for example as they relate to 2005 . 62 ( 16 ) : 1839 - 49 , the contents of each of which are bacteria in US Patent application publication US2010 / herein incorporated by reference in their entirety . ) Com - 0196983 and as it relates to other cell types in , for example , pounds and /or compositions of the present invention may US Patent application publication US2010 / 0009424 , the also be formulated to include cell penetrating agents , e . g . , 60 contents of each of which are incorporated herein by refer liposomes, which enhance delivery of the compositions to ence in their entirety . intracellular spaces. Compounds and /or compositions of the Electroporation techniques are also well known in the art present invention may be complexed with peptides and /or and are used to deliver nucleic acids in vivo and clinically proteins such as , but not limited to , peptides and/ or proteins (Andre , F . M . et al ., Nucleic acids electrotransfer in vivo : from Aileron Therapeutics ( Cambridge , Mass. ) and 65 mechanisms and practical aspects . Curr Gene Ther. 2010 Permeon Biologics (Cambridge , Mass . ) in order to enable 10 : 267 - 80 ; Chiarella , P . et al. , Application of electroporation intracellular delivery (Cronican , J . J . et al. , Potent delivery in DNA vaccination protocols . Curr Gene Ther. 2010 . US 9 , 758 ,577 B2 147 148 10 : 281 -6 ; Hojman , P ., Basic principles and clinical advance wetting agents , and / or suspending agents . Sterile injectable ments of muscle electrotransfer. Curr Gene Ther. 2010 preparations may be sterile injectable solutions , suspen 10 : 128 - 38 ; the contents of each of which are herein incor - sions, and /or emulsions in nontoxic parenterally acceptable porated by reference in their entirety ) . In some embodi diluents and / or solvents, for example , as a solution in ments , compounds and / or compositions of the present 5 1 , 3 - butanediol. Among the acceptable vehicles and solvents invention may be delivered by electroporation . that may be employed are water , Ringer' s solution , U . S . P ., Administration and Delivery and isotonic sodium chloride solution . Sterile , fixed oils are Compounds and / or compositions of the present invention conventionally employed as a solvent or suspending may be administered by any of the standard methods or routes known in the art. Such methods may include any 10 medium . For this purpose any bland fixed oil can be route which results in a therapeutically effective outcome . employed including synthetic mono - or diglycerides. Fatty These include , but are not limited to enteral, gastroenteral, acids such as oleic acid can be used in the preparation of epidural, oral, transdermal, epidural (peridural ) , intracere injectables . bral ( into the cerebrum ) , intracerebroventricular ( into the Injectable formulations may be sterilized , for example , by cerebral ventricles ), epicutaneous ( applicationplication onto thethe 1515 Tiltfiltration through a bacterial - retaining filter, and /or by incor skin ) , intradermal, ( into the skin itself ) , subcutaneous (under porating sterilizing agents in the form of sterile solid com the skin ) , nasal administration ( through the nose ) , intrave - positions which can be dissolved or dispersed in sterile nous ( into a vein ) , intraarterial ( into an artery ) , intramuscu water or other sterile injectable medium prior to use . lar ( into a muscle ), intracardiac ( into the heart ) , intraosseous In order to prolong the effect of active ingredients , it is infusion ( into the bone marrow ), intrathecal ( into the spinal 20 often desirable to slow the absorption of active ingredients canal) , intraperitoneal, ( infusion or injection into the peri from subcutaneous or intramuscular injections . This may be toneum ) , intravesical infusion , intravitreal, ( through the accomplished by the use of liquid suspensions of crystalline eye ), intracavernous injection , into the base of the penis ), or amorphous material with poor water solubility . The rate intravaginal administration , intrauterine , extra -amniotic of absorption of active ingredients depends upon the rate of administration , transdermal (diffusion through the intact 25 dissolution which , in turn , may depend upon crystal size and skin for systemic distribution ), transmucosal (diffusion crystalline form . Alternatively , delayed absorption of a par through a mucous membrane ), insufflation ( snorting ), sub - enterally administered drug form is accomplished by dis lingual, sublabial, enema, eye drops (onto the conjunctiva ), solving or suspending the drug in an oil vehicle . Injectable or in ear drops. In specific embodiments , compounds and / or compositions of the present invention may be administered 30 depot forms are made by forming microencapsule matrices in ways which allow them to cross the blood -brain barrier, of the drug in biodegradable polymers such as polylactide vascular barriers , or other epithelial barriers . Methods of polyglycolide . Depending upon the ratio of drug to polymer formulation and administration may include any of those and the nature of the particular polymer employed , the rate disclosed in US Pub . No . 2013 /0122007 , U . S . Pat. No . of drug release can be controlled . Examples of other biode 8 . 415 . 459 or International Pub . No. WO 2011/ 151432 . the 35 gradable polymers include poly (orthoesters ) and poly ( anhy contents of each of which are herein incorporated by refer drides) . Depot injectable formulations are prepared by ence in their entirety. Non - limiting routes of administration entrapping the drug in liposomes or microemulsions which for compounds and /or compositions of the present invention are compatible with body tissues . are described below . Rectal and Vaginal Administration Parenteral and Injectable Administration 40 In some embodiments , compounds and /or compositions In some embodiments , compounds and /or compositions of the present invention may be administered rectally and /or of the present invention may be administered parenterally . vaginally . Compositions for rectal or vaginal administration Liquid dosage forms for oral and parenteral administration are typically suppositories which can be prepared by mixing include , but are not limited to , pharmaceutically acceptable compositions with suitable non - irritating excipients such as emulsions, microemulsions , solutions, suspensions , syrups , 45 cocoa butter, polyethylene glycol or a suppository wax and / or elixirs . In addition to active ingredients , liquid dosage which are solid at ambient temperature but liquid at body formsmay comprise inert diluents commonly used in the art temperature and therefore melt in the rectum or vaginal such as , for example , water or other solvents , solubilizing cavity and release the active ingredient. agents and emulsifiers such as ethyl alcohol, isopropyl Oral Administration alcohol, ethyl carbonate , ethyl acetate , benzyl alcohol, ben - 50 In some embodiments , compounds and/ or compositions zyl benzoate , propylene glycol, 1 , 3 -butylene glycol, dim - of the present invention may be administered orally . Solid ethylformamide , oils ( in particular , cottonseed , groundnut, dosage forms for oral administration include capsules, tab corn , germ , olive, castor, and sesame oils) , glycerol, tetra - lets, pills , powders , and granules . In such solid dosage hydrofurfuryl alcohol, polyethylene glycols and fatty acid forms, an active ingredient is mixed with at least one inert, esters of sorbitan , and mixtures thereof. Besides inert 55 pharmaceutically acceptable excipient such as sodium cit diluents , oral compositions can include adjuvants such as rate or dicalcium phosphate and /or fillers or extenders ( e . g . wetting agents , emulsifying and suspending agents, sweet- starches, lactose , sucrose , glucose , mannitol, and silicic ening , flavoring , and / or perfuming agents . In certain acid ) , binders ( e . g . carboxymethylcellulose , alginates , gela embodiments for parenteral administration , compositions tin , polyvinylpyrrolidinone , sucrose, and acacia ) , humec are mixed with solubilizing agents such as CREMO - 60 tants ( e . g . glycerol) , disintegrating agents ( e . g . agar, calcium PHOR® , alcohols , oils , modified oils , glycols , polysorbates , carbonate , potato or tapioca starch , alginic acid , certain cyclodextrins, polymers , and /or combinations thereof . In silicates, and sodium carbonate ) , solution retarding agents other embodiments , surfactants are included such as ( e . g . paraffin ) , absorption accelerators ( e . g . quaternary hydroxypropylcellulose . ammonium compounds ), wetting agents ( e . g . cetyl alcohol Injectable preparations, for example , sterile injectable 65 and glycerol monostearate ) , absorbents ( e . g . kaolin and aqueous or oleaginous suspensions may be formulated bentonite clay ) , and lubricants ( e . g . talc , calcium stearate , according to the known art using suitable dispersing agents , magnesium stearate , solid polyethylene glycols , sodium US 9 ,758 ,577 B2 149 150 lauryl sulfate ), and mixtures thereof. In the case of capsules , or greater than 99. 99 % of the composition is retained in the tablets and pills, the dosage form may comprise buffering target tissues . Advantageously , retention is determined by agents . measuring the amount of compounds and / or compositions Topical or Transdermal Administration that enter one or more target cells . For example , at least 1 % , As described herein , compounds and / or compositions of 5 5 % , 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 85 % , the present invention may be formulated for administration 90 % , 95 % , 96 % , 97 % , 98 % , 99 % , 99. 9 % , 99. 99 % or greater topically . The skin may be an ideal target site for delivery as than 99 . 99 % of compounds and /or compositions adminis it is readily accessible . Three routes are commonly consid - tered to subjects are present intracellularly at a period of ered to deliver compounds and / or compositions of the time following administration . For example , intramuscular present invention to the skin : ( i ) topical application ( e . g . for 10 injection to mammalian subjects may be performed using local /regional treatment and/ or cosmetic applications ) ; ( ii ) aqueous compositions comprising compounds and / or com intradermal injection ( e . g . for local/ regional treatment and positions of the present invention and one or more transfec or cosmetic applications ) ; and ( iii ) systemic delivery ( e . g . tion reagent, and retention is determined by measuring the for treatment of dermatologic diseases that affect both amount of compounds and / or compositions present in cutaneous and extracutaneous regions ) . Compounds and/ or 15 muscle cells . compositions of the present invention can be delivered to the Certain aspects of the invention are directed to methods of skin by several different approaches known in the art . providing compounds and / or compositions of the present In some embodiments , the invention provides for a vari - invention to a target tissues of mammalian subjects , by ety of dressings ( e . g . , wound dressings ) or bandages ( e . g ., contacting target tissues ( comprising one or more target adhesive bandages ) for conveniently and/ or effectively car - 20 cells ) with compounds and/ or compositions under condi rying out methods of the present invention . Typically dress - tions such that they are substantially retained in such target ing or bandages may comprise sufficient amounts of com tissues . Compounds and / or compositions comprise enough pounds and /or compositions of the present invention active ingredient such that the effect of interest is produced described herein to allow users to perform multiple treat - in at least one target cell . In some embodiments , compounds ments . 25 and /or compositions generally comprise one or more cell Dosage forms for topical and /or transdermal administra penetration agents , although “ naked ” formulations (such as tion may include ointments , pastes, creams, lotions, gels , without cell penetration agents or other agents ) are also powders , solutions, sprays , inhalants and/ or patches. Gen contemplated , with or without pharmaceutically acceptable erally, active ingredients are admixed under sterile condi- carriers . tions with pharmaceutically acceptable excipients and / or 30 In some embodiments , the amount of a growth factor any needed preservatives and /or buffers . Additionally, the present in cells in a tissue is desirably increased . Preferably , present invention contemplates the use of transdermal this increase in growth factor is spatially restricted to cells patches, which often have the added advantage of providing within the target tissue . Thus, provided are methods of controlled delivery of compounds and/ or compositions of increasing the amount of growth factor of interest in tissues the present invention to the body . Such dosage formsmay be 35 of mammalian subjects . In some embodiments , formulations prepared , for example , by dissolving and / or dispensing are provided comprising compounds and / or compositions compounds and / or compositions in the proper medium . characterized in that the unit quantity provided has been Alternatively or additionally , rates may be controlled by determined to produce a desired level of growth factor of either providing rate controlling membranes and /or by dis - interest in a substantial percentage of cells contained within persing compounds and / or compositions in a polymer 40 predetermined volumes of target tissue . matrix and / or gel. In some embodiments , formulations comprise a plurality Formulations suitable for topical administration include , of different compounds and / or compositions , where one or but are not limited to , liquid and/ or semi liquid preparations more than one targets biomolecules of interest . Optionally , such as liniments , lotions, oil in water and / or water in oil formulations may also comprise cell penetration agents to emulsions such as creams, ointments and / or pastes, and / or 45 assist in the intracellular delivery of compounds and / or solutions and / or suspensions . compositions . In such embodiments , determinations are Topically - administrable formulations may, for example , made of compound and /or composition dose required to comprise from about 1 % to about 10 % ( w / w ) active ingre - target biomolecules of interest in substantial percentages of dient, although the concentration of active ingredient may be cells contained within predetermined volumes of the target as high as the solubility limit of the active ingredient in the 50 tissue ( generally , without targeting biomolecules of interest solvent. Formulations for topical administration may further in adjacent or distal tissues. ) Determined doses are then comprise oneor more of the additional ingredients described introduced directly into subject tissues . In some embodi herein . ments , the invention provides for compounds and / or com Depot Administration positions to be delivered in more than one administration or As described herein , in some embodiments , compounds 55 by split dose administration . and / or compositions of the present invention are formulated Pulmonary Administration in depots for extended release . Generally , specific organs or In some embodiments , compounds and /or compositions tissues (“ target tissues” ) are targeted for administration of the present invention may be prepared , packaged , and / or In some aspects of the invention , compounds and /or sold in formulations suitable for pulmonary administration . compositions of the present invention are spatially retained 60 In some embodiments , such administration is via the buccal within or proximal to target tissues . Provided are method of cavity . In some embodiments , formulations may comprise providing compounds and / or compositions to target tissues dry particles comprising active ingredients . In such embodi of mammalian subjects by contacting target tissues (which ments, dry particles may have a diameter in the range from comprise one or more target cells ) with compounds and /or about 0 . 5 nm to about 7 nm or from about 1 nm to about 6 compositions under conditions such that they are substan - 65 nm . In some embodiments , formulations may be in the form tially retained in target tissues, meaning that at least 10 , 20 , of dry powders for administration using devices comprising 30 , 40 , 50 , 60 , 70 , 80 , 85 , 90 , 95 , 96 , 97 , 98 , 99 , 99 . 9 , 99 . 99 dry powder reservoirs to which streams of propellantmay be