|HAO WAKATI WAKILUS009758576B2 MAARHET MONTH (12 ) United States Patent ( 10) Patent No. : US 9 ,758 , 576 B2 Schurpf et al. (45 ) Date of Patent: * Sep . 12 , 2017 (54 ) COMPOSITIONS AND METHODS FOR C12N 15 /1037 (2013 . 01 ) ; A61K 38 /00 GROWTH FACTOR MODULATION (2013 . 01 ) ; CO7K 2317 /21 (2013 . 01 ) ; CO7K 2317 / 24 (2013 .01 ) ; CO7K 2317/ 31 ( 2013 .01 ) ; (71 ) Applicant: SCHOLAR ROCK , INC ., Cambridge , CO7K 2317 /55 (2013 . 01 ); MA (US ) ( Continued ) (72 ) Inventors : Thomas Schurpf, Cambridge, MA (58 ) Field of Classification Search ( US) ; Nagesh K . Mahanthappa , None Cambridge , MA (US ) ; Michelle Marie See application file for complete search history . Straub , Brighton , MA (US ) (56 ) References Cited ( 73 ) Assignee: Scholar Rock , Inc ., Cambridge , MA (US ) U .S . PATENT DOCUMENTS 4 , 179 , 337 A 12 / 1979 Davis et al . ( * ) Notice : Subject to any disclaimer, the term of this 4 ,301 , 144 A 1112/ / 19791981 Iwashita et al . patent is extended or adjusted umdefunderthis 35 4: 301 , 324 A U . S . C . 154 ( b ) by 0 days. (Continued ) This patent is subject to a terminal dis FOREIGN PATENT DOCUMENTS claimer. 0239400 A2 9 / 1987 EI 0404097 12 / 1990 (21 ) Appl. No. : 14 /795 ,012 ( Continued ) (22 ) Filed : Jul. 9 , 2015 OTHER PUBLICATIONS (65 ) Prior Publication Data McFarlane et al ., Dev . Biol. , 2005, vol. 283( 1 ) : 58 - 69 . * US 2016 / 0031980 A1 Feb . 4 , 2016 (Continued ) Related U . S . Application Data Primary Examiner — Xiaozhen Xie (63 ) Continuation of application No. (74 ) Attorney , Agent, or Firm — McCarter & English , PCT/ US2014 /036933 , filed on May 6 , 2014 . LLP ; Marcie B . Clarke ; Maria Laccotripe Zacharakis (Continued ) (57 ) ABSTRACT (51 ) Int . CI. Provided herein are antibodies that bind growth factor COZK 16 / 22 (2006 . 01) prodomain complexes , for example , growth factor prodo A61K 39 / 395 ( 2006 .01 ) main complexes that include GDF- 8 growth factor and ( Continued ) GDF - 8 prodomain . Also included are antibodies that bind (52 ) U . S . CI. growth factor prodomain complexes that have been altered CPC ...... C07K 16 /22 (2013 .01 ) ; A61K 39/ 3955 by proteolytic processing . ( 2013 . 01 ) ; CO7K 14 /495 ( 2013 .01 ) ; COOK 16 / 005 ( 2013 .01 ); CO7K 16 /2863 (2013 .01 ) ; 10 Claims, 23 Drawing Sheets

Growth Factor -Prodomain Cornplex (GPC )

Free Latency Associated Pepode (LAP ) Oimer

Free Growth Factor Diner ROLET US 9 ,758 ,576 B2 Page 2

Related U . S . Application Data 5 , 821 , 047 A 10 / 1998 Garrard et al. 5 ,846 ,545 A 12 / 1998 Chari et al. (60 ) Provisional application No .61 /819 ,840 , filed on May 5 ,885 ,793 A 3 / 1999 Griffiths et al . 5 , 916 ,771 A 6 / 1999 Hori et al. 6 , 2013 , provisional application No. 61 / 823 , 552, filed 5 ,919 ,652 A 7 / 1999 Pang et al . on May 15 , 2013, provisional application No . 5 ,932 ,448 A 8 / 1999 Tso et al . 61/ 900 , 438 , filed on Nov . 6 , 2013 . 5 , 939 , 598 A 8 / 1999 Kucherlapati et al. 5 ,951 , 983 A 9 / 1999 Bazin et al. (51 ) Int. CI. 5 , 967 ,979 A 10 / 1999 Taylor et al . 5 , 969, 108 A 10 / 1999 McCafferty et al. CO7K 14 /495 ( 2006 .01 ) 5 , 972 , 335 A 10 / 1999 Ferguson et al . CO7K 16 / 00 ( 2006 .01 ) 6 ,075 , 181 A 6 / 2000 Kucherlapati et al. COZK 16 / 28 ( 2006 .01 ) 6 ,096 , 506 A 8 / 2000 Lee et al. C12N 15 / 10 ( 2006 .01 ) 6 , 114 , 148 A 9 / 2000 Seed et al. ( 2006 . 01 ) 6 , 114 , 598 A 9 /2000 Kucherlapati et al . 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mannen Alpha 1 helix Residues for extracellular protein associations

Free Growth Factor Dimer Finger ??????????gn U . S . Patent Sep. 12 , 2017 Sheet 5 of 23 US 9 ,758 ,576 B2

Figure 5 YRKYRKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK An embodiment of a recombinantGPC su{ fidebd C - terminus Growth factor domain Proprotein convertase cleavage site Latency - associated peptide ( LAP ) Cys residue muitos HSE S H N - terminus on Histidine tags U . S . Patent Sep. 12 , 2017 Sheet 6 of 23 US 9 ,758 ,576 B2

Figure 6 An embodiment of a proprotein convertase cleavage site mutant ( e .g . RXXR ? RXG ; D2G ) x L

An embodiment of an N -terminal cysteine mutant (e . g . C4S : Cys4 - -> Ser )

??? ??? U . S . Patent Sep. 12 , 2017 Sheet 7 of 23 US 9 ,758 ,576 B2

Figure 7 Embodiments of recombinant proteins

GARD 14HOOR LTBA_ 1 PIOTGF - 8 proprotein ProTGF - 8 proprotein proTGF - B proprotein convertase cleavage convertase cleavage convertase cleavage site mutant with N site mutant complexed site mutant complexed ??????a} st??? with LTBP with GARP 2fati 33

LD10 OH TGF- 8 LAP with N TGF- B LAP complexed terminal cysteine with GARA mutation U . S . Patent Sep. 12 , 2017 Sheet 8 of 23 US 9 ,758 ,576 B2

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Figure 8C

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Figure 8D

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COMPOSITIONS AND METHODS FOR regions and bowtie regions. In some embodiments , recom GROWTH FACTOR MODULATION binant proteins of the present invention may comprise one or more protein modules from a vertebrate species. In some CROSS REFERENCE TO RELATED embodiments , recombinant proteins of the present invention APPLICATIONS 5 may comprise one or more protein modules comprising one or more mutations. In some embodiments , recombinant This application is a continuation of International Appli - proteins of the present invention may comprise one or more cation No . PCT/ US2014 / 36933 , filed May 6 , 2014 entitled mutations comprising one or more furin cleavage site Compositions and Methods for Growth Factor Modulation , regions. In some embodiments , such mutations may prevent which claimspriority to U .S . Provisional Patent Application* 1010 enzymatic cleavage of recombinant proteins of the present No. 61 /819 ,840 filed May 6 , 2013 , entitled Compositions invention . In some embodiments , recombinant proteins of and Methods for Growth Factor Modulation , U . S . Provi the present invention may comprise one or more mutations sional Patent Application No . 61 /823 ,552 filed May 15 , comprising a mutation of the amino acid sequence RXXR to 2013 , entitled Compositions and Methods for Growth Factor the amino acid sequence RXG . In some embodiments , Modulation and U . S . Provisional Patent Application No . 15 recombinant proteins of the present invention may comprise 61 / 900 , 438 filed Nov. 6 , 2013 , entitled Compositions and one or more mutations comprising a mutation of the amino Methods for Growth Factor Modulation , the contents of acid sequence RXXR to the amino acid sequence AXXA . In each of which are herein incorporated by reference in their some embodiments, recombinant proteins of the present entireties. invention may comprise one or more mutations comprising 20 N - terminal regions for extracellular associations . In some embodiments , recombinant proteins of the present invention SEQUENCE LISTING 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 25 residues . In some embodiments , recombinant proteins of the copy . created on Jul. 9 . 2015 . is named present invention may comprise one or more substitution of at least one cysteine residue with at least one serine residue . 2035 _ 1001USCON2_ SL .txt and is 705 , 749 bytes in size . In some embodiments , recombinant proteins of the pres FIELD OF THE INVENTION ent invention may be complexed with a protein selected 30 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 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 ormore detectable labels . Such biology 35 detectable labels may comprise biotin labels , polyhistidine tags and /or flag tags. BACKGROUND OF THE INVENTION 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 40 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 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 45 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 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 50 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. 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 - ß family member and at least one growth factor 55 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 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 60 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 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 arm regions , fingers regions , N - terminal regions for extra - 65 family member, at least one C - terminal region is from a cellular associations , latency loops , alpha 1 helical regions , TGF - B family member and wherein the N - terminal region alpha 2 helical regions , RGD sequence regions, trigger loop and C - terminal region are from different TGF- B family US 9 , 758 ,576 B2 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 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 - 5 perlecan , decorin , elastin and collagen . In some cases , minal regions and inhibin beta A terminal regions . In some recombinant proteins may comprise a chimeric protein com embodiments , chimeric proteins of the present invention prising an amino acid sequence selected from the group may comprise a GPC from at least one TGF - B family consisting of SEQ ID NOs: 199 - 236 and 273 . member comprising at least one arm region from a different TGF - B family member. In some embodiments , chimeric 10 Other methods of selecting a desired antibody may com proteins of the present invention may comprise a GPC prise the steps of 1 ) providing a growth factor activity assay , comprising at least one TGF - ß family member comprising at 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 moduledule 1515 growthgrov 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 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 20 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 - responsive cells that yield gene expression data and / or tion may comprise one or more detectable labels. In some viability data . embodiments , such detectable labels may comprise at least In some embodiments , the present invention provides one biotin label, polyhistidine tag and /or flag tag. 25 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 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 - 30 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 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 . 35 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- 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 40 ( 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 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 - 45 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 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 50 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 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 55 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 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 60 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 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. ( ELISA ) and / or a fluorescence - associated cell sorting 65 FIG . 3 is a schematic of one embodiment of a growth ( FACS ) -based assay . In some cases, recombinant proteins of factor -prodomain complex (GPC ) as well as an embodiment such assays may be complexed with a protein selected from of a free growth factor dimer and a free latency associated US 9 , 758 ,576 B2 peptide (LAP ) dimer . The arrow indicates the ability of influence within biological systems, growth factor signaling proteins according to this embodiment to alter between free is tightly regulated , often through interactions with other and complexed forms. biomolecules , the extracellular and /or cellular matrix or FIG . 4 is a schematic of one embodiment of a free LAP within a particular cell environment or niche . These inter dimer and a free growth factor dimer with labeled features 5 actions may be direct or indirect. and / or protein modules . Growth factors of the transforming growth factor beta FIG . 5 is a schematic of an embodiment of a recombinant ( TGF - B ) family are involved in a variety of cellular pro GPC . cesses . Growth factor binding to type II receptors leads to FIG . 6 is a schematic of embodiments of mutant recom type I receptor phosphorylation and activation (Denicourt , binant GPCs. 10 C . et al. , Another twist in the transforming growth factor FIG . 7 depicts schematic representations of five recom - B - induced cell -cycle arrest chronicle . PNAS . 2003 . 100 ( 26 ) : binant proteins alone or in complex with LTBP or GARP . 15290 - 1 .) Activated type I receptors may in turn phospho FIGS. 8A - 8G show structure -based alignment between rylate receptor- associated SMADs (R - SMADs) promoting TGF - ß family member proteins (SEQ ID NOS 1 , 117 , 116 , co - SMAD (e . g . SMAD4) dimer/ trimer formation and 296 , 2 - 4 , 137 , 5 , 131, 125 , 6 , 14 , 21, 23 - 24 , 27 , 26 , 28 , and 15 nuclear translocation . SMAD complexes collaborate with 10 , respectively , in order of appearance ) [ adapted from Shi c ofactors to modulate expression of TGF - B family member et al (Shi , M . et al ., Latent TGF - B structure and activation . target genes. Nature . 2011 Jun . 15 ; 474 (7351 ) :343 - 9 , the contents of TGF -ß family member signaling cascades are involved in which are herein incorporated by reference in their a number of diverse biological pathways including , but not entirety .) ] Cysteine residues required for interaction with 20 limited to inhibition of cell growth , tissue homeostasis , LTBPs and /or GARPs are boxed . Residues mutated in extracellular matrix ( ECM ) remodeling , endothelial to mes Camurati - Engelmann syndrome are indicated with a star. enchymal transition (EMT ) in cell migration and invasion Protease cleavage sites are indicated with an up arrow . and immune modulation /suppression as well as in mesen Protein modules and secondary structural elements are indi- chymal to epithelial transition . TGF - B signaling related to cated with solid bars . Residues underlined at the N - terminus 25 growth inhibition and tissue homeostasis may affect epithe of GDF - 8 correspond to alternatively predicted signal pep - lial, endothelial, hematopoietic and immune cells through tide processing sites. “ Chimeric module breakpoints ” indi- the activation of p21 and p15/ NK to mediate cell cycle arrest cate regions where structural features are conserved and and repress myc . In relation to ECM remodeling , TGF - B provide modules for chimeric protein construction ( swap - signaling may increase fibroblast populations and ECM ping of modules between family members ) in all family 30 deposition ( e . g . collagen ) . TGF - B signaling related to cell members . N - terminal regions are shown in FIGS . 8A and migration and invasion may affect epithelial and/ or endothe 8B , internal regions are shown in FIGS . 8C and 8D and lial cells , inducing stem cell - like phenotypes . This aspect of C - terminal regions are shown in FIGS. 8E -8G . signaling may play a role in smooth muscle cell proliferation FIGS. 9A -9C present 3 tables showing the percent iden - following vascular surgery and /or stenting. In the immune tity between amino acid sequences found in the TGF - B 35 system , TGF - B ligand is necessary for T regulatory cell family . FIG . 9A demonstrates percent identity among pro - function and maintenance of immune precursor cell growth proteins (prodomain and growth factor. ) Percent identity and homeostasis . Nearly all immune cells comprise recep among growth factor domains is presented in FIG . 9B while tors for TGF - B and TGF - ß knockout mice die postnataly due percent identity among prodomains is presented in FIG . 9C . in part to inflammatory pathologies . Finally, TGF - B sup FIG . 10 presents an alignment conducted between GDF - 8 40 presses interferon gamma- induced activation of natural (myostatin , ) ( SEQ ID NO :GDF - 11 (SEQ ID NO : 4 ) , Inhibin killer cells ( Wi, J . et al. , 2011 . Hepatology . 53 ( 4 ) : 1342 - 51 , A (SEQ ID NO : 6 ) and a GDF -8 dimer (SEQ ID NO : 297) . the contents ofwhich are herein incorporated by reference in Arrows indicate cleavage sites . Regions involved in internal their entirety . ) interactions are boxed . Solid rectangles appear above resi The recent solution of the crystal structure of the latent dues predicted to be involved in steric clashes in chimeric 45 form of TGF- beta is a first for the entire TGF- beta family constructs . Stars denote important break points in protein and offers deep insights into these complexes ( Shi, M . et al. , 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 I 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 ,576 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 in FIG . precursor of, the prodomain and the growth factor. Shown in 1 and / or listed in Table 1 . These include , but are not limited the Table are the names of the originating TGF - B family to 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 ID TGF Member Prodomain and growth factor Sequence NO TGF - B1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPL PEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMV ETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRAELRL LRLKLKVEOHVELYOKYSNNSWRYLSNRLLAPSDSPEWLSF DVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFT TGRRGDLATIHGMNRPFLLLMATPLERAQHLOSSRHRRALD TNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFC LGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPL PIVYYVGRKPKVEOLSNMIVRSCKCS TGF - B2 SLSTCSTLDMDOFMRKRIEAIRGOILSKLKLTSPPEDYPEPEEV PPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKI DMPPFFPSENAIPPTFYRPYFRIVRFDV SAMEKNASNLVKAEF RVFRLONPKARVPEQRIELYQILKSKDLTSPTORYIDSKVVKT RAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVP SNNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIKSTRKKNSG KTPHLLLMLLPSYRLESOOTNRRKKRALDAAYCFRNVODN CCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSD TOHSRVLSLYNTINPEASASPCCVSODLEPLTILYYIGKTPKIE QLSNMIVKSCKCS TGF - B3 SLSLSTCTTLDFGHIKKKRVEAIRGOILSKLRLTSPPEPTVMTH 3 VPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIH KFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFR AEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKORYIGGKNL PTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFOP NGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHN US 9 ,758 ,576 B2 9 TABLE 1 - continued Pro - proteins of the TGF - beta family SEQ ID TGF Member Prodomain and growth factor Sequence NO PHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCC VRPLYIDFRODLGWKWVHEPKGYYANFCSGPCPYLRSADTT HSTVLGLYNTLNPEASASPCCVPODLEPLTILYYVGRTPKVE QLSNMVVKSCKCS GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPV CVWROHSRELRLESIKSOILSKLRLKEAPNISREVV KOLLPKA PPLOOILDLHDFOGDALOPEDFLEEDEYHATTETVISMAQET DPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRP ATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSG HWOSIDFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLG PGAEGLHPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYP LTVDFEAFGWDWIIAPKRYKANYCSGOCEYMFMOKYPHTH LVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGM VVDRCGCS GDF - 8 NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIQILSKLRL 5 ( myostatin ) ETAPNISKDVIROLLPKAPPLRELIDQYDVORDDSSDGSLEDD DYHATTETII TMPTESDFLMOVDGKPKCCFFKFSSKIOYNKV VKAOLWIYLRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKL DMNPGTGIWOSIDVKTVLONWLKOPESNLGIEIKALDENGH DLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHST ESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLO KYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKECHY GKIPAMVVDRCGCS Inhibin - beta A SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEMVEAVKKHI 6 LNMLHLKKRPDVTOPVPKAALLNAIRKLHVGKVGENGYVEI EDDIGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSD LSVVERAEVWLFLKVPKANRTRTKVTIRLFOOOKHPOGSLD TGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIOR LLDOGKSSLDVRIACEOCOESGASLVLLGKKKKKEEEGEGK KKGGGEGGAGADEEKEQSHRPFLMLQAROSEDHPHRRRRR GLECDGKVNICCKKOFFVSFKDIGWNDWIIAPSGYHANYCE GECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPT KLRPMSMLYYDDGONIIKKDIONMIVEECGCS Inhibin - beta B SPTPPPTPAAPPPPPPPGSPGGSODTCTSCGGFRRPEELGRVDG DFLEAVKRHILSRLQMRGRPNITHAVPKAAMVTALRKLHAG KVREDGRVEIPHLDGHASPGADGQERVSEIISFAETDGLASSR VRLYFFISNEGNONLFVVOASLWLYLKLLPYVLEKGSRRKV RVKVYFOEOGHGDRWNMVEKRVDLKRSGWHTFPLTEAIOA LFERGERRLNLDVQCDSCQELAVVPVFVDPGEESHRPFVW QARLGDSRHRIRKRGLECDGRTNLCCRQQFFIDFRLIGWND WIIAPTGYYGNYCEGSCPAYLAGVPGSASSFHTAVVNOYRM RGLNPGTVNSCCIPTKLSTMSMLYFDDEYNIVKRDVPNMIVE ECGCA Inhibin - beta C TPRAGGQCPACGGPTLELESQRELLLDLAKRSILDKLHLTOR PTLNRPVSRAALRTALOHLHGVPOGALLEDNREOECEIISFAE TGLSTINQTRLDFHFSSDRTAGDREVQQASLMFFVQLPSNTT WTLKVRVLVLGPHNTNLTLATQYLLEVDASGWHQLPLGPE AQAACSQGHLTLELVLEGQVAQSSVILGGAAHRPFVAARVR VGGKHQIHRRGIDCQGGSRMCCRQEFFVDFREIGWHDWIIQ PEGYAMNFCIGQCPLHIAGMPGIAASFHTAVLNLLKANTAAG TTGGGSCCVPTARRPLSLLYYDRDSNIVKTDI PDMVVEACGCS Inhibin - beta E QGTGSVCPSCGGSKLAPQAERALVLELAKQQILDGLHLTSRP 9 RI THPPPOAALTRALRRLOPGSVAPGNGEEVISFATVTDSTSA SLLTFHLSTPRSHHLYHARLWLHVLPTLPGTLCLRIFRWGP RRRROGSRTLLAEHHITNLGWHTLTLPSSGLRGEKSGVLKLO LDCRPLEGNSTVTGOPRRLLDTAGHOOPFLELKIRANEPGAG RARRRTPTCEPATPLCCRRDHYVDFQELGWRDWILQPEGYQ LNYCSGQCPPHLAGSPGIAASFHSAVFSLLKANNPWPASTSC CVPTARRPLSLLYLDHNGNVVKTDVPDMVVEACGCS Lefty 1 LTGEOLLGSLLROLOLKEVPTLDRADMEELVIPTHVRAQYV 10 ALLQRSHGDRSRGKRFSQSFREVAGRFLALEASTHLLVFGM EQRLPPNSELVQAVLRLFQEPVPKAALHRHGRLSPRSARAR VTVEWLRVRDDGSNRTSLIDSRLVSVHESGWKAFDVTEAVN FWOQLSRPROPLLLQVSVQREHLGPLASGAHKLVRFASQGA PAGLGEPOLELHTLDLGDYGAQGDCDPEAPMTEGTRCCRQE US 9 ,758 ,576 B2 11 TABLE 1 - continued Pro - proteins of the TGF - beta family SEQ ID TGF Member Prodomain and growth factor Sequence NO MYIDLQGMKWAENWVLEPPGFLAYECVGTCROPPEALAFK WPFLGPROCIAS ETDSLPMIVSI KEGGRTRPQWSLPNMRVO KCSCASDGALVPRRLQP Lefty2 LTEEQLLGSLLRQLQLSEVPVLDRADMEKLVIPAHVRAQYV 11 VLLRRSHGDRSRGKRFSOSFREVAGRFLAS EASTHLLVFGM EORLPPNSELVOAVLRLFOEPVPKAALHRHGRLSPRSAQAR VTVEWLRVRDDGSNRTSLIDSRLVSVHESGWKAFDVTEAVN FWQQLSRPROPLLLQVSVQREHLGPLASGAHKLVRFASQGA PAGLGEPOLELHTLDLRDYGAOGDCDPEAPMTEGTRCCROE MYIDLQGMKWAKNWVLEPPGFLAYECVGTCOQPPEALAFN WPFLGPROCIASETASLPMIVSIKEGGRTRPQVVSLPNMRVO KCSCASDGALVPRRLQP GDF - 15 LSLAEASRASFPGPSELHS EDSRFRELRKRYEDLLTRLRANOS 12 WEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLP EASRLHRALFRLSPTASRSWDVTRPLRROLSLARPOAPALHL RLSPPPSQSDOLLAESSSARPOLELHLRPQAARGRRRARARN GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVOVTM CIGACPSOFRAANMHAOIKTSLHRLKPDTVPAPCCVPASYNP MVLIQKTDTGVSLQTYDDLLAKDCHCI Anti -Mullerian LLGTEALRAEEPAVGTSGLIFREDLDWPPGIPQEPLCLVALGG 13 hormone DSNGSSSPLRVVGALSAYEOAFLGAVORARWGPRDLATFGV CNTGDROAALPSLRRLGAWLRDPGGORLVVLHLEEVTWEPT PSLRFOEPPPGGAGPPELALLVLYPGPGPEVTVTRAGLPGAOS LCPSRDTRYLVLAVDRPAGAWRGSGLALTLOPRGEDSRLST ARLQALLFGDDHRCFTRMTPALLLLPRSEPAPLPAHGQLDTV PFPPPRPSAELEESPPSADPFLETLTRLVRALRVPPARASAPRL ALDPDALAGFPOGLVNLSDPAALERLLDGEEPLLLLLRPTAA TTGDPAPLHDPTSAPWATALARRVAAELOAAAAELRSLPGL PPATAPLLARLLALCPGGPGGLGDPLRALLLLKALOGLRVE WRGRDPRGPGRAORSAGATAADGPCALRELSVDLRAERSV LIPETYOANNCOGVCGW POSDRNPRYGNHVVLLLKMOVRG AALARPPCCVPTAYAGKLLISLSEERISAHHVPNMVATECGCR Inhibin - alpha COGLELARELVLAKVRALFLDALGPPAVTREGGDPGVRRLP 14 RRHALGGFTHRGSEPEEEEDVSOAILFPATDASCEDKSAARG LAQEAEEGLFRYMFRPSQHTRSROVTSAQLWFHTGLDROGT AASNSSEPLLGLLALSPGGPVAVPMSLGHAPPHWAVLHLATS ALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSGG ERARRSTPLMSWPWSPSALRLLORPPEEPAAHANCHRVALN ISFOELGWERWIVYPPSFIFHYCHGGCGLHIPPNLSLPVPGAPP TPAOPYSLLPGAOPCCAALPGTMRPLHVRTTSDGGYSFKYET VPNLLTOHCACI

GDF - 1 PVPPGPAAALLOALGLRDEPOGAPRLRPVPPVMWRLFRRRD 15 POETRSGSRRTSPGVTLOPCHVEELGVAGNIVRHIPDRGAPTR ASEPASAAGHCPEWTVVFDLSAVEPAERPSRARLELRFAAAA AAAPEGGWELSVAQAGOGAGADPGPVLLROLVPALGPPVR AELLGAAWARNASWPRSLRLALALRPRAPAACARLAEASLL LVTLDPRLCHPLARPRRDAEPVLGGGPGGACRARRLYVSFR EVGWHRWVIAPRGFLANYCQGQCALPVALSGSGGPPALNH AVLRALMHAAAPGAADLPCCVPARLSPISVLFFDNSDNVVL RQYEDMVVDECGCR GDF - 3 QEYVFLOFLGLDKAPSPOKFOPVPYILKKIFODREAAATTGV 16 SRDLCYVKELGVRGNVLRFLPDOGFFLYPKKISQASSCLOKL LYFNLSAIKEREQLTLAQLGLDLGPNSYYNLGPELELALFLV QEPHVWGQTTPKPGKMFVLRSVPWPQGAVHFNLLDVAKD WNDNPRKNFGLFLEILVKEDRDSGVNFOPEDTCARLRCSLH ASLLVVTLNPDOCHPSRKRRAAIPVPKLSCKNLCHRHOLFIN FRDLGWHKWIIAPKGFMANYCHGECPFSLTISLNSSNYAFMO ALMHAVDPEIPQAVCIPTKLSPISMLYQDNNDNVILRHYEDM VVDECGCG GDF - 5 APDLGQRPQGTRPGLAKAEAKERPPLARNVFRPGGHSYGGG 17 ATNANARAKGGTGQTGGLTQPKKDEPKKLPPRPGGPEPKPG HPPOTRQATARTVTPKGOLPGGKAPPKAGSVPSSFLLKKARE PGPPREPKEPFRPPPITPHEYMLSLYRTLSDADRKGGNSSVKL EAGLANTITSFIDKGODDRGPVVRKORYVFDISALEKDGLLG AELRILRKKPSDTAKPAAPGGGRAAQLKLSSCPSGROPASLL DVRSVPGLDGSGWEVFDIWKLFRNFKNSAOLCLELEAWERG RAVDLRGLGFDRAAROVHEKALFLVFGRTKKRDLFFNEIKA US 9 ,758 ,576 B2 13 14 TABLE 1 - continued Pro - proteins of the TGF - beta family SEQ ID TGF Member Prodomain and growth factor Sequence NO RSGODDKTVYEYLFSORRKRRAPLATROGKRPSKNLKARCS RKALHVNFKDMGWDDWIIAPLEYEAFHCEGLCEFPLRSHLE PTNHAVIQTLMNSMDPESTPPTCCVPTRLSPISILFIDSANNVV YKQYEDMVVESCGCR GDF - 6 FQQASISSSSSSAELGSTKGMRSRKEGKMQRAPRDSDAGREG 18 QEPOPRPODEPRAQOPRAQEPPGRGPRVVPHEYMLSIYRTYSI AEKLGINASFFOSSKSANTITSFVDRGLDDLSHTPLRROKYLE DVSMLSDKEELVGAELRLFRQAPSAPWGPPAGPLHVQLFPCL SPLLLDARTLDPOGAPPAGWEVFDVWOGLRHOPWKOLCLE LRAAWGELDAGEAEARARGPOOPPPPDLRSLGFGRRVRPPO ERALLVVFTRSQRKNLFAEMREQLGSAEAAGPGAGAEGSWP PPSGAPDARPWLPSPGRRRRRTAFASRHGKRHGKKSRLRCS KKPLHVNFKELGWDDWIIAPLEYEAYHCEGVCDFPLRSHLEP TNHAIIQTLMNSMDPGSTPPSCCVPTKLTPISILYIDAGNNVV YKQYEDMVVESCGCR GDF - 7 RDGLEAAAVLRAAGAGPVRSPGGGGGGGGGGRTLAOAAGA 19 AAVPAAAVPRARAARRAAGSGFRNGSVV PHHFMMSLYRSL AGRAPAGAAAVSASGHGRADTITGFTDOATODESAAETGOS FLFDVSSLNDADEVVGAELRVLRRGSPESGPGSWTSPPLLLLS TCPGAARAPRLLYSRAAEPLVGORWEAFDVADAMRRHRRE PRPPRAFCLLLRAVAGPVPSPLALRRLGFGWPGGGGSAAEER AVLVVSSRTORKESLFREIRAQARALGAALASEPLPDPGTGT ASPRAVIGGRRRRRTALAGTRTAQGSGGGAGRGHGRRGRS RCSRKPLHVDFKELGWDDWIIAPLDYEAYHCEGLCDFPLRSH LEPTNHAIIQTLLNSMAPDAAPASCCVPARLSPISILYIDAANN VVYKQYEDMVVEACGCR BMP - 10 SPIMNLEQSPLEEDMSLFGDVFSEQDGVDFNTLLQSMKDEFL 20 KTLNLSDIPTODSAKVDPPEYMLELYNKFATDRTSMPSANIIR SFKNEDLFSOPVSFNGLRKYPLLFNVSIPHHEEVIMAELRLYT LVORDRMIYDGVDRKITIFEVLESKGDNEGERNMLVLVSGEI YGTNSEWETFDVTDAIRRWOKSGSSTHOLEVHIESKHDEAE DASSGRLEIDTSAONKHNPLLIVFSDDOSSDKERKEELNEMIS HEOLPELDNLGLDSFSSGPGEEALLOMRSNIIYDSTARIRRNA KGNYCKRTPLYIDFKEIGWDSWIIAPPGYEAYECRGVCNYPL AEHLTPTKHAIIQALVHLKNSQKASKACCVPTKLEPISILYLD KGWTYKFKYEGMAVSECGCR BMP - 9 (GDF - 2 ) KPLQSWGRGSAGGNAHSPLGVPGGGLPEHTFNLKMFLENVK 21 VDFLRSLNLSGVPSODKTRVEPPOYMIDLYNRYTSDKSTTPA SNIVRSFSMEDAISITATEDFPFOKHILLFNISIPRHEOITRAELR LYVSCONHVDPSHDLKGSVVIYDVLDGTDAWDSATETKTFL VSODIODEGWETLEVSSAVKRWVRSDSTKSKNKLEVTVESH RKGCDTLDISVPPGSRNLPFFVVFSNDHSSGTKETRLELREMI SHEQESVLKKLSKDGSTEAGESSHEEDTDGHVAAGSTLARR KRSAGAGSHCOKTSLRVNFEDIGWDSWIIAPKEYEAYECKG GCFFPLADDVTPTKHAIVOTLVHLKFPTKVGKACCVPTKLSPI SVLYKDDMGVPTLKYHYEGMSVAECGCR Nodal TVATALLRTRGOPSSPSPLAYMLSLYRDPLPRADIIRSLOAED 22 VAVDGONWTFAFDFSFLSOOEDLAWAELRLOLSSPVDLPTE GSLAIEIFHQPKPDTEQASDSCLERFQMDLFTVTLSQVTFSLG SMVLEVTRPLSKWLKRPGALEKOMSRVAGECWPRPPTPPAT NVLLMLYSNLSQEQRQLGGSTLLWEAESSWRAQEGQLSWE WGKRHRRHHLPDRSOLCRKVKFOVDFNLIGWGSWIIYPKO YNAYRCEGECPNPVGEEFHPTNHAYIQSLLKRYOPHRVPSTC CAPVKTKPLSMLYVDNGRVLLDHHKDMIVEECGCL BMP - 2 LVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLLSMFGLKOR 23 PTPSRDAVVPPYMLDLYRRHSGOPGSPAPDHRLERAASRAN TVRSFHHEESLEELPETSGKTTRRFFFNLSSIPTEEFITSAELOV FREQMODALGNNSSFHHRINIYEIIKPATANSKFPVTRLLDTR LVNQNASRWESFDVTPAVMRWTAQGHANHGFVVEVAHLE EKOGVSKRHVRISRSLHODEHSWSQIRPLLVTFGHDGKGHPL HKREKRQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIV APPGYHAFYCHGECPFPLADHLNSTNHAIVOTLVNSVNSKIP KACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR BMP - 4 GASHASLIPETGKKKVAEIQGHAGGRRSGQSHELLRDFEATL 24 LQMFGLRRRPQPSKSAVIPDYMRDLYRLQSGEEEEEQIHSTG LEYPERPASRANTVRSFHHEEHLENIPGTSENSAFRFLFNLSSI PENEVISSAELRLFREQVDQGPDWERGFHRINIYEVMKPPAE US 9 ,758 ,576 B2 15 TABLE 1 - continued Pro - proteins of the TGF - beta family SEQ ID TGF Member Prodomain and growth factor Sequence NO WPGHLITRLLDTRLVHHNVTRWETFDVSPAVLRWTREKQP NYGLAIEVTHLHOTRTHOGOHVRISRSLPOGSGNWAOLRPL LVTFGHDGRGHALTRRRRAKRSPKHHSQRARKKNKNCRRH SLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLNST NHAIVOTLVNSVNSSIPKACCVPTELSAISMLYLDEYDKVVL KNYQEMVVEGCGCR BMP - 5 DNHVHSSFIYRRLRNHERREIOREILSILGLPHRPRPFSPGKOA 25 SSAPLFMLDLYNAMTNEENPEESEYSVRASLAEETRGARKG YPASPNGYPRRIQLSRTTPLTTOSPPLASLHD TNFLNDADMV MSFVNLVERDKDFSHORRHYKEFRFDLTOIPHGEAVTAAEFR IYKDRSNNRFENETIKISIYQIIKEYTNRDADLFLLDTRKAQAL DVGWLVFDITVTSNHWVINPONNLGLOLCAETGDGRSINVK SAGLVGRQGPOSKQPFMVAFFKASEVLLRSVRAANKRKNQ NRNKSSSHQDSSRMSSVGDYNTSEQKQACKKHELYVSFRDL GWQDWIIAPEGYAAFYCDGECSFPLNAHMNATNHAIVOTLV HLMFPDHVPKPCCAPTKLNAISVLYFDDSSNVILKKYRNMV VRSCGCH BMP - 6 CCGPPPLRPPLPAAAAAAAGGOLLGDGGSPGRTEOPPPSPOS 26 SSGFLYRRLKTOEKREMOKEILSVLGLPHRPRPLHGLOOPOP PALRQQEEQQQQQQLPRGEPPPGRLKSAPLFMLDLYNALSA DNDEDGASEGEROOSWPHEAASSSORROPPPGAAHPLNRKS LLAPGSGSGGASPLTSAODSAFLNDADMVMSFVNLVEYDKE FSPRORHHKEFKFNLSQIPEGEVVTAAEFRIYKDCVMGSFKN QTFLISIYQVLQEHQHRDSDLFLLDTRVVWASEEGWLEFDIT ATSNLWVVTPOHNMGLOLSVVTRDGVHVHPRAAGLVGRD GPYDKQPFMVAFFKVSEVHVRTTRSASSRRROQSRNRSTOS QDVARVSSASDYNSSELKTACRKHELYVSFQDLGWQDWIIA PKGYAANYCDGECSFPLNAHMNATNHAIVOTLVHLMNPEY VPKPCCAPTKLNAISVLYFDDNSNVILKKYRNMVVRACGCH BMP - 7 DFSLDNEVHSSFIHRRLRSOERREMOREILSILGLPHRPRPHLO 27 GKHNSAPMFMLDLYNAMAVEEGGGPGGOGFSYPYKAVFST OGPPLASLODSHFLTDADMVMSFVNLVEHDKEFFHPRYHHR EFRFDLSKIPEGEAVTAAEFRIYKDYIRERFDNETFRISVYOVL OEHLGRESDLFLLDSRTLWASEEGWLVFDITATSNHWVVNP RHNLGLOLSVETLDGOSINPKLAGLIGRHGPONKOPFMVAFF KATEVHFRSIRSTGS KORSONRSKTPKNQEALRMANVAENS SSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGEC AFPLNSYMNATNHAIVOTLVHFINPETVPKPCCAPTOLNAISV LYFDDSSNVILKKYRNMVVRACGCH BMP - 8A GGGPGLRPPPGCPORRLGARERRDVOREILAVLGLPGRPRPR 28 APPAASRLPASAPLFMLDLYHAMAGDDDEDGAPAEORLGR ADLVMSFVNMVERDRALGHOEPHWKEFRFDLTOIPAGEAVT AAEFRIYKVPSIHLLNRTLHV SMFOVVOEOSNRESDLFFLDL OTLRAGDEGWLVLDVTAASDCWLLKRHKDLGLRLYVETED GHSVDPGLAGLLGORAPRSOOPFVVTFFRASPSPIRTPRAVR PLRRROPKKSNELPQANRLPGIFDDVRGSHGROVCRRHELYV SFQDLGWLDWVIAPQGYSAYYCEGECSFPLDS CMNATNHAI LQSLVHLMKPNAVPKACCAPTKLSATSVLYYDSSNNVILRK HRNMVVKACGCH BMP - 8B GGGPGLRPPPGCPORRLGARERRDVOREILAVLGLPGRPRPR 29 APPAASRLPASAPLFMLDLYHAMAGDDDEDGAPAERRLGRA DLVMSFVNMVERDRALGHOEPHWKEFRFDLTOIPAGEAVTA AEFRIYKVPSIHLLNRTLHVSMFOVVQEQSNRESDLFFLDLOT LRAGDEGWLVLDVTAASDCWLLKRHKDLGLRLYVETEDGH SVDPGLAGLLGQRAPRSQQPFVVTFFRASPSPIRTPRAVRPLR RROPKKSNELPOANRLPGIFDDVHGSHGROVCRRHELYVSF ODLGWLDWVIAPOGYSAYYCEGECSFPLDSCMNATNHAILO SLVHLMMPDAVPKACCAPTKLSATSVLYYDSSNNVILRKHR NMVVKACGCH BMP - 15 MEHRAOMAEGGOSSIALLAEAPTLPLIEELLEESPGEOPRKPR 30 LLGHSLRYMLELYRRSADSHGHPRENRTIGATMVRLVKPLTS VARPHRGTWHIQILGFPLRPNRGLYOLVRATVVYRHHLOLT RFNLSCHVEPWVOKNPTNHFPSSEGDSSKPSLMSNAWKEMD ITOLVOORFWNNKGHRILRLRFMCOOOKDSGGLELWHGTSS LDIAFLLLYFND THKSIRKAKFLPRGMEEFMERESLLRRTRO ADGI SAEVTASSSKHSGPENNOCSLHPFQISFRQLGWDHWIIA PPFYTPNYCKGTCLRVLRDGLNSPNHAIIONLINQLVDQSVPR PSCVPYKYVPISVLMIEANGSILYKEYEGMIAESCTCR US 9 ,758 ,576 B2 17 TABLE 1 - continued Pro - proteins of the TGF - beta family SEQ ID TGF Member Prodomain and growth factor Sequence NO

GDF - 9 SOASGGEAQIAASAELESGAMPWSLLOHIDERDRAGLLPALF 31 KVLSVGRGGSPRLQPDSRALHYMKKLYKTYATKEGIPKSNR SHLYNTVRLFTPCTRHKOAPGDOVTGILPSVELLFNLDRITTV EHLLKSVLLYNINNSVSFSSAVKCVCNLMIKEPKSSSRTLGRA PYSFTFNSOFEFGKKHKWIOIDVTSLLOPLVASNKRSIHMSIN FTCMKDOLEHPSAONGLFNMTLVSPSLILYLNDTSAOAYHS WYSLHYKRRPSOGPDOERSLSAYPVGEEAAEDGRSSHHRHR RGOETVSSELKKPLGPASFNLSEYFROFLLPONECELHDFRLS FSOLKWDNWIVAPHRYNPRYCKGDCPRAVGHRYGSPVHTM VONIIYEKLDSSVPRPSCVPAKYSPLSVLTIEPDGSIAYKEYED MIATKCTCR

BMP - 3 ERPKPPFPELRKAVPGDRTAGGGPDSELOPODKVSEHMLRLY 32 DRYSTVQAARTPGSLEGGSQPWRPRLLREGNTVRSFRAAAA ETLERKGLYIFNLTSLTKSENILSATLYFCIGELGNISLSCPVSG GCSHHAORKHIQIDLSAWTLKFSRNOSOLLGHLSVDMAKSH RDIMSWLSKDITOLLRKAKENEEFLIGFNITSKGROLPKRRLP FPEPYILVYANDAAISEPESVVSSLOGHRNFPTGTVPKWDSHI RAALSIERRKKRSTGVLLPLONNELPGAEYOYKKDEVWEER KPYKTLQAQAPEKSKNKKKQRKGPHRKSOTLOFDEQTLKK ARRKOWIEPRNCARRYLKVDFADIGWSEWIISPKSFDAYYCS GACOFPMPKSLKPSNHATIOSIVRAVGVVPGIPEPCCVPEKMS SLSILFFDENKNVLKVYPNMTVESCACR GDF - 10 SHRAPAWSALPAAADGLQGDRDLQRHPGDAAATLGPSAQD 33 MVAVHMHRLYEKYSROGARPGGGNTVRSFRARLEVDOK AVYFFNLTSMQDSEMILTATFHFYSEPPRWPRALEVLCKPRA KNASGRPLPLGPPTROHLLFRSLSONTATOGLLRGAMALAPP PRGLWOAKDISPIVKAARRDG?LLLSAOLDSEERDPGVPRPS PYAPYILVYANDLAISEPNSVAVTLORYDPFPAGDPEPRAAP NNSADPRVRRAAQATGPLODNELPGLDERPPRAHAOHFHKH QLWPSPFRALKPRPGRKDRRKKGQEVFMAASQVLDFDEKT MOKARRKOWDEPRVCSRRYLKVDFADIGWNEWIISPKSFDA YYCAGACEFPMPKIVRPSNHATIOSIVRAVGIIPGIPEPCCVPD KMNSLGVLFLDENRNVVLKVYPNMSVDTCACR

GDNF FPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDOF 34 DDVMDFIOATIKRLKRSPDKOMAVLPRRERNROAAAANPE NSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFR YCSGSCDAAETTYDKILKNLSRNRRLVSDKVGOACCRPIAFD DDLSFLDDNLVYHILRKHSAKRCGCI NRTN IWMCREGLLLSHRLGPALVPLHRLPRTLDARIARLAOYRALL 35 OGAPDAMELRELTPWAGRPPGPRRRAGPRRRRARARLGAR PCGLRELEVRVSELGLGYASDETVLFRY CAGACEAAARVYD LGLRRLRORRRLRRERVRAOPCCRPTAYEDEVSFLDAHSRY HTVHELSARECACV

PSPN WGPDARGVPVADGEFSSEQVAKAGGTWLGTHRPLARLRRA 36 LSGPCQLWSLTLSVAELGLGYASEEKVIFRYCAGSCPRGART QHGLALARLQGQGRAHGGPCCRPTRYTDVAFLDDRHRWOR LPOLSAAACGCGG ARTN SLGSAPRSPAPREGPPPVLASPAGHLPGGRTARWCSGRARRP 37 PPQPSRPAPPPPAPPSALPRGGRAARAGGPGSRARAAGARGC RLRSQLVPVRALGLGHRSDELVRFRFCSGSCRRARSPHDLSL ASLLGAGALRPPPGSRPVSOPCCRPTRYEAVSFMDVNSTWRT VDRLSATACGCLG

It is noted that some prodomains may be cleaved by porated by reference in their entirety . ) GDF - 11 may in , in proprotein convertase enzymes . As used herein , the term some cases, be cleaved by PC5 /6 . In some cases , proprotein " proprotein convertase ” refers to an enzyme that cleaves a convertases may cleave proproteins at additional sites , other prodomain from a translated protein to facilitate protein 60 than those indicated in Table 1 . In some embodiments , maturation . Some proprotein convertases of the present pro -proteins may be cleaved at a first cleavage site ( the first invention include the subtilisin - like proprotein convertase site being the site closest to the N - terminus ) . In other ( SPC ) family member enzymes. The SPC family comprises embodiments , pro -proteins may be cleaved at a cleavage site calcium -dependent serine endoproteases that include , but other than a first cleavage site . In some cases, proprotein are not limited to furin / PACE , PC1/ 3 , PC2, PC4, PC5/ 6 , 65 convertase cleavage may occur intracellularly . In some PACE4 and PC7 ( Fuller et al ., 2009 . Invest Ophthalmol Vis cases, proprotein convertase cleavage may occur extracel Sci. 50 ( 12 ) :5759 -68 , the contents of which are herein incor - lularly . US 9 ,758 ,576 B2 20 Many TGF - B family member proteins are synthesized in in length to about 250 amino acid residues in length , from conjunction with prodomains. Some prodomains may about 175 amino acid residues in length to about 400 amino remain associated with growth factors after cleavage. Such acid residues in length , from about 200 amino acid residues associations may form latent growth factor- prodomain com - in length to about 500 amino acid residues in length and /or plexes (GPCs ) that modulate the availability of growth 5 at least 500 amino acid residues in length . factors for cell signaling. Growth factors may be released in some embodiments , protein modules comprise one or from latency in GPCs through associations with one ormore more regions with known functional features ( e . g . protein extracellular proteins . In some cases , growth factor release binding domain , nucleic acid binding domain , hydrophobic may rely on force applied to GPCs through extracellular pocket , etc . ) Protein modules may comprise functional pro protein interactions . Such forces may pull from C - terminal 10 tein domains necessary for different aspects of growth factor and / or N -terminal regions of GPCs resulting in the release of signaling, secretion , latency and / or release from latent con associated growth factors. formations . In some TGF- B family members , the prodomain portion In some embodiments , protein modules may be derived of the GPC is responsible for growth factor retention and from TGF - b - related proteins. Such protein modules may blocking the interaction of retained growth factors with their 15 include, but are not limited to latency -associated peptides receptors. Prodomain portions ofGPCs that function in this (LAPs ), LAP - like domains , growth factor domains, fastener regard are referred to as latency associated peptides (LAPs ) . regions, proprotein convertase cleavage sites ( e . g . furin TGF -B1 , 2 and 3 are know to comprise LAPs. Some cleavage sites) , B / TP cleavage sites , arm regions, finger prodomains may comprise LAP - like domains . As used regions, residues (such as cysteine residues for example ) for herein , the term “ LAP - like domain ” refers to prodomain 20 extracellular protein [ e . g . latent TGF- B binding protein portions of GPCs and / or free prodomains that may be (LTBP ) , fibrillin and /or glycoprotein A repetitions predomi structurally similar or synthesized in a similar manner to nant (GARP ) protein ] associations , latency loops (also LAPs, but that may not function to prevent growth factor/ referred to herein as latency lassos, ) alpha 1 helical regions, receptor interactions. GDF - 8 and GDF - 11 prodomains com - alpha 2 helical regions, RGD sequences and bowtie regions . prise LAP - like domains . 25 FIG . 4 is a schematic diagram of an embodiment depicting Depending on a variety of factors , growth factors may be LAP and growth factor dimers comprising protein modules . free or associated with one or more LAP or LAP - like In some embodiments, protein modules may be derived domains. FIG . 3 is a schematic depicting an embodiment from one or more TGF- B isoform ( e . g . TGF -B1 , TGF- B2 wherein a growth factor dimer may associate with a LAP and /or TGF - B3 ) . Such protein modules may comprise the dimer . In some embodiments , GPCs comprise protein mod - 30 protein modules and / or amino acid sequences listed in Table ules necessary for different aspects of growth factor signal 2 . Some protein modules of the present invention may ing , secretion , latency and /or release from latent GPCs . As comprise amino acid sequences similar to those in Table 2 , used herein , the term " protein module ” refers to any com - but comprise additional or fewer amino acids than those ponent, region and / or feature of a protein . Protein modules listed . Such amino acid sequences may comprise about 1 may vary in length , comprising one or more amino acids. 35 more or fewer amino acids, about 2 more or fewer amino Protein modules may be from about 2 amino acid residues acids , about 3 more or fewer amino acids, about 4 more or in length to about 50 amino acid residues in length , from fewer amino acids, about 5 more or fewer amino acids, about about 5 amino acid residues in length to about 75 amino acid 6 more or fewer amino acids, about 7 more or fewer amino residues in length , from about 10 amino acid residues in acids, about 8 more or fewer amino acids, about 9 more or length to about 100 amino acid residues in length , from 40 fewer amino acids, about 10 more or fewer amino acids or about 25 amino acid residues in length to about 150 amino greater than 10 more or fewer amino acids on N -terminal acid residues in length , from about 125 amino acid residues and / or C - terminal ends. TABLE 2 TGF - B protein modules

TGF - B SEQ Family ID Member Protein Module Prodomain and growth factor Sequence NO TGF - Bi latency associated LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPP 38 peptide SOGEVPPGPLPEAVLALYNSTRDRVAGESAEP EPEPEADYYAKEVTRVLMVETHNEIYDKFKQS THSIYMFFNTSELREAVPEPVLLSRAELRLLRL KLKVEQHVELYQKYSNNSWRYLSNRLLAPSD SPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHC SCDSRDNTLOVDINGFTTGRRGDLATIHGMNR PFLLLMATPLERAQHLOSSRHRR TGF - B2 latency associated SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSP 39 peptide PEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAA ACERERSDEEYYAKEVYKIDMPPFFPSENAIPP TFYRPYFRIVRFDVSAMEKNASNLVKAEFRVF RLONPKARVPEORIELYOILKSKDLTSPTORYI DSKVVKTRAEGEWLSFDVTDAVHEWLHHKD RNLGFKISLHCPCCTFVPSNNYIIPNKSEELEAR FAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLL LMLLPSYRLESQQTNRRKKR US 9 ,758 ,576 B2 21 TABLE 2 - continued TGF - B protein modules

TGF - B SEO Family ID Member Protein Module Prodomain and growth factor Sequence NO

TGF - B3 latency associated SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLT 40 peptide SPPEPTVMTHVPYOVLALYNSTRELLEEMHGE REEGCTOENTESEYYAKEIHKFDMIOGLAEHN ELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEF RVLRVPNPSSKRNEORIELFOILRPDEHIAKOR YIGGKNLPTRGTAEWLSFDVTDTVREWLLRRE SNLGLEISIHCPCHTFQPNGDILENIHEVMEIKF KGVDNEDDHGRGDLGRLKKQKDHHNPHLILM MIPPHRLDNPGQGGQRKKR TGF - B1 straight jacket LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPP region SOGEVPPGPLP TGF - B2 straight jacket SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSP region PEDYPEPEEVP TGF - B3 straight jacket SLSLSTCTTLDFGHI KKKRVEAIRGOILSKLRLT region SPPEPTVMTHVP TGF - B1 growth factor ALDTNYCFSSTEKNCCVROLYIDFRKDLGWK domain WIHEPKGYHANFCLGPCPYIWSLDTOYSKVLA LYNOHNPGASAAPCCVPOALEPLPIVYYVGRK PKVEQLSNMIVRSCKCS TGF - B2 growth factor ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWK 45 domain WIHEPKGYNANFCAGACPYLWSSDTOHSRVL SLYNTINPEASASPCCVSODLEPLTILYYIGKTP KIEQLSNMIVKSCKCS TGF - B3 growth factor ALDTNYCFRNLEENCCVRPLYIDFRODLGWK 46 domain WVHEPKGYYANFCSGPCPYLRSADTTHS TVLG LYNTLNPEASASPCCVPODLEPLTILYYVGRTP KVEQLSNMVVKSCKCS TGF - B1 fastener region residues 74 - 76 , YYA TGF - B2 fastener region residues 79 - 81 , YYA residues 80 - 82 , YYA TGF - B3 fastener region l TGF - B1 furin cleavage site RHRR region TGF - B2 furin cleavage site RKKR ilcontro region TGF - B3 furin cleavage site RKKR con region TGF - B1 arm region EAVLALYNSTRDRVAGESAEPEPEPEADYYAK Theo EVTRVLMVETHNEIYDKFKOSTHSIYMFFNTSE LREAVPEPVLLSRAELRLLRLKLKVEOHVELY QKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGV VROWLSRGGEIEGFRLSAHCSCDSRDNTLOVD INGFTTGRRGDLATIHGMNRPFLLLMATPLER AQHLOSSRHRR TGF - B2 arm region PEVISIYNSTRDLLOEKASRRAAACERERSDEE 50 YYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVR FDVSAMEKNASNLVKAEFRVFRLONPKARVP EQRIELYQILKSKDLTSPTORYIDSKVVKTRAE GEWLSFDVTDAVHEWLHHKDRNLGFKISLHC PCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYT SGDOKTIKSTRKKNSGKTPHLLLMLLPSYRLES QQTNRRKKR TGF - B3 arm region YQVLALYNSTRELLE EMHGEREEGCTQENTES 51 EYYAKEIHKFDMIQGLAEHNELAVCPKGITSK VFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKR NEQRIELFQILRPDEHIAKORYIGGKNLPTRGT US 9 ,758 ,576 B2 23 24 TABLE 2 - continued TGF - B protein modules

TGF - B SEO Family ID Member Protein Module Prodomain and growth factor Sequence NO AEWLSFDVTDTVREWLLRRESNLGLEISIHCPC HTFOPNGDILENIHEVMEIKFKGVDNEDDHGR GDLGRLKKOKDHHNPHLILMMIPPHRLDNPGO GGQRKKR TGF - B1 fingers region 1 CVROLYIDFRKDLGWKWIHEPKGYHANFC 52 TGF -B2 fingers region 1 CLRPLYIDFKRDLGWKWIHEPKGYNANFCA ??? TGF - B3 fingers region 1 CVRPLYIDFRODLGWKWVHEPKGYYANFCS 54 TGF - B1 fingers region 2 CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSC 55 KCS TGF - B2 fingers region 2 CVSODLEPLTILYYIGKTPKIEOLSNMIVKSCKCS 56 TGF - B3 fingers region 2 CVPODLEPLTILYYVGRTPKVEOLSNMVVKSC 57 KCS

TGF - B1 residue for LTBP Cys 4 i association

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 . association

TGF - B3 residue for GARP Cys 7 I association TGF - B1 latency loop LASPPSOGEVPPGPL 58 TGF - B2 latency loop LTSPPEDYPEPEE 59 TGF - B3 latency loop LTSPPEPTVMTHV 60 TGF -B1 alpha 1 helical LSTCKTIDMELVKRKRIEAIRGQILSKLR 61 region TGF - B2 alpha 1 helical LSTCSTLDMDOFMRKRIEAIRGOILSKLK 62 region

63 TGF - B3 alpha 1 helical LSLSTCTTLDFGHIKKKRVEAIRGQILSKLR o region TGF - B1 trigger loop region NGFTTGRRGDLATIHGMNRP 64 TGF - B2 trigger loop region FAGIDGTSTYTSGDQKTIKSTRKKNSGKTP 65 ( long ) TGF - B3 trigger loop region GVDNEDDHGRGDLGRLKKQKDHHNP 66 I TGF - B1 RGD sequence residue 215 - 217 , RGD region

I TGF - B3 RGD sequence residue 241 - 243 , RGD region TGF - B1 bowtie region CSCDSRDNTLQVD 67 TGF - B2 bowtie region CPCCTFVPSNNYIIPNKSEELEAR 68 TGF - B3 bowtie region CPCHTFQPNGDILENIHEVMEIK 65 In some embodiments , LAPs or LAP - like domains com - and /or GPC . Some LAPs or LAP - like domains may asso prise the prodomain portion of a TGF - B - related protein ciate with growth factors in GPCs. Some LAPs may steri US 9 ,758 ,576 B2 25 26 cally prevent growth factor association with one or more LAP- like domains . Alpha 1 helical regions may also com cellular receptors . LAPs or LAP - like domains may comprise prise N -terminal regions for extracellular associations . Such arm regions and /or straight jacket regions. Some LAP or extracellular associations may comprise extracellular matrix proteins and / or proteins associated with the extracellular LAP- like domains may comprise C -terminal regions 5 matrix . Some extracellular associations may comprise asso referred to herein as “ bowtie regions. ” In some LAP or 5 ciations with proteins that may include , but are not limited LAP- like domain dimers , bowtie regions of each monomer to LTBPs ( e . g . LTBP1, LTBP2 , LTBP3 and / or LTBP4 ) , may associate and / or interact . Such associations may com fibrillins ( e . g . fibrillin - 1 , fibrillin - 2 , fibrillin - 3 and/ or fibril prise disulfide bond formation , as is found between mono - lin - 4 . ) perlecan , decorin and / or GARPs ( e . g . GARP and/ or mers of TGF - B isoform LAPs . LRRC33 ) . N - terminal extracellular associations may com In some embodiments, arm regions may comprise trigger prise disulfide bonds between cysteine residues . In some loop regions . Trigger loops may comprise regions that cases, extracellular matrix proteins and /or proteins associ associate with integrins. Such regions may comprise amino ated with the extraceullar matrix may comprise bonds with acid sequences comprising RGD ( Arg -Gly - Asp ) . Regions one or more regions of LAPs /LAP - like domains other than comprising RGD sequences are referred to herein as RGD N - terminal regions . sequence regions . In some embodiments , LAPs or LAP - like In some embodiments , growth factor domains comprise domains comprise latency loops ( also referred to herein as one or more growth factor monomers . Some growth factor latency lassos ). Some latency loops may maintain associa domains comprise growth factor dimers. Such growth factor tions between LAPs or LAP - like domains and growth fac domains may comprise growth factor homodimers or het tors present within GPCs. LAPs or LAP - like domains may erodimers (comprising growth factor monomers from dif also comprise fastener regions. Such fastener regions may ferent TGF - B - related proteins . ) Some growth factor domains maintain associations between LAPs or LAP - like domains may comprise fingers regions . Such fingers regions may and growth factors present within GPCs. Some fastener comprise B -pleated sheets . Fingers regions may associate regions may maintain LAP or LAP - like domain conforma with LAPs or LAP - like domains . Some fingers regions may tions that promote growth factor retention . maintain association between growth factor domains and In some cases , GPCsmay require enzymatic cleavage for » LAPs or LAP - like domains . dissociation of bound growth factors. Such cleavage may be In some embodiments, recombinant proteins of the pres carried out in some instances by members of the BMP - 1 ) ent invention may comprise protein modules from growth Tolloid - like proteinase ( B / TP ) family (Muir et al. , 2011 . J differentiation factor (GDF ) proteins. Such GDF protein Biol Chem . 286 (49 ): 41905 - 11 , the contents of which are modules may comprise the protein modules and /or amino herein incorporated by reference in their entirety . ) These acid sequences listed in Table 3 . In some embodiments , metaloproteinases may include , but are not limited to BMP - protein modules of the present invention may comprise 1 , mammalian tolloid protein (mTLD ,) mammalian tolloid amino acid sequences similar to those in Table 3 , but like 1 (mTLL1 ) and mammalian tolloid - like 2 (mTLL2 .) comprise additional or fewer amino acids than those listed . Exemplary GPCs that may be cleaved by such metallopro - . Some such amino acid sequences may comprise about 1 teinases may include , but are not limited to GDF - 8 and more or fewer amino acids, about 2 more or fewer amino GDF - 11. In some cases , GDF - 8 may be cleaved by mTLL2 . acids , about 3 more or fewer amino acids , about 4 more or In some cases, tolloid cleavage may occur intracellularly . In fewer amino acids, about 5 more or fewer amino acids, about some cases , tolloid cleavage may occur extracellularly . 6 more or fewer amino acids, about 7 more or fewer amino Straight jacket regions may comprise alpha 1 helical acids , about 8 more or fewer amino acids, about 9 more or regions. In some embodiments , alpha 1 helical regions may * fewer amino acids , about 10 more or fewer amino acids or be positioned between growth factor monomers . Some alpha greater than 10 more or fewer amino acids on N -terminal 1 helical regions comprise N - terminal regions of LAPs or and / or C - terminal ends. TABLE 3 GDF protein modules TGF - B SEQ Family ID Member Protein Module Prodomain and growth factor Sequence NO GDF - 8 prodomain NENSEQKENVEKEGLCNACTWRONTKSSRIEA 70 IKIQILSKLRLETAPNISKDVIROLLPKAPPLREL IDQYDVORDDSSDGSLEDDDYHATTETII TMPT ESDFLMOVDGKPKCCFFKFSSKIOYNKVVKAO LWIYLRPVETPTTVFVOILRLIKPMKDGTRYTG IRSLKLDMNPGTGIWOSIDVKTVLONWLKOPE SNLGIEIKALDENGHDLAVTFPGPGEDGLNPFL EVKVTDTPKRSRR GDF - 11 prodomain AEGPAAAAAAAAAAAAAGVGGERSSRPAPSV 71 APEPDGCPVCVWROHSRELRLESIKSQILSKLR LKEAPNISREVVKQLLPKAPPLQQILDLHDFQG DALQPEDFLEEDEYHATTETVISMAQETDPAV QTDGSPLCCHFHFSPKVMFTKVLKAQLWVYL RPVPRPATVYLQILRLKPLTGEGTAGGGGGGR RHIRIRSLKIELHSRSGHWQSIDFKOVLHSWFR QPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLH PFMELRVLENTKRSRR US 9 ,758 ,576 B2 27 28 TABLE 3 - continued GDF protein modules TGF - B SEO Family ID Member Protein Module Prodomain and growth factor Sequence NO

GDF - 8 straight jacket NENSEQKENVEKEGLCNACTWRONTKSSRIEA 72 region IKIQILSKLRLETAPNISKDVIROLLPKAPPL GDF - 11 straight jacket AEGPAAAAAAAAAAAAAGVGGERSSRPAPSV region APEPDGCPVCVWRQHSRELRLESIKSQILSKLR LKEAPNISREVVKQLLPKAPPL GDF - 8 growth factor DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWI 74 domain IAPKRYKANYCSGECEFVFLOKYPHTHLVHOA NPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGK IPAMVVDRCGCS GDF - 11 growth factor NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWI 75 domain IAPKRYKANYCSGQCEYMFMQKYPHTHLVQQ ANPRGSAGPCCTPTKMSPINMLYFNDKOOIIYG KIPGMVVDRCGCS GDF - 8 fastener region residues 87 - 89 , DYH

GDF - 11 fastener region residues 110 - 112 , EYH l

GDF - 8 furin cleavage site RSRR à region GDF - 11 furin cleavage site RSRR region ò GDF - 8 BMP / Tolloid between residues R75 and 176 cleavage site

GDF - 11 BMP / Tolloid between residues G97 and 198 L cleavage site GDF - 8 arm region RELIDOYDVORDDSSDGSLEDDDYHATTETIIT 77 MPTESDFLMOVDGKPKCCFFKFSSKIOYNKV KAQLWIYLRPVETPTTVFVQILRLIKPMKDGTR YTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLK QPESNLGIEIKALDENGHDLAVTFPGPGEDGLN PFLEVKVTDTPKRSRR GDF - 11 arm region QQILDLHDFQGDALQPEDFLEEDEYHATTETVI 78 SMAQETDPAVQTDGSPLCCHFHFSPKVMFTKV LKAQLWVYLRPVPRPATVYLQILRLKPLTGEG TAGGGGGGRRHIRIRSLKIELHSRSGHWOSIDF KOVLHSWFROPOSNWGIEINAFDPSGTD LAVT SLGPGAEGLHPFMELRVLENTKRSRR GDF - 8 fingers region 1 CRYPLTVDFEAFGWDWIIAPKRYKANYCS 79 GDF - 11 fingers region 1 CRYPLTVDFEAFGWDWIIAPKRYKANYCS 79 GDF - 8 fingers region 2 CTPTKMSPINMLYFNGKEOIIYGKIPAMVVDRC 80 GCS GDF - 11 fingers region 2 CTPTKMSPINMLYFNDKQQI IYGKIPGMVVDR 81 CGCS

ons GDF - 8 latency loop RLETAPNISKDVIROLLPKAPPL 2 GDF - 11 latency loop RLKEAPNISREVVKQLLPKAPP 83 GDF - 8 alpha 1 helical GLCNACTWRONTKSSRIEAIKIOILSK 84 region GDF - 11 alpha 1 helical DGCPVCVWRQHSRELRLESIKSQILSKL 85 region

GDF - 8 bowtie region DENGHDLAVTFPGP 86 GDF - 11 bowtie region DPSGTDLAVTSLG 87 US 9 ,758 , 576 B2 29 30 Some recombinant proteins of the present invention may j ects suffering from beta -thalassemia or dyserythropoietic comprise GDF - 15 , GDF - 15 signaling pathway - related pro anemias . teins and /or modules and /or portions thereof. GDF - 15 is a In some embodiments , recombinant proteins of the pres ent invention may comprise protein modules from activin TGF -B family protein that is highly expressed in liver. 5 subunits . Such protein modules may comprise the protein Expression ofGDF - 15 is dramatically upregulated follow - 5 modules and/ or amino acid sequences of the activin subunit ing liver injury (Hsiao et al. 2000 . Mol Cell Biol. 20 ( 10 ) : inhibin beta A , listed in Table 4 . In some embodiments , 3742 -51 . ) Additionally , its expression in macrophages may protein modules of the present invention may comprise serve a protective function in the context of atherosclerosis , amino acid sequences similar to those in Table 4 , but possibly through regulation of adhesion molecule expres comprise additional or fewer amino acids than those listed . sion ( Preusch et al. , 2013 . Eur J Med Res . 18 : 19 . ) While a 10 Some such amino acid sequences may comprise about 1 member of the TGF - B family , GDF - 15 comprises less than more or fewer amino acids, about 2 more or fewer amino 30 % homology with other members , making it the most acids , about 3 more or fewer amino acids , about 4 more or divergent member of the family ( Tanno et al. , 2010 . Curr fewer amino acids , about 5 more or fewer amino acids, about Opin Hematol. 17 ( 3 ) :184 - 90 , the contents of which are 6 more or fewer amino acids, about 7 more or fewer amino incorporated herein by reference in their entirety .) The 15 acids, about 8 more or fewer amino acids, about 9 more or mature form is soluble and can be found in the blood stream . fewer amino acids , about 10 more or fewer amino acids or Interestingly, GDF - 15 levels in circulation have been found greater than 10 more or fewer amino acids on N -terminal to negatively correlate with hepcidin levels , suggesting a and / or C -terminal ends. TABLE 4 Inhibin beta A protein modules SEQ Protein Module Prodomain and growth factor Sequence ID NO latency associated SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPE 88 peptide ( LAP ) MVEAVKKHILNMLHLKKRPDVTOPVPKAALL NAIRKLHVGKVGENGYVEIEDDIGRRAEMNEL MEOTSEIITFAESGTARKTLHFEISKEGSDLSVV ERAEVWLFLKVPKANRTRTKVTIRLFQQQKHP QGSLDTGEEAEEVGLKGERSELLLSEKVVDAR KSTWHVFPVSSSIQRLLDOGKSSLDVRIACEQC OESGASLVLLGKKKKKEEEGEGKKKGGGEGG AGADEEKEQSHRPFLMLQARQSEDHPHRRRRR straight jacket SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPE 89 region MVEAVKKHILNMLHLKKRPDVTQPVPKAALLN growth factor RGLECDGKVNICCKKQFFVSFKDIGWNDWIIA 90 domain PSGYHANYCEGECPSHIAGTSGSSLSFHSTVIN HYRMRGHSPFANLKSCCVPTKLRPMSMLYYD DGONIIKKDIONMIVEECGCS fastener region residues 89 - 91 , RRA furin cleavage site RRRR 91 region

arm region LNAIRKLHVGKVGENGYVEI EDDIGRRAEMNE 92 LMEOTSEIITFAESGTARKTLHFEISKEGSDLSV VERAEVWLFLKVPKANRTRTKVTIRLFQQQKH PQGSLDTGEEAEEVGLKGERSELLLSEKVVDA RKS TWHVFPVSSSIORLLDOGKSSLDVRIACEO COESGASLVLLGKKKKKEEEGEGKKKGGGEG GAGADEEKEQSHRPFLMLQAROSEDHPHRRR RR fingers region 1 KKOFFVSFKDIGWNDWIIAPSGYHANYC 93 fingers region 2 CVPTKLRPMSMLYYDDGONIIKKDIQNMIVEE 94 CGCS latency loop LKKRPDVTQPVPKAALL 95 alpha 1 helical ALAALPKDVPNSQPEMVEAVKKHILNML 96 region bowtie region OESGASLVLLGKKKKKEEEGEGKKKGGGEGG 97 AG role for GDF - 15 in iron load and /or metabolism (Finkenstedt Growth factor domains among TGF - B family members et al. , 2008 . British Journal of Haematology. 144 :789 - 93. ) are more highly conserved while prodomains comprise a Elevated GDF - 15 in the blood is also associated with much lower percent identity among family members (FIG . ineffective and /or apoptotic erythropoiesis , such as in sub 9. ) Table 5 demonstrates this trend among TGF- B isoforms . US 9, 758 ,576 B2 31 32 TABLE 5 tumorigenicity 1 (NBL1 ) , Norrin (NDP ) , Otogelin ( OTOG ) , Otogelin - like protein (OTOGL ), Protein CYR61 (CYR61 ) , Percent identity among TGF - B isoforms : LAP vs growth factor Protein NOV homolog (NOV ), Sclerostin (SOST ), Scle rostin domain - containing protein 1 (SOSTDC1 ) , SCO - spon TGF -B1 TGF- B2 TGF- B3 5 din (SSPO ) , Slit homolog 1 protein (SLIT1 ) , Slit homolog 2 TGF -B1 31. 2 % vs 71 . 2 % 31 . 9 % vs 76 . 7 % TGF -B2 31. 2 % vs 71. 2 % 44 . 4 % vs 79 . 4 % protein (SLIT2 ), Slit homolog 3 protein (SLIT3 ), von Wille TGF- B3 31. 9 % vs 76 . 7 % 44 . 4 % vs 79 . 4 % - brand factor (VWF ) , WNT1 - inducible - signaling pathway protein 1 (WISP1 ) and WNT1- inducible -signaling pathway Prodomains may vary in length from about 50 to about 10 protein 3 (WISP3 ) . 200 , from about 100 to about 400 or from about 300 to about Recombinant Proteins 500 amino acids residues . In some embodiments , prodo In some embodiments , the present invention provides mains range from about 169 to about 433 residues . Prodo recombinant proteins . As used herein , the term “ recombinant mains may be unrelated in sequence and / or low in homol protein ” refers to a protein produced by an artificial gene ogy . Some prodomains may have similar folds and /or three 15 and /or process ( e . g . genetic engineering ). Such recombinant dimensional structures . Prodomains of TGF - ß family mem proteins may comprise one or more protein modules from bers may comprise latency loops . Such loops may be pro - one or more TGF- b - related proteins . Some recombinant line - rich . Latency loop length may determine the ability of proteins disclosed herein may be useful as recombinant such loops to encircle growth factor finger regions. antigens . As used herein , the term “ recombinant antigen " In some embodiments , protein modules from some 20 refers to a recombinant protein that may be used to immu TGF -B family members comprise low sequence identity nize one or more hosts for the production of antibodies with protein modules from other TGF - B family members . directed toward one or more epitopes present on such Such low sequence identity may indicate specialized roles recombinant antigens. Some recombinant antigens may be for such family members with distinct protein modules . cell- based antigens. As used herein , the term " cell -based Association of GPCs with extracellular proteins may 25 antigen ” refers to recombinant antigens that are expressed in strengthen prodomain - growth factor interactions . In some cells for presentation of such antigens on the cell surface . embodiments , such extracellular proteins may include , but Such cells may be used to immunize hosts for the production are not limited to LTBPs, fibrillins and / or GARP. In some cases , extracellular protein associations are required to keep of antibodies directed to such cell -based antigens. growth factors latent in GPCs. In some embodiments , recombinant proteins disclosed GARP expression has been shown to be required for s herein may be used as therapeutics . Recombinant proteins surface expression of GPCs on the surface of cells of disclosed herein may modulate growth factor ( e . g . growth hematopoietic origin ( Tran , D . Q . et al. , GARP (LRRC32 ) is factors comprising TGF -B - related proteins ) levels and /or essential for the surface expression of latent TGF - B on activity (e . g . signaling ) upon administration and /or intro platelets and activated FOXP3 + regulatory T cells . PNASis . 35 duction to one or more subjects and /or niches. 2009 , Jun . 2 . 106 ( 32 ): 13445 - 50 . ) GARP may act as a tether In some embodiments, recombinant proteins disclosed to hold GPCs in place onon thethe surface of these cells , includ - herein may be used to assay growth factor ( e . g . growth ing, but not limited to regulatory T- cells and /or platelets . factors comprising TGF - B - related proteins) levels and /or In some embodiments , recombinant proteins of the pres activity (e .g . signaling ). Some recombinant proteins dis ent invention may comprise bone morphogenetic proteins 40 closed herein may be used in the isolation of antibodies (BMPs ) , a family of TGF - B -related proteins. Protein mod directed to TGF - B - related proteins . Recombinant proteins of ules comprising sequences from BMPs may comprise the present invention may also be used as recombinant sequences from any of those BMP modules disclosed in antigens in the development of stabilizing [ reducing or FIGS. 8A - 8 . While related to other TGF - B family member preventing dissociation between two agents , ( e . g . growth proteins , BMPs generally signal through SMAD1, 5 and 8 45 factor release from GPCs, GPC release from one or more proteins while TGF - B isoforms ( e . g . TGF -B1 , TGF -B2 and protein interactions ) ] and / or releasing ?enhancing the disso TGF - B3 ) signal through SMAD2 and SMAD3 . ciation between two agents ( e . g . growth - factor release from Some BMP receptors and /or co -receptors are also distinct GPCs, GPC release from one or more protein interactions) from other TGF - B family member proteins . Among these is antibodies . Recombinant proteins of the present invention the repulsive guidance molecule (RGM ) family of proteins . 50 may include TGF - ß family member proteins as well as RGM proteins act as co - receptors for BMP signaling . There components and /or protein modules thereof. Some recom are three RGM family members, RGMA , RGMB and binant proteins of the present invention may comprise RGMC falso known as hemojuvelin (Hjv . ) ] Recombinant prodomains without associated growth factors , furin cleav proteins of the present invention comprising one or more age -deficient mutants , mutants deficient in extracellular pro BMP protein module may be useful for the development of 55 tein associations and /or combinations thereof. antibodies and /or assays to study, enhance and /or perturb In some embodiments, recombinant proteins may com BMP interactions with RGM proteins. prise detectable labels . Detectable labels may be used to Another family ofGDF / BMP interacting proteins is C - ter allow for detection and /or isolation of recombinant proteins . minal cysteine knot- like (CTCK ) domain - containing pro - Some detectable labels may comprise biotin labels , poly teins . In some cases, CTCK domain -containing proteins may 60 histidine tags and / or flag tags . Such tags may be used to act antagonistically with regard to GDF /BMP signal trans - isolate tagged proteins. Proteins produced may comprise duction . CTCK domain -containing proteins include, but are additional amino acids encoding one or more 3C protease not limited to Cerberus , Connective tissue growth factor cleavage site . Such sites allow for cleavage at the 3C (CTGF ) , DAN domain family member 5 (DAND5 ) , Grem - protease cleavage site upon treatment with 3C protease , lin - 1 (GREM1 ) , Gremlin - 2 (GREM2 ) , Mucin - 19 (MUC19 ) , 65 including , but not limited to rhinovirus 3C protease . Such Mucin -2 (MUC2 ), Mucin -5AC (MUC5AC ) , Mucin - 5B cleavage sites are introduced to allow for removal of detect (MUC5B ), Mucin -6 (MUC6 ), Neuroblastoma suppressor of able labels from recombinant proteins . US 9 , 758 ,576 B2 33 34 Recombinant GPCs prodomains (see FIG . 6 . ) Some proprotein convertase cleav FIG . 5 is a schematic depicting an embodiment of a age sites comprising RXXR sequences may be mutated to recombinant GPC . Recombinant proteins according to FIG . RXG ( wherein X indicates a site where amino acid residues 5 comprising TGF - 3 - family member proteins may comprise may be variable ) . Such mutations are herein abbreviated as features including, but not limited to C - terminal regions of 5 “ D2G ” mutations and may be resistant to enzymatic cleav the mature growth factor , N - terminal regions of the prodo - age . In some embodiments , furin cleavage sites comprising main and /or proprotein cleavage sites. The proprotein cleav - RXXR sequences are mutated to AXXA . Such AXXA age site of recombinant TGF - B GPCs may , for example , sequences may also be resistant to enzymatic cleavage . comprise the furin consensus sequence RXXR wherein Ris In some embodiments , regions of proteolytic processing arginine and X indicates amino acid residues that may vary 10 by tolloid and/ or tolloid - like proteins may be mutated to among TGF - B family members . Furin cleavage site prevent such proteolytic processing . In some embodiments , sequences (although not limited to cleavage by furin alone tolloid processing regions on GDF - 8 and /or GDF - 11 may be and may include cleavage by other proprotein convertase mutated . In some embodiments , mutation of aspartic acid enzymes ) for each TGF - B family member are indicated in residues to alanine residues within tolloid processing regions Table 1 . Recombinant GPCs according to the embodiment 15 prevents tolloid processing . Mutation of aspartic acid resi depicted in FIG . 5 may also comprise one or more cysteine due 76 (D76 ) of the GDF - 8 (myostatin ) proprotein has been residues within and /or near the N -terminal region of the shown to prevent proteolytic activation of latent GDF - 8 prodomain . Such cysteine residues may be from about 1 to (Wolfman , N . M . et al. , PNAS . 2003 , Oct. 6 . 100 ( 26 ): 15842 about 10 amino acids, from about 4 to about 15 amino acids, 6 . ) In some embodiments , Asp 120 (D120 , residue number from about 5 to about 20 amino acids and /or from about 7 20 counted from the translated protein , D98 from the proprotein to about 50 amino acids from the N -terminus of the prodo - of SEQ ID NO : 4 ) in GDF - 11 may be mutated to prevent main . Recombinant GPCs may also comprise detectable tolloid processing (Ge et al ., 2005. Mol Cell Biol. 25 ( 14 ) : labels. Such detectable labels may be useful for detection 5846 -58 , the contents of which are herein incorporated by and / or isolation of recombinant GPCs. Detectable labels reference in their entirety . ) may comprise 2 or more histidine (His ) residues. Such 25 In some embodiments , one or more amino acids may be detectable labels may also be referred to herein as polyhis mutated in order to form recombinant GPCs with reduced tidine tags. Polyhistidine tags may include hexa histidine latency. Such mutations are referred to herein as “ activating tags (SEQ ID NO : 295 ) or HIS - TAGTM (EMD Biosciences , mutations. " These mutations may introduce one or more Darmstadt, Germany ) comprising a chain of six histidine regions of steric clash between complex prodomains and residues ( SEQ ID NO : 295 ). Some polyhistidine tags may be 30 growth factor domains. As used herein , the term " steric present at the N - terminus of recombinant proteins disclosed clash , ” when referring to the interaction between two pro herein . Some polyhistidine tags may be present at the teins or between two domains and / or epitopes within the C - terminus of recombinant proteins disclosed herein . Pro - same protein , refers to a repulsive interaction between such teins produced may comprise additional amino acids encod proteins , domains and / or epitopes due to overlapping posi ing one or more 3C protease cleavage site . Such sites allow 35 tion in three -dimensional space . Steric clash within GPCs for cleavage at the 3C protease cleavage site upon treatment may reduce the affinity between prodomains and growth with 3C protease , including , but not limited to rhinovirus 3C factor domains, resulting in elevated ratios of free growth protease . Some cleavage sites may be introduced to allow factor to latent growth factor. In some embodiments , one or for removal of detectable labels from recombinant proteins. more amino acids may be mutated in order to form recom In some embodiments of the present invention , recombi - 40 binant GPCs with increased latency . Such mutations are nant GPCs may comprise mutations in one or more amino referred to herein as “ stabilizing mutations. " These muta acids as compared to wild type sequences . In some cases , tions may increase the affinity between prodomains and one or more regions of proteolytic processing may be growth factor domains , resulting in decreased ratios of free mutated . Such regions may comprise proprotein convertase growth factor to latent growth factor. cleavage sites . Proprotein convertase ( e . g . furin ) cleavage 45 In some embodiments, recombinant proteins of the pres site mutations prevent enzymatic cleavage at that site and /or ent invention may comprise any of the sequences listed in prevent enzymatic cleavage of growth factors from their Table 6 or fragments thereof. TABLE 6 Recombinant proteins

SEO ID Protein Sequence NO proTGF - B1 LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPPGP 1 LPEAVLAL YNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTOYSKVLAL YNOHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF -B1 C4S LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPPGP 98 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEOHVELYOKY SNNSWRYLSNRLLAPSDSP US 9 ,758 , 576 B2 35 36 TABLE 6 - continued Recombinant proteins SEQ ID Protein Sequence NO EWLSFDVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNTLQ DINGFTTGRRGDLATIHGMNRPFLLLMATPLERAOHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF -B1 C4S LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 99 ( LAP ) LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEOHVELYOKY SNNSWRYL SNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHRR proTGF - B1 D2G LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPPGP 100 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHGALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEPK GYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPC CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF - B1 C4S D2G LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 101 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVRQWLSRGGEIEGFRLSAHCS CDSRDNTLQ VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAOHLOSS RHGALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWI HEPK GYHANFCLGPCPYIWSLDTOYSKVLALYNOHNPGASAAPC CVPOALEPLPIVYYVGRKPKVEOLSNMIVRSCKCS proTGF - B1 LAP LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPPGP 38 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQ VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHRR proTGF - B2 SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPE 2 EVPPEVISIYNSTRDLLOEKASRRAAACERERSDEEYYAKE VYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNL VKAEFRVFRLONPKARVPEORIELYOILKSKDLTSPTORYID SKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLH CPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIK STRKKNSGKTPHLLLMLLPSYRLESOOTNRRKKRALDAAY CFRNVODNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCA GACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPL TILYYIGKTPKIEQLSNMIVKSCKCS proTGF - B2 C5S SLSTSSTLDMDOFMRKRIEAIRGOILSKLKLTSPPEDYPEPEE 102 VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEV YKIDMPPFFPSENAIPPTFYRPYFRIVRFDV SAMEKNASNLV KAEFRVFRLONPKARV PEORIELYOILKSKDLTSPTORYIDS KVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHC PCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIKS TRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYC FRNVODNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAG ACPYLWSSDTOHSRVLSLYNTINPEASASPCCVSODLEPLTI LYYIGKTPKI EOLSNMIVKSCKCS proTGF -B2 LAP C5S SLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 103 VPPEVISIYNSTRDLLOEKASRRAAACERERSDEEYYAKEV YKIDMPPFFPSENAIPPTFYRPYFRIVRFDV SAMEKNASNLV KAEFRVFRLONPKARV PEORIELYOILKSKDLTSPTORYIDS KVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHC PCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIKS TRKKNSGKTPHLLLMLLPSYRLESOOTNRRKKR proTGF - B2 C5S D2G SLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 104 VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEV US 9 ,758 , 576 B2 37 38 TABLE 6 - continued Recombinant proteins SEQ ID Protein Sequence NO YKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLV KAEFRVFRLONPKARVPEORIELYQILKSKDLTSPTORYIDS KVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHC PCCTFVPSNNYI IPNKSEELEARFAGIDGTSTYTSGDOKTIKS TRKKNSGKTPHLLLMLLPSYRLESQOTNRRKGALDAAYCF RNVODNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGA CPYLWSSDTOHSRVLSLYNTINPEASASPCCVSODLEPLTIL YYIGKTPKIEOLSNMIVKSCKCS proTGF - B2 D2G SLSTCSTLDMDOFMRKRIEAIRGOILSKLKLTSPPEDYPEPE 105 EVPPEVISIYNSTRDLLOEKASRRAAACERERSDEEYYAKE VYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNL VKAEFRVFRLONPKARVPEORIELYOILKSKDLTSPTORYID SKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLH CPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIK STRKKNSGKTPHLLLMLLPSYRLESOOTNRRKGALDAAYC FRNVODNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAG ACPYLWSSDTOHSRVLSLYNTINPEASASPCCVSODLEPLTI LYYIGKTPKIEQLSNMIVKSCKCS proTGF - B2 LAP SLSTCSTLDMDOFMRKRIEAIRGOILSKLKLTSPPEDYPEPE 39 EVPPEVISIYNSTRDLLOEKASRRAAACERERSDEEYYAKE VYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNL VKAEFRVFRLONPKARVPEORIELYOILKSKDLTSPTORYID SKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLH CPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIK STRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKR proTGF - B3 SLSLSTCTTLDFGHIKKKRVEAIRGOILSKLRLTSPPEPTVMT 3 HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAK EIHKFDMIOGLAEHNELAVCPKGITSKVFRFNVSSVEKNRT NLFRAEFRVLRVPNPSSKRNEORIELFOILRPDEHIAKORYI GGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHC PCHTFOPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRL KKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNY CFRNLEENCCVRPLYIDFRODLGWKWVHEPKGYYANFCS GPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPODLEPL TILYYVGRTPKVEQLSNMVVKSCKCS proTGF - B3 07s SLSLSTSTTLDFGHIKKKRVEAIRGOILSKLRLTSPPEPTVMT 106 HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAK EIHKFDMIOGLAEHNELAVCPKGITSKVFRFNVSSVEKNRT NLFRAEFRVLRVPNPSSKRNEORIELFOILRPDEHIAKORYI GGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHC PCHTFOPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRL KKOKDHHNPHLILMMIPPHRLDNPGOGGORKKRALDTNY CFRNLEENCCVRPLYIDFRODLGWKWVHEPKGYYANFCS GPCPYLRSADTTHSTVLGLYNTLNPEASASPCCV PODLEPL TILYYVGRTPKVEOLSNMVVKSCKCS proTGF - B3 LAP C7S SLSLSTSTTLDFGHIKKKRVEAIRGOILSKLRLTSPPEPTVMT 107 HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAK EIHKFDMICGLAEHNELAVCPKGITSKVFRFNVSSVEKNRT NLFRAEFRVLRVPNPSSKRNEORIELFOILRPDEHIAKORYI GGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHC PCHTFOPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRL KKOKDHHNPHLILMMIPPHRLDNPGOGGORKKR proTGF -B3 C7S D2G SLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT 108 HVPYOVLALYNSTRELLEEMHGEREEGCTOENTESEYYAK EIHKFDMIOGLAEHNELAVCPKGITSKVFRFNVSSVEKNRT NLFRAEFRVLRVPNPSSKRNEORIELFOILRPDEHIAKORYI GGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHC PCHTFOPNGDILENIHEVMEIKF KGVDNEDDHGRGDLGRL KKOKDHHNPHLILMMIPPHRLDNPGOGGORKGALDTNYC FRNLEENCCVRPLYIDFRODLGWKWVHEPKGYYANFCSGP CPYLRSADTTHSTVLGLYNTLNPEASASPCCVPODLEPLTIL YYVGRTPKVEQLSNMVVKSCKCS proTGF - B3 D2G SLSLSTCTTLDFGHIKKKRVEAIRGOILSKLRLTSPPEPTVMT 109 HVPYOVLALYNSTRELLEEMHGEREEGCTOENTESEYYAK EIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRT NLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKORYI GGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCmi US 9 ,758 , 576 B2 39 TABLE 6 - continued Recombinant proteins SEQ ID Protein Sequence NO PCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRL KKOKDHHNPHLILMMIPPHRLDNPGOGGORKGALDTNYC FRNLEENCCVRPLYIDFRODLGWKWVHEPKGYYANFCSGP CPYLRSADTTHSTVLGLYNTLNPEASASPCCVPODLEPLTIL YYVGRTPKVEOLSNMVVKSCKCS proTGF - B3 LAP SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT 40 HVPYOVLALYNSTRELLEEMHGEREEGCTOENTESEYYAK EIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRT NLFRAEFRVLRVPNPSSKRNEORIELFOILRPDEHIAKORYI GGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHC PCHTFOPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRL KKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKR

In some embodiments , activating mutations may com - 20 tion . Mutations to 8218 , H222 , C223 and /or C225 may lead prise residues critical for LAP or LAP - like protein dimeriza - to weakened or disrupted disulfide bond formation and LAP tion . Some activating mutations may comprise TGF - B iso dimerization . In some embodiments , CED mutations lead to forms ( TGF -B1 , TGF -B2 and /or TGF -B3 ). Mutant GPCs with activating mutations may comprise mutations that elevated release of TGF - B and / or increased TGF - B activity . correspond to mutations identified in Camurati - Engelmann 25 In some embodiments , recombinant GPCs comprising TGF disease (CED ) . Subjects suffering from CED typically have B1 with CED mutations comprise sequences listed in Table genetic defects in TGF -B1 . Mutations identified in such 7 . The amino acid substitutions indicated in these proteins subjects include , but are not limited to mutations in residues reflect the residue number as counted from the start of the Y81 , 8218 , H222 , C223 and C225 . Residues C223 and C225 translated protein (before removal of the secretion signal are necessary for disulfide bond formation in LAP dimeriza sequence ) . TABLE 7 Recombinant GPCs with Camurati - Engelmann mutations

SEQ ID Protein Sequence NO proTGF - Bi Y81H LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 110 LPEAVLALHNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYOKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAOHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF - B1 R2180 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 111 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFCLSAHCSCDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAOHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTOYSKVLALYNQHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF -B1 H222D LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 112 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSADCSCDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTOYSKVLAL YNOHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF - B1 C223R LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 113 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHRSCDSRDNTLO US 9 ,758 , 576 B2 41 TABLE 7 - continued Recombinant GPCs with Camurati - Engelmann mutations

SEO ID Protein Sequence NO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTQYSKVLALYNCHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF - B1 C225R LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPPGP 114 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKF KOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHCSRDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTOYSKVLALYNOHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF - B1 C223R LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 115 C225R LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEOHVELYOKY SNNSWRYLSNRLLAPSDSP EWLSFDVTGVVROWLSRGGEIEGFRLSAHRSRDSRDNTLO VDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLOSS RHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTOYSKVLALYNOHNPGASAAP CCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

GPCs comprising CED mutations may find several uses in nal regions for extracellular associations. As used herein , the the context of the present invention . In some embodiments , 30 term “ N - terminal region for extracellular association ” refers such GPCs may be used to produce recombinant proteins to regions at or near protein N - termini thatmay be necessary comprising LAPs or LAP - like domains complexed with for extracellular associations with one or more N -terminal GARP. Coexpression of the entire GPC with GARP may be regions . Such regions may comprise at least the first N - ter necessary in some embodiments , for proper association and minal residue, at least the first 5 N - terminal residues , at least folding . Through expression of GPCs comprising CED the first 10 N - terminal residues, at least the first 20 amino mutations , growth factors may be able to dissociate leaving acid residues and /or at least the first 50 amino acid residues . the desired GARP -LAP complex . Y81H mutations may be Some mutations may comprise from about 1 amino acid useful in this regard . Y81H mutations lead to growth factor residue to about 30 amino acid residues, from about 5 amino release , but do not disrupt disulfide bonding between LAP an acid residues to about 40 amino acid residues and /or from monomers at residues C223 and C225 . Therefore , GARP - about 10 amino acid residues to about 50 amino acid LAP complexes formed through expression of Y81H GPC residues at or near protein N - termini. Such regions may mutants may comprise intact LAP dimers wherein growth comprise residues for LTBP, fibrillin and / or GARP associa factors have become dissociated . In some embodiments, tion . In some cases , one or more cysteine residues present additional co -expression or addition of excess furin during 45 within and / or near N -terminal regions for extracellular asso the production process may enhance growth factor disso - ciations may be necessary for such associations . In some ciation as well . embodiments , cysteine residues present within and / or near GPCs comprising CED mutations may be expressed to N -terminal regions for extracellular associations are present allow for the production and release ofmature growth factor within about the first 2 N - terminal residues, about the first 3 Some GPC - free growth factors expressed according to this 50 N -terminal residues , about the first 4 N - terminal residues , method may be used to assess antibody reactivity , for about the first 5 N - terminal residues, about the first 6 example in enzyme- linked immunosorbent assays (ELI - N - terminal residues, about the first 7 N -terminal residues SAs .) Some GPCs comprising CED mutations may be and /or at least the first 30 N -terminal residues . Some muta expressed to allow for the production and release of GPC - tions in one or more N - terminal regions for extracellular bound growth factors. GPCs comprising CED mutations 55 associations comprise substitution and / or deletion of such may be expressed to allow for the production and release of cysteine residues . Such mutations may modulate the asso chimeric proteins comprising the TGF -B1 LAP (or protein ciation of GPCs and /or prodomains with one or more modules or fragments thereof ) expressed with one or more extracellular proteins , including, but not limited to LTBPs, protein modules from other TGF - B family members . Such fibrillins and / or GARP. These mutations may also comprise chimeric proteins may comprise TGF -B1 LAP and TGF - B2 60 substitution of one or more cysteine with another amino or TGF -B3 growth factor domains . acid . Cysteine residue substitutions are abbreviated herein as Furin cleavage of recombinant proteins of the invention “ C # X ” wherein # represents the residue number [ counting may in some cases occur intracellularly . In some cases furin from the N -terminus of the pro -protein (without the signal cleavage of recombinant proteins of the invention may occur peptide ) ] of the original cysteine residue and X represents extracellularly . 65 the one letter amino acid code for the amino acid that is used In some embodiments , recombinant GPCs of the present for substitution . Any amino acid may be used for such invention may comprise mutations in one or more N -termi - substitutions. In some cases , serine ( S ) residues are used to US 9 , 758 ,576 B2 43 44 substitute cysteine residues . Nonlimiting examples of such cases, such cysteines may include those present in one or mutations may include C4S , C5S and/ or C7S . In recombi more of mature growth factors , alpha 2 helices, fasteners , nant GPCs comprising N - terminal prodomain regions from latency lassos and /or bow -tie regions. TGF -B1 , cysteine residues residing at amino acid position number 4 may be mutated . In recombinantGPCs comprising 5 In some embodiments , recombinant proteins of the pres N - terminal prodomain regions from TGF -B2 , cysteine resi ent invention may comprise protein modules derived from dues residing at amino acid position number 5 may be one or more species , including mammals , including, but not mutated In recombinant GPCs comprising N - terminal pro - limited to mice , rats , rabbits , pigs ,monkeys and /or humans . domain regions from TGF -B3 , cysteine residues at position Recombinant proteins may comprise one or more amino 7 may be mutated . 10 acids from one or more amino acid sequences derived from In some cases, one or more cysteine in one or more other one or more non -human protein sequences listed in Table 8 . region of GPCs may be substituted or deleted . In some In some cases , recombinant proteins of the present invention embodiments , such GPC modifications may promote the may comprise such sequences with or without the native release of mature growth factor from prodomains. In some signal peptide. TABLE 8 Non - human proteins

SEQ ID Protein Species Sequence NO proTGF - 62 1 Mouse LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPP 116 GPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEV TRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPP LLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRL LTPTDTPEWLSFDVTGVVROWLNOGDGIOGFRFSAHCS CDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMAT PLERAQHLHSSRHRRALDTNYCFSSTEKNCCVROLYIDF RKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTOYSKVL ALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCS proTGF - P1 Cyno LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPP 117 GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVT 1 RVLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPV LLSRAELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRL LAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAHCSC DSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMAT PLERAOHLOSSRHRRALDTNYCFSSTEKNCCVROLYIDF RKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTOYSKVL ALYNOHNPGASAAPCCVPOALEPLPIVYYVGRKPKVEO LSNMIVRSCKCS proTGF - B1 Mouse LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 118 C4S (LAP ) GPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEV 1 TRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPP LLSRAELRLORLKSSVEOHVELYOKYSNNSWRYLGNRL LTPTDTPEWLSFDVTGVVROWLNOGDGIOGFRFSAHCS CDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMAT PLERAQHLHSSRHRR proTGF -B1 Cyno LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 119 C4S (LAP ) GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVT RVLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPV LLSRAELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRL LAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAHCSC DSKDNTLOVDINGFTTGRRGDLATIHGMNRPFLLLMAT PLERAOHLOSSRHRR proTGF - B1 Mouse LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 120 C4S D2G GPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEV TRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPP LLSRAELRLORLKSSVEQHVELYOKYSNNSWRYLGNRL LTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCS CDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMAT PLERAOHLHSSRHGALDTNYCFSSTEKNCCVROLYIDFR KDLGWKWIHEPKGYHANFCLGPCPYIWSLDTOYSKVLA LYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLS NMIVRSCKCS proTGF -B1 Mouse LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 121 C4S GPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEV TRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPP LLSRAELRLORLKSSVECHVELYQKYSNNSWRYLGNRL LTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCS CDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMAT PLERAOHLHSSRHRRALDTNYCFSSTEKNCCVROLYIDF US 9 ,758 ,576 B2 45 46 TABLE 8 - continued Non - human proteins SEQ ID Protein Species Sequence NO RKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVL ALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCS proTGF - B1 Cyno LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPP 122 C4S GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVT RVLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPV LLSRAELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRL LAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAHCSC DSKDNTLOVDINGFTTGRRGDLATIHGMNRPFLLLMAT PLERAOHLOSSRHRRALDTNYCFSSTEKNCCVROLYIDF RKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVL ALYNOHNPGASAAPCCVPOALEPLPIVYYVGRKPKVEO LSNMIVRSCKCS proTGF - B1 Cyno LSTSKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPP 123 C4S D2G GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVT RVLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPV LLSRAELRLLRLKLKVEOHVELYOKYSNNSWRYLSNRL LAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAHCSC DSKDNTLOVDINGFTTGRRGDLATIHGMNRPFLLLMAT PLERAOHLOSSRHGALDTNYCFSSTEKNCCVROLYIDFR KDLGWKWIHEPKGYHANFCLGPCPYIWSLDTOYSKVLA LYNOHNPGASAAPCCVPOALEPLPIVYYVGRKPKVEOL SNMIVRSCKCS

LRRC32 Cyno MSPOILLLLALLTLGLAAQHODKVACKMVDKKVSCOG 124 LGLLQVPLVLPPDTETLDLSGNQLRSILASPLGFYTALRH LDLSTNEINFLQPGAFQALTHLEHLSLAHNRLAMATALS AGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSL AENSLTRLTRHTFRDMPALEOLDLHSNVLMDIEDGAFE GLPHL THLNLSRNSLTCI SDFSLOOLRVLDLSCNSIEAFO TASOPOAEFOLTWLDLRENKLLHFPDLAALPRLIYLNLS NNLIRLPTGPPODSKGIHAPSEGWSALPLSTPNGNVSARP LSOLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTF EARRSGSLPCLMLLDLSHNALETLELGARALGSLRTLLL QGNALRDLPPYTFANLASLQRLNLQGNRVSPCGGPNEP GPASCVAFSGIASLRSLSLVDNEIELLRAGAFLHTPLTEL DLSSNPGLEVATGALTGLEASLEVLALOGNGLTVLOVD LPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSF SLLPGSAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLH OGRVDVDATODLICRFSSOEEVSLSHVRPEDCEKGGLK NINLIIILTFILVSAILLTTLATCCCVRROKFNQQYKA proGDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIOILS 125 KLRLETAPNISKDAIROLLPRAPPLRELIDQYDVORDDSS DGSLEDDDYHATTETIITMPTESDFLMOADGKPKCCFFK FSSKIOYNKVVKAOLWIYLRPVKTPTTVFVOILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLONWLKO PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGW DWIIAPKRYKANYCSGECEFVFLOKYPHTHLVHOANPR GSAGPCCTPTKMSPINMLYFNGKEOIIYGKIPAMVDRC GCS proGDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIOILS 126 AXXA KLRLETAPNISKDAIROLLPRAPPLRELIDOYDVORDDSS DGSLEDDDYHATTETIITMPTESDFLMOADGKPKCCFFK FSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLQNWLKQ PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGW DWIIAPKRYKANYCSGECEFVFLOKYPHTHLVHOANPR GSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRC GCS proGDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIQILS 127 D76A KLRLETAPNISKDAIROLLPRAPPLRELIDOYDVORADSS DGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFK FSSKIOYNKVVKAOLWIYLRPVKTPTTVFVOILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLONWLKO PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGW DWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPR US 9 ,758 ,576 B2 47 48 TABLE 8 - continued Non - human proteins SEQ ID Protein Species Sequence NO GSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRC GCS

proGDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIQILS 128 AxxA D76A KLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRADSS DGSLEDDDYHATTETIITMPTESDFLMOADGKPKCCFFK FSSKIOYNKVVKAOLWIYLRPVKTPTTVFVOILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLONWLKO PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGW DWIIAPKRYKANYCSGECEFVFLOKYPHTHLVHOANPR GSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRC GCS

GDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIQILS 129 prodomain KLRLETAPNISKDAIROLLPRAPPLRELIDOYDVORDDSS DGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFK FSSKIOYNKVVKAOLWIYLRPVKTPTTVFVOILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLONWLKO PESNLGIEIKALDENGHD LAVTFPGPGEDGLNPFLEVKV TDTPKRSRR

GDF - 8 Mouse NEGSEREENVEKEGLCNACAWRONTRYSRIEAIKIQILS 130 prodomain KLRLETAPNISKDAIROLLPRAPPLRELIDOYDVORADSS D76A DGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFK FSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPM KDGTRYTGIRSLKLDMSPGTGIWOSIDVKTVLQNWLKO 1illii PESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKRSRR profDF - 8 Cyno NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 131 LRLETAPNISKDAIROLLPKAPPLRELIDOYDVORDDSSD GSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKF SSKIQYNKWVKAQLWIYLRPVETPTTVFVQILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLONWLKOP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWD WIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRG SAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS proGDF - 8 Cyno NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 132 ???? LRLETAPNISKDAIRQLLPKAPPLRELIDQYDVORDDSSD GSLEDDDYHATTETII TMPTESDFLMOVDGKPKCCFFKF SSKIOYNKVV KAOLWIYLRPVETPTTVFVOILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLONWLKOP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGWD WIIAPKRYKANYCSGECEFVFLOKYPHTHLVHOANPRG SAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS proGDF - 8 Cyno NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 133 D76A LRLETAPNISKDAIRQLLPKAPPLRELIDQYDVORADSSD GSLEDDDYHATTETII TMPTESDFLMOVDGKPKCCFFKF SSKIOYNKVVKAQLWIYLRPVETPTTVFVOILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLONWLKOP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWD WIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRG SAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS proGDF - 8 Cyno NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 134 AxxA D76A LRLETAPNISKDAIRQLLPKAPPLRELIDQYDVORADSSD GSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKF SSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLONWLKOP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGWD WIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRG SAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS US 9 ,758 ,576 B2 49 50 TABLE 8 - continued Non - human proteins SEQ ID Protein Species Sequence NO GDF - 8 Cyno NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIQILSK 135 prodomain LRLETAPNISKDAIROLLPKAPPLRELIDOYDVORDDSSD GSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKF SSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLONWLKOP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKRSRR

GDF - 8 Cyno NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIOILSK 136 prodomain LRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRADSSD D76A GSLEDDDYHATTETII TMPTESDFLMOVDGKPKCCFFKF SSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMK DGTRYTGIRSLKLDMNPGTGIWOSIDVKTVLONWLKOP ESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKRSRR proGDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 137 CVWROHSRELRLESIKSOILSKLRLKEAPNISREVV KOLL PKAPPLQQILDLHD FOGDALOPEDFLEEDEYHATTETVIS MAQETDPAVOTDGSPLCCHFHFSPKVMFTKVLKAQLW VYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRN LGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKA NYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPT KMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 138 AXXA CVWROHSRELRLESIKSQILSKLRLKEAPNISREWKOLL PKAPPLOOILDLHD FOGDALOPEDFLEEDEYHATTETVIS MAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLW VYLRPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKASRA NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYK ANYCSGOCEYMFMOKYPHTHLVOOANPRGSAGPCCTP TKMSPINMLYFNDKOQIIYGKIPGMVVDRCGCS proGDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 139 AXXA D96A CVWROHSRELRLESIKSQILSKLRLKEAPNISREW KOLL PKAPPLQQILDLHDFQGAALOPEDFLEEDEYHATTETVIS MAQETDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOLW VYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKASRA NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYK ANYCSGQCEYMFMOKYPHTHLVQQANPRGSAGPCCTP TKMSPINMLYFNDKOOLIYGKIPGMVVDRCGCS proGDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 140 D96A CVWROHSRELRLESIKSOILSKLRLKEAPNISREVV KOLL PKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVIS MAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLW VYLRPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRN LGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKA NYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPT KMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS

GDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 141 prodomain CVWROHSRELRLESIKSOILSKLRLKEAPNISREVV KOLL PKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVIS MAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLW VYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRR

GDF - 11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 142 prodomain CVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKOLL D96A PKAPPLQQILDLHD FOGAALOPEDFLEEDEYHATTETVIS MAQETDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOLW US 9 ,758 , 576 B2 51 52 TABLE 8 - continued Non - human proteins SEQ ID Protein Species Sequence NO VYLRPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHIR IRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSNWGIEIN AFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRR LTBP3 CYNO MPGPRGAPGGLAPEMRGAGAAGLLALLLLLGLGGRVE 143 GGPAGERGAGGGGALARERFKVVFAPVICKRTCLKGQC RDSCOOGSNMTLIGENGHSTDTLTGSGFRVVVCPLPCM NGGQCSSRNQCLCPPDFTGRFCQVPAGGAGGGTGGS GP GLSRAGALSTGALPPLAPEGDSVAS KHAIYAVQVIADPP GPGEGPPAOHAAFLVPLGPGOISAEVOAPPPVVNVRVH HPPEASVOVHRIESSNAEGAAPSOHLLPHPKPSHPRPPTO KPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWG OSKCHKCPOLOYTGVOKPGPVRGEVGADCPOGYKRLN STHCODINECAMPGVCRHGDCLNNPGSYRCVCPPGHSL GPSRTQCIADKPEEKSLCFRLVSPEHQCQHPLTTRLTROL CCCSVGKAWGARCORCPADGTAAFKEICPAGKGYHILT SHOTLTIOGESDFSLFLHPDGPPKPOOLPESPSOAPPPEDT EEERGVTTDSPVSEERSVOOSHPTATTSPARPYPELISRPS PPTMRWFLPDLPPSRSAVEIAPTOVTETDECRLNONICG HGECVPGPPDYSCHCNPGYRSHPOHRYCVDVNECEAEP CGPGRGI CMNTGGSYNCHCNRGYRLHVGAGGRSCVDL NECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPP VCEDIDECRDPSSCPDGKCENKPGSFKCIACQPGYRSQG GGACRDVNECAEGSPCSPGWCENLPGSFRCTCAOGYAP APDGRSCVDVDECEAGDVCDNGICTNTPGSFQCQCLSG YHLSRDRSHCEDIDECDFPAACIGGDCINTNGSYRCLCP QGHRLVGGRKCODIDECTODPGLCLPHGACKNLOGSYV CVCDEGFTPTQDQHGCEEVEQPHHKKECYLNFDDTVFC DSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYS SAEF HSLCPDGKGYTODNNIVNYGIPAHRDIDECMLFGAEICK EGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECLDE SNCRNGVCENTRGGYRCACTPPAEYSPAOROCLSPEEM DVDECOD PAACRPGRCVNLPGSYRCECRPPWVPGPSGR DCOLPESPAERAPERRDVCWSORGEDGMCAGPOAGPA LTFDDCCCROGRGWGAOCRPCPPRGAGSOCPTSOSESN SFWDTSPLLLGKPRRDEDSSEEDSDECRCVSGRCVPRPG GAVCECPGGFOLDASRARCVDIDECRELNQRGLLCKSE RCVNTSGSFRCVCKAGFARSRPHGACVPQRRR

LTBP3 Mouse MPGPRGAAHGLAPAMHQAGALGLLALLLLALLGPGGG 144 AEGGPAGERGTGGGGALARERFKVVFAPVICKRTCLKG QCRDSCQQGSNMTLIGENGHSTDTLTGSAFRWVCPLPC MNGGQCSSRNOCLCPPDFTGRFCQVPAAGTGAGTGS SG PGLARTGAMSTGPLPPLAPEGESVASKHAIYAVQVIADP PGPGEGPPAQHAAFLVPLGPGQISAEVQAPPPVVNVRVH HPPEASVQVHRIEGPNAEGPAS SQHLLPHPKPPHPRPPTQ KPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWG QSKCHKCPQLQYTGVQKPVPVRGEVGADCPQGYKRLN STHCODINECAMPGNVCHGDCLNNPGSYRCVCPPGHSL GPLAAOCIADKPEEKSLCFRLVSTEHOCOHPLTTRLTRO LCCCSVGKAWGARCORCPADGTAAFKEICPGKGYHILT SHQTLTIQGESDFSLFLHPDGPPKPQOLPESPSRAPPLEDT EEERGVTMDPPVSEERSVOO SHPTTTTSPPRPYPELISRPS PPTFHRFLPDLPPSRSAVEIAPTOVTETDECRLNONICGH GQCVPGPSDYSCHCNAGYRSHPQHRYCVDVNECEAEPC GPGKGICMNTGGSYNCHCNRGYRLHVGAGGRSCVDLN ECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPPIC EDIDECRDPSTCPDGKCENKPGSFKCIACQPGYRSQGGG ACRDVNECSEGTPCSPGWCENLPGSYRCTCAQYEPAQD GLSCIDVDECEAGKVCODGICTNTPGSFOCOCLSGYHLS RDRSRCEDIDECDFPAACIGGDCINTNGSYRCLCPLGHR LVGGRKCKKDIDECSODPGLCLPHACENLOGSYVCVCD EGFTLTQDQHGCEEVEQPHHKKECYLNFDDTVFCDSVL ATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLV PDGKRLHSGOOHCELCIPAHRDIDECILFGAEICKEGKCV NTOPGYECYCKOGFYYDGNLLECVDVDECLDESNCRN GVCENTRGGYRCACTPPAEYSPAQAQCLIPERWSTPOR DVKCAGASEERTACVWGPWAGPALTFDDCCCROPRLG TOCRPCPPRGTGSQCPTSQSESNSFWDTSPLLLGKSPRDE DSSEEDSDECRCVSGRCVPRPGGAVCECPGGFQLDASR ARCVDIDECRELNORGLLCKSERCVNTSGSFRCVCKAGF TRSRPHGPACLSAAADDAAIAHTSVIDHRGYFH US 9 ,758 , 576 B2 53 54 TABLE 8 - continued Non - human proteins SEQ ID Protein Species Sequence NO

LTBP1 Cyno MAGAWLRWGLLLWAGLLASSAHGRLRRITYVVHPGPG 145 LAAGALPLSGPPRSRTFNVALNARYSRSSAAAGAPSRAS PGVPSERTRRTSKPGGAALOGLRPPPPPPPEPARPAAPGG QLHPKPGGHPAAAPFAKQGRQVVRSKVPQETOSSGGSR LQVHQKQQLQGVNVCGGRCCHGWSKAPGSQRCTKRSC VPPCONGGMCLRPOLCVCKPGTKGKACETIAAQDTS SP VFGGOSPGAASSWGPPEQAAKHTSSKKADTLPRVSPVA QMTLTLKPKPSVGLPQQIHSQVTPLSSQSVMIHHSQTQE YVLKPKYFPAOKGISGEQSTEGSFPLRYVODOVAAPFOL SNHTGRIKVVFTPSICKVTCTKGSCONSCEKGNTTTLISE NGHAADTLTATNFRWLCHLPCMNGGQCSSRDKCQCPP NFTGKLCOIPVHGASVPKLYOHSOOPGKALGTHVIHSTH TLPLTVTSOOGVKVKFPPNIVNIHVKHPPEASVOIHOVSR IDGPTGQKTKEAQPGQSQVSYQGLPVQKTQTIHSTYSHQ OVI PHVYPVAAKTOLGRCFQETIGSOCGKALPGLSKOED CCGTVGTSWGFNKCQKCPKKPSYHGYNQMMECLPGYK RVNNTFCQDINECQLQGVCPNGECLNTMGSYRCTCKIG FGPDPTFSSCVPDPPVISEEKGPCYRLVSSGROCMHPLSV HLTKOLCCCSVGKAWGPHCEKCPLPGTAAFKEICPGGM GYTVSGVHRRRPIHHHVGKGPVFVKPKNTOPVAKSTHP PPLPAKEEPVEALTFSREHGPGVAEPEVATAPPEKEIPSL DOEKTKLEPGOPOLSPGISTIHLHPOFPVVIEKTSPPVPVE VAPEASTSSASOVIAPTOVTEINECTVNPDICGAGHCINL PVRYTCICYEGYKFSEQQRKCVDIDECTQVQHLCSQGRC ENTEGSFLCICPAGFMASEEGTNCIDVDECLRPDVCGEG HCVNTVGAFRCEYCDSGYRMTORGRCEDIDECLNPSTC PDEOCVNSPGSYOCVPCTEGFRGWNGOCLDVDECLEPN VCTNGDCSNLEGSYMCSCHKGYTRTPDHKHCKDIDECO QGNLCVNGOCKNTEGSFRCTCGQGYQLSAAKDQCEDID ECQHHHLCAHGQCRNTEGSFQCVCDQGYRASGLGDHC EDINECLEDKSVCORGDCINTAGSYDCTCPDGFOLDDN KTCODINECEHPGLCGPOGECLNTEGSFHCVCOOGFSIS ADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYOG FOAPODGOGCVDVNECELLSGVCGEAFCENVEGSFLCV CADENOEYSPMTGOCRSRTSTDLDVEOPKEEKKECYYN LNDASLCDNVLAPNVTKOECCCTSGAGWGDNCEIFPCP VLGTAEFTEMCPKGKGFV PAGESSSEAGGENYKDADEC LLFGOEICKNGFCLNTRPGYECYCKOGTYYDPVKLOCF DMDECODPS SCIDGQCVNTEGSYNCFCTHPMVLDASEK RCIRPAESNEQIEETDVYQDLCWEHLSDEYVCSRPLVGK OTTYTECCCLYGEAWGMOCALCPMKDSDDYAQLCNIP VTGRROPYGRDALVDFSEOYAPEADPYFIODRFLNSFEE LOAEECGILNGCENGRCVRVOEGYTCDCFDGYHLDTAK MTCVDVNECDELNNRMSLCKNAKCINTEGSYKCLCLPG YVPSDKPNYCTPLNTALNLEKDSDLE

LTBP1S mouse NHTGRIKVVFTPSICKVTCT KGNCONSCOKGNTTTLISE 146 NGHAADTLTATNFRVVICHLPCMNGGOCSSRDKCOCPP NFTGKLCQIPVLGASMPKLYQHAQQQGKALGSHVIHST HTLPLTMTSQQGVKVKFPPNIVNIHVKHPPEASVQIHQV SRIDSPGGQKVKEAQPGQSQVSYQGLPVOKTQTVHSTY SHQQLIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSK QEDCCGTVGTSWGFNKCQKCPKKQSYHGYTQMMECL QGYKRVNNTFCQDINECOLOGVCPNGECLNTMGSYRCS CKMGFGPDPTFSSCVPDPPVISEEKGPCYRLVSPGRHCM HPLSVHLTKOICCCSVGKAWGPHCEKCPLPGTAAFKEIC PGGMGYTVSGVHRRRPIHOHIGKEAVYVKPKNTOPVAK STHPPPLPAKEEPVEALTSSWEHGPRGAEPEVVTAPPEK EIPSLDQEKTRLEPGQPQLSPGVSTIHLHPQFPVVVEKTSP PVPVEVAPEASTSSASOVIAPTOVTEINECTVNPDICGAG HCINLPVRYTCICYEGYKFSEOLRKCYDIDECAOVRHLC SOGRCENTEGSFLCVCPAGFMASEEGTNCIDVDECLRPD MCRDGRCINTAGAFRCEYCDSGYRMSRRGYCEDIDECL KPSTCPEEQCVNTPGSYQCVPCTEGFRGWNGOCLDVDE CLOPKVCTNGSCTNLEGSYMCSCHRGYSPTPDHRHCQD IDECQQGNLCMNGQCRNTDGSFRCTCGQGYQLSAAKD QCEDIDECEHHHLCSHGQCRNTEGSFQCVCNQGYRASV LGDHCEDINECLEDSSVCOGGDCINTAGSYDCTCPDGFO LNDNKGCODINECAOPGLCGSHGECLNTOGSFHCVCEO GFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCL CYQGFQAPQDGQGCVDVNECELLSGVCGEAFCENVEGS FLCVCADENCEYSPMTGQCRSRVTEDSGVDRQPREEKK ECYYNLNDASLCDNVLAPNVTKQECCCTSGAGWGDNC US 9 ,758 ,576 B2 5555 56 TABLE 8 - continued Non - human proteins SEQ ID Protein Species Sequence NO EIFPCPVOGTAEFTEMCPRGKGLVPAGESSYDTGGENYK DADECLLFGEEICKNGYCLNTOPGYECYCKOGTYYDPV KLQCFDMDECODPNSCIDGQCVNTEGSYNCFCTHPMVL DASEKRCVQPTESNEQIEETDVYQDLCWEHLSEEYVCSR PLVGKOTTYTECCCLYGEAWGMOCALCPMKDSDDYA OLCNIPVTGRRRPYGRDALVDFSEOYGPETDPYFIODRF LNSFEELOAEECGILNGCENGRCVRVOEGYTCDCFDGY HLDMAKMTCVDVNECSELNNRMSLCKNAKCINTEGSY KCLCLPGYIPSDKPNYCTPLNSALNLDKESDLE

GARP mouse ISORREQVPCRTVNKEALCHGLGLLQVPSVLSLDIQALY 147 LSGNQLQSILVSPLGFYTALRHLDLSDNQISFLQAGVFQA LPYLEHLNLAHNRLATGMALNSGGLGRLPLLVSLDLSG NSLHGNLVERLLGETPRLRTLSLAENSLTRLARHTFWG MPAVEQLDLHSNVLMDIEDGAFEALPHLTHLNLSRNSL TCI SDFSLQQLQVLDLSCNSIEAFOTAPEPOAOFOLAWL DLRENKLLHFPDLAVFPRLIYLNVSNNLIOLPAGLPRGSE DLHAPSEGWSASPLSNPSRNASTHPLSOLLNLDLSYNEIE LVPASFLEHLTSLRFLNLSRNCLRSFEAROVDSLPCLVLL DLSHNVLEALELGTKVLGSLOTLLLODNALQELPPYTFA SLASLORLNLOGNOVSPCGGPAEPGPPGCVDFSGIPTLH VLNMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATG ALVGLEASLEVLELOGNGLTVLRVDLPCFLRLKRLNLAE NOLSHLPAWTRAVSLEVLDLRNNSFSLLPGNAMGGLET SLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDL ICRFGSQEELSLSLVRPEDCEKGGLKNVNLILLLSFTLVS AIVLTTLATICFLRROKLSQQYKA SGARP mouse ISORREOVPCRTVNKEALCHGLGLLOVPSVLSLDIOALY 148 LSGNOLOSILVSPLGFYTALRHLDLSDNOISFLOAGVFOA LPYLEHLNLAHNRLATGMALNSGGLGRLPLLVSLDLSG NSLHGNLVERLLGETPRLRTLSLAENSLTRLARHTFWG MPAVEOLDLHSNVLMDIEDGAFEALPHLTHLNLSRNSL TCISDFSLOOLOVLDLSCNSIEAFOTAPEPQAQFOLAWL DLRENKLLHFPDLAVFPRLIYLNVSNNLIOLPAGLPRGSE DLHAPSEGWSASPLSNPSRNASTHPLSOLLNLDLSYNEIE LVPASFLEHLTSLRFLNLSRNCLRSFEAROVDSLPCLVLL DLSHNVLEALELGTKVLGSLOTLLLODNALQELPPYTFA SLASLQRLNLQGNQVSPCGGPAEPGPPGCVDFSGIPTLH VLNMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATG ALVGLEASLEVLELQGNGLTVLRVDLPCFLRLKRLNLAE NOLSHLPAWTRAVSLEVLDLRNNSFSLLPGNAMGGLET SLRRLYLOGNPLSCCGNGWLAAOLHOGRVDVDATODL ICRFGSQEELSLSLVRPEDCEKGGLKNVN LRRC33 mouse WRSGPGTATAASQGGCKVVDGVADCRGLNLASVPSSLP 149 PHSRMLILDANPLKDLWNHSLOAYPRLENLSLHSCHLD RISHYAFREOGHLRNLVLADNRLSENYKESAAALHTLL GLRRLDLSGNSLTEDMAALMLONLSSLEVVSLARNTLM RLDDSIFEGLEHLVELDLORNYIFEIEGGAFDGLTELRRL NLAYNNLPCIVDFSLTOLRFLNVSYNILEWFLAAREEVA FELEILDLSHNOLLFFPLLPQCGKLHTLLLODNNMGFYR ELYNTSSPQEMVAQFLLVDGNVTNITTVNLWEEFSSSDL SALRFLDMSQNQFRHLPDGFLKKTPSLSHLNLNONCLK MLHIREHEPPGALTELDLSHNOLAELHLAPGLTGSLRNL RVFNLSSNQLLGVPTGLFDNASSITTIDMSHNQISLCPQM VPVDWEGPPSCVDFRNMGSLRSLSLDGCGLKALODCPF QGTSLTHLDLSSNWGVLNGSISPLWAVAPTLQVLSLRD VGLGSGAAEMDFSAFGNLRALDLSGNSLTSFPKFKGSLA LRTLDLRRNSLTALPQRVVSEQPLRGLQTIYLSONPYDC CGVEGWGALOOHFKTVADLSMVTCNLSSKIVRVVELPE GLPQGCKWEQVDTGLFYLVLILPSCLTLLVACTVVFLTF KKPLLQVIKSRCHWSSIY

SLRRC33 mouse WRSGPGTATAASOGGCKVVDGVADCRGLNLASVPSSLP 150 PHSRMLILDANPLKDLWNHSLQAYPRLENLSLHSCHLD RISHYAFREQGHLRNLVLADNRLSENYKESAAALHTLL GLRRLDLSGNSLTEDMAALMLQNLSSLEVVSLARNTLM RLDDSIFEGLEHLVELDLORNYIFEIEGGAFDGLTELRRL NLAYNNLPCIVDFSLTQLRFLNVSYNILEWFLAAREEVA FELEILDLSHNOLLFFPLLPQCGKLHTLLLQDNNMGFYR ELYNTSSPQEMVAQFLLVDGNVTNITTVNLWEEFSSSDL SALRFLDMSQNQFRHLPDGFLKKTPSLSHLNLNQNCLK MLHIREHEPPGALTELDLSHNOLAELHLAPGLTGSLRNL US 9 ,758 ,576 B2 57 58 TABLE 8 - continued Non - human proteins SEQ ID Protein Species Sequence NO RVFNLSSNOLLGVPTGLFDNASSITTIDMSHNOISLCPOM VPVDWEGPPSCVDFRNMGSLRSLSLDGCGLKALODCPF QGTSLTHLDLSSNWGVLNGSISPLWAVAPTLQVLSLRD VGLGSGAAEMDFSAFGNLRALDLSGNSLTSFPKFKGSLA LRTLDLRRNSLTALPQRVVSEQPLRGLQTIYLSONPYDC CGVEGWGALQQHFKTVADLSMVTCNLSSKIVRVVELPE GLPQGCKWEQVDTGL LRRC33 Cyno WRDRSVTATAASQRGCKLVGGDTDCRGOSLASVPSSLP 151 PHARTLILDANPLKALWNHSLQPYPLLESLSLHSCHLERI GRGAFQEQGHLRSLVLGDNCLSENYKETAAALHTLPGL QTLDLSGNSLTEDMAALMLQNLSSLOSVSLARNTIMRL DDSVFEGLERLRELDLORNYIFEIEGGAFDGLTELRHLNL AYNNLPCIVDFGLTOLRSLNVSYNVLEWFLAAGGEAAF ELETLDLSHNOLLFFPLLPQYSKLHTLLLRDNNMGFYRD LYNTSSPREMVAOFLLVDGNVTNITTVNLWEEFSSSDLA DLRFLDMSONOFOYLPDGFLRKMPSLSHLNLNONCLMT LHIREHEPPGALTELDLSHNOLSELHLTPGLASCLGSLRL FNLSSNOLLGVPPGLFANARNITTLDMSHNOISLCPLPAA SDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFOGT SLTSLDLSSNWGVLNGSLAPLRDVAPMLQVLSLRNMGL HSNFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALET LDLRRNSLTALPOKAVSEOLSRGLRTIYLSONPYDCCGV DGWGALOOGOTVADWATVTCNLSSKIIRLAELPGGVPR DCKWERLDLGLLYLVLILPSCLTLLVACTLIVLTFKKPLL QVIKSRCHWSSVY SLRRC33 Cyno WRDRSVTATAASORGCKLVGGDTDCRGOSLASVPSSLP 152 PHARTLILDANPLKALWNHSLQPYPLLESLSLHSCHLERI GRGAFOEOGHLRSLVLGDNCLSENYKETAAALHTLPGL QTLDLSGNSLTEDMAALMLQNLSSLOSVSLARNTIMRL DDSVFEGLERLRELDLORNYIFEIEGGAFDGLTELRHLNL AYNNLPCIVDFGLTOLRSLNVSYNVLEWFLAAGGEAAF ELETLDLSHNOLLFFPLLPOYSKLHTLLLRDNNMGFYRD LYNTSSPREMVAOFLLVDGNVTNITTVNLWEEFSSSDLA DLRFLDMSONOFOYLPDGFLRKMPSLSHLNLNONCLMT LHIREHEPPGALTELDLSHNQLSELHLTPGLASCLGSLRL FNLSSNQLLGVPPGLFANARNITTLDMSHNQISLCPLPAA SDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFOGT SLTSLDLSSNWGVLNGSLAPLRDVAPMLQVLSLRNMGL HSNFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALET LDLRRNSLTALPOKAVSEOLSRGLRTIYLSONPYDCCGV DGWGALQQGQTVADWATVTCNLSSKIIRLAELPGGVPR DCKWERLDLGL

In some embodiments , recombinant proteins may be 45 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 mutations. Some recombinant forms of LTBPs complexed proteins , follistatin , follistatin - related gene ( FLRG ) , decorin 50 with recombinant GPCsmay 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

SEO ID Protein Sequence NO LTBP1 1265 - 1443 NECELLSGVCGEAFCENVEGSFLCVCADENQEYSPMTGQC 153 RSRTSTDLDVDVDOPKEEKKECYYNLNDASLCDNVLAPNV TKQECCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGF US 9 ,758 , 576 B2 59 60 TABLE 9 - continued LTBP sequences

SEO ID Protein Sequence NO VPAGESSSEAGGENYKDADECLLFGQEICKNGFCLNTRPGY ?????QGTYYDPVKLQCF LTBP1 1265 - 1698 NECELLSGVCGEAFCENVEGSFLCVCADENOEYSPMTGOC 154 RSRTSTDLDVDVDOPKEEKKECYYNLNDASLCDNVLAPNV TKOECCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGF VPAGESSSEAGGENYKDADECLLFGOEICKNGFCLNTRPGY ECYCKOGTYYDPVKLOCFDMDECODPSSCIDGOCVNTEGS YNCFCTHPMVLDASEKRCIRPAESNEQIEETDVYQDLCWE HLSDEYVCSRPLVGKOTTYTECCCLYGEAWGMOCALCPL KDSDDYAQLCNIPVTGRROPYGRDALVDFSEQYTPEADPY FIODRFLNSFEELQAEECGILNGCENGRCVRVOEGYTCDCF DGYHLDTAKMTCVDVNECDELNNRMSLCKNAKCINTDGS YKCLCLPGYVPSDKPNYCTPLNTALNLEKDSDLE LTBP1 809 - 1698 PSLDOEKTKLEPGOPOLSPGISTIHLHPOFPVVIEKTSPPVPV 155 EVAPEASTSSASQVIAPTQVTEINECTVNPDICGAGHCINLP VRYTCICYEGYRFSEOORKCYDIDECTOVOHLCSOGRCEN TEGSFLCICPAGFMASEEGTNCIDVDECLRPDVCGEGHCVN TVGAFRCEYCDSGYRMTORGRCEDIDECLNPSTCPDEOCV NSPGSYOCVPCTEGFRGWNGOCLDVDECLEPNVCANGDC SNLEGSYMCSCHKGYTRTPDHKHCRDIDECQQGNLCVNG OCKNTEGSFRCTCGOGYOLSAAKDOCEDIDECOHRHLCAH GQCRNTEGSFQCVCDQGYRASGLGDHCEDINECLEDKSVC QRGDCINTAGSYDCTCPDGFOLDDNKTCODINECEHPGLC GPQGECLNTEGSFHCVCQQGFSISADGRTCEDIDECVNNTV CDSHGFCDNTAGSFRCLCYOGFQAPQDGQGCVDVNECEL LSGVCGEAFCENVEGSFLCVCADENOEYSPMTGOCRSRTS TDLDVDVDQPKEEKKECYYNLNDASLCDNVLAPNVTKQE CCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGFVPAG ESSSEAGGENYKDADECLLFGOEICKNGFCLNTRPGYECYC KOGTYYDPVKLOCFDMDECODPSSCIDGOCVNTEGSYNCF CTHPMVLDASEKRCIRPAESNEQIEETDVYQDLCWEHLSDE YVCSRPLVGKOTTYTECCCLYGEAWGMOCALCPLKDSDD YAOLCNIPVTGRROPYGRDALVDFSEOYTPEADPYFIODRF LNSFEELOAEECGILNGCENGRCVRVOEGYTCDCFDGYHL DTAKMTCVDVNECDELNNRMSLCKNAKCINTDGSYKCLC LPGYVPSDKPNYCTPLNTALNLEKDSDLE LTBP1S NHTGRIKVVFTPSICKVTCTKGSCONSCEKGNTTTLISENGH 156 AADTLTATNFRVVICHLPCMNGGQCSSRDKCQCPPNFTGK LCQIPVHGASVPKLYQHSQQPGKALGTHVIHSTHTLPL TVT SQQGVKVKFPPNIVNIHVKHPPEASVQIHOVSRIDGPTGOK TKEAQPGQSQVSYQGLPVOKTQTIHSTYSHOQVIPHVYPVA AKTOLGRCFOETIGSOCGKALPGLS KOEDCCGTVGTSWGF NKCOKCPKKPSYHGYNOMMECLPGYKRVNNTFCODINEC OLOGVCPNGECLNTMGSYRCTCKIGFGPDPTFSSCVPDPPV ISEEKGPCYRLVSSGROCMHPLSVHLTKOLCCCSVGKAWG PHCEKCPLPGTAAFKEICPGGMGYTVSGVHRRRPIHHHVG KGPVFVKPKNTOPVAKSTHPPPLPAKEEPVEALTFSREHGP GVAEPEVATAPPEKEIPSLDQEKTKLEPGQPQLSPGISTIHLH POFPVVIEKTSPPVPVEVAPEASTSSASQVIAPTOVTEINECT VNPDICGAGHCINLPVRYTCICYEGYRFSEQQRKCVDIDEC TQVQHLCSQGRCENTEGSFLCICPAGFMASEEGTNCIDVDE CLRPDVCGEGHCVNTVGAFRCEYCDSGYRMTORGRCEDI DECLNPSTCPDEQCVNSPGSYQCVPCTEGFRGWNGQCLDV DECLEPNVCANGDCSNLEGSYMCSCHKGYTRTPDHKHCR DIDECOOGNLCVNGOCKNTEGSFRCTCGOGYOLSAAKDO CEDIDECQHRHLCAHGQCRNTEGSFQCVCDQGYRASGLGD HCEDINECLEDKSVCORGDCINTAGSYDCTCPDGFOLDDN KTCODINECEHPGLCGPQGECLNTEGSFHCVCQQGFSISAD GRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYOGFOAP ODGOGCVDVNECELLSGVCGEAFCENVEGSFLCVCADEN QEYSPMTGOCRSRTSTDLDVDVDOPKEEKKECYYNLNDAS LCDNVLAPNVTKQECCCTSGVGWGDNCEIFPCPVLGTAEF TEMCPKGKGFVPAGESSSEAGGENYKDADECLLFGQEICK NGFCLNTRPGYECYCKOGTYYDPVKLOCFDMDECODPSS CIDGQCVNTEGSYNCFCTHPMVLDASEKRCIRPAESNEQIE ETDVYODLCWEHLSDEYVCSRPLVGKOTTYTECCCLYGEA WGMQCALCPLKDSDDYAQLCNIPVTGRROPYGRDALVDF SEOYTPEADPYFIQDRFLNSFEELQAEECGILNGCENGRCVR VQEGYTCDCFDGYHLDTAKMTCVDVNECDELNNRMSLCK NAKCINTDGSYKCLCLPGYVPSDKPNYCTPLNTALNLE KDS DLE US 9 ,758 , 576 B2 61 62 TABLE 9 - continued LTBP sequences

SEO ID Protein Sequence NO

LTBP3 GPAGERGAGGGGALARERFKVVFAPVICKRTCLKGQCRDS 157 COQGSNMTLIGENGHSTDTLTGSGFRVVVCPLPCMNGGQC SSRNQCLCPPDFTGRFCQVPAGGAGGGTGGSGPGLSRTGA LSTGALPPLAPEGDSVASKHAIYAVQVIADPPGPGEGPPAQ HAAFLVPLGPGQISAEVQAPPPVVNVRVHHPPEASVQVHRI ESSNAESAAPSOHLLPHPKPSHPRPPTOKPLGRCFODTLPKO PCGSNPLPGLTKQEDCCGSIGTAWGQSKCHKCPQLQYTGV QKPGPVRGEVGADCPQGYKRLNSTHCQDINECAMPGVCR HGDCLNNPGSYRCVCPPGHSLGPSRTOCIADKPEEKSLCFR LVSPEHQCQHPLTTRLTROLCCCSVGKAWGARCORCPTDG TAAFKEICPAGKGYHILTSHOTLTIQGESDFSLFLHPDGPPK POOLPESPSOAPPPEDTEEERGVTTDSPVSEERSVOOSHPTA TTTPARPYPELISRPSPPTMRWFLPDLPPSRSAVEIAP TOVTE TDECRLNQNICGHGECVPGPPDYSCHCNPGYRSHPQHRYC VDVNECEAEPCGPGRGICMNTGGSYNCHCNRGYRLHVGA GGRSCVDLNECAKPHLCGDGGFCINFPGHYKCNCYPGYRL KASRPPVCEDIDECRDPSSCPDGKCENKPGSFKCIACOPGY RSOGGGACRDVNECAEGSPCSPGWCENLPGSFRCTCAOGY APAPDGRSCLDVDECEAGDVCDNGICSNTPGSFOCOCLSG YHLSRDRSHCEDIDECDFPAACIGGDCINTNGSYRCLCPOG HRLVGGRKCODIDECSQDPSLCLPHGACKNLQGSYVCVCD EGFTPTODOHGCEEVEOPHHKKECYLNFDDTVFCDSVLAT NVTOOECCCSLGAGWGDHCEIYPCPVYSSAEFHSLCPDGK GYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKCVNTQPG YECYCKOGFYYDGNLLECVDVDECLDESNCRNGVCENTR GGYRCACTPPAEYSPAQRQCLSPEEMDVDECODPAACRPG RCVNLPGSYRCECRPPWVPGPSGRDCOLPESPAERAPERRD VCWSQRGEDGMCAGPLAGPALTFDDCCCROGRGWGAQC RPCPPRGAGSHCPTSOSESNSFWDTSPLLLGKPPRDEDSSEE DSDECRCVSGRCVPRPGGAVCECPGGFOLDASRARCVDID ECRELNQRGLLCKSERCVNTSGSFRCVCKAGFARSRPHGA CVPQRRR 35 In some embodiments , LTBPs may comprise detectable repeat containing 32 (LRRC32 . ) ] Such LRRC33 fragments labels . Detectable labels may be used to allow for detection and /or mutants may comprise one or more regions from the and / or isolation of recombinant proteins comprising LTBPs. LRRC33 sequence listed in Table 10 below . Recombinant GARPs may also comprise mutants and / or GARP frag Some detectable labels may comprise biotin labels , poly - 40 ments . Some recombinant GARPs may be soluble ( referred histidine tags and / or flag tags. Such tags may be used to to herein as sGARP ) . isolate tagged proteins. Proteins produced may comprise In some embodiments, recombinant GARPs may com additional amino acids encoding one or more 3C protease prise one or more amino acid sequences listed in Table 10 . cleavage site . Such sites allow for cleavage at the 3C Some recombinant GARPs used herein may be expressed protease cleavage site upon treatment with 3C protease , 45 without the N -terminal residues AQ . Expressed GARPs may including , but not limited to rhinovirus 3C protease . Such comprise detectable labels . Such detectable labels may be cleavage sites may be introduced to allow for removal of used to allow for detection and / or isolation . Some detectable detectable labels from recombinant proteins. labels may comprise biotin labels , polyhistidine tags and / or In some embodiments, GARPs, including , but not limited flag tags. Such tags may be used to isolate tagged proteins . to recombinant forms of GARP, may be complexed with 50 Proteins produced may comprise additional amino acids recombinant GPCs. Some recombinant GPCs of the present encoding one or more 3C protease cleavage site . Such sites invention may be co - expressed with GARPs to ensure allow for cleavage at the 3C protease cleavage site upon proper folding and /or expression . In other embodiments , the treatment with 3C protease , including , but not limited to GARP homologue , leucine rich repeat containing 33 rhinovirus 3C protease . 3C protease cleavage sites may be (LRRC33 , ) or fragments and /or mutants thereof may be introduced to allow for removal of detectable labels from substituted for GARP ( also referred to herein as leucine rich recombinant proteins . TABLE 10 GARP sequences Protein Sequence SEQ ID NO GARP AQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTETLDLS 158 GNQLRSILASPLGFYTALRHLDLSTNEISFLQPGAFQALTHL EHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSG LLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEOLDL HSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLOOLRV US 9 , 758 ,576 B2 63 64 TABLE 10 - continued GARP sequences Protein Sequence SEQ ID NO LDLSCNSIEAFQTASQPQAEFQLTWLDLRENKLLHFPDLAA LPRLIYLNLSNNLIRLPTGP PODSKGIHAPSEGWSALPLSAPS GNASGRPLSOLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRN CLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRT LLLOGNALRDLPPYTFANLASLORLNLOGNRVSPCGGPDEP GPSGCVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLS SNPGLEVATGALGGLEASLEVLALOGNGLMVLOVDLPCFI CLKRLNLAENRLSHLPAWTOAVSLEVLDLRNNSFSLLPGSA MGGLETSLRRLYLOGNPLSCCGNGWLAAOLHOGRVDVDA TODLICRFSSOEEVSLSHVRPEDCEKGGLKNINLIIILTFILVS AILLTTLAACCCVRROKFNQQYKA SGARP AQHQDKVPCKMVDKKVSCOVLGLLQVPSVLPPDTETLDLS 159 GNOLRSILASPLGFYTALRHLDLSTNEISFLOPGAFOAL THL EHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSG LLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEQLDL HSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLQQLRV LDLSCNSIEAFOTASQPQAEFOLTWLDLRENKLLHFPDLAA LPRLIYLNLSNNLIRLPTGP PODSKGIHAPSEGWSALPLSAPS GNASGRPLSOLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRN CLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRT LLLOGNALRDLPPYTFANLASLORLNLOGNRVSPCGGPDEP GPSGCVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLS SNPGLEVATGALGGLEASLEVLALOGNGLMVLOVDLPCFI CLKRLNLAENRLSHLPAWTOAVSLEVLDLRNNSFSLLPGSA MGGLETSLRRLYLOGNPLSCCGNGWLAAOLHOGRVDVDA TODLICRFSSQEEVSLSHVRPEDCEKGGLKNIN LRRC33 WRNRSGTATAASQGVCKLVGGAADCRGOSLASVPSSLPPH 160 ARMLTLDANPLKTLWNHSLQPYPLLESLSLHSCHLERISRG AFOEOGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDL SGNALTEDMAALMLONLSSLRSVSLAGNTIMRLDDSVFEG LERLRELDLORNYIFEIEGGAFDGLAELRHLNLAFNNLPCIV DFGLTRLRVLNVSYNVLEWFLATGGEAAFELETLDLSHNO LLFFPLLPQYSKLRTLLLRDNNMGFYRDLYNTSSPREMVA OFLLVDGNVTNITTVSLWEEFSSSDLADLRFLDMSONOFOY LPDGFLRKMPSLSHLNLHONCLMTLHIREHEPPGALTELDL SHNOLSELHLAPGLASCLGSLRLFNLSSNOLLGVPPGLFAN ARNITTLDMSHNQISLCPLPAASDRVGPPSCVDFRNMASLR SLSLEGCGLGALPDCPFOGTSL TYLDLSSNWGVLNGSLAPL ODVAPMLQVLSLRNMGLHSSFMALDFSGFGNLRDLDLSG NCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGL RTIYLSQNPYDCCGVDGWGALQHGQTVADWAMVTCNLSS KIIRVTELPGGVPRDCKWERLDLGLLYLVLILPSCLTLLVAC TVIVLTFKKPLLQVIKSRCHWSSVY SLRRC33 WRNRSGTATAASOGVCKLVGGAADCRGOSLASVPSSLPPH 161 ARMLTLDANPLKTLWNHSLQPYPLLESLSLHSCHLERISRG AFOEOGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDL SGNALTEDMAALMLONLSSLRSVSLAGNTIMRLDDSVFEG LERLRELDLORNYIFEIEGGAFDGLAELRHLNLAFNNLPCIV DFGLTRLRVLNVSYNVLEWFLATGGEAAFELETLDLSHNO LLFFPLLPOYSKLRTLLLRDNNMGFYRDLYNTSSPREMVA OFLLVDGNVTNITTVSLWEEFSSSDLADLRFLDMSONOFOY LPDGFLRKMPSLSHLNLHONCLMTLHIREHEPPGALTELDL SHNOLSELHLAPGLASCLGSLRLFNLSSNOLLGVPPGLFAN ARNITTLDMSHNOISLCPLPAASDRVGPPSCVDFRNMASLR SLSLEGCGLGALPDCPFOGTSLTYLDLSSNWGVLNGSLAPL ODVAPMLOVLSLRNMGLHSSFMALDFSGFGNLRDLDLSG NCLTTFPRFGGSLALETLDLRRNSLTALPOKAVSEQLSRGL RTIYLSONPYDCCGVDGWGALQHGQTVADWAMVTCNLSS KIIRVTELPGGVPRDCKWERLDLGL

GPCs bound to LTBPs may adopt three dimensional invention are directed to such conformation -dependent conformations that are distinct from conformations found epitopes . Such antibodies may function selectively to acti with GPCs bound to GARP or other matrix proteins. This 60 vate or inhibit growth factor activity depending on the may be due , in some cases , to the presence of cysteines identity of bound protein ( e . g . LTBP or GARP. ) In some available on LTBP for disulfide bond formation with GPCs cases , different conformation - dependent epitopes may be that comprise a different distance from one another than present on N -terminal alpha helices of proTGF- B when corresponding cysteines available for disulfide bond forma - bound to LTBP or GARP. tion on GARP . Such differences in three dimensional con - 65 Recombinant proteins of the present invention may be formations may provide unique conformation - dependent coexpressed with GDF - associated serum protein (GASP ) 1 epitopes on GPCs. In some embodiments , antibodies of the and /or GASP -2 . Such recombinant proteinsmay include, but US 9 ,758 ,576 B2 65 66 are not limited to GDF - 8 and / or GDF - 11. GASPs are cir one or more features and /or combinations of protein mod culating proteins that bind and prevent activity of GDF - 8 ules from the embodiments depicted in FIG . 7 . and GDF - 11 (Hill , J. J. et al. , 2003 . Mol Endocrinology. Recombinant Growth Differentiation Factors (GDFs , ) 17 ( 6 ): 1144 -54 and Hill , J. J. et al. , 2002 . JBC . 277 (43 ): Activins and Inhibins 40735 - 41, the contents of each of which are herein incor- 5 Growth differentiation factors (GDFs ), activins and inhib ins are TGF - B family member proteins involved in a number porated by reference in their entirety . ) Interestingly , GDF - 8 of cellular and / or developmental activities . In some embodi and GDF - 11 growth factors are not found free in serum . ments of the present invention , recombinant proteins may About 70 % are in GPCs with the remaining 30 % associated comprise one or more protein modules from one or more with GASPs as well as other proteins ( e . g . follistatin , GDFs , activins and / or inhibins. In further embodiments , follistatin - like related gene and decorin . ) Studies using mice GDF protein modules may comprise GDF - 8 and /or GDF - 11 lacking expression of GASP - 1 and /or GASP -2 display phe protein modules. notypes indicative of myostatin and / or GDF - 11 overactivity GDF - 8 and GDF - 11 , which are secreted as latent com (Lee et al. , 2013 . PNAS . 110 ( 39 ) :E3713 -22 . ) GASP bound plexes (Sengle et al. , 2011 . J Biol Chem . 286 ( 7 ) :5087 - 99 ; Ge et al. , 2005 . Mol Cel Biol. 25 ( 14 ) : 5846 -58 , ) show GDF - 8 and / or GDF - 11 are unable to bind type II receptors 15 conservation of the fastener residues (Lys 27 and Tyr 75 of and transmit related cellular signals . TGF - B ; see FIGS. 8A - 8G . ) GDF - 8 (also referred to herein as Some recombinant proteins may be coexpressed with myostatin ) is involved in regulating muscle mass , and its perlecan . Such recombinant proteins may include, but are deficiency increases muscle mass in multiple species , not limited to GDF - 8 . Studies by Sengle et al ( Sengle et al. , including humans (Rodino -Klapac . L . R . et al. , 2009 . 2011 . J Biol Chem . 286 ( 7 ) : 5087 - 99 , the contents of which 20 Muscle Nerve . 39( 3 ) :283 - 96 ) . GDF - 8 may be found in the are herein incorporated by reference in their entirety ) found circulation in latent form , but may also be stored in the that the GDF - 8 prodomain associates with perlecan . Further extracellular matrix , bound to LTBP3 (Anderson et al. , 2007 . studies indicate that perlecan knockout leads to muscular J Biol Chem . 283 ( 11 ) : 7027 -35 ) or perlecan ( Sengle et al. , hypertrophy , suggesting that the interaction between GDF - 8 2011. J Biol Chem . 286 ( 7 ) :5087 - 99 . ) While complexed with and perlecan may contribute to GDF - 8 activity (Xu et al. 25 its prodomain , GDF - 8 is unable to participate in receptor 2010 . Matrix Biol. 29 (6 ) :461 - 70 . ) binding with the type II receptor , ActRIIB (Sengle et al. , In some cases , recombinant proteins of the invention may 2008 . J Mol Biol. 381 ( 4 ) : 1025 - 39. ) While GDF - 8 is be coexpressed with follistatin and /or FLRG . Such recom - expressed primarily in muscle , GDF - 11 expression is more binant proteins may include , but are not limited to GDF - 8 . systemic and its activity is thought to be involved in multiple Both follistatin and FLRG are known to antagonize some 30 processes (Lee et al ., 2013 . PNAS . 110 ( 39 ) : E3713 - 22 . ). It is TGF - ß family member proteins, including, but not limited to believed to be involved in development of multiple tissues , GDF - 8 (Lee , S - J . et al. , 2010 . Mol Endocrinol. 24 ( 10 ) : 1998 - including , but not limited to the retina , kidney, pancreas and 2008 , Takehara -Kasamatsu , Y . et al ., 2007. J Med Invest. olfactory system . It is also believed to be a circulating factor 54 ( 3 - 4 ) :276 - 88 , the contents of each of which are herein in the blood ( Sinha , M . et al. , 2014 . Science Express . incorporated by reference in their entirety . ) Follistatin has 35 10 . 1126 / science . 1251152 , p2 - 6 and Katsimpardi, L . et al . , been shown to block GDF - 8 activity by binding to the free 2014 . Science Express. 10 . 1126 / science . 1251141, the con growth factor and preventing receptor binding. Both fol- tents of each of which are herein incorporated by reference listatin and FLRG are implicated in modulating growth in their entirety . ) factor activity during development. GDF - 8 and GDF - 11 also share considerable homology . In some embodiments , recombinant proteins of the inven - 40 While the prodomains only share 48 % homology , GDF - 8 tion may be coexpressed with decorin . Such recombinant and GDF - 11 growth factor domains share 90 % homology proteins may include , but are not limited to TGF - B and (60 % homology when prodomains and growth factors are GDF - 8. Decorin is a known antagonist of TGF- B activity taken together .) ( Zhu , J . et al ., 2007 . J Biol Chem . 282 : 25852- 63 , the Release ofGDF - 8 and GDF - 11 from latent GPCs requires contents of which are herein incorporated by reference in 45 cleavage of the prodomains at the BMP/ tolloid cleavage site their entirety ) and may also antagonize other TGF - B family (located between Arg 75 and Asp 76 in GDF - 8 and between members, including , but not limited to GDF - 8 . Decorin - Gly 97 and Asp 98 in GDF - 11 ) by BMP1 / tolloid metallo dependent inhibition of TGF -B and GDF -8 activity has been proteinases. This cleavage is between the a2 helix and the shown to reduce fibrosis in various tissues. Decorin expres fastener. Thus at least two different methods of unfastening sion has also been shown to increase the expression of 50 the straitjacket, force and proteolysis , can release family follistatin , a known inhibitor of free GDF - 8 . members from latency . In some embodiments , recombinant proteins of the pres In some embodiments, recombinant proteins of the pres ent invention may comprise those depicted in FIG . 7 . Some ent invention comprising GDFs may comprise sequences recombinant proteins of the present invention may comprise listed in Table 11 or fragments thereof. TABLE 11 Recombinant GDFs SEQ ID Protein Sequence NO proGDF - 8 NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIOILSKLRLE 5 TAPNISKDVIROLLPKAPPLRELIDOYDVORDDSSDGSLEDDDY HATTETIITMPTESDFLMOVDGKPKCCFFKFSSKIOYNKVVKA OLWIYLRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKOPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRY US 9 , 758 ,576 B2 67 68 TABLE 11 - continued Recombinant GDFs

SEO ID Protein Sequence NO PLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKI PAMW DRCGCS GDF - 8 prodomain NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIOILSKLRLE 70 TAPNISKDVIRQLLPKAPPLRELIDQYDVORDDSSDGSLEDDDY HATTETIITMPTESDFLMOVDGKPKCCFFKFSSKIOYNKVVKA QLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKQPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKRSRR GDF - 8 prodomain NENSEQKENVEKEGLCNACTWRONNENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSKLRLE 162 D76A TAPNISKDVIROLLPKAPPLRELIDOYDVORADSSDGSLEDDDY HATTETII TMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKA QLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKQPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKRSRR proGDF - 8 AXXA NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSKLRLE 163 TAPNISKDVIROLLPKAPPLRELIDOYDVORDDSSDGSLEDDDY HATTETIITMPTESDFLMOVDGKPKCCFFKFSSKIOYNKVVKA OLWIYLRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKOPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKASRADFGLDCDEHSTESRCCRY PLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLOKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMW DRCGCS proGDF - 8 076A NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIOILSKLRLE 164 TAPNISKDVIROLLPKAPPLRELIDOYDVORADSSDGSLEDDDY HATTETIITMPTESDFLMOVDGKPKCCFFKFSSKIOYNKVVKA OLWIYLRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKOPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRY PLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLOKYPHTHL VHOANPRGSAGPCCTPTKMSPINMLYFNGKEOIIYGKI PAMVV DRCGCS proGDF - 8 AXXA NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQILSKLRLE 165 D76A TAPNISKDVIROLLPKAPPLRELIDQYDVQRADSSDGS LEDDDY HATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKA OLWIYLRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLDMNP GTGIWOSIDVKTVLONWLKOPESNLGIEIKALDENGHD LAVTF PGPGEDGLNPFLEVKVTDTPKASRADFGLDCDEHSTESRCCRY PLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLOKYPHTHL VHOANPRGSAGPCCTPTKMSPINMLYFNGKEOIIYGKI PAMW DRCGCS progDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 4 VWROHSRELRLESIKSCILSKLRLKEAPNISREVVKOLLPKAPP LQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPA VOTDGSPLCCHFHFSPKVMFTKVLKAOLWVYLRPVPRPATVY LQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWOSI DFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGOCEYMFMOKYPHTHLVQQANPR GSAGPCCTPTKMSPINMLYFNDKOOIIYGKIPGMVVDRCGCS proGDF - 11 D98A AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 166 VWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPP LOOILDLHDFOGAALOPEDFLEEDEYHATTETVISMAQETDPA VOTDGSPLCCHFHFSPKVMFTKVLKAOLWVYLRPVPRPATVY LOILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWOSI KOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGP GAEGL HPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPR GSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF - 11 D2G AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 167 VWROHSRELRLESIKSOILSKLRLKEAPNISREVVKOLLPKAPP LOOILDLHDFOGDALOPEDFLEEDEYHATTETVISMAQETDPA VQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVY LQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSI DFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGPGAEGL US 9 , 758 ,576 B2 69 TABLE 11 - continued Recombinant GDFs

SEO ID Protein Sequence NO HPFMELRVLENTKRSGNLGLDCDEHSSESRCCRYPLTVDFEAF GWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRG SAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF - 11 AXXA AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 168 VWROHSRELRLESIKSCILSKLRLKEAPNISREVVKOLLPKAPP LOQILDLHDFOGDALOPEDFLEEDEYHATTETVISMAQETDPA VQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVY LQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSI DFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKASRANLGLDCDEHSSESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPR GSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF - 11 AxxA AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 169 D98A VWROHSRELRLESIKSQILSKLRLKEAPNISREVVKOLLPKAPP LQQILDLHDFQGAALQPEDFLEEDEYHATTETVISMAQETDPA VOTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVY LOILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWOSI DFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKASRANLGLDCDEHSSESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPR GSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 170 prodomain D9 8A VWROHSRELRLESIKSOILSKLRLKEAPNISREVVKOLLPKAPP LQQILDLHDFQGAALQPEDFLEEDEYHATTETVISMAQETDPA VQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVY LOILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWOSI DFKOVLHSWFROPOSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKRSRR GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 71 prodomain VWROHSRELRLESIKSOILSKLRLKEAPNISREVVKOLLPKAPP LOQILDLHDFOGDALOPEDFLEEDEYHATTETVISMAQETDPA VOTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVY LQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWOSI DFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKRSRR

40 Activins and inhibins are TGF- B family member proteins, together to form the ligand binding site . Recombinant pro the activity of each of which often results in opposing teins of the present invention may comprise integrins and /or functions (Bilezikjian et al 2012. ) Like other family mem integrin subunits . Such integrins and /or integrin subunits bers , these proteins occur physiologically as dimers . may comprise any of those disclosed in U . S . Provisional Activins and inhibins are constructed in part from the same 45 Patent Application No. 61 / 722, 919 filed Nov . 6 , 2012 , the B -subunits , that may include inhibin -beta A , inhibin -beta B , contents of which are herein incorporated by reference in inhibin - beta C and inhibin - beta E ( referred to herein as their entirety . B - subunit A , B , C and E , respectively .) The difference Recombinant proteins of the invention may include inter between activins and inhibins , structurally , is that activins so cellular adhesion molecule 1 (ICAM - 1 ) . In some cases , are B - subunit dimers while inhibins are heterodimers , ICAM -1 proteins of the present invention may be used as wherein the second subunit is inhibin -a . Activins are named control proteins during antibody development and /or anti for their subunit pairs , such that activin A comprises a body testing . In some cases, ICAM - 1 may be used as a homodimer of two A subunits , activin AB comprises a dimer control during selection of binding molecules using phage of A and B subunits , B comprises a dimer of B subunits , etc . 55 display technologies . In some cases , ICAM - 1 proteins of the (Muenster et al 2011 . ) Activins are involved in a variety of invention comprise one or more detectable label. Detectable functions that may include , but are not limited to cell labels may include , for example , histidine tags . growth , differentiation , programmed cell death , endocrine Chimeric Proteins functions, cellular metabolism , bone growth , etc . They are In some embodiments , recombinant proteins of the pres especially recognized for their control of reproductive hor - 60 ent invention may comprise chimeric proteins. As used mone cycles . Activin and inhibin signaling often functions herein , the term “ chimeric protein ” refers to a protein antagonistically in this regard . comprising one or more protein modules from at least two In some embodiments , recombinant proteins of the pres - different proteins [formed from the same gene ( e . g . variants ent invention may comprise integrins. Integrins are cell arising from alternative splicing ) or from different genes ] . surface heterodimers formed by alpha and beta subunits , 65 Chimeric proteins may comprise protein modules from two each of which has a transmembrane domain and in the or more TGF - B family member proteins . Such chimeric N - terminal portion of the extracellular domain come proteins may comprise protein modules from TGF- B1 , TGF US 9 ,758 ,576 B2 72 B2 and /or TGF -B3 . Some chimeric proteins of the present or fewer amino acids , about 2 more or fewer amino acids, invention may comprise protein modules including , but not limited to the protein modules and /or amino acid sequences about 3 more or fewer amino acids , about 4 more or fewer listed in Table 12 ( residue numbers correspond to the amino acids, about 5 more or fewer amino acids , about 6 pro - protein sequences listed in Table 1. ) Some chimeric 5 more or fewer amino acids , about 7 more or fewer amino proteins of the present invention may comprise protein acids , about 8 more or fewer amino acids, about 9 more or modules comprising amino acid sequences similar to those fewer amino acids , about 10 more or fewer amino acids or in Table 12 , but comprising additional or fewer amino acids greater than 10 more or fewer amino acids on N -terminal than those listed . Such modules may comprise about 1 more and / or C - terminal ends. TABLE 12 Protein modules

SEO Protein Residues Sequence ID NO TGF - B1 1 - 74 LSTCKTIDMELVKRKRIEAIRGQIL SKLRLASPPSQGEV 171 PPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADY

TGF - B1 1 - 207 LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEV 172 PPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYA KEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTSELRE AVPEPVLLSRAELRLLRLKLKVEOHVELYOKYSNNS WRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGEIE GFRLSAHCSCDSRDNTLQVDI TGF - B1 46 - end EAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTR 173 VLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEP VLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLS NRLLAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLS AHCSCDSRDNTLOVDINGFTTGRRGDLATIHGMNRPF LLLMATPLERAOHLO S SRHRRALDTNYCFSSTEKNCC VROLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIW SLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIV YYVGRKPKVEQLSNMIVRSCKCS TGF - B1 47 - end AVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRV 174 LMVETHNEIYDKFKOSTHSIYMFFNTSELREAVPEPVL LSRAELRLLRLKLKVEOHVELYOKYSNNSWRYLSNR LLAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLSAH CSCDSRDNTLOVDINGFTTGRRGDLATIHGMNRPFLL LMATPLERAQHLOSSRHRRALDTNYCFSSTEKNCCVR QLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSL DTQYSKVLALYNCHNPGASAAPCCVPQALEPLPIVYY VGRKPKVEQLSNMIVRSCKCS TGF - B1 74 - 249 YYAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTSE 175 LREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSN NSWRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGG EIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATI HGMNRPFLLLMATPLERAQHLOSSRHRR TGF - B1 74 - end YYAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTSE 176 LREAVPEPVLLSRAELRLLRLKLKVEOHVELYOKYSN NSWRYL SNRLLAPSDSPEWLSFDVTGVVROWLSRGG EIEGFRLSAHCSCDSRDNTLOVDINGFTTGRRGDLATI HGMNRPFLLLMATPLERAQHLQS SRHRRALDTNYCF SSTEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFC LGPCPYIWSLDTOYSKVLALYNOHNPGASAAPCCVP QALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS TGF - B1 75 - 249 YAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTSEL 177 REAVPEPVLLSRAELRLLRLKLKVEOHVELYOKYSNN SWRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGEI EGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIH GMNRPFLLLMATPLERAQHLOSSRHRR TGF - B1 75 - end YAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTSEL 178 REAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNN SWRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGEI EGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIH GMNRPFLLLMATPLERAQHLOSSRHRRALDTNYCFSS TEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFCLG PCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQA LEPLPIVYYVGRKPKVEOLSNMIVRSCKCS US 9 , 758 ,576 B2 73 74 TABLE 12 - continued Protein modules

SEO Protein Residues Sequence ID NO TGF - B1 228 - 361 FLLLMATPLERAQHLOSSRHRRALDTNYCFSSTEKNC 179 CVROLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYI WSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPI VYYVGRKPKVEQLSNMIVRSCKCS TGF - B1 250 - 361 ALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWIHEP 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 1 - 79 SLSLSTCTTLDFGHIKKKRVEAIRGOILSKLRLTSPPEP 181 TVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQEN TESE TGF - B3 80 - 280 YYAKEIHKFDMIQLAEHNELAVCPKGITSKVFRFNV 182 SSVEKNRTNLFRAEFRVLRVPNPSSKRNEORIELFOIL RPDEHIAKORYIGGKNLPTRGTAEWLSFDVTDTVRE WLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIK FKGVDNEDDHGRGDLGRLKKOKDHHNPHLILMMIPP HRLDNPGOGGORKKR

TGF - B3 281 - 392 ALDTNYCFRNLEENCCVRPLYIDFRODLGWKWVHEP 46 KGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEAS ASPCCVPODLEPLTILYYVGRTPKVEQLSNMVVKSCK CS GDF - 8 1 - 75 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKIQIL 183 SKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVOR GDF - 8 1 - 64 NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKIOIL 72 SKLRLETAPNISKDVIROLLPKAPPL GDF - 8 75 - end RDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGK 184 PKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFV OILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDVK h TVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGE DGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCR YPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLOK YPHTHLVHOANPRGSAGPCCTPTKMSPINMLYFNGK EQIIYGKIPAMVVDRCGCS GDF8 65 - end RELIDQYDVORDDSSDGSLEDDDYHATTETII TMPTES 185 DFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIYLR PVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLDMNPG TGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHD LAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCD EHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCS GECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSP INMLYFNGKEQI IYGKIPAMVVDRCGCS GDF8 65 - 243 RELIDQYDVORDDSSDGSLEDDDYHATTETIITMPTES 77 DFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIYLR PVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLDMNPG TGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHD LAVTFPGPGEDGLNPFLEVKVTDTPKRSRR GDF - 8 76 - 243 DDSSDGSLEDDDYHATTETII TMPTESDFLMQVDGKP 186 KCCFFKFSSKIOYNKVVKAOLWIYLRPVETPTTVFVOI LRLIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDVKT VLONWLKQPESNLGIEIKALDENGHDLAVTFPGPGED GLNPFLEVKVTDTPKRSRR

GDF - 8 244 - 352 DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKR 74 YKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPC CTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS US 9 , 758 ,576 B2 75 TABLE 12 - continued Protein modules

SEO Protein Residues Sequence ID NO

GDF - 11 1 - 86 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 73 GCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISRE VVKOLLPKAPPL GDF - 11 1 - 96 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 187 GCPVCVWROHSRELRLESIKSCILSKLRLKEAPNISRE VVKQLLPKAPPLQQILDLHDFQ GDF - 11 1 - 108 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 188 GCPVCVWROHSRELRLESIKSOILSKLRLKEAPNISRE VVKOLLPKAPPLOOILDLHDFOGDALOPEDFLEE

GDF - 11 97 - 274 GDALOPEDFLEEDEYHATTETVISMAQETDPAVOTDG 189 SPLCCHFHFSPKVMFTKVLKAOLWVYLRPVPRPATV YLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSR i SGHWOSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTD LAVTSLGPGAEGLHPFMELRVLENTKRSRR GDF - 11 87 - 274 QQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQ 78 ETDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOLWVY LRPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHIRI RSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEI NAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRS RR GDF - 11 275 - 383 NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKR 75 YKANYCSGOCEYMFMQKYPHTHLVQQANPRGSAGP CCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS Inhibin 1 - 64 SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEA 190 Beta A VKKHILNMLHLKKRPDVTQPVPKAALL Inhibin 1 - 76 SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEA 191 Beta A VKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVG KVG

Inhibin 65 - 288 NAIRKLHVGKVGENGYVEI EDDIGRRAEMNELMEOT 192 Beta A SEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFL KVPKANRTRTKVTIRLFOOOKHPOGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDO GKSSLDVRIACEOCOESGASLVLLGKKKKKEEEGEGK KKGGGEGGAGADEEKEQSHRPFLMLQARQS EDHPHR RR Inhibin 65 - 289 NAIRKLHVGKVGENGYVEI EDDIGRRAEMNELMEQT 193 Beta A SEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFL KVPKANRTRTKVTIRLFOOOKHPOGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIORLLDO GKSSLDVRIACEOCOESGASLVLLGKKKKKEEEGEGK KKGGGEGGAGADEEKEQSHRPFLMLQAROSEDHPHR RRR Inhibin 65 - 290 NAIRKLHVGKVGENGYVEI EDDIGRRAEMNELMEOT 194 Beta A SEIITFAESGTARKTLHFEISKEGSDLSVERAEVWLFL KVPKANRTRTKVTIRLFOOOKHPOGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDO GKSSLDVRIACEOCOESGASLVLLGKKKKKEEEGEGK KKGGGEGGAGADEEKEQSHRPFLMLQAROSEDHPHR RRRR

Inhibin 77 - 289 ENGYVEIEDDIGRRAEMNELMEOTSEIITFAESGTARK 195 Beta A TLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKV TIRLFOOOKHPOGSLDTGEEAEEVGLKGERSELLLSEK VVDARKSTWHVFPVSSSIQRLLDOGKSSLDVRIACEQ CQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGA DEEKEQSHRPFLMLQARQS EDHPHRRRR Inhibin 77 - 290 ENGYVEIEDDIGRRAEMNELMEOTSEIITFAESGTARK 196 Beta A TLHFEISKEGSDLSVERAEVWLFLKVPKANRTRTKV TIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEK VVDARKSTWHVFPVSSSIQRLLDOGKSSLDVRIACEQ CQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGA lilililililili DEEKEQSHRPFLMLQAROSEDHPHRRRRR US 9 , 758 ,576 B2 77 TABLE 12 - continued Protein modules

SEO Protein Residues Sequence ID NO Inhibin 77 - end ENGYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARK 197 Beta A TLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKV TIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEK VVDARKSTWHVFPVSSSIQRLLDOGKSSLDVRIACEQ COESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGA DEEKEQSHRPFLMLQAROSEDHPHRRRRRGLECDGK VNICCKKOFFVSFKDIGWNDWIIAPSGYHANYCEGEC PSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCV PTKLRPMSMLYYDDGONIIKKDIONMIVEECGCS Inhibin 291 - 406 GLECDGKVNICCKKOFFVSFKDIGWNDWIIAPSGYHA 198 Beta A NYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFA NLKSCCVPTKLRPMSMLYYDDGONIIKKDIQNMIVEE CGCS

In some embodiments , chimeric proteins of the present 20 proteins contains the a2 helix . Furthermore , F95 may be an invention may comprise combinations of any of the protein 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 GPCs may 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 . 23 Alanine , may be carried out to promote dissociation of the In some embodiments, chimeric proteins may comprise 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 s 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 . 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 of GDF - 11 . Such chime- 40 more or fewer amino acids, about 6 more or fewer amino rics may be unstable due to steric clash between residue F95 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 SEO Protein Protein Protein ID module 1 module 2 module 3 Chimeric Sequence NO TGF - B2 TGF - B1 N / A SLSTCSTLDMDOFMRKRIEAIRGOILSKLKLTSPPE 199 LAP growth DYPEPEEVPPEVISIYNSTRDLLOEKASRRAAACE factor RERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPY FRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKAR VPEQRIELYQILKSKDLTSPTORYIDSKVVKTRAE GEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPC CTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQ KTIKSTRKKNSGKTPHLLLMLLPSYRLESOOTNRR KKRALDTNYCFS STEKNCCVROLYIDFRKDLGWK WIHEPKGYHANFCLGPCPYIWSLDTOYSKVLALY NQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVE QLSNMIVRSCKCS TGF - B3 TGF - B1 N / A SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 200 LAP growth PEPTVMTHVPYOVLALYNSTRELLEEMHGEREEG factor CTOENTESEYYAKEIHKFDMIOGLAEHNELAVCP KGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNP SSKRNEORIELFOILRPDEHIAKORYIGGKNLPTRG US 9 ,758 , 576 B2 79 80 TABLE 13 - continued Protein module combinations

SEO Protein Protein Protein ID module 1 module 2 module 3 Chimeric Sequence NO TAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCH TFOPNGDILENIHEVMEIKFKGVDNEDDHGRGDL GRLKKOKDHHNPHLILMMIPPHRLDNPGQGGQR KKRALDTNYCFS STEKNCCVRQLYIDFRKDLGWK WIHEPKGYHANFCLGPCPYIWSLDTOYSKVLALY NOHNPGASAAPCCVPOALEPLPIVYYVGRKPKVE QLSNMIVRSCKCS TGF - B3 TGF - B1 N / A SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 201 ( 1 - 46 ) (47 - end ) PEPTVMTHVPAVLAL YNSTRDRVAGESAEPEPEP EADYYAKEVTRVLMVETHNEIYDKFKOSTHSIYM FFNTSELREAVPEPVLLSRAELRLLRLKLKVEOHV ELYOKYSNNSWRYLSNRLLAPSDSPEWLSFDVTG WRQWLSRGGEIEGFRLSAHCSCDSRDNTLOVDI NGFTTGRRGDLATIHGMNRPFLLLMATPLERAQH LOSSRHRRALDTNYCFSSTEKNCCVROLYIDFRK DLGWKWIHEPKGYHANFCLGPCPYIWSLDTOYS KVLALYNOHNPGASAAPCCVPOALEPLPIVYYVG RKPKVEQLSNMIVRSCKCS TGF - B3 TGF - B1 N / A SLSLSTCTTLDFGHIKKKRVEAIRGOILSKLRLTSP 202 ( 1 - 79 ) ( 75 - end ) PEPTVMTHVPYQVLALYNSTRELLEEMHGEREEG CTOENTESEYAKEVTRVLMVETHNEIYDKF KOST HSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLK VEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLS FDVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNT LOVDINGFTTGRRGDLATIHGMNRPFLLLMATPL ERAQHLOSSRHRRALDTNYCFSSTEKNCCVROLY IDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLD TOYSKVLALYNCHNPGASAAPCCVPQALEPLPIV YYVGRKPKVEQLSNMIVRSCKCS TGF - B1 TGF - B3 TGF - B1 LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPPSO 203 ( 1 - 74 ) (80 - 280 ) ( 250 - 361 ) GEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPE ADYYYAKEIHKFDMIOGLAEHNELAVCPKGITSK VFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNE QRIELFQILRPDEHIAKORYIGGKNLPTRGTAEWL SFDVTDTVREWLLRRESNLGLEISIHCPCHTFOPN GDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKK QKDHHNPHLILMMIPPHRLDNPGQGGQRKKRAL DTNYCFS STEKNCCVRQLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTOYSKVLAL YNOHNP GASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNM IVRSCKCS TGF - B3 TGF - B1 TGF - B3 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 204 ( 1 - 79 ) ( 75 - 249 ) ( 281 - 392 ) PEPTVMTHVPYOVLALYNSTRELLEEMHGEREEG CTOENTESEYAKEVTRVLMVETHNEIYDKF KOST HSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLK VEOHVELYOKYSNNSWRYLSNRLLAPSDSPEWLS FDVTGVVROWLSRGGEIEGFRLSAHCSCDSRDNT LOVDINGFTTGRRGDLATIHGMNRPFLLLMATPL ERACHLOSSRHRRALDTNYCFRNLEENCCVRPLY IDFRODLGWKWVHEPKGYYANFCSGPCPYLRSA DTTHSTVLGLYNTLNPEASASPCCVPODLEPLTIL YYVGRTPKVEQLSNMVVKSCKCS TGF - B1 TGF - B2 TGF - B1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQ 205 ( 1 - 207 ) trigger ( 228 - 361 ) GEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPE loop ADYYAKEVTRVLMVETHNEIYDKFKOSTHSIYMF Short FNTSELREAVPEPVLLSRAELRLLRLKLKVEOHVE ( 236 - 254 ) LYOKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGV VROWLSRGGEIEGFRLSAHCSCDSRDNTLOVDIG TSTYTSGDQKTIKSTRKKFLLLMATPLERAQHLOS SRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLG WKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVL ALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKP KVEQLSNMIVRSCKCS TGF - B1 TGF - B2 TGF - B1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQ 206 ( 1 - 207 ) trigger ( 228 - 361 ) GEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPE loop ADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMF Long FNTSELREAVPEPVLLSRAELRLLRLKLKVEOHVE ( 232 - 260 ) LYOKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGV US 9 , 758 ,576 B2 81 TABLE 13 - continued Protein module combinations

SEO Protein Protein Protein ID module 1 module 2 module 3 Chimeric Sequence NO VROWLSRGGEIEGFRLSAHCSCDSRDNTLOVDIA GIDGTSTYTSGDOKTIKSTRKKNSGKTPFLLLMAT PLERAQHLOSSRHRRALDTNYCFSSTEKNCCVRO LYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWS LDTOYSKVLALYNOHNPGASAAPCCVPOALEPLP IVYYVGRKPKVEQLSNMIVRSCKCS GDF - 11 GDF - 8 GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 207 ( 1 - 96 ) ( 76 - 243 ) ( 275 - 383 ) EPDGCPVCVWROHSRELRLESIKSQILSKLRLKEA PNISREVV KOLLPKAPPLOOILDLHD FODDSSDGS LEDDDYHATTETIITMPTESDFLMOVDGKPKCCFF KFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILR LIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDVKT VLONWLKOPESNLGIEIKALDENGHDLAVTFPGP GEDGLNPFLEVKVTDTPKRSRRNLGLDCDEHSSE SRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQ CEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKM SPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF - 11 GDF - 8 GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 208 ( 1 - 86 ) (65 - 243 ) ( 275 - 383 ) EPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEA PNISREVVKQLLPKAPPLRELIDQYDVORDDSSDG SLEDDDYHATTETIITMPTESDFLMOVDGKPKCCF FKFSSKIOYNKVVKAOLWIYLRPVETPTTVFVOIL RLIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDV KTVLONWLKOPESNLGIEI KALDENGHD LAVTFP GPGEDGLNPFLEVKVTDTPKRSRRNLGLDCDEHS SESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCS GQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPT KMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF - 11 GDF - 8 N / A AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 209 ( 1 - 96 ) ( 76 - 243 ) EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA PNISREVV KOLLPKAPPLOOILDLHD FODDSSDGS LEDDDYHATTETIITMPTESDFLMOVDGKPKCCFF KFSSKIOYNKVVKAOLWIYLRPVETPTTVFVOILR LIKPMKDGTRYTGIRSLKLDMNPGTGIWOSIDVKT VLONWLKQPESNLGIEIKALDENGHDLAVTFPGP GEDGLNPFLEVKVTDTPKRSRR GDF - 11 GDF - 8 NA AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 210 ( 1 - 86 ) (65 - 243 ) EPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEA PNISREVV KOLLPKAPPLRELIDOYDVORDDSSDG SLEDDDYHATTETIITMPTESDFLMOVDGKPKCCF FKFSSKIOYNKVVKAOLWIYLRPVETPTTVFVOIL RLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDV KTVLONWLKOPESNLGIEI KALDENGHDLAVTFP GPGEDGLNPFLEVKVTDTPKRSRR GDF - 11 Inhibin GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 211 ( 1 - 96 ) Beta A ( 275 - 383 ) EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA ( 77 - 290 ) PNISREVV KOLLPKAPPLQQILDLHD FQENGYVEI EDDIGRRAEMNELMEOTSEIITFAESGTARKTLHF EISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTI RLFOOOKHPOGSLDTGEEAEEVGLKGERSELLLS EKVVDARKSTWHVFPVSSSIQRLLDOGKSSLDVRI ACEOCOESGASLVLLGKKKKKEEEGEGKKKGGG EGGAGADEEKEQSHRPFLMLQAROSEDHPHRRR RRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWI IAPKRYKANYCSGQCEYMFMQKYPHTHLVQQAN PRGSAGPCCTPTKMSPINMLYFND KOOIIYGKIPG MVVDRCGCS GDF - 11 Inhibin GDF - 11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 212 ( 1 - 86 ) Beta A ( 275 - 383 ) EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA ( 65 - 290 ) PNISREVVKOLLPKAPPLNAIRKLHVGKVGENGY VEIEDDIGRRAEMNELMEQTSEIITFAESGTARKTL HFEISKEGSDLSVVERAEVWLFLKVPKANRTRTK VTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELL LSEKVVDARKS TWHVFPVSSSIORLLDOGKSSLD VRIACEOCOESGASLVLLGKKKKKEEEGEGKKKG GGEGGAGADEEKEQSHRPFLMLQARQSEDHPHR RRRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWD WIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQ US 9 , 758 ,576 B2 83 84 TABLE 13 - continued Protein module combinations

SEO Protein Protein Protein ID module 1 module 2 module 3 Chimeric Sequence NO ANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKI PGMVVDRCGCS GDF - 11 Inhibin N / A AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 213 ( 1 - 96 ) Beta A EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA ( 77 - 290 ) PNISREVVKOLLPKAPPLQQILDLHDFQENGYVEI EDDIGRRAEMNELMEOTSEIITFAESGTARKTLHF EISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTI RLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLS EKVVDARKSTWHVFPVSSSIQRLLDOGKSSLDVRI ACEOCOESGASLVLLGKKKKKEEEGEGKKKGGG EGGAGADEEKEQSHRPFLMLQAROSEDHPHRRR RR GDF - 11 Inhibin NA AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 214 ( 1 - 86 ) Beta A EPDGCPVCVWROHSRELRLESIKSOILSKLRLKEA ( 65 - 290 ) PNISREVV KOLLPKAPPLNAIRKLHVGKVGENGY VEIEDDIGRRAEMNELMEOTSEIITFAESGTARKTL HFEISKEGSDLSVVERAEVWLFLKVPKANRTRTK VTIRLFOOOKHPOGSLDTGEEAEEVGLKGERSELL LSEKVVDARKS TWHVFPVSSSIQRLLDOGKSSLD VRIACEOCOESGASLVLLGKKKKKEEEGEGKKKG GGEGGAGADEEKEQSHRPFLMLQARQSEDHPHR RRRR GDF - 8 GDF - 11 GDF - 8 NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKI 215 ( 1 - 75 ) ( 97 - 274 ) ( 244 - 352 ) QILSKLRLETAPNISKDVIROLLPKAPPLRELIDQY DVQRGDALQPEDFLEEDEYHATTETVISMAQETD PAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVY LRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRR HIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQS NWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELR VLENTKRSRRDFGLDCDEHSTESRCCRYPLTVDFE AFGWDWIIAPKRYKANYCSGECEFVFLOKYPHTH LVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQII YGKIPAMVVDRCGCS GDF - 8 GDF - 11 GDF - 8 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKI 216 ( 1 - 64 ) ( 87 - 274 ) ( 244 - 352 ) QILSKLRLETAPNISKDVIROLLPKAPPLQQILDLH DFOGDALQPEDFLEEDEYHATTETVISMAQETDP AVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYL RPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHI RIRSLKIELHSRSGHWOSIDFKOVLHSWFROPOSN WGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRV LENTKRSRRDFGLDCDEHSTESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGECEFVFLOKYPHTHL VHOANPRGSAGPCCTPTKMSPINMLYFNGKEOIIY GKIPAMVVDRCGCS GDF - 8 GDF - 11 N / A NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKI 217 ( 1 - 75 ) ( 97 - 274 ) QILSKLRLETAPNISKDVIROLLPKAPPLRELIDOY DVORGDALOPEDFLEEDEYHATTETVISMAQETD PAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVY LRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRR HIRIRSLKIELHSRSGHWQSIDFKQVLHSWFROPOS NWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELR VLENTKRSRR GDF - 8 GDF - 11 GDF - 8 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKI 218 ( 1 - 64 ) (87 - 274 ) ( 244 - 352 ) QILSKLRLETAPNISKDVIROLLPKAPPLQQILDLH DFOGDALOPEDFLEEDEYHATTETVISMAQETDP AVOTDGSPLCCHFHFSPKVMFTKVLKAOLWVYL RPVPRPATVYLOILRLKPLTGEGTAGGGGGGRRHI RIRSLKIELHSRSGHWOSIDF KOVLHSWFROPOSN WGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRV LENTKRSRRDFGLDCDEHSTESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIY GKIPAMVVDRCGCS GDF - 8 Inhibin GDF - 8 NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKI 219 ( 1 - 75 ) Beta A ( 244 - 352 ) QILSKLRLETAPNISKDVIROLLPKAPPLRELIDQY ( 77 - 289 ) DVQRENGYVEI EDDIGRRAEMNELMEQTSEIITFA ESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVP US 9 , 758 ,576 B2 85 86 TABLE 13 - continued Protein module combinations

SEO Protein Protein Protein ID module 1 module 2 module 3 Chimeric Sequence NO KANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIORLL DOGKSSLDVRIACEQCQESGASLVLLGKKKKKEE EGEGKKKGGGEGGAGADEEKEQSHRPFLMLQAR QSEDHPHRRRRDFGLDCDEHSTESRCCRYPLTVD FEAFGWDWIIAPKRYKANYCSGECEFVFLOKYPH THLVHOANPRGSAGPCCTPTKMSPINMLYFNGKE OLIYGKIPAMVVDRCGCS GDF - 8 Inhibin GDF - 8 NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKI 220 ( 1 - 64 ) Beta A ( 244 - 352 ) OILSKLRLETAPNISKDVIROLLPKAPPLNAIRKLH ( 65 - 290 ) VGKVGENGYVEIEDDIGRRAEMNELMEOTSEIITF AESGTARKTLHFEISKEGSDLSVVERAEVWLFLK VPKANRTRTKVTIRLFOOOKHPOGSLDTGEEAEE VGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQR LLDOGKSSLDVRIACEQCQESGASLVLLGKKKKK EEEGEGKKKGGGEGGAGADEEKEOSHRPFLMLO AROSEDHPHRRRRRDFGLDCDEHSTESRCCRYPL TVDFEAFGWDWIIAPKRYKANYCS GECEFVFLOK YPHTHLVHOANPRGSAGPCCTPTKMSPINMLYFN GKEQIIYGKIPAMVVDRCGCS GDF - 8 Inhibin N / A NENSEQKENVEKEGLCNACTWRONTKSSRIEAIKI 221 ( 1 - 75 ) Beta A OILSKLRLETAPNISKDVIROLLPKAPPLRELIDOY ( 77 - 290 ) DVORENGYVEI EDDIGRRAEMNELMEQTSEIITFA ESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVP KANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLL DOGKSSLDVRIACEQCQESGASLVLLGKKKKKEE EGEGKKKGGGEGGAGADEEKEQSHRPFLMLQAR QSEDHPHRRRRR GDF - 8 Inhibin NA NENSEOKENVEKEGLCNACTWRONTKSSRIEAIKI 222 ( 1 - 64 ) Beta A QILSKLRLETAPNISKDVIROLLPKAPPLNAIRKLH ( 65 - 290 ) VGKVGENGYVEIEDDIGRRAEMNELMEOTSEIITF AESGTARKTLHFEISKEGSDLSVVERAEVWLFLK VPKANRTRTKVTIRLFOOOKHPOGSLDTGEEAEE VGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQR LLDOGKSSLDVRIACEQCQESGASLVLLGKKKKK EEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQ AROSEDHPHRRRRR Inhibin GDF - 8 Inhibin SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 223 Beta A ( 76 - 243 ) Beta A VEAVKKHILNMLHLKKRPDVTOPVPKAALLNAIR ( 1 - 76 ) ( 291 - 406 ) KLHVGKVGDDSSDGSLEDDDYHATTETIITMPTES DFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIY LRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWOSIDVKTVLONWLKOPESNLGIEIKA LDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKR SRRGLECDGKVNICCKKOFFVSFKDIGWNDWIIAP SGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYR MRGHSPFANLKSCCVPTKLRPMSMLYYDDGONII KKDIQNMIVEECGCS Inhibin GDF - 8 Inhibin SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 224 Beta A (65 - 243 ) Beta A VEAVKKHILNMLHLKKRPDVTOPVPKAALLRELI ( 1 - 64 ) ( 291 - 406 ) DOYDVORDDSSDGSLEDDDYHATTETIITMPTES DFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIY LRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWOSIDVKTVLONWLKOPESNLGIEIKA LDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKR SRRGLECDGKVNICCKKOFFVSFKDIGWNDWIIAP SGYHANYCEGECP SHIAGTSGSSLSFHSTVINHYR MRGHSPFANLKSCCVPTKLRPMSMLYYDDGONII KKDIQNMIVEECGCS Inhibin GDF - 8 NA SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 225 Beta A ( 76 - 243 ) VEAVKKHI LNMLHLKKRPDVTOPVPKAALLNAIR ( 1 - 76 ) KLHVGKVGDDSSDGSLEDDDYHATTETIITMPTES DFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIY LRPVETPTTVFVOILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWQSIDVKTVLONWLKQPESNLGIEIKA LDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKR SRR US 9 , 758 ,576 B2 87 88 TABLE 13 - continued Protein module combinations

SEO Protein Protein Protein ID module 1 module 2 module 3 Chimeric Sequence NO

Inhibin GDF - 8 NA SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 226 Beta A ( 65 - 243 ) VEAVKKHI LNMLHLKKRPDVTOPVPKAALLRELI ( 1 - 64 ) DOYDVORDDSSDGSLEDDDYHATTETII TMPTES DFLMOVDGKPKCCFFKFSSKIOYNKVVKAOLWIY LRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWOSIDVKTVLONWLKOPESNLGIEIKA LDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKR SRR Inhibin GDF - 11 Inhibin SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 227 Beta A ( 97 - 274 ) Beta A VEAVKKHILNMLHLKKRPDVTOPVPKAALLNAIR ( 1 - 76 ) ( 291 - 406 ) KLHVGKVGGDALOPEDFLEEDEYHATTETVISMA QETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQL WVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGG GRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFR OPOSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPF MELRVLENTKRSRRGLECDGKVNICCKKOFFVSF KDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSS LSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMS MLYYDDGONIIKKDIONMIVEECGCS Inhibin GDF - 11 Inhibin SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 228 Beta A (87 - 274 ) BetaA VEAVKKHILNMLHLKKRPDVTOPVPKAALLQQIL ( 1 - 64 ) ( 291 - 406 ) DLHDFQGDALQPEDFLEEDEYHATTETVISMAQE TDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOLW VYLRPVPRPATVYLOILRLKPLTGEGTAGGGGGG RRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRO POSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFM ELRVLENTKRSRRGLECDGKVNICCKKOFFVSFK DIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSL SFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMS MLYYDDGONIIKKDIONMIVEECGCS Inhibin GDF - 11 N / A SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 229 Beta A ( 97 - 274 ) VEAVKKHI LNMLHLKKRPDVTOPVPKAALLNAIR ( 1 - 76 ) KLHVGKVGGDALQPEDFLEEDEYHATTETVISMA OETDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOL WVYLRPVPRPATVYLOILRLKPLTGEGTAGGGGG GRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFR OPOSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPF MELRVLENTKRSRR Inhibin GDF - 11 NA SPTPGSEGHSAAPDCPSCALAALPKDVPNSOPEM 230 Beta A ( 87 - 274 ) VEAVKKHILNMLHLKKRPDVTOPVPKAALLOOIL ( 1 - 64 ) DLHDFOGDALOPEDFLEEDEYHATTETVISMAQE TDPAVOTDGSPLCCHFHFSPKVMFTKVLKAOLW VYLRPVPRPATVYLOILRLKPLTGEGTAGGGGGG RRHIRIRSLKIELHSRSGHWOSIDFKOVLHSWFRO POSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFM ELRVLENTKRSRR

Chimeric proteinsmay be used to characterize and /or map 50 tein and /or protein module .) Some chimeric proteins may be epitopes associated with GPCs. As used herein , the terms used to characterize functions associated with one or more " map " or " mapping ” refer to the identification , character ization and /or determination of one or more functional proteins and /or protein modules. regions of one or more proteins. Such characterizations may In some embodiments , chimeric proteins of the present be necessary for determining interactions between one or invention may comprise the sequences listed in Table 14 or more protein modules and another agent ( e . g . another pro - fragments thereof. TABLE 14 Chimeric proteins SEQ ID Protein Sequence NO proTGF - Blarm3 C4S LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP 231 PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE US 9 ,758 ,576 B2 89 90 TABLE 14 - continued Chimeric proteins SEQ ID Protein Sequence NO IHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKN RTNLFRAEFRVLRVPNPSSKRNEORIELFOILRPDEHIA KORYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESN LGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNED DHGRGDLGRLKKOKDHHNPHLILMMIPPHRLDNPGOG GORKKRALDTNYCFSSTEKNCCVROLYIDFRKDLGWK WIHEPKGYHANFCLGPCPYIWSLDTOYSKVLAL YNOH NPGASAAPCCV POALEPLPIVYYVGRKPKVEOL SNMIV RSCKCS proTGF - B1 Trigger Loop LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP 232 ( short ) B2 C4S PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE VTRVLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVP EPVLLSRAELRLLRLKLKVEOHVELYOKYSNNSWRYL SNRLLAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLS AHCSCDSRDNTLQVDINGFTGTSTYTSGDQKTIKSTRK KHGMNRPFLLLMATPLERAOHLOSSRHRRALDTNYCF SSTEKNCCVROLYIDFRKDLGWKWIHEPKGYHANFCL GPCPYIWSLDTOYSKVLALYNOHNPGASAAPCCVPOA LEPLPIVYYVGRKPKVEOLSNMIVRSCKCS proTGF - B3 arml C7S SLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPT 233 VMTHVPYOVLALYNSTRELLEEMHGEREEGCTOENTE SEYYAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTS ELREAVPEPVLLSRAELRLLRLKLKVEOHVELYOKYSN NSWRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGE IEGFRLSAHCSCDSRDNTLOVDINGFTTGRRGD LATIHG MNRPFLLLMATPLERAQHLOSSRHRRALDTNYCFRNL EENCCVRPLYIDFRODLGWKWVHEPKGYYANFCSGPC PYLRSADTTHS TVLGLYNTLNPEASASPCCVPODLEPL TILYYVGRTPKVEQLSNMVVKSCKCS TGF - Blarm3 C4S (LAP ) LSTSKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVP 234 PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE IHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKN RTNLFRAEFRVLRVPNPSSKRNEORIELFOILRPDEHIA KORYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESN LGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNED DHGRGDLGRLKKOKDHHNPHLILMMIPPHRLDNPGOG GORKKR

TGF - B3armi C7S (LAP ) SLSLSTSTTLDFGHIKKKRVEAIRGOILSKLRLTSPPEPT 235 VMTHVPYOVLALYNSTRELLEEMHGEREEGCTOENTE SEYYAKEVTRVLMVETHNEIYDKFKOSTHSIYMFFNTS ELREAVPEPVLLSRAELRLLRLKLKVEOHVELYOKYSN NSWRYLSNRLLAPSDSPEWLSFDVTGVVROWLSRGGE IEGFRLSAHCSCDSRDNTLOVDINGFTTGRRGDLATIHG MNRPFLLLMATPLERAQHLOSSRHRR TGF - B1 Trigger Loop LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP 236 ( short ) B2 C4S (LAP ) PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE VTRVLMVETHNEIYDKFKOSTHSIYMFFNTSELREAVP EPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYL SNRLLAPSDSPEWLSFDVTGVVROWLSRGGEIEGFRLS AHCSCDSRDNTLOVDINGFTGTSTYTSGDOKTIKSTRK KHGMNRPFLLLMATPLERAQHLOSSRHRR

In some embodiments , chimeric proteins may comprise 55 According to mouse tissue staining , integrin subunit a9 is one or more protein modules from TGF -B2 . Although the widely expressed in skeletal and cardiac muscle , visceral crystal structure for the TGF -B2 growth factor has been smooth muscle , hepatocytes, airway epithelium , squamous elucidated (Daopin , S . et al. , Crystal structure of transform epithelium , choroid plexus epithelium and also on neutro ing growth factor- B2 : an unusual fold for the superfamily . phils (Palmer , E . L . et al. , Sequence and tissue distribution ms 60 of the integrin ag subunit , a novel partner of ( 31 that is Science. 1992 . 257 ( 5068 ): 369 - 73 , ) activation mechanisms widely distributed in epithelia and muscle . Journal of Cell remain to be fully understood . Activation may be dependent Biology . 1993. 123 ( 5 ) : 1289 - 97 . ) Expression of A , is not upon one or more interactions between the TGF -B2 trigger detected earlier than E12 . 5 , suggesting that it does not play loop and doß , integrin . The TGF - B2 trigger loop may a major role in the earliest tissue morphogenesis (Wang , A . comprise similar structural and / or functional features asso - 65 et al. , Expression of the integrin subunit al in the murine ciated with RGD sequences. TGF -B2 trigger loops may bind embryo . Developmental Dynamics. 1995 . 204 :421 - 31. ) In integrins, including , but not limited to aß , integrins. vivo functions of a9 are unclear . Phenotypes observed in US 9 , 758 ,576 B2 knockout mice suggest a role in lymphatic valve develop TABLE 15 - continued ment (Bazigou , E . et al ., Integrin - d , is required for fibronec tin matrix assembly during lymphatic valve morphogenesis . Trigger loop sequences Dev Cell . 2009 August . 17 ( 2 ) : 175 - 86 . ) Reported interaction SEQ partners of integrin agß , include VCAM - 1, the third FnIII 5 ID domain on tenascin C , osteopontin , polydom /SVEP1 , Source protein Trigger loop sequence NO VEGF- A and NGF ( Yokasaki, Y . et al . , Identification of the ligand binding site for the integrin ayß , in the third fibronec tin type III repeat of tenascin C . The Journal of Biological GDF - 8 PGEDGLNP 245 Chemistry . 1998 . 273 ( 19 ) : 11423 - 8 ; Marcinkiewicz , C . et al ., 10 GDF - 11 PGAEGLHP 246 Inhibitory effects of MLDG - containing heterodimeric dis integrins reveal distinct structural requirements for interac Inhibin A RPEATP 247 tion of the integrin aoß , with VCAM - 1 , tenascin - C , and BMP - 9 SHRKGCDTLDISVPPGSRNLP 248 osteopontin . JBC . 2000 . 275 (41 ) :31930 - 7 ; Oommen , S . et al. , Vacular endothelial growth factor A ( VEGF - A ) induces 15 BMP RHVRISRSLHQDEHSWSQIRP 249 endothelial and cancer cell migration through direct binding BMP - 4 250 to integrin aoß . JBC . 2011. 286 ( 2 ) : 1083 - 92 ; Sato -Nishi QHVRISRSLPQGSGNWAQLRP uchi, R . et al. , Polydom /SVEP1 is a ligand for integrin ayßi. BMP - 7 IGRHGPONKOP 251 JBC . 2012 . 287 ( 30 ) :25615 -30 ; Staniszewska, I . et al. , Inte grin ayß , is a receptor for nerve growth factor and other 20 BMP - 6 VGRDGPYDKOP 252 neurotrophins. Journal of Cell Science . 2007 . 121 ( Pt 4 ) : 504 BMP - 8 LGQRAPRSQQP 253 13 ; Yokosaki, Y . et al. , The integrin agß , binds to a novel recognition sequence (SVVYGLR ; SEQ ID NO : 238 ) in the Lefty1 RFASQGAPAGLGEP 254 thrombin - cleaved amino -terminal fragment of osteopontin . osteopontin SVVYGLR 238 JBC . 1999 . 274 (51 ) : 36328 - 34 . ) 25 Binding sites on proteins that interact with coß , have been tenascinc AEIDGIEL 237 mapped using linear peptides . These sites include binding sites on tenascin C ( AEIDGIEL ; SEQ ID NO : 237) , osteo polydom / SVEP1 EDDMMEVPY 239 pontin (SVVYGLR ; SEQ ID NO : 238 ) , polydom /SVEP1 VEGF - A EYP ( EDDMMEVPY; SEQ ID NO : 239 ) and VEGF - A (EYP ) . 30 - Unlike aaß , and as?, aoß , does not require a canonical RGD sequence motif . Some, but not all reported targets have In some embodiments , chimeric proteins of the present an acidic residue /hydrophobic residue /proline motif . Some invention may comprise one or more TGF -B2 trigger loops . also comprise a tyrosine residue . Such chimeric proteins may exhibit activation ( e . g . growth The trigger loop of TGF- B1 and TGF -13 carries an RGD 35 factor release ) regulated in a manner similar to that of sequence where a , ßo and / or aß , bind to enable growth TGF -B2 . Some chimeric proteins of the present invention factor release. The TGF- B2 trigger loop region is different may comprise TGF - B - related proteins wherein one or more from those of TGF - B1 and TGF -B3 , comprising the protein modules are substituted with one or more protein sequence FAGIDGTSTYTSGDQKTIKSTRKKNSGKTP modules comprising one or more TGF - B2 trigger loops . ( SEQ ID NO : 65) , without an RGD trimer . Of this region , 40 Some chimeric proteins comprise TGF -B -related proteins residues AGIDGTST ( SEQ ID NO : 240 ) align with the wherein one or more protein modules comprising at least peptide on the third FnIII domain of tenascin - C that has been one RGD sequence are substituted with one or more protein mapped as an agß , binding site . Also , the tyrosine following modules comprising one or more TGF -B2 trigger loops. In this region may play a role in potential aoß? binding . other embodiments , chimeric proteins may comprise TGF Therefore, aoß , binding to TGF- B2 could be physiologically 45 B1 and / or TGF- B3 proteins wherein one or more protein relevant. In some embodiments, chimeric proteins of the modules comprising at least one RGD sequence are substi present invention may comprise trigger loop sequences tuted with one or more protein modules comprising one or comprising any of the sequences listed in Table 15 . more TGF -B2 trigger loops. Such chimeric proteins may exhibit TGF -B1 activity . TABLE 15 50 In some embodiments, chimeric proteins of the present invention may comprise one or more protein modules from Trigger loop sequences BMPs . Protein modules comprising sequences from BMPs SEQ may comprise sequences from any of those BMP modules ID disclosed in FIGS. 8A - 8G . Chimeric proteins of the present Source protein Trigger loop sequence NO 55 invention comprising one or more BMP protein module may be useful for the development of antibodies and / or assays to TGF - B2 FAGIDGTSTYTSGDOKTIKSTRKKNSGKTP 65 study , enhance and/ or perturb BMP interactions with other TGF - B2 AGIDGTST 240 proteins, including, but not limited to RGM proteins. Chimeric proteins may comprise detectable labels . TGF - B2 ( short ) GTSTYTSGDQKTIKSTRKK 180 60 Detectable labels may be used to allow for detection and / or 241 isolation of chimeric proteins . Such detectable labels may TGF - B1 INGFTTGRRGDLATIHGMNRP comprise biotin labels , polyhistidine tags and / or flag tags . TGF - B1 SGRRGDLATI 242 Tags may be used to identify and / or isolate tagged proteins . Proteins produced may comprise additional amino acids TGF - B1 TGRRGDLATI 243 65 encoding one or more 3C protease cleavage site . Such sites TGF - B3 FKGVDNEDDHGRGDLGRLKKOKDHHNP 244 allow for cleavage at the 3C protease cleavage site upon treatment with 3C protease, including , but not limited to US 9 ,758 ,576 B2 93 94 rhinovirus 3C protease . 3C protease cleavage sites may be TGF -B - related proteins described herein . In some cases, Fc introduced to allow for removal of detectable labels from fusion proteins may comprise TGF- B , GDF - 8 and / or GDF chimeric proteins. 11 . Protein Expression Sequences encoding recombinant proteins of the present In some embodiments , synthesis of recombinant proteins 5 invention may be inserted into any number of DNA vectors of the present invention may be carried out according to any known in the art for expression . Such vectors may include method known in the art . Some protein synthesis may be plasmids. In some embodiments , sequences encoding recombinant proteins of the present invention are cloned into carried out in vitro . Some protein synthesis may be carried PTT5 vectors (NRC Biotechnology Research Institute , Mon out using cells . Such cells may be bacterial and / or eukary 10 tréal, Québec . ) In other embodiments pTT22 , PTT28 , PYD5 , otic . In some embodiments , eukaryotic cells may be used for pYD7, pYD11 (NRC Biotechnology Institute , Montréal, protein synthesis . Some such cells may be mammalian . Québec ) and /or PMA vectors (Life Technologies, Carlsbad , Some mammalian cells used for protein expression may Calif . ) may be used . Vectors may comprise promoter include , but are not limited to mouse cells , rabbit cells , rat sequences to modulate expression of sequences encoding cells , monkey cells , hamster cells and human cells. Such 15 recombinant proteins of the present invention . Such promot cells may be derived from a cell line . In other embodiments , ers may be constitutively active and / or may be regulated by human cells may be used . In further embodiments , cell lines extrinsic and/ or intrinsic factors . Some extrinsic factors may may include , but are not limited to HEK293 cells , CHO be used to enhance or suppress expression of sequences cells , HeLa cells , Sw - 480 cells , EL4 T lymphoma cells , encoding recombinant proteins of the present invention . TMLC cells, 293T / 17 cells , Hs68 cells , CCD1112sk cells , 20 Some vectors may encode nuclear localization signals that HFF - 1 cellscells , KeloidKeloid fibroblastsfibroblasts , A204 cellscells , L17 RIB cells may be incorporated into recombinant proteins of the pres and C2C12 cells . ent invention upon translation . Some vectors may produce In some embodiments , 293 cells are used for synthesis of mRNA transcripts that comprise nuclear export signals . recombinant proteins of the present invention . These cells RNA transcribed from a modified pTT5 vector (PTT5 are human cells that post- translationally modify proteins 25 WPRE ) contains an element that facilitates nuclear export of with human - like structures ( e . g . glycans ) . Such cells are the transcripts. Some vectors may be modified by insertion easily transfectable and scalable and are able to grow to high of one or more ligation - independent cloning ( LIC ) cassettes to provide for simpler cloning. densities in suspension culture . 293 cells may include 293E Vectors encoding recombinant proteins of the present cells . 293E cells are HEK293 cells stably expressing 30 invention may be delivered to cells according to any method EBNA1 (Epstein - Barr virus nuclear antigen - 1 ) . In some known in the art , including , but not limited to transfection , cases, 293E cells may be grown in serum - free medium to electroporation and /or transduction . In some embodiments , simplify down -stream purification . In some cases, 293 -6E vectors may comprise one or more elements to enhance cells (NRC Canada, Ottawa, Calif. ) may be used . Such cells vector replication in host cells . In some embodiments , express truncated EBNA1 (EBNAlt ) and may comprise 35 vectors may comprise oriP sites for episomal replication in enhanced production of recombinant proteins and may be cells that express EBNA - 1 . optimized for growth and / or protein expression in serum In some cases , cells are stably transfected to produce free medium to simplify down - stream purification . In some recombinant proteins of the present invention . Stably trans cases, insect cells may be used to express recombinant fected cells pass transfected genes to daughter cells during proteins of the invention . In some cases, insect cell expres- 40 cell division , thus eliminating the need for repeated trans sion may be carried out using Spodoptera frugiperda cells fection . In some cases , the transfected genes are stably including, but not limited to Sf9 and / or Sf -21 cells . In some inserted into the genome of the transfected cells . Transfected cases , insect cell cultures may comprise Trichoplusia ni genes may comprise genes for cell selection , such as genes cells , including, but not limited to Tn - 368 and /or HIGH that confer resistance to one or more toxic or repressive FIVETM BTI- TN -5B1 - 4 cells. A further list of exemplary 45 compounds . Such genes may be used to support the growth insect cell lines can be found in U . S . Pat . No . 5 , 024 , 947 , the of only cells with stable incorporation of the transfected contents of which are herein incorporated by reference in genes when grown in the presence of such one or more toxic their entirety. or repressive compounds ( e . g . puromycin , kenomycin , etc . ) In some embodiments , recombinant proteins of the inven - Cell selection may also comprise selecting cells based on tion may comprise an antibody Fc domain to create an Fc 50 overall recombinant protein expression levels . Determina fusion protein . The formation of an Fc fusion protein with tion of such levels may be carried out, for example , by any of the recombinant proteins described herein may be Western Blot and / or ELISA . carried out according to any method known in the art , In some embodiments , nucleotide sequences encoding including as described in Czajkowsky, D . M . et al ., 2012 . recombinant proteins of the present invention may comprise EMBO MolMed . 4 ( 10 ) : 1015 - 28 and U . S . Pat. Nos. 5 , 116 , 55 one or more woodchuck hepatitis virus posttranscriptional 964, 5 ,541 , 087 and 8 ,637 ,637 , the contents of each of which regulatory element (WPRE ) . RNA nucleic acids comprising are herein incorporated by reference in their entirety . Fc such elements may comprise the sequence GCCACGGCG fusion proteins of the invention may be linked to the hinge GAACUCAUCGCCGCCUGCCUUGCCCGCUGCUG region of an IgG Fc via cysteine residues in the Fc hinge GACAGGGGCUCGGC UGUUGGGCACUGACAAUUC region . Resulting Fc fusion proteins may comprise an anti - 60 CGUGGU (SEQ ID NO : 255 ). RNA comprising WPRES body - like structure , but without Cy? domains or light chains . may be transcribed from DNA comprising the sequence In some cases , Fc fusion proteins may comprise pharma AATCAACCTCTGGATTACAAAATTTGTGAAAGATT 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 65 GCTATTGCTT CCCGTATGGCTTTCATTTTCTCCT activity . In some cases, Fc fusion proteins of the invention CCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAG may be prepared using any of the TGF - B family proteins or GAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTG US 9 , 758 ,576 B2 95 96 GTGTGCACTGTGTTTGCTGACGCA ACCCCCACTG - Some proteins produced may comprise additional amino GTTGGGGCATTGCCACCACCTGTCAGCTCCTTTC acids encoding one or more detectable labels for purification CGGGACTTTCGCTT TCCCCCTCCCTATTGCCACG [ e . g . polyhistidine tag, flag tag , etc . ] In some embodiments , GCGGAACTCATCGCCGCCTGCCTTGCCCGCT proteins are N - terminally labeled . In some embodiments , GCTGGA CAGGGGCTCGGCTGTTGGGCACT- 5 proteins are C - terminally labeled . In some embodiments , GACAATTCCGTGGTGTTGTCGGGGAAGCTGACGT proteins are biotinylated . In some embodiments , recombi CCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTG - nant proteins of the present invention are N -terminally GATTCTGCGCGGGACGTCCTTCTG CTACGTCCCT- biotinylated . TCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCG Proteins produced may comprise additional amino acids GCCTGCTGCCGGCT CTGCGGCCTCTTCCGCGTC - " encoding one or more 3C protease cleavage site . Such sites TTCGCCTTCGCCCTCAGACGAGTCGGATCTC allow for cleavage between residues Q and G of the 3C CCTTTGGG CCGCCTCCCCGCCTG (SEQ ID NO : 256 ) . protease cleavage site upon treatment with 3C protease , WPREs may enhance translation of nucleic acids compris including, but not limited to rhinovirus 3C protease . In some ing WPREs. Such enhanced translation may be due to 15 embodiments , such cleavage sites are introduced to allow increased cytoplasmic export of newly transcribed mRNA . for removal of detectable labels from recombinant proteins . In some embodiments , recombinant proteins may com In some embodiments , modification of expressed growth prise one or more secretion signal sequences . As used factor proproteins may be carried out by enzymatic cleav herein , the term “ secretion signal sequence ” refers to a chain age . In some cases, proprotein convertases may be used . of amino acids ( or nucleotides that encode them at the 20 Such proprotein convertases may include , but are not limited nucleic acid level ) that when part of a protein , modulate to furin /PACE3 , PC1/ 3 , PC2, PC4 , PC5 /6 , PACE4 and PC7. secretion of such proteins from cells . Some secretion signal Proprotein convertase cleavage may be caried out in solution sequences may be located at protein termini. In other or in tissue culture . In some cases, proprotein convertases embodiments , secretion signal sequences may be N - terminal are expressed in cells expressing proproteins to be cleaved . amino acid sequences. Other secretions signal sequences 25 In some cases, proprotein convertases are added to tissue may comprise the secretion signal of the Ig kappa chains. cultures of cells expressing proproteins to be cleaved . Such Ig kappa chains may be human Ig kappa chains. In Antibodies some embodiments , secretion signal sequences may com In some embodiments , compounds and / or compositions prise the amino acid sequence MDMRVPAQLLGLLLLWF of the present invention may comprise antibodies or frag SGVLG ( SEQ ID NO : 257 ) . 30 In some embodiments , recombinant proteins of the pres ments thereof. As used herein , the term “ antibody” is ent invention may require coexpression with one or more referred to in the broadest sense and specifically covers other proteins for proper expression , folding, secretion , various embodiments including , but not limited to mono activity and /or function . Some recombinant GPCs of the clonal antibodies , polyclonal antibodies , multispecific anti present invention may be coexpressed with LTBPs, fibrillinsna 35 bodies ( e . g . bispecific antibodies formed from at least two and / or GARP. intact antibodies ) , and antibody fragments such as diabodies In some embodiments , recombinant proteins of the pres so long as they exhibit a desired biological activity . Anti ent invention may be biotinylated . As used herein , the term bodies are primarily amino - acid based molecules but may “ biotinylating ” refers to the attaching of one or more biotin also comprise one or more modifications ( including , but not labels . Such biotin labels may facilitate interactions of 40 limited to the addition of sugar moieties , fluorescent moi biotinylated recombinant proteins with avidin and /or eties, chemical tags, etc . ) streptavidin coated surfaces and /or proteins. As used herein , Recombinant and Chimeric Protein Use in Antibody Gen a “ biotin label” refers to a detectable label comprising one eration or more biotin molecules. The term “ biotinylated ” refers to In some embodiments , recombinant and / or chimeric pro a molecule or protein that comprises one or more biotin 45 teins described herein may be used as antigens ( referred to labels. Biotin molecules bind with high affinity to avidin and herein as antigenic proteins ) to generate antibodies . Such streptavidin molecules. This property may be used to capture antigenic proteins may comprise epitopes that may be less biotinylated proteins using avidin and / or stretavidin coated accessible for antibody generation in similar wild type materials . Some recombinant GPCs of the present invention proteins. Some antibodies directed to antigenic proteins of may be biotinylated near the N - terminus. Such recombinant 50 the present invention may modulate the release of one or GPCs may be introduced to avidin / streptavidin coated cell more growth factors from one or more GPCs . ) Some such culture surfaces , allowing biotinylated recombinant GPCs to adhere to the surface in a manner such that the orientation antibodies may be stabilizing [ reducing or preventing dis and bonding of such bound GPCs mimics the orientation and sociation between two agents, ( e . g . growth - factor release bonding of GPCs to LTBPs, fibrillins and /or GARPs. 55 from GPCs, GPC release from one or more protein interac In some embodiments, recombinant proteins produced tions and / or releasing ?enhancing the dissociation between may be analyzed for quality control purposes to assess both two agents (e . g . growth -factor release from GPCs , GPC biophysical properties as well as bioactive properties . Bio release from one or more protein interactions )] antibodies . physical characterization may include assessing protein Antigenic proteins of the present invention may comprise migration patterns after reducing and /or non - reducing SDS 60 TGF -b -related proteins as well as components and /or pro PAGE . Biophysical characterization may also comprise gel tein modules thereof. In some cases , antigenic proteins of filtration , mass spectrometric analysis and /or analysis of the present invention may comprise prodomains without association / dissociation between LAPs or LAP - like associated growth factors , furin cleavage- deficient mutants , domains and growth factor domains. Bioactive properties mutants deficient in extracellular protein associations and /or may be analyzed by assessing reactivity with antibodies 65 combinations thereof. and /or signaling activity of dissociated growth factors and /or In some embodiments , antigenic proteins may comprise latent GPCs. TGF- B -related proteins and /or modules thereof. Such anti US 9 ,758 ,576 B2 97 98 genic proteins may comprise epitopes from regions where ments, recombinantGARPs are modified to comprise one or growth factors associate with or comprise stereological more detectable labels . In further embodiments , such detect proximity with prodomain regions. Antibodies of the present able labels may include , but are not limited to biotin labels , invention directed to such epitopes may bind overlapping polyhistidine tags, flag tags , myc tags, HA tags and /or regions between growth factors and prodomains . Such anti- 5 fluorescent tags . In some embodiments , antigenic proteins bodies may stereologically inhibit the dissociation of growth may comprise one or more recombinant protein and / or factors from GPCs. chimeric protein complexed with one or more recombinant In some embodiments , antigenic proteins comprise only GARP . In some embodiments , antigenic proteins comprise the prodomain or only the growth factor from a particular LAPs ( e . g . TGF - B LAPs ) and /or LAP -like domains com GPC . Epitopes present on such antigenic proteins may be 10 plexed with recombinant GARP . In some embodiments , shielded or unexposed in intact GPCs. Some antibodies of antigenic proteins comprise D2G mutants ( e . g . TGF - B D2G the present invention may be directed to such epitopes. Such mutants ) complexed with recombinant GARP. In some antibodies may be releasing antibodies , promoting growth embodiments , complexed recombinant GARPs may be factor dissociation from GPCs. Further antibodies may soluble forms of GARP ( SGARP ). In some embodiments , compete with free growth factor for prodomain binding, 15 SGARPs comprises one or more biotin labels , polyhistidine thereby promoting growth factor dissociation from GPCs . tags and / or flag tags . In some embodiments, antigenic proteins may comprise In some embodiments , GARPs complexed with LAP proprotein convertase ( e . g . furin ) cleavage site mutations. and /or LAP - like domains are desired as antigens , in assays Such mutations may prevent enzymatic cleavage of growth and / or for antibody development. In such embodiments , factors from their prodomains. Some antibodies of the 20 LAPs and/ or LAP - like domains may comprise CED muta present invention may be directed to epitopes present on tions. Such LAPs and / or LAP - like domains may be such mutant proteins. Such antibodies may stabilize the expressed as GPCs to facilitate proper protein folding, association between prodomains and growth factors . In conformation and / or expression , but the CED mutations some embodiments , furin cleavage site mutants comprise present may enhance growth factor release , leaving the D2G mutants as described herein . 25 desired GARP -LAP (or LAP - like domain ) complex behind . In some embodiments , antigenic proteins comprising pro GARP - LAP ( or LAP - like domain ) complexes may be useful domains may comprise N -terminal mutations that lead to as antigens in the production of releasing antibodies that decreased prodomain association with LTBPs and/ or GARP specifically target GARP -associated GPCs. and therefore may present epitopes in the N -terminal region In some embodiments , GPCs comprising CED mutations that may otherwise be shielded by those associations . Some 30 may act to stabilize a natively populated conformation of antibodies of the present invention may be directed to such LAP (or LAP - like domain ) characterized by reduced growth epitopes . Some antigenic proteins comprising TGF- B1 pro - factor association ( both as a free LAP or LAP - like domains domains may comprise C4S mutations. Such mutations may and /or as a GARP and /or LTBP /LAP complex ), thereby prevent association of antigenic proteins with LTBPs and /or exposing epitopes that may be less exposed in wild -type GARP , making these proteins useful for presenting N - ter - 35 proteins . Such mutations may shift the conformational equi minal epitopes . Antibodies directed to C4S mutants may librium of LAP or LAP - like domains to facilitate the pro prevent GPC association with LTBPs and / or GARP. Some duction of activating antibodies . antibodies directed to C4S mutants may reduce growth In some embodiments , antigenic proteins of the present factor signaling in a particular niche . Some such antibodies invention may comprise one or more protein modules from may reduce or prevent the release of growth factor by 40 GDFs ( e . g . GDF - 11 and /or GDF - 8 ) . In some embodiments , blocking the ability of the GPCs to associate securely with antibodies of the present invention may be directed toward the extracellular matrix . antigenic proteins comprising GDF - 8 protein modules. In In some embodiments , antigenic proteins may comprise some embodiments, such antibodies may modulate GDF - 8 one or more recombinant LTBP . Such recombinant LTBPs levels and / or activity in one or more niches. In some may comprise LTBP1, LTBP2 , LTBP3 , LTBP4 , alternatively 45 embodiments , antibodies of the present invention may pre spliced variants and / or fragments thereof. Recombinant vent the release of GDF - 8 growth factors from GPCs. In LTBPs may also be modified to comprise one or more some embodiments , antibodies of the present invention may detectable labels . Such detectable labels may include, but be used to repair and /or enhance muscle tissues . are not limited to biotin labels, polyhistidine tags, myc tags , In some embodiments , recombinant proteins ( including , HA tags and / or fluorescent tags . 50 but not limited to chimeric proteins ) described herein may In some embodiments , antigenic proteins may comprise be used in studies to identify and map epitopes that may be one or more recombinant protein and / or chimeric protein important targets for antibody development. Such studies complexed with one or more recombinant LTBP . Some may be used to identify epitopes that may promote growth antigenic proteins may comprise proprotein convertase factor release or stabilization of GPCs upon antibody bind cleavage site mutants ( e . g . D2G mutants , AXXA mutants ) 55 ing . complexed with one or more recombinant LTBP. Some such Releasing Antibodies recombinant LTBPs may comprise LTBP1 S . Some recom As used herein , the term " releasing antibody ” refers to an binant LTBPs may comprise one or more detectable labels , antibody that increases the ratio of active and / or free growth including , but not limited to biotin labels , polyhistidine tags factor relative to inactive and/ or prodomain -associated and / or flag tags. 60 growth factor upon the introduction of the antibody to a In some embodiments , antigenic proteins may comprise GPC , cell , niche , natural depot or any other site of growth GARP ( or homologues thereof, including , but not limited to factor sequestration . In this context, releasing antibodies 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 65 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- natural depot for one or more growth factors . US 9 , 758 ,576 B2 99 100 The contact necessary for growth - factor release may be context of cellular events , as used herein , the term " down defined as direct or indirect contact of antibody with a GPC stream ” refers to any signaling or cellular event that happens or a component thereof or with a cellular structure such as after the action , binding or targeting by compounds and / or an extracellular and/ or cellular matrix protein and /or protein compositions of the present invention . associated with the extracellular and /or cellular matrix ?e . g . 5 Contact necessary for inhibition or stabilization may be LTBPs ( e . g . LTBP1, LTBP2 , LTBP3 and/ or LTBP4 ) , direct or indirect contact between antibody and GPC or fibrillins ( e . g . fibrillin - 1 , fibrillin - 2 , fibrillin - 3 and /or fibril - components thereof or with cellular structures such as an lin - 4 , ) perlecan , decorin , elastin , collagen and / or GARPs extracellular and / or cellular matrix protein and / or protein ( e . g . GARP and / or LRRC33 ) ] for release of growth factor. associated with the extracellular and /or cellular matrix ( e . g . Release of at least 5 % , 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , 10 LTBPs ( e . g . LTBP1 , LTBP2 , LTBP3 and/ or LTBP4 ) , 70 % , 80 % , 90 % or more of growth factor is sufficient to fibrillins ( e . g . fibrillin - 1 , fibrillin - 2 , fibrillin - 3 and /or fibril characterize antibodies of the present invention as releasing lin - 4 ,) perlecan , decorin , elastin , collagen and/ or GARPs antibodies . It is understood that growth factor release after (e . g . GARP and /or LRRC33 ) ] whereby release of growth antibody administration may be local and may occur over a factor is inhibited Inhibition of release of at least 5 % , 10 % , sustained period of time and may include peaks or spikes of 15 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % or more of release . Antibodies of the present invention may act to growth factors may be sufficient, in some cases, to charac release one or more growth factor over minutes, hours , days terize antibodies of the present invention as inhibitory or or longer . stabilizing . Inhibitory antibodies may stabilize GPCs and Release profiles may have an initial peak or burst within trap them as heterodimers . from about 4 hours to about 7 days of contacting in vivo or 20 It is understood that inhibition of growth factor release shorter periods in vitro . For example , initial peak or burst after contact with one or more antibodies of the present may occur from about 4 hours to about 5 hours , or from invention may be local and may occur over a sustained about 4 hours to about 6 hours , or from about 4 hours to period of time and may include peaks , troughs or spikes . about 7 hours , or from about 4 hours to about 8 hours , or Inhibitory antibodies which may also function to stabilize from about 4 hours to about 9 hours , or from about 4 hours 25 GPCs may be defined by their release kinetics . Release of to about 10 hours , or from about 4 hours to about 11 hours, growth factor and corresponding release kinetics, even or from about 4 hours to about 12 hours, or from about 4 locally, may be directly measured or inferred by downstream hours to about 24 hours , or from about 4 hours to about 36 signaling events . In some embodiments , changes in protein hours, or from about 4 hours to about 48 hours, or fromim or nucleic acid concentrations or phenotypic responses may about 1 day to about 7 days , or from about 1 day to about 30 be indicative of the effects of compounds and / or composi 2 days , or from about 1 day to about 3 days , or from about tions of the present invention . 1 day to about 4 days , or from about 4 days to about 5 days , Antibodies of the present invention may act to inhibit or from about 4 days to about 6 days , or from about 4 days release of a growth factor over minutes , hours or days to about 7 days. Compounds and / or compositions of the Inhibition and / or stabilization profiles may have an initial present invention may stimulate the release of 5 to 100 % of 35 trough within from about 4 hours to about 7 days of the growth factor present . For example , the percent of introduction in vivo or shorter periods in vitro . For example , growth factor release may be from about 5 % to about 10 % , initial trough of inhibition or stabilization may occur from or from about 5 % to about 15 % , or from about 5 % to about about 4 hours to about 5 hours , or from about 4 hours to 20 % , or from about 5 % to about 25 % , or from about 10 % to about 6 hours , or from about 4 hours to about 7 hours, or about 30 % , or from about 10 % to about 40 % , or from about 40 from about 4 hours to about 8 hours , or from about 4 hours 10 % to about 50 % , or from about 10 % to about 60 % , or from to about 9 hours , or from about 4 hours to about 10 hours , about 20 % to about 70 % , or from about 20 % to about 80 % , or from about 4 hours to about 11 hours , or from about 4 or from about 40 % to about 90 % , or from about 40 % to hours to about 12 hours , or from about 4 hours to about 24 about 100 % . hours , or from about 4 hours to about 36 hours , or from Releasing antibodies generated according to methods 45 about 4 hours to about 48 hours, or from about 1 day to about described herein may be generated to release growth factors 7 days , or from about 1 day to about 2 days, or from about from GPCs comprising any of the pro -proteins listed in 1 day to about 3 days , or from about 1 day to about 4 days, Table 1 . In some cases , releasing antibodies are directed to or from about 4 days to about 5 days , or from about 4 days GPCs comprising TGF - B isoforms and/ or one or more to about 6 days , or from about 4 days to about 7 days . modules of such isoforms. In some cases , releasing antibod - 50 Introduction of compounds and / or compositions of the pres ies are directed to GPCs comprising GDFs and / or one or e nt invention may lead to inhibition and / or stabilization of more modules from GDFs. 5 % to 100 % of growth factor present. For example , the Stabilizing Antibodies percent of growth factor inhibition or stabilization may be As used herein , the term " stabilizing antibody ” refers to from about 5 % to about 10 % , from about 5 % to about 15 % , an antibody that decreases the ratio of active and /or free 55 from about 5 % to about 20 % , from about 5 % to about 25 % , growth factor relative to inactive and /or prodomain -associ from about 10 % to about 30 % , from about 10 % to about ated growth factor upon the introduction of the antibody to 40 % , from about 10 % to about 50 % , from about 10 % to one or more GPC , cell, niche , natural depot and / or any other about 60 % , from about 20 % to about 70 % , from about 20 % site of growth factor sequestration . In this context, antibod to about 80 % , from about 40 % to about 90 % or from about ies may be characterized as antagonists . As used herein , an 60 40 % to about 100 % . " antagonist” is one which interferes with or inhibits the Stabilizing antibodies generated according to methods physiological action of another. Antagonist action may even described herein may be generated to block the release of 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 65 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 cleavage sites. In some cases, stabilizing antibodies are US 9 ,758 , 576 B2 101 102 directed to GPCs comprising TGF - B isoforms and /or one or In some embodiments , antibodies of the present invention more modules of such isoforms. In some cases , stabilizing may be selected from a larger pool of two or more candidate antibodies are directed to GPCs comprising GDFs and / or antibodies based on their ability to modulate growth factor one or more modules from GDFs. levels and /or activity . In some cases, growth factor activity Stabilizing antibodies directed to GPCs comprising 5 assaysmay be used to test the ability of candidate antibodies GDF - 8 may block metalloproteinase cleavage of such com to modulate growth factor activity . Growth factor activity plexes . Such agentsmay bind to GPCs comprising GDF -8 in assays may include, cell- based assays as described herein such a way as to physically prevent interactions between below . Additional assays that may be used to determine the such GPCs and metalloproteinases targeting such GPCs . effect of candidate antibodies on growth factor activity may 10 include, but are not limited to enzyme- linked immunosor Agents that actually target metalloproteinases themselves bent assay (ELISA ) , Western blotting , reporter assays (e . g . have been described previously (see U . S . Pat . No . 7 ,572 , luciferase -based reporter assays or other enzyme -based 599 , the contents of which are herein incorporated by reporter assays ) , PCR analysis , RT- PCR analysis and /or reference in their entirety . ) other methods known in the art including any of the methods Antibody Selection 15 described in U . S . Provisional Patent Applications 61 / 722 , A desired antibody may be selected from a larger pool of 919 , filed Nov. 6 , 2012 and 61 /722 , 969 , filed Nov . 6 , 2012 , two or more candidate antibodies based on the desired the contents of each of which are herein incorporated by antibody ' s ability to associate with desired antigens and /or reference in their entireties . epitopes . Such antigens and /or epitopes may include , but are In some embodiments , one or more recombinant proteins not limited to any of those described herein , including , but 20 or antibodies disclosed herein may be used in assays to test, not limited to recombinant proteins, chimeric proteins, develop and /or select antibodies. Recombinant GPCs may GPCs, prodomains ( e . g . LAPs or LAP - like domains ) , be expressed to test releasing and / or stabilizing abilities of growth factors, protein modules, LTBPs, fibrillins , GARP, one or more antibodies being assayed . In some embodi TGF - B - related proteins and / or mutants and / or variants and ments , recombinant proteins may be expressed as positive or or complexes and / or combinations thereof. Selection of 25 negative control components of assays . In some embodi desired antibodies may be carried out using an antibody ments , multiple recombinant proteins may be expressed at binding assay , such as a surface Plasmon resonance - based once to modulate growth factor release and / or activity , assay , an enzyme- linked immunosorbent assay (ELISA ) or wherein such recombinant proteins may act synergistically fluorescence -associated cell sorting ( FACS ) -based assay. or antagonistically in such modulation . Such assays may utilize a desired antigen to bind a desired 30 In some embodiments GPCs comprising CED mutations antibody and then use one or more detection methods to may provide a baseline level of growth factor activity in detect binding . assays designed to test releasing antibodies, as these mutant In some embodiments , antibodies of the present invention proteins are sufficient for producing a biological effect in may be selected from a larger pool of two or more candidate humans. In some embodiments , GPCs comprising CED antibodies based on their ability to associate with desired 35 mutations may be used as positive controls in activity assays antigens and / or epitopes from multiple species (referred to geared toward screening for releasing antibodies. In some herein as “ positive selection .” ) embodiments , GPCs comprising CED mutations may be In some embodiments , such species may comprise verte - used for screening for stabilizing antibody activity, as they brate species. In some embodiments , such species may can be presumably activated in the absence of integrins . In comprise mammalian species . In some embodiments , such 40 such assays, GPCs comprising CED mutations may be species may include , but are not limited to mice , rats , expressed in cell lines ( e . g . 293 cells or others ) and growth rabbits , goats , sheep , pigs, horses, cows and / or humans . factor activity and /or release may be assessed in the pres In some embodiments , negative selection is used to ence or absence of antibodies being tested . In some embodi remove antibodies from a larger pool of two or more ments , co - expression of GPCs comprising CED mutation candidate antibodies . As used herein the term " negative 45 with wild type GPCs ( including, but not limited to TGF - B1, selection ” refers to the elimination of one or more factors TGF -B2 , or TGF - B3 ) could also be used to regulate free from a group based on their ability to bind to one or more growth factor levels . In such embodiments , modulation of undesired antigens and / or epitopes . In some embodiments , free growth factor levels may accomplished by co - transfec undesired antigens and / or epitopes may include, but are not tion of different ratios of wild type and mutant GPCs ( e . g . limited to any of those described herein , including , but not 50 1 : 1 , 1 : 2 , 1 : 3 , 1 : 4 , 1 : 5 , 1 : 10 ) . In some embodiments , further limited to recombinant proteins, chimeric proteins, GPCs , co - expression of LTBPs, fibrillins or GARPs may be carried prodomains ( e . g . LAPs or LAP - like domains ) , growth fac out to add one or more additional levels of free growth factor tors , protein modules , LTBPs , fibrillins, GARPs, TGF - B - modulation . related proteins and / or mutants and /or variants and / or com - Antibody Development binations and /or complexes thereof. 55 In some embodiments , compounds and / or compositions In some embodiments , antibodies of the present invention of the present invention comprising antibodies , antibody may be directed to prodomains ( e . g . the prodomain portion fragments , their variants or derivatives as described above of a GPC and / or free LAP or LAP - like domains ) that are specifically immunoreactive with antigenic proteins as decrease growth factor signaling and / or levels ( e . g . TGF- B described herein . growth factor signaling and /or levels ) in a given niche. In 60 Antibodies of the present invention may be characterized some embodiments , antibodies of the present invention may by their target molecule ( s ) , by the antigens used to generate directed to LAPs or LAP - like domains that increase growth them , by their function (whether as agonists, antagonists , factor signaling and / or levels in a given niche . In some growth - factor releasing , GPC stabilizing , activating and /or embodiments , antibodies of the present invention may be inhibitory ) and /or by the cell niche in which they function . directed to prodomains ( e . g . LAPs or LAP - like domains ) 65 As used herein the term , " antibody fragment” refers to and /or GPCs only when complexed with LTBPs, fibrillins any portion of an intact antibody. In some embodiments , and / or GARP. antibody fragments comprise antigen binding regions from US 9 , 758 ,576 B2 103 104 intact antibodies . Examples of antibody fragments may chain . By using a linker that is too short to allow pairing include , but are not limited to Fab , Fab ', F ( ab ') , and Fv between the two domains on the same chain , the domains are fragments ; diabodies; linear antibodies ; single -chain anti - forced to pair with the complementary domains of another body molecules ; and multispecific antibodies formed from chain and create two antigen -binding sites. Diabodies are antibody fragments . Papain digestion of antibodies produces 5 described more fully in , for example , EP 404 ,097 ; WO two identical antigen -binding fragments , called “ Fab ” frag - 93 / 11161 ; and Hollinger et al. (Hollinger , P . et al. , “ Diabod ments, each with a single antigen -binding site . Also pro - ies ” : Small bivalent and bispecific antibody fragments . duced is a residual “ Fc” fragment, whose name reflects its PNAS . 1993 . 90 :6444 - 8 ) the contents of each of which are ability to crystallize readily . Pepsin treatment yields an incorporated herein by reference in their entirety . F ( ab ') 2 fragment that has two antigen - binding sites and is 10 As used herein , the term “ monoclonal antibody ” refers to still capable of cross - linking antigen . Compounds and / or an antibody obtained from a population of substantially compositions of the present invention may comprise one or homogeneous cells (or clones ) , i . e . , the individual antibodies more of these fragments . For the purposes herein , an “ anti- comprising the population are identical and / or bind the same body ” may comprise a heavy and light variable domain as epitope , except for possible variants that may arise during well as an Fc region . 15 production of the monoclonal antibodies , such variants As used herein , the term “ native antibody ” refers to a generally being present in minor amounts . In contrast to usually heterotetrameric glycoprotein of about 150 ,000 dal - polyclonal antibody preparations that typically include dif tons, composed of two identical light (L ) chains and two ferent antibodies directed against different determinants identical heavy ( H ) chains. Each light chain is linked to a (epitopes ) , each monoclonal antibody is directed against a heavy chain by one covalent disulfide bond , while the 20 single determinant on the antigen number of disulfide linkages varies among the heavy chains The modifier “ monoclonal ” indicates the character of the of different immunoglobulin isotypes . Each heavy and light antibody as being obtained from a substantially homoge chain also has regularly spaced intrachain disulfide bridges. neous population of antibodies, and is not to be construed as Each heavy chain has at one end a variable domain ( V ) requiring production of the antibody by any particular followed by a number of constant domains . Each light chain 25 method . The monoclonal antibodies herein include " chime has a variable domain at one end ( VL) and a constant domain ric ” antibodies ( immunoglobulins ) in which a portion of the at its other end ; the constant domain of the light chain is heavy and /or light chain is identical with or homologous to aligned with the first constant domain of the heavy chain , corresponding sequences in antibodies derived from a par and the light chain variable domain is aligned with the ticular species or belonging to a particular antibody class or variable domain of the heavy chain . 30 subclass , while the remainder of the chain (s ) is identical As used herein , the term “ variable domain ” refers to with or homologous to corresponding sequences in antibod specific antibody domains that differ extensively in sequence ies derived from another species or belonging to another among antibodies and are used in the binding and specificity antibody class or subclass , as well as fragments of such of each particular antibody for its particular antigen . antibodies . As used herein , the term “ Fv ” refers to antibody fragments 35 As used herein , the term “ humanized antibody ” refers to comprising complete antigen -recognition and antigen -bind - a chimeric antibody comprising a minimal portion from one ing sites . These regions consist of a dimer of one heavy or more non - human ( e . g . , murine ) antibody source with the chain and one light chain variable domain in tight, non - remainder derived from one or more human immunoglobu covalent association . lin sources. For the most part, humanized antibodies are As used herein , the term “ light chain ” refers to a com - 40 human immunoglobulins ( recipient antibody ) in which resi ponent of an antibody from any vertebrate species assigned dues from the hypervariable region from an antibody of the to one of two clearly distinct types , called kappa and lambda recipient are replaced by residues from the hypervariable based on amino acid sequences of constant domains . region from an antibody of a non -human species (donor Depending on the amino acid sequence of the constant antibody ) such as mouse , rat, rabbit or nonhuman primate domain of their heavy chains, antibodies can be assigned to 45 having the desired specificity , affinity , and / or capacity . different classes . There are five major classes of intact As used herein , the term “ hypervariable region ” refers to antibodies : IgA , IgD , IgE , IgG , and IgM , and several of regions within the antigen binding domain of an antibody these may be further divided into subclasses (isotypes ) , e . g ., comprising amino acid residues responsible for antigen IgG1, IgG2, IgG3 , IgG4, IgA , and IgA2 . As used herein , the binding . The amino acids present within the hypervariable term " Single - chain Fy” or “ scFv ” refers to a fusion protein 50 regions determine the structure of the complementarity of Vy and V , antibody domains , wherein these domains are determining region (CDR ) . As used herein , the term “ CDR ” linked together into a single polypeptide chain . In some refers to regions of antibodies comprising a structure that is embodiments , the Fv polypeptide linker enables the scFv to complimentary to its target antigen or epitope . form the desired structure for antigen binding. In some embodiments , compounds and /or compositions As used herein , the term “ bispecific antibody ” refers to an 55 of the present invention may be antibody mimetics . As used antibody capable of binding two different antigens . Such herein , the term “ antibody mimetic ” refers to any molecule antibodies typically comprise regions from at least two which mimics the function or effect of an antibody and different antibodies. Bispecific antibodies may include any which binds specifically and with high affinity to their of those described in Riethmuller, G . 2012 . Cancer Immu- molecular targets. In some embodiments , antibody mimetics nity . 12 : 12 - 18 , Marvin , J . S . et al. , 2005 . Acta Pharmaco - 60 may be monobodies , designed to incorporate the fibronectin logica Sinica . 26 ( 6 ) :649 - 58 and Schaefer, W . et al. , 2011 . type III domain (Fn3 ) as a protein scaffold ( U . S . Pat. No. PNAS . 108 ( 27 ) : 11187 - 92 , the contents of each of which 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 65 anticalins, avimers , Centyrins, DARPINSTM , Fynomers and comprise a heavy chain variable domain Vy connected to a 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 . US 9 ,758 ,576 B2 105 106 As used herein , the term “ antibody variant” refers to a may include CD56 + cells, CD3 , natural killer (NK ) cells , biomolecule resembling an antibody in structure and /or monocytes and neutrophils (Strohl , W . R . Therapeutic Anti function comprising some differences in their amino acid body Engineering . Woodhead Publishing , Philadelphia Pa . sequence, composition or structure as compared to a native 2012 . Ch . 8 , p 186 , the contents of which are herein antibody . 5 incorporated by reference in their entirety . ) The preparation of antibodies , whether monoclonal or In some cases , antibodies of the present invention may be polyclonal, is known in the art. Techniques for the produc - engineered to comprise a given isotype depending on tion of antibodies are well known in the art and described , whether or not ADCC or ADCP is desired upon antibody e . g . in Harlow and Lane “ Antibodies, A Laboratory binding . Such antibodies , for example , may be engineered Manual ” , Cold Spring Harbor Laboratory Press , 1988 ; Har - 10 according to any of the methods disclosed by Alderson , K . low and Lane “ Using Antibodies : A Laboratory Manual” L . et al. , J Biomed Biotechnol. 2011 . 2011 : 379123 . ) In the Cold Spring Harbor Laboratory Press, 1999 and “ Therapeu case of mouse antibodies, different isotypes of antibodies are tic Antibody Engineering : Current and Future Advances more effective at promoting ADCC . IgG2 , for example , is Driving the Strongest Growth Area in the Pharmaceutical more effective at inducing ADCC than is IgG2b . Some Industry ” Woodhead Publishing , 2012 . 15 antibodies of the present invention , comprising mouse Standard Monoclonal Antibody Generation IgG2b antibodies may be reengineered to comprise IgG2a In some embodiments , antibodies are generated in knock - antibodies. Such reengineered antibodies may be more out mice , lacking the gene that encodes for desired target effective at inducing ADCC upon binding cell- associated antigens. Such mice may not be tolerized to target antigens antigens. and therefore may be better suited for generating antibodies 20 In some embodiments , genes encoding variable regions of against such antigens that may cross react with human and antibodies developed according to methods of the present mouse forms of the antigen . For the production of mono - invention may be cloned into mammalian expression vectors clonal antibodies , host mice may be immunized with recom - encoding human Fc regions. Such Fc regions may comprise binant proteins to elicit lymphocytes that specifically bind Fc regions from human IgGlk . IgGlk Fc regions may such proteins. Resulting lymphocytes may be collected and 25 comprise amino acid mutations known to enhance Fc fused with immortalized cell lines. Resulting hybridoma receptor binding and antibody -dependent cell - mediated cells may be cultured in suitable culture medium with cytotoxicity ADCC . selection agents to support the growth of only fused cells. In some cases , antibodies may be engineered to reduce Desired hybridoma cell lines may be identified through ADCC . Antibodies that do not activate ADCC or that are binding specificity analysis of secreted antibodies for target 30 associated with reduced levels of ADCC may be desireable peptides and clones of such cells may be subcloned through for antibody embodiments of the present invention , in some limiting dilution procedures and grown by standard meth cases due to no or limited immune -mediated clearance , ods . Antibodies produced by subcloned hybridoma cells allowing longer half - lives in circulation . may be isolated and purified from culture medium by Antibody Fragment Display Library Screening Techniques stastandard immunoglobulin purification procedures 35 In some embodiments , antibodies of the present invention Recombinant Antibodies may be produced and /or optimized using high throughput Recombinant antibodies of the present invention may be methods of discovery . Such methods may include any of the generated according to any of the methods disclosed in U .S . display techniques ( e .g . display library screening tech Provisional Patent Applications 61/ 722 , 919 , filed Nov . 6 , niques ) disclosed in U . S . Provisional Patent Applications 2012 and 61 /722 , 969, filed Nov. 6 , 2012 , the contents of 40 61 /722 ,919 , filed Nov. 6 , 2012 and 61/ 722 , 969, filed Nov . 6 , each of which are herein incorporated by reference in their 2012 , the contents of each of which are herein incorporated entireties . In some embodiments , recombinant antibodies by reference in their entireties . In some embodiments , may be produced using hybridoma cells produced according synthetic antibodies may be designed , selected or optimized to methods described herein . Heavy and light chain variable by screening target antigens using display technologies ( e . g . region cDNA sequences of antibodies may be determined 45 phage display technologies . ) Phage display libraries may using standard biochemical techniques . Total RNA may be comprise millions to billions of phage particles , each extracted from antibody - producing hybridoma cells and expressing unique antibody fragments on their viral coats . In converted to cDNA by reverse transcriptase (RT ) poly - some cases, cDNA encoding each fragment may contain the merase chain reaction (PCR ) . PCR amplification may be same sequence with the exception of unique sequences carried out on resulting cDNA to amplify variable region 50 encoding variable loops of the complementarity determining genes. Such amplification may comprise the use of primers regions (CDRs ) . V , chains of CDRs may be expressed as a specific for amplification of heavy and light chain fusion protein , linked to viral coat proteins ( e . g . the N -ter sequences . Resulting PCR products may then be subcloned minus of the viral pIII coat protein . ) V , chains may be into plasmids for sequence analysis . Once sequenced , anti - expressed separately for assembly with Vh chains in the body coding sequences may be placed into expression 55 periplasm prior to complex incorporation into viral coats . vectors . For humanization , coding sequences for human For selection , target antigens may be incubated , in vitro , heavy and light chain constant domains may be used to with phage display library particles for precipitation of substitute for homologous murine sequences. The resulting positive binding partners. This process is referred to herein constructs may then be transfected into mammalian cells for as “ phage enrichment. ” In some cases , phage enrichment large scale translation . 60 comprises solid -phase phage enrichment. According to such 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 65 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 US 9 , 758 ,576 B2 107 108 such methods, target antigens may comprise detectable associated structures to either enhance or inhibit growth labels ( e . g . biotin labels) to facilitate retrieval from solution factor signaling. Such epitopes may include any and all and recovery of bound phage . possible sites for altering, enhancing or inhibiting GPC After selection , cDNA encoding CDRs of precipitated function . In some embodiments , such epitopes include, but library members may be sequenced from the bound phage . 5 are not limited to epitopes on or within growth factors , Such sequences may be directly incorporated into antibody regulatory elements , GPCs , GPC modulatory factors, sequences for recombinant antibody production , or mutated growth factor receiving cells or receptors, LAPs or LAP - like and utilized for further optimization through in vitro affinity domains , fastener regions , furin cleavage sites , arm regions , maturation . fingers regions , LTBP binding domains, fibrillin binding In some cases phage display screening may be used to 10 domains , glycoprotein A repetitions predominant (GARP ) generate broadly diverse panels of antibodies . Such diversity binding domains, latency lassos , alpha 1 regions, RGD may be measured by diversity of antibody sequences and / or sequences , bowtie regions, extracellular matrix and/ or cel diversity of epitopes targeted . lular matrix components and /or epitopes formed by com Affinity Maturation Techniques bining regions or portions of any of the foregoing . Affinity maturation techniques of the present invention 15 Compounds and /or compositions of the present invention may comprise any of those disclosed in U . S . Provisional exert their effects via binding ( reversibly or irreversibly ) to Patent Applications 61/ 722 , 919 , filed Nov. 6 , 2012 and one ormore epitopes and / or regions of antibody recognition . 61/ 722 , 969, filed Nov . 6 , 2012, the contents of each of which While not wishing to be bound by theory , such binding sites are herein incorporated by reference in their entireties. After for antibodies , are most often formed by proteins, protein antibody fragments capable of binding target antigens are 20 domains or regions . Binding sites may ; however, include identified ( e . g . through the use of phage display libraries as biomolecules such as sugars , lipids , nucleic acid molecules described above, ) high affinity mutants may be derived from or any other form of binding epitope. these through the process of affinity maturation . Affinity In some embodiments, antagonist antibodies of the pres maturation technology is used to identify sequences encod - ent invention may bind to TGF - B prodomains, stabilizing ing CDRs that have the highest affinity for target antigens . 25 and preventing integrin -mediated release , for example , by Using such technologies , select CDR sequences ( e . g . ones blocking the RGD site or by stabilizing the structure . Such that have been isolated or produced according to processes antibodies would be useful in the treatment of Camurati described herein ) may be mutated randomly as a whole or at Engelmann disease , in which mutations in the prodomain specific residues to create millions to billions of variants . cause excessive TGF - B activation . Such antibodies would Such variants may be subjected to repeated rounds of affinity 30 also be useful in Marfan ' s syndrome, in which mutations in screening ( e . g . display library screening ) for their ability to fibrillins or LTBPs alter TGF - B and BMP activation . bind target antigens . Such repeated rounds of selection , In some embodiments , antibodies of the present invention mutation and expression may be carried out to identify selectively inhibit the release of TGF - B from GPCs associ antibody fragment sequences with the highest affinity for ated with LTBPs but not those associated with GARP. Such target antigens . Such sequences may be directly incorpo - 35 antibodies function as anti - fibrotic therapeutics but exhibit rated into antibody sequences for recombinant antibody minimal inflammatory effects . In some embodiments , GPC production . LTBP complex -binding antibodies do not bind GPC -GARP Antibody Characterization complexes. In some embodiments, such antibodies, may not Compounds and /or compositions of the present invention be specific to a particular LTBP or GPC , but may bind to comprising antibodies may act to decrease local concentra - 40 GPCs close to or overlapping with GARP binding sites, such tion of one or more GPC through removal by phagocytosis , that binding is impeded by GARP, but not by LTBPs . In pinocytosis , or inhibiting assembly in the extracellular some embodiments , antibodies are provided that selectively matrix and /or cellular matrix . Introduction of compounds bind one or more combinatorial epitopes between GARP and and / or compositions of the present invention may lead to the proTGF - ß . In some embodiments of the present invention , removal of 5 % to 100 % of the growth factor present in a 45 compounds and / or compositions are provided which induce given area . For example, the percent of growth factor release of TGF - B from GARP -proTGF - B complexes . Such removal may be from about 5 % to about 10 % , from about antibodies may be selected for their ability to bind to GARP 5 % to about 15 % , from about 5 % to about 20 % , from about prodomain binary complexes but not GARP - proTGF -B ter 5 % to about 25 % , from about 10 % to about 30 % , from about nary complexes, GARPs alone , or prodomains alone . 10 % to about 40 % , from about 10 % to about 50 % , from 50 Alternatively or additionally , antibodies of the present about 10 % to about 60 % , from about 20 % to about 70 % , invention may function as ligand mimetics which would from about 20 % to about 80 % , from about 40 % to about induce internalization of GPCs. Such antibodies may act as 90 % or from about 40 % to about 100 % . nontraditional payload carriers , acting to deliver and / or ferry Measures of release , inhibition or removal of one or more bound or conjugated drug payloads to specific GPC and / or growth factors may be made relative to a standard or to the 55 GPC -related sites . natural release or activity of growth factor under normal Changes elicited by antibodies of the present invention physiologic conditions, in vitro or in vivo . Measurements may result in neomorphic changes in the cell . As used may also be made relative to the presence or absence of herein , the term “ neomorphic change ” refers to a change or antibodies. Such methods ofmeasuring growth factor levels , alteration that is new or different. For example , an antibody release , inhibition or removal include standard measurement 60 that elicits the release or stabilization of one or more growth 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 . 65 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 US 9 ,758 , 576 B2 109 110 ments , such proteolytic events may include the alteration of to successfully produce anti- LAP antibodies . In some furin cleavage and /or other proteolytic processing events . In embodiments of the present invention , these methods may some embodiments , such proteolytic events may comprise be used to generate antibodies . In some embodiments , such proteolytic processing of growth factor signaling molecules methods may comprise the use of human antigens . In some or downstream cascades initiated by growth factor signaling 5 embodiments , cells used for immunization may express molecules . TGF - B and GARP. In such embodiments , GARPs may be In some embodiments, compounds and / or compositions expressed with native transmembrane domains to allow for of the present invention may induce or inhibit dimerization GARP - TGF - B complexes to remain tethered to the cell or multimerization of growth factors ( ligands ) or their surface of the transfected cells used from immunization . receptors . In some embodiments , such actions may be 10 Some antigens may comprise proTGF - B1 tethered to LTBP through stabilization of monomeric , dimeric or multimeric (e . g . LTBP1S . ) In some cases, recombinant proteins related forms or through the disruption of dimeric or multimeric to other TGF - B family members may be used as antigens . complexes. Methods of the present invention may also comprise one In some embodiments, compounds and /or compositions or more steps of the immunization methods described by of the present invention may act on homo and / or heterodi- 15 Oida et al combined with one or more additional and /or mers of the monomeric units comprising either receptor modified steps. Modified steps may include , but are not groups or GPCs or other signaling molecule pairs . limited to the use of alternate cell types for fusions, the Antibodies of the present invention may be internalized pooling of varying number of spleen cells when performing into cells prior to binding target antigens. Upon internaliza fusions , altering the injection regimen , altering the date of tion , such antibodies may act to increase or decrease one or 20 spleen cell harvest, altering immunogen and / or altering more signaling events , release or stabilize one or more immunogen dose . Additional steps may include the harvest GPCs, block or facilitate growth factor release and /or alter ing of other tissues ( e . g . lymph nodes ) from immunized one or more cell niche . mice . In some embodiments , compounds and /or compositions Activating and Inhibiting Antibodies of the present invention may also alter the residence time of 25 Antibodies of the present invention may comprise acti one or more growth factor in one or more GPC and / or alter v ating or inhibiting antibodies . As used herein , the term the residence time of one or more GPC in the extracellular " activating antibody ” refers to an antibody that promotes matrix and /or cellular matrix . Such alterations may result in growth factor activity . Activating antibodies include anti irreversible localization and / or transient localization . bodies targeting any epitope that promotes growth factor Antibodies of the present invention may be designed , 30 activity . Such epitopes may lie on prodomains ( e . g . LAPS manufactured and / or selected using any methods known to and LAP - like domains , ) growth factors or other epitopes one of skill in the art. In some embodiments , antibodies that when bound by antibody , lead to growth factor activity . and / or antibody producing cells of the present invention are Activating antibodies of the present invention may include , produced according to any of the methods listed in U . S . but are not limited to TGF - B - activating antibodies , GDF - 8 Provisional Patent Applications 61/ 722 , 919 , filed Nov . 6 , 35 activating antibodies, GDF - 11 - activating antibodies and 2012 and 61 / 722 , 969, filed Nov. 6 , 2012 , the contents of BMP -activating antibodies. each of which are herein incorporated by reference in their As used herein , the term “ inhibiting antibody ” refers to an entireties . antibody that reduces growth factor activity . Inhibiting anti Antibody Generation in Knockout Mice bodies include antibodies targeting any epitope that reduces In some embodiments , antibodies of the current invention 40 growth factor activity when associated with such antibodies. may be generated in knockout mice that lack a gene encod - Such epitopes may lie on prodomains ( e . g . LAPs and ing one or more desired antigens. Such mice would not be LAP - like domains, ) growth factors or other epitopes that tolerized to such antigens and therefore may be able to lead to reduced growth factor activity when bound by generate antibodies against them that could cross react with antibody . Inhibiting antibodies of the present invention may human and mouse forms of the antigen . For the production 45 include , but are not limited to TGF - B - inhibiting antibodies , of monoclonal antibodies , hostmice are immunized with the GDF - 8 - inhibiting antibodies , GDF - 11 - inhibiting antibodies target peptide to elicit lymphocytes that specifically bind and BMP- inhibiting antibodies . that peptide . Lymphocytes are collected and fused with an Embodiments of the present invention include methods of immortalized cell line . The resulting hybridoma cells are using activating and / or inhibiting antibodies in solution , in cultured in a suitable culture medium with a selection agent 50 cell culture and / or in subjects to modify growth factor to support the growth of only the fused cells . signaling. In some embodiments , knocking out one or more growth Anti- LAP and Anti -LAP -Like Domain Antibodies factor gene may be lethal and /or produce a fetus or neonate In some embodiments , compounds and /or compositions that is non - viable . In some embodiments , neonatal animals of the present invention may comprise one or more antibody may only survive for a matter of weeks ( e . g . 1 , 2 , 3 , 4 or 5 55 targeting a prodomain , including LAP and / or LAP - like weeks ) . In such embodiments , immunizations may be car - domains. Such antibodies may reduce or elevate growth ried out in neonatal animals shortly after birth . Oida et al factor signaling depending on the specific LAP or LAP - like ( Oida , T . et al. , TGF - B induces surface LAP expression on domain that is bound and /or depending on the specific Murine CD4 T cells independent of FoxP3 induction . PLOS epitope targeted by such antibodies . Anti -LAP and / or anti One. 2010 . 5 (11 ) :e15523 ) demonstrate immunization of 60 LAP - like protein antibodies of the invention may promote neonatal TGF- B knockout mice 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 gth day after birth and spleen cells were harvested on day 22 65 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 US 9 , 758 ,576 B2 111 112 some cases through by releasing TGF -B free growth factor referring to an original molecule against which a comparison from latent GPCs and / or preventing the reassociation of free may be made . Native or starting sequences should not be TGF - B growth factor with LAP. In some cases, anti- TGF- B confused with wild type sequences. Native sequences or LAP antibodies may increase TGF - B activity more favorably molecules may represent the wild - type ( that sequence found when proTGF - B is associated with LTBP. In some cases , 5 in nature ) but do not have to be identical to the wild - type anti - TGF- B LAP antibodies may increase TGF -B activity sequence . more favorably when proTGF - B is associated with GARP . In Ordinarily , variants will possess at least about 70 % some cases , anti- TGF - B LAP antibodies may function syn - homology to a native sequence, and preferably , they will be ergistically with other TGF -B activators ( e . g . avß6 and / or at least about 80 % , more preferably at least about 90 % außg) to increase TGF - ß activity . 10 homologous to a native sequence . Variations As used herein , the term “ homology ” as it applies to Compounds and / or compositions of the present invention amino acid sequences is defined as the percentage of resi may exist as a whole polypeptide, a plurality of polypeptides dues in the candidate amino acid sequence that are identical or fragments of polypeptides, which independently may be with the residues in the amino acid sequence of a second encoded by one or more nucleic acids, a plurality of nucleic 15 sequence after aligning the sequences and introducing gaps, acids , fragments of nucleic acids or variants of any of the if necessary , to achieve the maximum percent homology . aforementioned . As used herein , the term “ polypeptide” Methods and computer programs for the alignment are well refers to a polymer of amino acid residues (natural or known in the art. It is understood that homology depends on unnatural) linked together most often by peptide bonds . The a calculation of percent identity but may differ in value due term , as used herein , refers to proteins, polypeptides, and 20 to gaps and penalties introduced in the calculation . peptides of any size , structure , or function . In some instances As used herein , the term “ homolog ” as it applies to amino the polypeptide encoded is smaller than about 50 amino acid sequences is meant the corresponding sequence of other acids and the polypeptide is then termed a peptide . If the species having substantial identity to a second sequence of polypeptide is a peptide , it will be at least about 2 , 3 , 4 , or a second species. at least 5 amino acid residues long . Thus, polypeptides 25 As used herein , the term “ analog ” is meant to include include gene products , naturally occurring polypeptides, polypeptide variants which differ by one or more amino acid synthetic polypeptides , homologs , orthologs, paralogs, frag - alterations, e . g ., substitutions, additions or deletions of ments and other equivalents , variants , and analogs of the amino acid residues that still maintain the properties of the foregoing . A polypeptide may be a single molecule or may parent polypeptide . be a multi -molecular complex such as a dimer, trimer or 30 As used herein , the term " derivative” is used synony tetramer . They may also comprise single chain or multichain mously with the term “ variant” and refers to a molecule that polypeptides and may be associated or linked . The term has been modified or changed in any way relative to a polypeptide may also apply to amino acid polymers in which reference molecule or starting molecule . one or more amino acid residues are an artificial chemical The present invention contemplates several types of com analogue of a corresponding naturally occurring amino acid . 35 pounds and / or compositions which are amino acid based As used herein , the term " polypeptide variant” refers to including variants and derivatives . These include substitu molecules which differ in their amino acid sequence from a tional, insertional, deletional and covalent variants and native or reference sequence . The amino acid sequence derivatives . As such , included within the scope of this variants may possess substitutions, deletions, and / or inser - invention are compounds and /or compositions comprising tions at certain positions within the amino acid sequence , as 40 substitutions, insertions, additions , deletions and/ or covalent compared to a native or reference sequence . Ordinarily , modifications . For example , sequence tags or amino acids , variants will possess at least about 50 % identity ( homology ) such as one or more lysines, can be added to peptide to a native or reference sequence , and preferably , they will sequences of the invention ( e . g . , at the N -terminal or C - ter be at least about 80 % , more preferably at least about 90 % minal ends) . Sequence tags can be used for peptide purifi identical (homologous ) to a native or reference sequence . 45 cation or localization . Lysines can be used to increase In some embodiments “ variant mimics” are provided . As peptide solubility or to allow for biotinylation . Alternatively , used herein , the term " variant mimic ” refers to a variant amino acid residues located at the carboxy and amino which contains one or more amino acids which would mimic terminal regions of the amino acid sequence of a peptide or an activated sequence . For example , glutamate may serve as protein may optionally be deleted providing for truncated a mimic for phospho -threonine and / or phospho - serine . 50 sequences . Certain amino acids ( e . g . , C - terminal or N - ter Alternatively , variantmimics may result in deactivation or in minal residues ) may alternatively be deleted depending on an inactivated product containing the mimic , e . g . , phenyl- the use of the sequence , as for example , expression of the alanine may act as an inactivating substitution for tyrosine ; sequence as part of a larger sequence which is soluble, or or alanine may act as an inactivating substitution for serine . linked to a solid support . The amino acid sequences of the compounds and / or com - 55 “ Substitutional variants " when referring to proteins are positions of the invention may comprise naturally occurring those that have at least one amino acid residue in a native or amino acids and as such may be considered to be proteins, starting sequence removed and a different amino acid peptides , polypeptides, or fragments thereof. Alternatively, inserted in its place at the same position . The substitutions the compounds and /or compositions may comprise both may be single , where only one amino acid in the molecule naturally and non - naturally occurring amino acids . 60 has been substituted , or they may be multiple , where two or As used herein , the term “ amino acid sequence variant” more amino acids have been substituted in the same mol refers to molecules with some differences in their amino acid ecule . sequences as compared to a native or starting sequence . The As used herein , the term " conservative amino acid sub amino acid sequence variants may possess substitutions, stitution ” refers to the substitution of an amino acid that is deletions , and / or insertions at certain positions within the 65 normally present in the sequence with a different amino acid amino acid sequence . As used herein , the terms “ native " or of similar size , charge , or polarity . Examples of conservative " starting ” when referring to sequences are relative terms substitutions include the substitution of a non - polar (hydro US 9 , 758 ,576 B2 113 114 phobic ) residue such as isoleucine , valine and leucine for fall within the scope of this invention , e . g. polyvinylalcohol another non - polar residue. Likewise , examples of conserva - and polyvinylpyrrolidone . Particularly useful are polyvinyl tive substitutions include the substitution of one polar (hy - alkylene ethers such a polyethylene glycol, polypropylene drophilic ) residue for another such as between arginine and glycol . The proteins may be linked to various non -proteina lysine , between glutamine and asparagine, and between 5 ceous polymers , such as polyethylene glycol, polypropylene glycine and serine . Additionally , the substitution of a basic glycol or polyoxyalkylenes, in the manner set forth in U . S . residue such as lysine , arginine or histidine for another , or Pat. Nos. 4 ,640 ,835 ; 4 ,496 ,689 ; 4 ,301 , 144 ; 4 ,670 ,417 ; the substitution of one acidic residue such as aspartic acid or 4 ,791 , 192 or 4 , 179 ,337 . glutamic acid for another acidic residue are additional As used herein , the term " features” when referring to examples of conservative substitutions. Examples of non - 10 proteins are defined as distinct amino acid sequence -based conservative substitutions include the substitution of a non - components of a molecule . Features of the proteins of the polar (hydrophobic ) amino acid residue such as isoleucine , present invention include surface manifestations , local con valine , leucine, alanine ,methionine for a polar (hydrophilic ) formational shape , folds, loops, half - loops, domains , half residue such as cysteine, glutamine, glutamic acid or lysine domains, sites, termini or any combination thereof. and / or a polar residue for a non - polar residue . 15 As used herein , the term “ surface manifestation " when As used herein , the term “ insertional variants ” when referring to proteins refers to a polypeptide based compo referring to proteins are those with one or more amino acids n ent of a protein appearing on an outermost surface . inserted immediately adjacent to an amino acid at a particu - As used herein , the term “ local conformational shape ” lar position in a native or starting sequence . As used herein , when referring to proteins refers to a polypeptide based the term “ immediately adjacent” refers to an adjacent amino 20 structuralmanifestation of a protein which is located within acid that is connected to either the alpha - carboxy or alpha - a definable space of the protein . amino functional group of a starting or reference amino acid . As used herein , the term " fold ” , when referring to pro As used herein , the term “ deletional variants " when teins , refers to the resultant conformation of an amino acid referring to proteins, are those with one or more amino acids sequence upon energy minimization . A fold may occur at the in the native or starting amino acid sequence removed . 25 secondary or tertiary level of the folding process. Examples Ordinarily , deletional variants will have one or more amino of secondary level folds include beta sheets and alpha acids deleted in a particular region of the molecule . helices. Examples of tertiary folds include domains and As used herein , the term " derivatives , " as referred to regions formed due to aggregation or separation of energetic herein includes variants of a native or starting protein forces. Regions formed in this way include hydrophobic and comprising one or more modifications with organic pro - 30 hydrophilic pockets, and the like . teinaceous or non -proteinaceous derivatizing agents , and As used herein , the term “ turn ” as it relates to protein post - translational modifications. Covalent modifications are conformation , refers to a bend which alters the direction of traditionally introduced by reacting targeted amino acid the backbone of a peptide or polypeptide and may involve residues of the protein with an organic derivatizing agent one, two, three or more amino acid residues. that is capable of reacting with selected side - chains or 35 As used herein , the term “ loop , " when referring to pro terminal residues , or by harnessing mechanisms of post- teins , refers to a structural feature of a peptide or polypeptide translational modifications that function in selected recom which reverses the direction of the backbone of a peptide or binant host cells. The resultant covalent derivatives are polypeptide and comprises four or more amino acid resi useful in programsdirected at identifying residues important dues. Oliva et al. have identified at least 5 classes of protein for biological activity , for immunoassays , or for the prepa - 40 loops (Oliva , B . et al. , An automated classification of the ration of anti -protein antibodies for immunoaffinity purifi - structure of protein loops . J Mol Biol. 1997 . 266 ( 4 ) :814 - 30 . ) cation of the recombinant glycoprotein . Such modifications As used herein , the term " half -loop , " when referring to are within the ordinary skill in the art and are performed proteins , refers to a portion of an identified loop having at without undue experimentation . least half the number of amino acid resides as the loop from Certain post- translational modifications are the result of 45 which it is derived . It is understood that loops may not the action of recombinant host cells on the expressed poly - always contain an even number of amino acid residues . peptide . Glutaminyl and asparaginyl residues are frequently Therefore , in those cases where a loop contains or is post - translationally deamidated to the corresponding gluta - identified to comprise an odd number of amino acids , a myl and aspartyl residues. Alternatively , these residues are half -loop of the odd - numbered loop will comprise the whole deamidated under mildly acidic conditions . Either form of 50 number portion or next whole number portion of the loop these residues may be present in the proteins used in ( number of amino acids of the loop / 2 + / - 0 . 5 amino acids ) . accordance with the present invention . For example , a loop identified as a 7 amino acid loop could Other post- translational modifications include hydroxy - produce half - loops of 3 amino acids or 4 amino acids lation of proline and lysine , phosphorylation of hydroxyl (7 /2 = 3 .5 + / - 0 .5 being 3 or 4 ). groups of seryl or threonyl residues, methylation of the 55 As used herein , the term " domain ,” when referring to alpha - amino groups of lysine, arginine , and histidine side proteins , refers to a motif of a polypeptide having one or chains ( T . E . Creighton , Proteins : Structure and Molecular more identifiable structural or functional characteristics or Properties , W . H . Freeman & Co . , San Francisco , pp . 79 - 86 properties ( e . g . , binding capacity, serving as a site for ( 1983 ) ) . protein -protein interactions. ) Covalent derivatives specifically include fusion mol- 60 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 , not always contain an even number of amino acid residues . polymers which exist in nature and are produced by recom - 65 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 US 9 , 758 ,576 B2 115 116 whole number portion or next whole number portion of the allow compounds and / or compositions to be used in diag domain (number of amino acids of the domain / 2 + / - 0 . 5 nostic and / or experimental applications . amino acids) . For example , a domain identified as a 7 amino Conjugates and Combinations acid domain could produce half -domains of 3 amino acids or It is contemplated by the present invention that the 4 amino acids ( 7 / 2 = 3 . 5 + / - 0 . 5 being 3 or 4 ). It is also 5 compounds and /or compositions of the present invention understood that sub - domains may be identified within may be complexed , conjugated or combined with one or domains or half -domains , these subdomains possessing less more homologous or heterologous molecules. As used than all of the structural or functional properties identified in herein , the term “ homologous molecule ” refers to a mol the domains or half domains from which they were derived . ecule which is similar in at least one of structure or function It is also understood that the amino acids that comprise any 10 relative to a starting molecule while a " heterologous mol of the domain types herein need not be contiguous along the ecule ” is one that differs in at least one of structure or backbone of the polypeptide ( i. e ., nonadjacent amino acids function relative to a starting molecule . Structural homologs may fold structurally to produce a domain , half- domain or are therefore molecules which may be substantially struc subdomain ) . turally similar. In some embodiments , such homologs may As used herein , the terms " site , " as it pertains to amino 15 be identical. Functional homologs are molecules which may acid based embodiments is used synonymously with “ amino be substantially functionally similar. In some embodiments , acid residue ” and “ amino acid side chain " . A site represents such homologs may be identical. a position within a peptide or polypeptide that may be compounds and/ or compositions of the present invention modified , manipulated , altered , derivatized or varied within may comprise conjugates. Such conjugates of the invention the polypeptide based molecules of the present invention . 20 may include naturally occurring substances or ligands, such As used herein , the terms " termini” or “ terminus ," when as proteins ( e . g . , human serum albumin (HSA ) , low - density referring to proteins refers to an extremity of a peptide or lipoprotein (LDL ) , high - density lipoprotein (HDL ) , or polypeptide . Such extremity is not limited only to the first or globulin ) ; carbohydrates (e . g. , a dextran , pullulan , chitin , final site of the peptide or polypeptide but may include chitosan , inulin , cyclodextrin or hyaluronic acid ) ; or lipids. additional amino acids in the terminal regions . The poly - 25 Conjugates may also be recombinant or synthetic molecules , peptide based molecules of the present invention may be such as synthetic polymers , e . g ., synthetic polyamino acids, characterized as having both an N - terminus ( terminated by an oligonucleotide ( e . g . an aptamer ) . Examples of an amino acid with a free amino group (NH2 )) and a polyamino acids may include polylysine (PLL ) , poly L - as C - terminus ( terminated by an amino acid with a free car - partic acid , poly L - glutamic acid , styrene -maleic acid anhy boxyl group (COOH ) ) . Proteins of the invention are in some 30 dride copolymer, poly ( L - lactide- co - glycolied ) copolymer , cases made up of multiple polypeptide chains brought divinyl ether- maleic anhydride copolymer , N - ( 2 - hydroxy together by disulfide bonds or by non -covalent forces (mul - propyl)methacrylamide copolymer (HMPA ) , polyethylene timers , oligomers ) . These sorts of proteins will have mul - glycol (PEG ), polyvinyl alcohol (PVA ), polyurethane , poly tiple N - and C - termini. Alternatively , the termini of the ( 2 - ethylacryllic acid ) , N - isopropylacrylamide polymers , or polypeptides may be modified such that they begin or end , 35 polyphosphazine . Example of polyamines include: polyeth as the case may be , with a non -polypeptide based moiety ylenimine, polylysine (PLL ), spermine , spermidine , such as an organic conjugate . polyamine , pseudopeptide- polyamine , peptidomimetic Once any of the features have been identified or defined polyamine , dendrimer polyamine , arginine , amidine , as a component of a molecule of the invention , any of protamine, cationic lipid , cationic porphyrin , quaternary salt several manipulations and / or modifications of these features 40 of a polyamine , or an alpha helical peptide . may be performed by moving , swapping , inverting, deleting , In some embodiments , conjugates may also include tar randomizing or duplicating . Furthermore , it is understood geting groups. As used herein , the term “ targeting group ” that manipulation of features may result in the same out- refers to a functional group or moiety attached to an agent come as a modification to the molecules of the invention . that facilitates localization of the agent to a desired region , For example , a manipulation which involved deleting a 45 tissue, cell and / or protein . Such targeting groups may domain would result in the alteration of the length of a include , but are not limited to cell or tissue targeting agents molecule just as modification of a nucleic acid to encode less or groups ( e . g . lectins, glycoproteins, lipids , proteins, an than a full length molecule would . antibody that binds to a specified cell type such as a kidney Modifications and manipulations can be accomplished by cell or other cell type ) . In some embodiments , targeting methods known in the art such as site directed mutagenesis . 50 groups may comprise thyrotropins , melanotropins , lectins , The resulting modified molecules may then be tested for glycoproteins, surfactant protein A , mucin carbohydrates , activity using in vitro or in vivo assays such as those multivalent lactose , multivalent galactose , N -acetyl - galac described herein or any other suitable screening assay tosamine , N - acetyl - gulucosamine , multivalent mannose , known in the art . multivalent fucose , glycosylated polyaminoacids, multiva In some embodiments, compounds and /or compositions 55 lent galactose , transferrin , bisphosphonate , polyglutamate , of the present invention may comprise one or more atoms polyaspartate , lipids, cholesterol, steroids, bile acids, folates, that are isotopes. As used herein , the term “ isotope ” refers to vitamin B12 , biotin , an RGD peptide, an RGD peptide a chemical element that has one or more additional neutrons. mimetic or an aptamer. In some embodiments , compounds of the present invention In some embodiments , targeting groups may be proteins , may be deuterated . As used herein , the term " deuterate " 60 e . g . , glycoproteins , or peptides , e . g . , molecules having a refers to the process of replacing one or more hydrogen specific affinity for a co - ligand , or antibodies e . g . , an anti atoms in a substance with deuterium isotopes . Deuterium body , that binds to a specified cell type such as a cancer cell, isotopes are isotopes of hydrogen . The nucleus of hydrogen endothelial cell , or bone cell. Targeting groups may also contains one proton while deuterium nuclei contain both a comprise hormones and /or hormone receptors. proton and a neutron . The compounds and/ or compositions 65 In some embodiments, targeting groups may be any of the present invention may be deuterated in order to ligand capable of targeting specific receptors . Examples change one or more physical property , such as stability , or to include, without limitation , folate , GalNAc, galactose , man US 9 ,758 ,576 B2 117 118 nose , mannose- 6 -phosphate , apatamers , integrin receptor limited to biomolecules , including, but not limited to recom ligands , chemokine receptor ligands, transferrin , biotin , binant proteins , protein complexes and / or antibodies serotonin receptor ligands, PSMA , endothelin , GCPII , described herein . somatostatin , LDL , and HDL ligands . In some embodi In some embodiments , methods of the present invention ments , targeting groups are aptamers. Such aptamers may be 5 may be used to initiate or increase growth factor activity, unmodified or comprise any combination of modifications termed “ activating methods” herein . Some such methods disclosed herein . may comprise growth factor release from a GPC and/ or inhibition of growth factor reassociation into a latent GPC . In still other embodiments , compounds and /or composi In some cases , activating methods may comprise the use of tions of the present invention may be covalently conjugated 10 an antibody, a recombinant protein and /or a protein com to cell penetrating polypeptides . In some embodiments , plex . According to some activating methods, one or more cell- penetrating peptides may also include signal sequences . activating antibody is provided . In such methods, one or In some embodiments , conjugates of the invention may be more growth factor may be released or prevented from being designed to have increased stability , increased cell transfec drawn back into a GPC . In one, non - limiting example , an tion and / or altered biodistribution ( e . g ., targeted to specific 15 anti -LAP antibody may be provided that enhances dissocia tissues or cell types. ) tion between a growth factor and a GPC and /or prevents In some embodiments , conjugating moieties may be reformation of a GPC . added to compounds and/ or compositions of the present Embodiments of the present invention include methods of invention such that they allow the attachment of detectable using anti -LAP and /or anti- LAP - like domain antibodies to labels to targets for clearance . Such detectable labels 20 modify growth factor activity . In some cases, such methods include , but are not limited to biotin labels , ubiquitins, may include the use of anti - TGF - B -LAP antibodies as TGF fluorescent molecules , human influenza hemaglutinin (HA ), B -activating antibodies . In some cases , methods of using c -myc , histidine (His ) , flag , glutathione S -transferase (GST ), and /or testing such antibodies may include any of the V5 ( a paramyxovirus of simian virus 5 epitope ), biotin , methods taught in Tsang , M . et al . 1995 . Cytokine 7 ( 5 ) : 389 avidin , streptavidin , horse radish peroxidase (HRP ) and 25 97 , the contents of which are herein incorporated by refer digoxigenin . ence in their entirety . In some embodiments , compounds of the invention may In some embodiments , methods of the present invention be conjugated with an antibody Fc domain to create an Fc may be used to reduce or eliminate growth factor activity , fusion protein . The formation of an Fc fusion protein with termed " inhibiting methods ” herein . Some such methods any of the compounds described herein may be carried out 30 may comprise growth factor retention in a GPC and / or promotion of reassociation of growth factor into a latent according to any method known in the art , including as GPC . In some cases, inhibiting methods may comprise the described in U . S . Pat. Nos. 5 , 116 , 964 , 5 ,541 , 087 and 8 ,637 , use of an antibody 637 , the contents of each of which are herein incorporated Therapeutics by reference in their entirety . Fc fusion proteins of the 35 In some embodiments , compositions and methods of the invention may comprise a compound of the invention linked invention may be used to treat a wide variety of diseases , to the hinge region of an IgG Fc via cysteine residues in the disorders and / or conditions. In some cases , such diseases, Fc hinge region . Resulting Fc fusion proteins may comprise disorders and /or conditions may be TGF- B - related indica an antibody - like structure , but without Chi domains or light tions. As used herein , the term “ TGF - B - related indication ” chains . In some cases, Fc fusion proteins may comprise 40 refers to any disease, disorder and / or condition related to pharmacokinetic profiles comparable to native antibodies . In expression , activity and / or metabolism of a TGF - B family some cases , Fc fusion proteins of the invention may com - member protein or any disease, disorder and /or condition prise extended half - life in circulation and / or altered biologi - that may benefit from modulation of the activity and / or cal activity . levels of one or more TGF - B family member protein . In some embodiments, compounds and /or compositions 45 TGF - B -related indications may include , but are not limited of the present invention may be combined with one another to , fibrosis , anemia of the aging , cancer ( including, but not or other molecules in the treatment of diseases and / or limited to colon , renal, breast, malignant melanoma and conditions . glioblastoma, ) facilitation of rapid hematopoiesis following Nucleic Acids chemotherapy , bone healing , endothelial proliferation syn In some embodiments , compounds and /or compositions 50 dromes, asthma and allergy, gastrointestinal disorders , aortic of the present invention may be encoded by nucleic acid aneurysm , orphan indications (such as Marfan ' s syndrome molecules. Such nucleic acid molecules include , without and Camurati -Engelmann disease ,) obesity , diabetes, arthri limitation , DNA molecules , RNA molecules , polynucle - tis, multiple sclerosis, muscular dystrophy, amyotrophic otides, oligonucleotides, mRNA molecules , vectors, plas - lateral sclerosis ( ALS , Parkinson ' s disease , osteoporosis , mids and the like . In some embodiments , the present inven - 55 osteoarthritis , osteopenia , metabolic syndromes, nutritional tion may comprise cells programmed or generated to express disorders , organ atrophy , chronic obstructive pulmonary nucleic acid molecules encoding compounds and / or com disease (COPD ) and anorexia . Additional indications may positions of the present invention . include any of those disclosed in US Pub . No . 2013 / Methods of Use 0122007 , U . S . Pat. No . 8 , 415 ,459 or International Pub . No . Methods of the present invention include methods of 60 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 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 65 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 US 9 , 758 ,576 B2 119 120 treated or targeted for prevention . It is well within the ability between GPCs and GARP unaffected . Such compounds of one skilled in the art to monitor efficacy of treatment or and / or compositions of the present invention may act as prevention by measuring any one of such parameters , or any inhibitory antibodies , preventing growth factor signaling combination of parameters . In connection with the admin and inhibiting fibrosis . In some embodiments , compounds istration of compositions of the present invention , " effective 5 and / or compositions of the present invention are designed to against” for example a cancer , indicates that administration target one or more of TGF -B1 , 2 and 3 or chimeric antigens in a clinically appropriate manner results in a beneficial thereof. effect for at least a statistically significant fraction of Fibrotic indications for which compounds and / or compo patients , such as an improvement of symptoms, a cure, a sitions of the present invention may be used therapeutically reduction in disease load , reduction in tumor mass or cell 10 include , but are not limited to lung indications ( e . g . Idio numbers , extension of life , improvement in quality of life , or pathic Pulmonary Fibrosis ( IPF ) , Chronic Obstructive Pul other effect generally recognized as positive by medical monary Disorder ( COPD ) , Allergic Asthma , Acute Lung doctors familiar with treating the particular type of cancer. injury , Eosinophilic esophagitis , Pulmonary arterial hyper A treatment or preventive effect is evident when there is tension and Chemical gas -injury , ] kidney indications [ e .g . a statistically significant improvement in one or more 15 Diabetic glomerulosclerosis , Focal segmental glomerulocle parameters of disease status, or by a failure to worsen or to r osis (FSGS ) , Chronic kidney disease , Fibrosis associated develop symptoms where they would otherwise be antici - with kidney transplantation and chronic rejection , IgA neph pated . As an example , a favorable change of at least 10 % in ropathy and Hemolytic uremic syndrome, ] liver fibrosis [ e .g . a measurable parameter of disease , and preferably at least Non - alcoholic steatohepatitis (NASH ) , Chronic viral hepa 20 % , 30 % , 40 % , 50 % or more can be indicative of effective 20 titis , Parasitemia , Inborn errors of metabolism , Toxin -medi treatment. Efficacy for a given composition or formulation ated fibrosis , such as alcohol fibrosis , Non -alcoholic steato of the present invention can also be judged using an experi - hepatitis -hepatocellular carcinoma (NASH -HCC ) , Primary mental animal model for the given disease as known in the biliary cirrhosis and Sclerosing cholangitis , ] cardiovascular art. When using an experimental animal model , efficacy of fibrosis ( e . g . cardiomyopathy, hypertrophic cardiomyopathy , treatment is evidenced when a statistically significant 25 atherosclerosis and restenosis , ) systemic sclerosis , skin change is observed . fibrosis ( e g Skin fibrosis in systemic sclerosis , Diffuse Therapeutics for Fibrosis cutaneous systemic sclerosis , Scleroderma, Pathological In some embodiments, compounds and /or compositions skin scarring , Keloid , Post surgical scarring, Scar revision of the present invention may be useful for altering fibrosis . surgery , Radiation - induced scarring and Chronic wounds ) In some embodiments , such compounds and / or composi - 30 and cancers or secondary fibrosis ( e . g . Myelofibrosis , Head tions are antagonists of TGF - B . TGF - B is recognized as the and Neck Cancer, M7 acute Megakaryoblastic Leukemia central orchestrator of the fibrotic response . Antibodies and Mucositis . ) Other diseases, disorders or conditions targeting TGF - ß decrease fibrosis in numerous preclinical related to fibrosis that may be treated using compounds models. Such antibodies and /or antibody - based compounds and / or compositions of the present invention , include , but include LY2382770 ( Eli Lilly , Indianapolis , Ind . ). Also 35 are not limited to Marfan ' s Syndrome, Stiff Skin Syndrome, included are those described in U . S . Pat . No. 6 ,492 ,497 , U . S . Scleroderma, Rheumatoid arthritis , bone marrow fibrosis , Pat. No. 7 , 151 , 169 and U . S . Pat. No. 7 ,723 ,486 and U . S . Crohn ' s disease , Ulcerative colitis , Systemic lupus erythe publication US2011 / 0008364, the contents of each of which matosus, Muscular Dystrophy , Dupuytren ' s contracture , are herein incorporated by reference in their entirety . Camurati- Engelmann Disease , Neural scarring , Proliferative Fibrosis is a common sequela of many types of tissue 40 vitreoretinopathy, corneal injury, complications after glau destructive diseases . When new space is created by the coma drainage surgery and Multiple Sclerosis . disruption of differentiated cells , progenitors or stem cells Assays useful in determining the efficacy of the com that normally occupy a niche in the tissue , the default pounds and /or compositions of the present invention for the pathway appears to be the proliferation of connective tissue alteration of fibrosis include , but are not limited to , histo cells , e . g . fibroblasts , to fill in the empty space . This is 45 logical assays for counting fibroblasts and basic immuno accompanied by the production of extracellular matrix con - histochemical analyses known in the art . stituents including collagens that result in scarring and Animal models are also available for analysis of the permanent effacement of the tissue . efficacy of compounds and /or compositions of the present A difficult aspect of fibrosis is its chronicity , which may invention in altering fibrosis . Examples of animal fibrosis require continued therapy until the underlying destruction of 50 models useful for such analysis may include, for example , parenchymal cells is terminated or the cells are replaced by any of those taught by Schaefer , D . W . et al. , 2011 . Eur stem cell pools , or by transplantation . Fibrosis is thought to Respir Rev . 20 : 120 , 85 - 97 , the contents of which are herein be much easier to arrest than to reverse . The TGF -beta incorporated by reference in their entirety . Such models may family is of central importance in regulating the growth of include , but are not limited to those described in Table 1 of fibroblastic cells and the production of extracellular matrix 55 that publication , including lung models , renal models , liver constituents including collagen . Integrins a ,ßo and a , ß models , cardiovascular models and/ or collagen - induced (and possibly auß , ) may participate in activation of TGF- models . Schaefer et al also teach the use of pirfenidone in the betal and 3 . The integrin VLA - 1 is a receptor for collagen treatment of fibrosis . In some cases , compounds and / or 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 60 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 a , ßo, 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 65 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 US 9 , 758 ,576 B2 121 122 be carried out as described by Schaefer et al, and also by signaling pathway. In some cases, cyclosporine A - induced Horan et al. (Horan G . S . et al. , 2008. Am J Respir Crit Care nephropathy models may be used . Such models may be Med , 177 ( 1 ) : 56 -65 . Epub 2007 Oct. 4 , the contents of each carried out as described in Ling , H . et al. , 2003 . J Am Soc of which are herein incorporated by reference in their Nephrol. 14 : 377 -88 , the contents of which are herein incor entirety . ) According to the Horan study , SV129 mice are 5 porated by reference in their entirety . In some cases , renal tracheally exposed to bleomycin which results in the devel- models of Alport Syndrome may be used . Transgenic mice opment of lung fibrosis . With this model , potential thera - with collagen III knockout may be used in Alport syndrome peutics are administered through intraperitoneal injections studies . These mice develop progressive fibrosis in their while postmortem lung tissue or bronchoalveolar lavage kidneys . Alport syndrome models may be carried out as collections can be assayed for levels of hydroxyproline as an 10 described in Koepke , M . L . et al. , 2007 . Nephrol Dial indicator of fibrotic activity. Using the same technique , mice Transplant. 22 ( 4 ) : 1062- 9 and /or Hahm , K . et al. , 2007 . Am carrying a luciferase reporter gene , driven by the collagen J Pathol. 170 ( 1 ) : 110 - 5 , the contents of each of which are la2 gene promoter may be used in the model so that fibrotic herein incorporated by reference in their entirety . activity may be determined by luciferase activity assay as a In some cases, models of cardiovascular fibrosis may be function of collagen gene induction . Additional bleomycin 15 used to develop and/ or test compounds and/ or compositions induced lung models may be carried out according to those of the invention for treatment of cardiovascular fibrotic described by Thrall et al ( Thrall, R . S . et al. , 1979 . Am J indications. In some cases, vascular injury models may be Pathol. 95 : 117 - 30 , the contents of which are herein incor- used . Such models may include balloon injury models . In porated by reference in their entirety . ) Additional lung some cases , these may be carried out as described in Smith models may include the mouse asthma models . Airway 20 et al . , 1999 . Circ Res. 84 ( 10 ): 1212 - 22 , the contents of which remodeling ( lung fibrosis ) may be a serious problem in are herein incorporated by reference in their entirety. Block subjects with chronic asthma. Asthma models may include ing TGF- B in this model was shown to block neointima any of those described by Nials et al (Nials , A . T . et al. , 2008 . formation . Accordingly , TGF- B inhibiting antibodies of the Disease Models and Mechanisms. 1 :213 - 20 , the contents of present invention may be used to reduce and /or block which are herein incorporated by reference in their entirety. ) 25 neointima formation . Models of chronic obstructive pulmonary disease ( COPD ) In some embodiments , models of liver fibrosis may be may be used . Such models may include any of those used to develop and /or test compounds and /or compositions described by Vlahos et al (Vlahos , R . et al. , 2014 . Clin Sci. of the invention for treatment of liver fibrotic indications . 126 : 253 -65 , the contents of which are herein incorporated Liver models may include any of those described in Iredale , by reference in their entirety. ) Models of cigarette smoking 30 J . P . 2007 . J Clin Invest . 117 ( 3 ) : 539- 48 , the contents of emphysema may be used . Such models may be carried out which are herein incorporated by reference in their entirety . as described in Ma et al. 2005 . J Clin Invest. 115 : 3460 - 72 , These include , but are not limited to , any of the models listed the contents of which are herein incorporated by reference in in Tables 1 and / or 2 . In some cases , liver models may their entirety . Models of chronic pulmonary fibrosis may be include carbon tetrachloride induced liver fibrosis models . used . Such models in rodents may be carried out according 35 Such models may be carried out according to the methods to the intratracheal fluorescein isothiocyanate (FITC ) instil- described in Fujii , T . et al. , 2010 . BMC Gastroenterology . lation model described in Roberts , S . N . et al. 1995 . J Pathol. 10 : 79 , the contents of which are herein incorporated by 176 ( 3 ) : 309 - 18 , the contents of which are herein incorpo - reference in their entirety. rated by reference in their entirety . Models of asbestos and In some embodiments, models of wound healing may be silica induced lung injury may also be used . Such models 40 used to develop and/ or test compounds and / or compositions may be carried out as described in Coin , P . G . et al. , 1996 . of the invention for treatment of fibrotic wound indications . Am J Respir Crit Care Med . 154 ( 5 ) : 1511 - 9 , the contents of Wound models may include chronic wound models . which are herein incorporated by reference in their entirety. In some cases, models ofGI injury - related fibrosis may be In some cases , models of lung irradiation may be used . Such used to develop and /or test compounds and / or compositions models may be carried out as described in Pauluhn , J . et al . 45 of the invention for treatment of GI- related fibrosis . Such 2001 . Toxicology . 161 : 153 -63 , the contents of which are injury models may include, but are not limited to 2 , 4 , 6 herein incorporated by reference in their entirety . In some trinitrobenzenesulfonic acid ( TNBS ) induced colitis models . cases, phorbol myristate acetate (PMA ) - induced lung injury Such models may be carried out as described in Scheiffele , models may be used . Such models may be carried out as F . et al. , 2002 . Curr Protoc Immunol. Chapter 15 :Unit 15 . 19 , described in Taylor , R . G . et al ., 1985 . Lab Invest . 52 ( 1 ): 50 the contents of which are herein incorporated by reference in 61- 70 , the contents of which are herein incorporated by their entirety . reference in their entirety . In some embodiments , compounds and /or compositions Renal fibrosis models may be utilized to develop and/ or of the invention may be used to treat diseases , disorders test compounds and /or compositions of the present inven - and / or conditions related to bone marrow fibrosis . In some tion . In some embodiments , a well established model of 55 cases , bone marrow fibrosis models may be used to develop renal fibrosis , unilateral ureteral obstruction (UUO ) model , and /or test such compounds and / or compositions. Models may be used . In this model , mice are subjected to proximal may include the marrow cell adoptive transfer model ureteral ligation . After a period of hours to days , fibrosis is described in Lacout, C . et al. , 2006 . Blood . 108 ( 5 ) : 1652 -60 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 60 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 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 65 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. US 9 , 758 ,576 B2 123 124 In some embodiments , compounds and / or compositions entirety , ) simtuzumab (Gilead Biosciences, Foster City , of the invention may be used to treat diseases , disorders Calif. ) treatment to block lysyl oxidase activity and collagen and /or conditions related to muscular dystrophy (MD ) cross -linking and Pentraxin - 2 (Promedior , Lexington , including , but not limited to Duchenne M D and Becker M Mass . ) treatment to stimulate regulatory macrophages and D . In some cases MD models may be used to develop and / or 5 inhibit myelofibroblasts . In some cases , models of myelo test such compounds and / or compositions . Such models may proliferative disorders may be used to develop and / or test include those described in Ceco , E . et al. , 2013 . FEBS J. such compounds and/ or compositions of the invention 280 ( 17 ) :4198 - 209 , the contents of which are herein incor intended for the treatment of myelofibrosis. Models may porated by reference in their entirety . include the marrow cell adoptive transfermodel described in Compounds and/ or compositions of the invention may , in 10 Lacout, C . et al. , 2006 . Blood . 108 ( 5 ) : 1652 -60 and trans some cases, be combined with one or more other therapeu - genic mouse models , including , but not limited to the model tics for the treatment of one or more fibrotic indication . described in Vannucchi, A . M . et al ., 2002. Blood . 100 ( 4 ) : Examples of such other therapeutics may include , but are not 1123 - 32 , the contents of each of which are herein incorpo limited to LPA1 receptor antagonists , lysyl oxidase 2 inhibi- rated by reference in their entirety . Myelofibrosis models tors , hedgehog inhibitors , IL -3 /IL - 4 inhibitors , CTGF 15 may include thrombopoietin - induced myelofibrosis . Such inhibitors , anti -avßo antibodies and anti - IL - 13 antibodies . models may be carried out as described in Chagraoui, H . et In some cases, compounds and / or compositions of the al. , 2002 . Blood . 100 ( 10 ) :3495 - 503 , the contents of which present invention are designed to increase TGF - B growth are herein incorporated by reference in their entirety . TGF factor activity to promote fibrosis to treat diseases , disorders B1 has been shown to be the primary agonist of fibrosis and / or conditions where fibrosis may be advantageous . Such 20 according to this model. Further myelofibrosis models may compounds may include activating antibodies. be carried out as described in Mullally , A . et al. , 2010 . Therapeutics for Myelofibrosis Cancer Cell. 17 : 584 - 96 , the contents of which are herein Myelofibrosis is a chronic blood cancer caused by muta incorporated by reference in their entirety . tions in bone marrow stem cells . Disease is characterized by Therapeutics for Scarring and Wound Healing an impaired ability to make normal blood cells . Patients 25 In some embodiments , compounds and /or compositions develop splenomegaly and hepatomegaly and excessive of the present invention may be useful in altering wound fibrosis occurs in the bone marrow . Myeloproliferative neo - healing and /or scar formation . In some cases , compounds plasms (MPNs ) are the collective name for three related and/ or compositions of the invention may ensure proper types of myelofibrosis with different clinical features : pri- wound healing ( including , but not limited to chronic mary myelofibrosis (PMF ), essential thrombocythemia and 30 wounds. ) In some cases, compounds and /or compositions of polycythemia vera . All three have overactive signaling of the invention may be used for reducing , treating and or the JAK -STAT cell signaling pathway (Klampfi , et al. , 2013 . preventing scar formation . Such compounds and/ or compo NEJM 369: 2379 - 90 , the contents of which are herein incor - sitions may comprise anti- TGF - B antibodies . In some cases , porated by reference in their entirety .) Primary myelofibrosis TGF- B -activating antibodies may be used to promote heal (PMF ) is characterized by increased angiogenesis , reticulin 35 ing in wounds . and collagen fibrosis . As the disease advances, the number Therapeutics for Disorders of Iron Metabolism of osteoclasts increase and bone marrow becomes unaspi- In some embodiments, methods , compounds and / or com rable . Some fibrosis of PMF may be reversed by stem cell positions of the present invention may be used to treat transplantation ( SCT. ) 98 % of individuals with poly - disorders of iron metabolism . Such disorders may include cythemia vera have mutated JAK2 leading to overactive 40 disorders comprising reduced iron levels ( e . g . anemias ) or JAK - STAT signaling. disorders comprising elevated iron levels ( e . g . hemochro Current therapeutics for MPNs include allogeneic matosis . ) BMP -6 and hemojuvelin interact to modulate hematopoietic cell transplantation (HCT ) and Janus kinase hepcidin expression . Some methods , compounds and / or ( JAK ) inhibition . Allogeneic HCT is associated with up to compositions of disclosed herein may be used to alter 10 % mortality as well as graft failure and significant side 45 hepcidin levels , thereby regulating bodily iron levels. effects and toxicity. JAK inhibition therapy comprises the Some embodiments of the present invention may com use of Ruxolitinib (Rux , ) a small molecule inhibitor of JAK2 prise hepcidin agonists or hepcidin antagonists . Hepcidin that was approved in 2011 to treat MPNs. Rux is marketed agonists may activate or promote the expression and /or under the names JAKAFI? and JAKAVI® by Incyte phar - physiological action of hepcidin . Such agonists may be maceuticals (Wilmington , Del. ) and Novartis ( Basel , Swit - 50 useful in the treatment or prevention of iron overload due to zerland ) . Although able to improve splenomegaly and low hepcidin levels and /or activity . In some cases, agonists hepatomegaly , Rux is not curative and some studies do not may not reverse established iron overload , butmay diminish show much benefit (Odenike , O ., 2013 . Hematology . 2013 iron damage to tissues. Some hepcidin agonists of the ( 1 ) : 545 - 52 , the contents of which are herein incorporated by present invention may elevate production of hepcidin reference in their entirety . ) 55 through activating and /or enhancing BMP - 6 /hemojuvelin In some cases, compounds and / or compositions of the signaling . invention may be used to treat myeloproliferative disorders, Hepcidin antagonists may block or reduce the expression including , but not limited to primary myelofibrosis , second - and/ or physiological action of hepcidin . Such antagonists ary myelofibrosis , essential thrombocythemia , polycythemia may be useful in the case of iron deficiency due to high vera , idiopathic myelofibrosis and chronic myeloid leuke - 60 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 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 . 65 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 US 9 ,758 ,576 B2 125 126 referred to as anemia of inflammation (AI ) . Subjects with load in mouse model of B - thalassemia and a mouse model of ACD , may suffer from chronic renal failure or acute inflam - hemochromatosis ( Viatte et al. , 2006 , Blood. 107: 2952. ) mation due to rheumatoid arthritis , cancer , infection , etc . GDF - 15 levels in circulation have been found to nega Subjects suffering from ACD typically comprise elevated tively correlate with hepcidin levels , suggesting a role for levels of hepcidin and impaired erythropoiesis . In a study by 5 GDF - 15 in iron loading and /or metabolism (Finkenstedt et Sasu et al (Sasu et al. , 2010 . Blood . 115 ( 17 ) :3616 - 24 , ) an al. , 2008 . British Journal of Haematology . 144 : 789 - 93 , the antibody with high affinity for hepcidin was effective in contents of which are herein incorporated by reference in treating murine anemia in a mouse model of inflammation . their entirety . ) Transcription of the gene encoding GDF - 15 The studies found that the most effective treatments may be upregulated under stress and /or hypoxic conditions. involved combining the antibody with an erythropoiesis - 10 In some cases, compounds and / or compositions of the stimulating agent (ESA . ) Accordingly, some compounds present invention may be used to treat subjects suffering and / or compositions of the present invention may be used in from iron disorders and /or anemias by altering GDF - 15 combination with ESAs to increase efficacy . Current anti signaling activity . Such compounds and / or compositions hepcidininclude Ab1239antibodies (Amgen being testedThousand for treatmentOaks, Calif of. ) ACDand 15 may comprise antibodies capable of stabilizing or destabi LY2787106 (Eli Lilly , Indianapolis , Ind .) FG4592 (Fibro lizing the GDF - 15 GPC or through modulation of one or Gen , San Francisco , Calif . ) is a small molecule inhibitor of more interaction between GDF - 15 and one or more co hypoxia - inducible factor (HIF ) that is also currently used to factor. treat anemia . Hemochromatosis is a disease characterized by iron over In some cases, compounds and /or compositions of the 20 load due to hyperabsorption of dietary iron . In hereditary present invention may be used to treat subjects with iron hemochromatosis (HH , ) this overload is caused by inheri deficiency anemia ( IDA ) associated with gastric bypass tance of a common autosomal recessive copy of the HFE surgery and / or inflammatory bowel disease ( IBD . ) Gastric gene from both parents . In such cases, iron may be over bypass surgery leaves subjects with a reduced ability to loaded in plasma as well as in organs and tissues, including, metabolize iron due to bypass of the proximal gastric pouch 25 but not limited to the pancreas, liver and skin , leading to and duodenum (Warsh et al. , 2013 , the contents of which are damage caused by iron deposits ( Tussing -Humphreys et al , herein incorporated by reference in their entirety .) IBD 2013 .) Current therapies for HH may include phlebotomy, patients often suffer from iron deficiency due to intestinal multiple times per year . In some embodiments , compounds blood loss and decreased absorption due to inflammation and /or compositions of the present invention may be used to Some compounds and / or compositions of the present 30 treat HH by modulating subject iron levels . invention may be used to treat subjects suffering from Mutations in the hepcidin (HAMP ) and /or hemojuvelin iron - refractory iron deficiency anemia ( IRIDA . ) IRIDA is a (HFE2 ) genes are responsible for a severe form of genetic disease caused by a defect in the enzyme hemochromatosis known as juvenile hemochromatosis Matriptase - 2 (De Falco , L . et al. , 2013 , the contents of which (Roetto et al ., 2003; Papanikolauou et al. , 2004 . ) Some are herein incorporated by reference in their entirety . ) 35 mutations of hemojuvelin associated with juvenile Matriptase- 2 , a transmembrane serine protease , is an impor- hemochromatosis lead to protein misfolding and reduce tant hepcidin regulator. Matriptase - 2 is capable of enzymatic hemojuvelin secretion from the cell, thus decreasing overall cleavage of hemojuvelin . Subjects with defective hemojuvelin signaling activity . Other mutations affect Matriptase - 2 activity have elevated levels of hemojuvelin , hemojuvelin interactions with other signaling molecules . due to lack of degradation , and therefore hepcidin expres - 40 Hemojuvelin comprising the mutation G99R , for example , sion remains high and iron levels are reduced . Characteris - is unable to bind BMP- 2 . Hemojuvelin comprising the tics of the disease include , but are not limited to microcytic mutation L101P is unable to associate with either BMP - 2 or hypochromic anemia , low saturation of transferrin and nor - neogenin . Some therapeutic embodiments of the present mal to high levels of hepcidin . Some subjects with IRIDA invention may comprise the modulation of hemojuvelin are diagnosed soon after birth , but many are not diagnosed 45 signaling . until adulthood . Treatments described herein may be used to During chemotherapy , cell division is temporarily halted modulate irregular hepcidin levels associated with IRIDA . to prevent the growth and spread of cancerous cells . An Iron overloading anemias can occur as a result of blood unfortunate side effect is the loss of red blood cells which transfusion . Excess iron associated with transfused blood depend on active cell division of bone marrow cells . In some cannot be secreted naturally and requires additional treat - 50 embodiments , compounds and / or compositions of the pres ments for removal, such as chelation therapy. Such therapy ent invention may be used to treat anemia associated che is generally not well tolerated and may comprise many side motherapy . effects. Thus , there is a clinical need for new , better tolerated In some cases, compounds and/ or compositions of the therapies . Additional therapies include EXJADE? , for the present invention may be combined with any of the thera treatment of patients , age 10 and older , with non - transfu - 55 peutics described herein to increase efficacy . sion -dependent thalassemia (NTDT ) syndromes . Also Therapeutics for Anemia , Thrombocytopenia and Neutrope included is ACE - 536 , a ligand trap that blocks TGF - B nia superfamily members . Both EXJADE and ACE -536 are During chemotherapy , cell division is temporarily halted known to elevate erythropoiesis . In some embodiments , to prevent the growth and spread of cancerous cells. An compounds and / or compositions of the present invention 60 unfortunate side effect is the loss of red blood cells, platelets may be used to control iron overloading . Some such embodi- and white blood cells which depend on active cell division ments may function to redistribute iron from parenchyma to of bone marrow cells . In some embodiments , compounds macrophages where iron is better tolerated . In some cases and /or compositions of the present invention may be this may be carried out through elevation of hepcidin levels . designed to treat patients suffering from anemia ( the loss of In studies by Gardenghi et al (Gardenghi et al. , 2010 , JCI. 65 red blood cells ) , thrombocytopenia ( a decrease in the num 120 ( 12 ) :4466 -77 , ) overexpression of murine hepcidin was ber of platelets ) and /or neutropenia ( a decrease in the able to increase hemoglobin levels and decrease iron over - number of neutrophils ) . US 9 , 758 ,576 B2 127 128 Therapeutics for Cancer nant hemangioendothelioma, malignant schwannoma, Various cancers may be treated with compounds and /or osteosarcoma, and chondrosarcoma. compositions of the present invention . As used herein , the In some embodiments , compositions and methods of the term “ cancer ” refers to any of various malignant neoplasms invention may be used to treat one or more types of cancer characterized by the proliferation of anaplastic cells that 5 or cancer- related conditions that may include , but are not tend to invade surrounding tissue and metastasize to new limited to colon cancer , renal cancer , breast cancer , malig body sites and also refers to the pathological condition nant melanoma and glioblastomas (Schlingensiepen et al ., 2008 ; Ouhtit et al ., 2013. ) characterized by such malignant neoplastic growths. Can High - grade gliomas ( e . g . anaplastic astrocytomas and cers may be tumors or hematological malignancies , and 10 glioblastomas ) make up around 60 % of malignant brain include but are not limited to , all types of lymphomas/ tumors . TGF - B2 has been found to be overexpressed in over leukemias, carcinomas and sarcomas , such as those cancers 90 % of such gliomas and expression levels correlate with or tumors found in the anus , bladder, bile duct, bone, brain , tumor progression . Further, studies using TGF- B2 reduction breast , cervix , colon /rectum , endometrium , esophagus, eye , at the mRNA level in cancer patients showed significant gallbladder , head and neck , liver, kidney , larynx , lung , 15 improvement in tumor outcome ( Bogdahn et al ., 2010 . ) In mediastinum ( chest ) , mouth , ovaries , pancreas, penis , pros - light of these studies , some compositions of the present tate , skin , small intestine , stomach , spinal marrow , tailbone , invention may be used therapeutically to treat individuals testicles, thyroid and uterus. with high - grade gliomas. Such compositions may act to In cancer, TGF - B may be either growth promoting or lower the levels of free TGF -B2 and /or the levels of TGF -B2 growth inhibitory . As an example , in pancreatic cancers , 20 activity . SMAD4 wild type tumors may experience inhibited growth In some cases, TGF - B2 activity may contribute to tumor in response to TGF - B , but as the disease progresses , con - development through modulation of metastasis, angiogen stitutively activated type II receptor is typically present. esis , proliferation and / or immunosuppressive functions that Additionally , there are SMAD4 -null pancreatic cancers . In impair immunological tumor surveillance ( Schlingensiepen some embodiments , compounds and/ or compositions of the 25 et al. , 2008 . ) A study by Reed et al (Reed et al. , 1994 ) present invention are designed to selectively target compo - demonstrated TGF -B2 mRNA expression in a large percent nents of TGF - B signaling pathways that function uniquely in age ofmelanocytic lesions including primary invasive mela one or more forms of cancer . Leukemias , or cancers of the nomas and metastatic melanomas . Some compounds and /or blood or bone marrow that are characterized by an abnormal compositions of the present invention may be used to proliferation of white blood cells i . e ., leukocytes , can be 30 modulate TGF -B2 activity and /or levels in such lesions and divided into four major classifications including Acute lym - or prevent lesion formation . Melanoma cell growth in the phoblastic leukemia (ALL ), Chronic lymphocytic leukemia brain parenchyma has also been shown to be influenced by ( CLL ) , Acute myelogenous leukemia or acute myeloid leu - TGF - B2 activity ( Zhang et al. , 2009 . ) Some compounds kemia ( AML ) ( AML with translocations between chromo and /or compositions of the present invention may be used to some 10 and 11 ?t ( 10 , 11 ) , chromosome 8 and 21 ?t( 8 ; 21 ) ], 35 prevent or control such cell growth through modulation of chromosome 15 and 17 ?t ( 15 ; 17 ) ] , and inversions in chro - TGF -B2 activity and / or levels . mosome 16 ?inv ( 16 ) ); AML with multilineage dysplasia , Among females worldwide , breast cancer is the most which includes patients who have had a prior myelodys - prevalent form of cancer. Breast cancer metastasis is medi plastic syndrome (MDS ) or myeloproliferative disease that ated in part through interactions between cancer cells and transforms into AML , AML and myelodysplastic syndrome 40 extracellular matrix components , such as hyaluronic acid (MDS ), therapy - related , which category includes patients (HA .) CD44 has been shown to be the major receptor for HA who have had prior chemotherapy and / or radiation and on cancer cells (Ouhtit et al . , 2013 . ) The interaction between subsequently develop AML or MDS ; d ) AML not otherwise CD44 and HA leads to modulation of cell motility , survival categorized , which includes subtypes ofAML that do not fall adhesion and proliferation . TGF -B2 transcription is also into the above categories ; and e ) Acute leukemias of 45 upregulated by CD44 signaling activity and is believe to ambiguous lineage , which occur when the leukemic cells contribute to resulting changes in cell motility . Unfortu cannot be classified as either myeloid or lymphoid cells , or nately , current therapies have limited efficacy and many where both types of cells are present ); and Chronic myel carry adverse effects due to a lack of specificity . In some ogenous leukemia (CML ) . cases , compounds and / or compositions of the present inven The types of carcinomas include, but are not limited to , 50 tion may be used to alter cellular activities induced by papilloma/ carcinoma, choriocarcinoma, endodermal sinus TGF -B2 upregulation . tumor, teratoma , adenoma /adenocarcinoma , melanoma, The invention further relates to the use of compounds fibroma, lipoma , leiomyoma , rhabdomyoma, mesothelioma , and /or compositions of the present invention for treating one angioma, osteoma, chondroma, glioma, lymphoma/ leuke or more forms of cancer, in combination with other phar mia , squamous cell carcinoma, small cell carcinoma, large 55 maceuticals and /or other therapeutic methods , e . g ., with cell undifferentiated carcinomas , basal cell carcinoma and known pharmaceuticals and / or known therapeutic methods , sinonasal undifferentiated carcinoma. such as, for example , those which are currently employed The types of sarcomas include , but are not limited to , soft for treating these disorders . For example , the compounds tissue sarcoma such as alveolar soft part sarcoma, angiosar - and /or compositions of the present invention can also be coma, dermatofibrosarcoma, desmoid tumor, desmoplastic 60 administered in conjunction with one or more additional 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 - 65 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) , US 9 ,758 ,576 B2 129 130 rituximab , interferon -a , anthracycline drugs ( such as dauno - Biological therapies use the body ' s immune system , rubicin or idarubicin ), L - asparaginase , doxorubicin , cyclo either directly or indirectly , to fight cancer or to lessen the phosphamide , doxorubicin , bleomycin , fludarabine , etopo side effects that may be caused by some cancer treatments . side , pentostatin , or cladribine ), bone marrow transplant, In some embodiments , compounds and / or compositions of stem cell transplant, radiation therapy, anti- metabolite drugs 5 the present invention may be considered biological therapies (methotrexate and 6 -mercaptopurine ), or any combination in that they may stimulate immune system action against one thereof. or more tumor, for example . However, this approach may also be considered with other such biological approaches , Radiation therapy (also called radiotherapy, X -ray e . g ., immune response modifying therapies such as the therapy, or irradiation ) is the use of ionizing radiation to kill 10 administration of interferons, interleukins, colony -stimulat cancer cells and shrink tumors . Radiation therapy can be ing factors , other monoclonal antibodies , vaccines, gene administered externally via external beam radiotherapy therapy , and nonspecific immunomodulating agents are also (EBRT ) or internally via brachytherapy . The effects of envisioned as anti- cancer therapies to be combined with the radiation therapy are localized and confined to the region compounds and /or compositions of the present invention . being treated . Radiation therapy may be used to treat almost 15 Small molecule targeted therapy drugs are generally every type of solid tumor, including cancers of the brain , inhibitors of enzymatic domains on mutated , overexpressed , breast, cervix , larynx , lung , pancreas, prostate , skin , stom - or otherwise critical proteins within the cancer cell, such as ach , uterus, or soft tissue sarcomas. Radiation is also used to tyrosine kinase inhibitors imatinib (Gleevec / Glivec ) and treat leukemia and lymphoma. gefitinib (Iressa ) . Examples of monoclonal antibody thera Chemotherapy is the treatment of cancer with drugs that 20 pies that can be used with compounds and / or compositions can destroy cancer cells . In current usage , the term “ che - of the present invention include, but are not limited to , the motherapy ” usually refers to cytotoxic drugs which affect anti -HER2 / neu antibody trastuzumab (Herceptin ) used in rapidly dividing cells in general , in contrast with targeted breast cancer, and the anti -CD20 antibody rituximab , used in therapy . Chemotherapy drugs interfere with cell division in a variety of B -cell malignancies . The growth of some various possible ways , e . g . with the duplication of DNA or 25 cancers can be inhibited by providing or blocking certain the separation of newly formed chromosomes . Most forms hormones . Common examples of hormone - sensitive tumors of chemotherapy target all rapidly dividing cells and are not include certain types of breast and prostate cancers . Remov specific to cancer cells, although some degree of specificity i ng or blocking estrogen or testosterone is often an important may come from the inability of many cancer cells to repair additional treatment. In certain cancers, administration of DNA damage , while normal cells generally can . 30 hormone agonists, such as progestogens may be therapeu Most chemotherapy regimens are given in combination . tically beneficial. Exemplary chemotherapeutic agents include, but are not Cancer immunotherapy refers to a diverse set of thera limited to , 5 -FU Enhancer, 9 - AC , AG2037 , AG3340 , Aggre - peutic strategies designed to induce the patient' s own canase Inhibitor , Aminoglutethimide, Amsacrine immune system to fight the tumor, and include, but are not (m -AMSA ), Asparaginase , Azacitidine , Batimastat (BB94 ) , 35 limited to , intravesical BCG immunotherapy for superficial BAY 12 - 9566 , BCH - 4556 , Bis - Naphtalimide, Busulfan , bladder cancer , vaccines to generate specific immune Capecitabine, Carboplatin , Carmustaine + Polifepr Osan , responses, such as for malignant melanoma and renal cell cdk4 /cdk2 inhibitors, Chlorombucil , CI- 994 , Cisplatin , carcinoma, and the use of Sipuleucel- T for prostate cancer, Cladribine , CS -682 , Cytarabine HC1, D2163 , Dactinomycin , in which dendritic cells from the patient are loaded with Daunorubicin HCI, DepoCyt , Dexifosamide, Docetaxel , 40 prostatic acid phosphatase peptides to induce a specific Dolastain , Doxifluridine, Doxorubicin , DX8951f, E 7070 , immune response against prostate -derived cells . EGFR , Epirubicin , Erythropoietin , Estramustine phosphate In some embodiments , compounds and / or compositions sodium , Etoposide ( VP16 - 213 ) , Farnesyl Transferase Inhibi- of the present invention are designed to prevent T cell tor, FK 317 , Flavopiridol, Floxuridine , Fludarabine , Fluo - inhibition . Such compounds and / or compositions may pre rouracil ( 5 -FU ) , Flutamide , Fragyline , Gemcitabine , Hex - 45 vent the dissociation of growth factors from the prodomain amethylmelamine (HMM ) , Hydroxyurea of the GPC or from extracellular matrix and / or cellular (hydroxycarbamide ) , Ifosfamide , Interferon Alfa - 2a , Inter - matrix components including, but not limited to GARPs , feron Alfa - 2b , Interleukin - 2 , Irinotecan , ISI 641, Krestin , fibrillins or LTBPs. Lemonal DP 2202, Leuprolide acetate (LHRH -releasing Therapeutics for Bone Healing factor analogue ), Levamisole , LiGLA ( lithium - gamma lino - 50 Compounds and /or compositions of the present invention lenate ) , Lodine Seeds , Lometexol, Lomustine (CCNU ) , may be used to treat bone disorders and /or improve bone Marimistat , Mechlorethamine HCl (nitrogen mustard ), healing or repair . Cellular remodeling of bone is a lifelong Megestrol acetate , Meglamine GLA , Mercaptopurine, process that helps to maintain skeletal integrity . This process Mesna , Mitoguazone (methyl -GAG ; methyl glyoxal bis involves cycles of osteoclastic bone resorption and new guanylhydrazone; MGBG ) , Mitotane ( 0 .p ’ - DDD ) , Mitoxan - 55 bone formation that function to repair defects and areas of trone , Mitoxantrone HC1, MMI 270 , MMP, MTA /LY weakness in bone. TGF - beta family members , preferably 231514 , Octreotide , ODN 698 , OK -432 , Oral Platinum , Oral BMPs, are thought to be important factors in coupling the Taxoid , Paclitaxel ( TAXOL® ), PARP Inhibitors, PD processes of resorption and formation by osteoclasts . TGF 183805 , Pentostatin ( 2 ' deoxycoformycin ) , PKC 412 , Pli - beta family members are prevalent in the bone matrix and camycin , Procarbazine HCI, PSC 833 , Ralitrexed , RAS 60 upregulated by bone injury. TGF -beta family members are Farnesyl Transferase Inhibitor, RAS Oncogene Inhibitor, also believed to impart strength to the fully formed bone 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- 65 and disease . fate , Vindesine sulfate , VX -710 , VX -853 , YM 116 , ZD 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 , US 9 ,758 ,576 B2 131 132 genetic and / or acquired . Such diseases and / or disorders Such conditions include, but are not limited to coronary include, but are not limited to , bone cysts , infectious arthri artery disease , stroke , diabetes and chronic wounds. tis , Paget 's disease of the bone , Osgood -Schlatter disease, Therapeutics for Orphan Indications and Diseases Kohler 's bone disease , bone spurs ( osteophytes ), bone The compounds and / or compositions of the present inven tumors, craniosynostosis , fibrodysplasia ossificans progres - 5 tion may be used to treat orphan indications and /or diseases. sive, fibrous dysplasia , giant cell tumor of bone, hypophos Such diseases includeMarfan ' s syndrome. This syndrome is a connective tissue disorder , effecting bodily growth and phatasia , Klippel- Feil syndrome, metabolic bone disease, development. Tissues and organs that are most severely osteoarthritis , osteitis deformans, osteitis fibrosa cystica , compromised include the heart, blood vessels , bones , eyes , osteitis pubis , condensing osteitis , osteitis condensans ilii , 10 lungs and connective tissue surrounding the spinal cord . osteochondritis dissecans, osteochondroma, osteogenesis Unfortunately , the effects can be life threatening . Marfan ' s imperfecta , osteomalacia , osteomyelitis , osteopenia , osteo syndrome is caused by a genetic mutation in the gene that petrosis , osteoporosis , osteosarcoma, porotic hyperostosis , produces fibrillin , a major component of bodily connective primary hyperparathyroidism , renal osteodystrophy and tissue . Latent TGF - B binding protein (LTBP ) is an important water on the knee. 15 regulator of TGF - B signaling that exhibits close identity to Mouse models for evaluating the effectiveness of thera fibrillin protein family members . Functional LTBP is peutics on bone development and repair are well known in required for controlling the release of active TGF - B (Oklu . the art . In one such model demonstrated by Mohammad , et R . et al. , The latent transforming growth factor beta binding al. (Mohammad , K . S . et al. , Pharmacologic inhibition of the protein (LTBP ) family . Biochem J . 2000 Dec . 15 ; 352 Pt TGF -beta type I receptor kinase has anabolic and anti - 20 3 :601 - 10 ) . In some embodiments , compounds and /or com catabolic effects on bone . PLoS One . 2009 ; 4 ( 4 ) : e5275 . positions of the present invention are designed to alter the Epub 2008 Apr. 16 ) , inhibition of the TGF - beta type I release profile of TGF - B . In such embodiments, compounds receptor was carried out in C57B1/ 6 mice through twice and /or compositions may comprise antibodies characterized daily administration of a potent inhibitor, SD - 208 , by gav - as inhibitory antibodies . age . Subsequently , bone mineral density (BMD ) was ana - 25 In some embodiments , compounds and / or compositions lyzed using a PIXImus mouse densitometer (GE Lunar II , of the present invention may be useful in the treatment of Faxitron Corp . , Wheeling , Ill . ). Changes in BMD are Camurati- Engelmann disease (CED ) . This disease primarily expressed as a percentage change in the area scanned . The affects the bones, resulting in increased bone density . Espe study found that after 6 weeks of treatment , male mice cially affected are the long bones of the legs and arms ; exhibited a 4 . 12 % increase in bone accrual while female 30 however , the bones of the skill and hips can also be affected . mice exhibited a 5 . 2 % increase . The disease results in leg and arm pain as well as a variety Compounds and / or compositions of the present invention of other symptoms. CED is very rare , reported in approxi may be useful as therapies for simple or complex bone mately 200 individuals worldwide and is caused by a fractures and /or bone repair . In such treatments , compounds mutation in the TGF - B gene . TGF - ß produced in the bodies and/ or compositions of the present invention may be intro - 35 of these individuals has a defective prodomain , leading to duced to the site of injury directly or through the incorpo - overactive TGF - B signaling (Janssens , K . et al. , Transform ration into implantation devices and coated biomatrices . ing growth factor -beta 1 mutations in Camurati - Engelmann Additionally , treatments are contemplated in which com disease lead to increased signaling by altering either acti pounds and / or compositions of the present invention are vation or secretion of the mutant protein . J Biol Chem . 2003 supplied together with one or more GPC in a treatment area , 40 Feb . 28 ; 278 ( 9 ): 7718 - 24 . Epub 2002 Dec . 18 ). As described facilitating the slow release of one or more growth factors by Shi et al. , (Shi , M . et al. , Latent TGF - beta structure and from such GPCs. activation . Nature . 2011 Jun . 15 ; 474 ( 7351) : 343 - 9 , ) among Therapeutics for Angiogenic and Endothelial Proliferation CED mutations, Y81H disrupts an a2 -helix residue that Conditions cradles the TGF - B fingers . The charge - reversal E169K and The compounds and/ or compositions of the present inven - 45 H222D mutations disrupt a pH - regulated salt bridge tion may be used to treat angiogenic and endothelial prolif- between Glu 169 and His 222 in the dimerization interface eration syndromes , diseases or disorders . The term “ angio - of the prodomain . Residue Arg 218 is substantially buried : genesis ” , as used herein refers to the formation and /or it forms a cation - ot bond with Tyr 171 and salt bridges across reorganization of new blood vessels . Angiogenic disease the dimer interface with residue Asp 226 of the “ bowtie ' involves the loss of control over angiogenesis in the body. In 50 region of the growth factor prodomain complex (GPC ) . such cases, blood vessel growth , formation or reorganization Moreover, CED mutations in Cys 223 and Cys 225 demon may be overactive ( including during tumor growth and strate the importance of disulphide bonds in the bowtie cancer where uncontrolled cell growth requires increased region for holding TGF - B in inactive form . In this embodi blood supply ) or insufficient to sustain healthy tissues. Such ment , compounds and / or compositions of the present inven conditions may include , but are not limited to angiomas, 55 tion comprising one or more inhibitory antibodies would angiosarcomas, telangiectasia , lymphangioma, congenital serve to alleviate symptoms. In some embodiments, admin vascular anomalies, tumor angiogenesis and vascular struc - istration would be to the neonate subject. tures after surgery . Excessive angiogenesis is noted in can - Therapeutics for Immune and Autoimmune Diseases and cer , macular degeneration , diabetic blindness , rheumatoid Disorders arthritis , psoriasis as well as many other conditions. Exces - 60 Compounds and/ or compositions of the present invention sive angiogenesis is often promoted by excessive angiogenic may be used to treat immune and autoimmune disorders . growth factor expression . Compounds and / or compositions Such disorders include , but are not limited to Acute Dis 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 65 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 . lipid syndrome (APS ), Autoimmune angioedema, Autoim US 9 , 758 ,576 B2 133 134 mune aplastic anemia , Autoimmune dysautonomia , Autoim - (UCTD ), Uveitis , Vesiculobullous dermatosis , Vasculitis , mune hepatitis , Autoimmune hyperlipidemia , Autoimmune Vitiligo and Wegener' s granulomatosis ( also known as immunodeficiency, Autoimmune inner ear disease (AIED ), Granulomatosis with Polyangiitis (GPA ) ) . Autoimmune myocarditis , Autoimmune pancreatitis, Auto TGF - B plays an active role in leukocyte differentiation , immune retinopathy , Autoimmune thrombocytopenic pur - 5 proliferation and activation making it an important factor in pura (ATP ) , Autoimmune thyroid disease, Autoimmune urti - immune and autoimmune diseases . Additionally , TGF - B caria , Axonal & neuronal neuropathies, Balo disease , promotes chemotaxis of leukocytes and influences adhesion Behcet' s disease , Bullous pemphigoid , Cardiomyopathy, molecule- mediated localization . A role for TGF- B in cardiac , Castleman disease , Celiac disease , Chagas disease , Chronic pulmonary and gastric inflammation has been demonstrated . fatigue syndrome, Chronic inflammatory demyelinating 10 Furthermore , SMAD3 - deficient mice are prone to chronic polyneuropathy (CIDP ), Chronic recurrent multifocal osto - mucosal infections as a result of T -cell activation impair myelitis (CRMO ), Churg - Strauss syndrome, Cicatricialm ent and reduced mucosal immunity (Blobe , G . C . et al. , pemphigoid /benign mucosal pemphigoid , Crohn ' s disease , Role of transforming growth factor beta in human disease . Cogans syndrome, Cold agglutinin disease , Congenital heart N Engl J Med . 2000 May 4 ; 342 ( 18 ): 1350 - 8 ) . As an immu block , Coxsackie myocarditis , CREST disease , Essential 15 nosuppressant, TGF - B has been shown to both inhibit the mixed cryoglobulinemia , Demyelinating neuropathies , Der - function of inflammatory cells as well as enhance the matitis herpetiformis, Dermatomyositis , Devic ' s disease function of regulatory T cells . Recent studies have shown ( neuromyelitis optica ), Diabetes Type I , Discoid lupus , that the latent TGF - B growth factor prodomain complex Dressler ' s syndrome, Endometriosis , Eosinophilic esoph - (GPC ) binds to regulatory T cells through an interaction with agitis, Eosinophilic fasciitis , Erythema nodosum , Experi - 20 the Glycoprotein - A repetitions anonymous protein (GARP ) . mental allergic encephalomyelitis , Evans syndrome, Fibro - In fact , GARP is necessary for TGF - B association with T myalgia , Fibrosing alveolitis, Giant cell arteritis ( temporal cells ( Tran , D . Q . et al. , GARP (LRRC32 ) is essential for the arteritis ) , Glomerulonephritis , Goodpasture 's syndrome, surface expression of latent TGF- B on platelets and activated Granulomatosis with Polyangiitis (GPA ) see Wegener ’ s , FOXP3+ regulatory T cells . PNAS . 2009 . 106 ( 32 ) : 13445 Graves' disease , Guillain - Barre syndrome, Hashimoto ' s 25 50 ) . This interaction provides the platform necessary to encephalitis , Hashimoto ' s thyroiditis , Hemolytic anemia , release active TGF - ß from the GPC in an integrin -dependent Henoch - Schonlein purpura , Herpes gestationis , Hypogam - manner (Wang , R . et al . , GARP regulates the bioavailability maglobulinemia , Idiopathic thrombocytopenic purpura and activation of TGF - B . Mol Biol Cell . 2012 March ; (ITP ) , IgA nephropathy, IgG4- related sclerosing disease , 23 (6 ): 1129 - 39 . Epub 2012 Jan . 25 ) . In some embodiments , Immunoregulatory lipoproteins , Inclusion body myositis , 30 compounds and / or compositions of the present invention Insulin -dependent diabetes ( typel) , Interstitial cystitis , Juve modulate the interaction between GARP and TGF - B . Such nile arthritis , Juvenile diabetes, Kawasaki syndrome, Lam - modulation may selectively modulate T cell activity for bert- Eaton syndrome, Large vessel vasculopathy, Leukocy - treatment of disease ( e . g . autoimmune disease and / or can toclastic vasculitis , Lichen planus, Lichen sclerosus , cer . ) In some embodiments , compounds and / or composi Ligneous conjunctivitis, Linear IgA disease (LAD ) , Lupus 35 tions of the present invention may be used for the treatment ( SLE ) , Lyme disease , chronic , Meniere ' s disease , Micro - of immune and / or autoimmune disorders . In some embodi scopic polyangiitis , Mixed connective tissue disease ments, compounds and / or compositions of the present (MCTD ) , Mooren ’ s ulcer, Mucha -Habermann disease , Mul- invention may specifically target GARP -bound GPC , GARP tiple endocrine neoplasia syndromes, Multiple sclerosis , or the interaction site between GARP and the GPC . In some Myositis , Myasthenia gravis , Narcolepsy, Neuromyelitis 40 embodiments , compounds and /or compositions of the pres optica (Devic ' s ), Neutropenia , Ocular cicatricial pemphig ent invention comprising antibodies are designed to promote oid , Optic neuritis , Palindromic rheumatism , PANDAS (Pe - release of growth factors ( including, but not limited to diatric Autoimmune Neuropsychiatric Disorders Associated TGF - B ) from GARP- bound GPCs while not affecting with Streptococcus ) , Paraneoplastic cerebellar degeneration , growth factor release from LTBP -bound GPCs. Treatment of Paroxysmal nocturnal hemoglobinuria (PNH ) , Parry Rom - 45 immune and autoimmune disorders with compounds and/ or berg syndrome, Parsonnage - Turner syndrome, Pars planitis compositions of the present invention may be in combina (peripheral uveitis ) , Pemphigus , Peripheral neuropathy, tion with standard of care ( SOC ) or synergistic combinations Perivenous encephalomyelitis , Pernicious anemia , POEMS or with companion diagnostics . syndrome, Polyarteritis nodosa , Type I , II , & III autoim - Therapeutics for Infectious Agents mune polyglandular syndromes, Polyendocrinopathies , 50 In some embodiments , compounds and/ or compositions Polymyalgia rheumatica , Polymyositis , Postmyocardial of the present invention may be useful for treatment of infarction syndrome, Postpericardiotomy syndrome, Proges - infectious diseases and / or disorders, for example , in subjects terone dermatitis , Primary biliary cirrhosis , Primary scleros- with one or more infections. In some embodiments , subjects ing cholangitis , Psoriasis , Psoriatic arthritis , Idiopathic Pul- have one or more infection or are at risk of developing one monary fibrosis , Pyoderma gangrenosum , Pure red cell 55 or more infection . As used herein , the term “ infection ” refers aplasia , Raynauds phenomenon , Reactive arthritis , Reflex to a disease or condition in a host attributable to the presence sympathetic dystrophy , Reiter ' s syndrome , Relapsing poly - of one or more foreign organism or agent capable of chondritis , Restless legs syndrome, Retroperitoneal fibrosis , reproduction within the host . Infections typically comprise Rheumatic fever, Rheumatoid arthritis , Sarcoidosis , breaching of one or more normal mucosal or other tissue Schmidt syndrome, Scleritis , Scleroderma, Sjogren 's syn - 60 barriers by one or more infectious organisms or agents . drome, Small vessel vasculopathy , Sperm & testicular auto - Subjects having one or more infection are subjects that immunity , Stiff person syndrome, Subacute bacterial endo - comprise one or more objectively measurable infectious 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, 65 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 exposure to one or more infectious organisms or agents . In US 9 , 758 ,576 B2 135 136 some embodiments , subjects at risk of having infections al. , Inhibitory effect of blocking TGF -beta / Smad signal on may also include subjects with conditions associated with injury - induced fibrosis of corneal endothelium . Mol Vis . impaired abilities to mount immune responses to infectious 2008 ; 14 :2272 -81 . Epub 2008 Dec. 11 ; Carrington , L . M . et organisms and / or agents , e . g ., subjects with congenital and / al. , Differential regulation of key stages in early corneal or acquired immunodeficiency , subjects undergoing radia - 5 wound healing by TGF - beta isoforms and their inhibitors . tion therapy and /or chemotherapy, subjects with burn inju Invest Ophthalmol Vis Sci. 2006 May ; 47 ( 5 ) : 1886 - 94 . ) ries, subjects with traumatic injuries and subjects Compounds and /or compositions of the present invention undergoing surgery or other invasive medical or dental may be used to modulate TGF -B -related proteins in the procedures . cornea to enable and /or enhance wound healing . Such com Infections are broadly classified as bacterial, viral, fungal, 10 pounds and / or compositions would be welcomed in the field and/ or parasitic based on the category of infectious organ - where previous attempts have been unsuccessful. Mead et al isms and / or agents involved . Other less common types of (Mead , A . L . et al. , Evaluation of anti - TGF -beta2 antibody infection are also known in the art , including , e . g . , infections as a new postoperative anti - scarring agent in glaucoma involving rickettsiae, mycoplasmas, and agents causing surgery. Invest Ophthalmol Vis Sci. 2003 August ; 44 (8 ): scrapie , bovine spongiform encephalopathy (BSE ) , and 15 3394 -401 ) developed anti - TGF -B2 antibodies to prevent prion diseases ( e . g ., kuru and Creutzfeldt -Jacob disease ) . scarring in eye tissues ; however, results of clinical trials Examples of bacteria , viruses, fungi , and parasites which were inconclusive . In some embodiments , compounds and / cause infection are well known in the art . An infection can or compositions of the present invention may be used to be acute , subacute , chronic , or latent, and it can be localized modulate TGF - B2 levels ( free versus GPC - bound ) thereby or systemic . As used herein , the term " chronic infection " 20 providing an alternate method of approaching anti - scarring refers to those infections that are not cleared by the normal therapy . actions of the innate or adaptive immune responses and Therapeutics for Cardiovascular Indications persist in the subject for a long duration of time, on the order I n some embodiments , compounds and /or compositions of weeks, months, and years . A chronic infection may reflect of the present invention may be used to treat one or more latency of the infectious agent, and may include periods in 25 cardiovascular indications , including , but not limited to which no infectious symptoms are present, i . e ., asymptom - cardiac hypertrophy. Cardiac hypertrophy comprises atic periods. Examples of chronic infections include , but are enlargement of the heart due , typically due to increased cell not limited to , HIV infection and herpesvirus infections. volume of cardiac cells (Aurigemma 2006 . N Engl J Med . Furthermore , an infection can be predominantly intracellular 355 ( 3 ) : 308 - 10 . ) Age - related cardiac hypertrophy may be or extracellular during at least one phase of the infectious 30 due , in part , to reduced circulating levels of GDF - 11 . A study organism ' s or agent' s life cycle in the host . by Loffredo et al (Loffredo et al. , 2013 . Cell . 153 : 828 -39 ) Compounds and / or compositions of the present invention found that fusion of the circulatory system between young and additional therapeutic agents may be administered in and old mice had a protective effect with regard to cardiac combination in the same composition ( e . g ., parenterally ) , as hypertrophy. The study identified GDF - 11 as a circulating part of a separate composition or by another method 35 factor that decreased with age in mice and was able to show described herein . that its administration could also reduce cardiac hypertro Therapeutics for Eye Related Diseases, Disorders and / or phy . Some compounds and / or compositions of the present Conditions invention may be used to treat and /or prevent cardiac In some embodiments , compounds and / or compositions atrophy. Such compounds and / or compositions may com of the present invention may be useful in the treatment of 40 prise GDF - 11 agonists that elevate levels of circulating diseases , disorders and / or conditions related to eyes . These GDF - 11 , in some cases through enhancing the dissociation may include, but are not limited to glaucoma, dry eye and / or of GDF - 11 growth factor from latent GPCs . corneal wound healing . In some embodiments , compounds In some embodiments , compositions and methods of the and / or compositions may be useful in the treatment of invention may be used to treat one or more types of arterial glaucoma . Evidence suggests that TGF - B2 is upregulated in 45 disorders . Such disorders may include , but are not limited to glaucoma ( Picht, G . et al. , Transforming growth factor beta the development of aortic aneurysms. Aortic aneurysmsmay 2 levels in the aqueous humor in different types of glaucoma arise from a variety of causes, but most result ultimately in and the relation to filtering bleb development. Graefes Arch the overexpression of TGF -B2 . A study by Boileau et al Clin Exp Ophthalmol. 2001 March 239 ( 3 ): 199 - 207 ; Trip ( Boileau et al. , Nature Genetics Letters . 2012 . 44 ( 8 ): 916 - 23 , athi, R . C . et al. , Aqueous humor in glaucomatous eyes 50 the contents of which are herein incorporated by reference in contains an increased level of TGF -B2 . Exp Eye Res . 1994 their entirety ) uncovered causative mutations in TGF - B2 that December 59 ( 6 ) :723 - 7 .) This includes primary open - angle were associated with some inherited forms of susceptibility glaucoma and juvenile glaucoma . There is also evidence that to thoracic aortic disease . Interestingly , although the muta TGF - B2 may induce senescence - like effects in human tra - tions were predicted to cause haploinsufficiency for TGF - B2 , becular meshwork cells , which control intraocular pressure 55 the aortic tissues of individuals with such mutations com ( often dysfunctional in glaucoma) ( Yu , A . L . et al. , TGF -B2 prised increased levels of TGF - B2 , as determined by immu induces senescence -associated changes in human trabecular nostaining Similar findings were found in aortic tissues from meshwork cells . Invest Ophthalmol Vis Sci . 2010 November individuals suffering from Marfans syndrome (Nataatmadja 51( 11 ) : 5718 - 23 .) In some embodiments , compounds and / or et al ., 2006 . ) In some cases, compounds and / or compositions compositions of the present invention may be used to 60 of the present invention may be used to reduce or prevent decrease the ratio of free TGF -B2 to GPC -bound ( inactive ) elevated TGF -B2 signaling in such instances thereby limit TGF -B2 in or around eye tissues affected by or related to ing aneurysm development and / or progression . 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 65 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 lar injury models may be used . Such models may include US 9 , 758 ,576 B2 137 138 balloon injury models . In some cases , these may be carried Therapeutics for Diabetes out as described in Smith et al ., 1999 . Circ Res. 84 ( 10 ) : Skeletal muscle uses and stores glucose for fuel. Due to 1212 - 22 , the contents of which are herein incorporated by this , skeletalmuscle is an important regulator of circulating reference in their entirety . glucose levels . Uptake of glucose by muscle can be stimu Therapeutics Related to Muscle Disorders and / or Injuries 5 lated by either contraction or by insulin stimulation In some embodiments , compounds and /or compositions (McPherron et al . , 2013 . Adipocyte . 2 ( 2 ) : 92 - 8 , herein incor of the present invention may be used to treat one or more porated by reference in its entirety ) . A recent study by Guo muscle disorders and /or injuries. In some cases, such com - et al (Guo , et al. , 2012 . Diabetes 61 ( 10 ): 2414 - 23 ) found that pounds and /or composition may include , but are not limited when GDF - 8 receptor - deficient mice were crossed with to antibodies that modulate GDF - 8 , GDF - 11 and /or activin 10 A - ZIP /F1 mice ( a lipodistrophic mouse strain , used as a activity . Muscle comprises about 40 - 50 % of total body diabetic model ,) hybrid off - spring showed reduced levels of weight, making it the largest organ in the body . Muscle blood glucose and improved sensitivity to insulin . Hyper disorders may include cachexia ( e . g . muscle wasting . ) phagia ( excessive eating ) was also reduced in these mice . In Muscle wasting may be associated with a variety of diseases some embodiments , compound and/ or compositions of the and catabolic disorders ( e . g . HIV /AIDS , cancer, cancer 15 present invention may be used to treat diabetes and /or cachexia , renal failure , congestive heart failure , muscular hyperphagia . Some such treatments may be used to reduce dystrophy, disuse atrophy , chronic obstructive pulmonary blood glucose and / or improve insulin sensitivity . In some disease , motor neuron disease , trauma, neurodegenerative cases , such treatments may comprise GDF - 8 signaling disease , infection , rheumatoid arthritis , immobilization , dia - antagosists , such as one or more antibodies that prevent betes , etc . ) In such disorders , GDF - 8 and / or activin signal - 20 dissociation of GDF - 8 from its prodomain . ing activity may contribute to muscle catabolism (Han et al. , Therapeutics for Gastro - Intestinal Diseases , Disorders and / 2013 . Int J Biochem Cell Biol . 45 ( 10 ) :2333 - 47 ; Lee . , 2010 . or Conditions Immunol Endocr Metab Agents Med Chem . 10 : 183 - 94 , the In some embodiments , compositions and methods of the contents of each of which are herein incorporated by refer - invention may be used to treat one or more types of ence in their entirety . ) Other muscle disorders may comprise 25 gastro - intestinal (GI ) disorders . Such disorders may include , sarcopenia . Sarcopenia is the progressive loss ofmuscle and but are not limited to inflammatory bowel disease (IBD ) function associated with aging . In the elderly , sarcopenia can ( e . g . Crohn ' s disease and ulcerative colitis . ) cause frailty , weakness , fatigue and loss ofmobility (Morely . TGF - B2 may play a role in gut homeostasis and may have 2012 . Family Practice . 29 : 144 - 148 .) With the aged popula - an anti - inflammatory role , protecting against GI- related dis tion increasing in numbers , sarcopenia is progressively 30 orders such as mucositis and certain forms of colitis . In one becoming a more serious public health concern . A study by study , TGF -B2 was shown to suppress macrophage inflam Hamrick et al (Hamrick et al. , 2010 . 69 ( 3 ) :579 - 83 ) demon - matory responses in the developing intestine and protect strated that GDF - 8 inhibition could repair muscle in a mouse against inflammatory mucosal injury Maheshwari et al. , model of fibula osteotomoy comprising lateral compartment 2011. ) Interestingly , levels of TGF -B2 are high in breast muscle damage . Administration of GDF - 8 propeptides was 35 milk , suggesting that TGF -B2 may function , in some cases , sufficient to increase muscle mass by nearly 20 % as well as topically. Indeed , TGF - B2 in breast milk may attenuate improve fracture healing . Some compounds and / or compo - inflammatory responses (Rautava et al. , 2011 . ) Some com sitions of the present invention may be used to treat muscle pounds , compositions and / or methods of the present inven diseases , disorders and / or injuries by modulating GDF - 8 tion may be used to modulate GI TGF - B2 levels and /or activity . In some cases , compounds of the present invention 40 activity in the maintenance of homeostasis and/ or in the may be GDF - 8 signaling antagonists, preventing or reducing management of GI- related disorders . GDF -8 signaling activity . In some cases , models of GI- related diseases , disorders Inclusion body myositis (IBM ) is a disease characterized and /or conditions may be used to develop and /or test com by progressive muscle loss , typically occurring in mid - to pounds and /or compositions of the invention for treatment of late - life . The disease is thought to occur due to an autoim - 45 GI- related diseases , disorders and / or conditions. In some mune response to autoantigens in the muscle causing T -cell cases , GI injury models may be used . Such injury models invasion of the muscle fiber and resulting in myofiber may include , but are not limited to 2 , 4 ,6 - trinitrobenzenesul destruction (Greenberg 2012. Curr Opin Neurol. 25 ( 5 ): 630 fonic acid ( TNBS ) induced colitis models . Such models may 9 . ) Therapeutic compounds are being investigated , including be carried out as described in Scheiffele, F . et al . , 2002 . Curr Bimagrumab (BYM338 ; Novartis , Basel, Switzerland , ) an 50 Protoc Immunol . Chapter 15 : Unit 15 . 19 , the contents of antibody that targets type II activin receptors , preventing which are herein incorporated by reference in their entirety . GDF - 8 and /or activin signal transduction , thereby stimulat - Veterinary Applications ing muscle production and strengthening (see clinical trial In some embodiments , it is contemplated that composi number NCT01925209 entitled Efficacy and Safety of tions and methods of the invention will find utility in the area Bimagrumab / BYM338 at 52 Weeks on Physical Function , 55 of veterinary care including the care and treatment of Muscle Strength , Mobility in sIBM Patients (RESILIENT . ) ] non - human vertebrates . As described herein , the term “ ver Some compounds and /or compositions of the present inven tebrate ” includes all vertebrates including , but not limited to tion may be used to treat subjects with IBM . In some cases , fish , amphibians , birds , reptiles and mammals ( including, such compounds and/ or compositions may block GDF - 8 but not limited to alpaca , banteng , bison , camel , cat , cattle , activity ( e . g . through stabilization of GDF - 8 GPCs. ) In 60 deer, dog , donkey, gayal, goat, guinea pig , horse , llama, addition to IBM , BYM338 is being investigated for treat- mice , monkeys , mule , pig , rabbit , rats , reindeer, sheep water ment of chronic obstructive pulmonary disease (COPD . ) In buffalo , yak and humans . ) As used herein the term " non 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 - 65 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 . ticated animals raised in an agricultural setting to produce