US 20110159588A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0159588 A1 Lin (43) Pub. Date: Jun. 30, 2011 (54) METHODS FOR MODULATING A PDGF-AA Publication Classification MEDIATED BIOLOGICAL RESPONSE (51) Int. Cl. CI2N 5/02 (2006.01) (76) Inventor: Kui Lin, Foster City, CA (US) (52) U.S. Cl. ........................................................ 435/375 (57) ABSTRACT (21) Appl. No.: 12/980,074 The present invention is directed towards methods and means for modulating PDGF-AA mediated biological responses and is based, at least in part, on examining the association of (22) Filed: Dec. 28, 2010 various proteins with TMEFF2 and identification of PDGF AA as a major growth factor that interacts specifically with Related U.S. Application Data TMEFF2. The invention provides the first evidence that TMEFF2 can function to regulate PDGF signaling, to help (60) Provisional application No. 61/291,030, filed on Dec. illuminate the seemingly conflicting biological roles of 30, 2009. TMEFF2 in human cancers. Patent Application Publication Jun. 30, 2011 Sheet 1 of 15 US 2011/O159588A1 .. 50 100 150 200 250 300 350 8 EGF is: 4 69 135 16 22, 26; 31 319 342 374 F. A h;2 -EGF-7-" TMEF2FL 374 YYYY YYYY a FA ECD-FLAG i 303 : FS is FS i? ex-EGFr fifty TECD-FC i 303 F. i-fift EC kia s: 9. 200 F. C. SPYMattire Protein 40 f if///ESPROCSS//f CEGFC//iiiffiti/ARP/KAAFPISSDCOi PGA/C (SEQ ID AO: 10) F. Patent Application Publication Jun. 30, 2011 Sheet 2 of 15 US 2011/O159588A1 TECD-FLAG, (PDGFor blank Coat IECD-FLAG Coat -e-O-PDGFIPDGF-AA -e-o-FLAG -E-TECD-FLAGPDGF-AA -e- HER2-FC --TECD-FLAGIPDGF-AB ---- HER3-FC -e-IECD-FLAGPDGF-BB . HERA-FC - E3 - BlankiPDGFAA -E- EGFR-FC 3. A. BlankiPDGF-AB -a-TNFR-FC to BlaikiPDG-BE a IECD-FC - 10 0.01 0 1 1 0 100 000 PDGFConcentration (ngiml) PDGFConcentration (nglm) FIG. 2A FIG. 2B 2 PDGF Coat PDGFAA Coat -- PDGF-AA/IECD-FC -- IECD-FC 2- PDGF-ABTECD-FC -- CD4-FC -o- PDGF-BBITECD-FC -- PDGFRB-FC s -- OX40-FC S Of 10 100 7000 O O 100 TECD-Fc Conc. (nglm) AnalyteConc. (nglm) FIG. 2C FIG. 2D Patent Application Publication Jun. 30, 2011 Sheet 3 of 15 US 2011/O159588A1 Iliff 2-f Tiff2-AfC) 343 PDGF-AA -- PDGF-AB kiva 38 - to-Eff pi . ig C | IB: a-PDGFApAb PDGF. A ff Fi. 3 & 38 - \ iP. cy-PDGF path ig iC iB: o-PDGF. Apah 2. PDGF-A 2: - if EFF3-Fi. ii.3 M iPo-TMEFF2pAb '" ififi 2-ACD i8: C-liff2 nAi. 64 5: ... 36 - Patent Application Publication Jun. 30, 2011 Sheet 4 of 15 US 2011/O159588A1 faig offix:)T 374 -- gD-TRAFFF2-Fl giFair + giD-IMEFF2-AFS fill giFS if x:EGF: A?ia: -- giD-IMEFF2-AFS i62 374 -- - - FDGF-AA kia 36 iP. grgi mah 22 - ig iC IB: a-PDGFApAb , fil-A i FIG. 4 ( 36 \ iso-PorpAbiPo-PDGF pi 2 igiC 6 PDGFA 25. gD-TEFF2-Fi i48 8 gi-IRAEFF2. AFS ; iP. (gi mith 84 i: orgi inAi. 5 36. 22 gD-TMEFF2-AFS fill Patent Application Publication Jun. 30, 201 1. Sheet 5 of 15 US 2011/0159588A1 PDGF-AA alone PDGF-AA (10 ng/ml) with TECD-Fc or sRo. f 0.55 0.9 0.5 is 0.8 is 0.7 0.45 s 0.6 0.4 S 0.5 0.35 0.4 0.3 st 0.3 0.2 0.25 0.1 -- O2 0.001 0.01 0 1 1 10 100 0 0 64 3.2 16 80 400 2000 PDGFAA (ng/ml) ng/ml FG. A F.G. 5B PDGF-AB alone PDGF-AB (10 mg/ml) with TECD-Fc or sRo. 1 0.7 0.9 is 0.8 0.6 is 0.7 s 0.6 0.5 S 0.5 0.4 0.4 r 0.3 0.3 0.2. 0. 0.2 0.001 001 0 1 1 10 100 PDGFAB (ng/ml) ng/ml F.G. 5C FIG. 5D Patent Application Publication Jun. 30, 2011 Sheet 6 of 15 US 2011/O159588A1 No proState Cao co o Normal Adjacent to proState Cao o co o Oligodendroglioma o os Benign hypertrophy coop o Glioma or oce Aden0Carcinoma ooooooooooo co e g o Other CanCerS oo ---|--|-- Oe--00 4e+04 8e-04 O 2000 6000 10000 4000 Affymetrix signal intensity Affymetrix signal intensity F.G. 6A FIG. 6B 8000 7000 6000 5000 4000 s 3000 2000 1000 PN Prolif MES FIG. 60 O 1000 2000 3000 4000 O 1000 2000 3000 PDGFA PDGFA FIG. 6E FIG. 6F Patent Application Publication Jun. 30, 2011 Sheet 7 of 15 US 2011/O159588A1 Ovarian Ca Rectalade/IOCa 60 50 is 40 3 30 S. 20 10 ex Si3.33. O one 0.02005 0200.50 OC2005 0.200.50 0.02005 O2OC50 Methylation (average beta) Methylation (average beta) Methylation (average beta) Colonaden0ca Ling Squamous cell Ca ling aden0ca 60 50 is 40 330 S. 20 IO c exigi O sigg is O occo o cle 8 0.02005 0200.50 0.02005 C.200.50 OC2005 0200.50 Methylation (average beta) Alethylation (average beta) Methylation (average beta) Renal papillary cellca essee OC2005 0200.50 Methylation (average beta) FIG. 7 Patent Application Publication Jun. 30, 2011 Sheet 8 of 15 US 2011/O159588A1 mvil WesproC SSWtl Cegfc Willip VIIll iVarp Viklaa fptSlsdcot ptgWinCS gyd 61 drendliflcd tintCKfcgec lirigdtvtCV CCfkCnndyV pVCCSnces y CineCylrdaa 121 CkgQSeil VV Segs Catdag SCSgdgwheg Sget sqkets to di CCfgae Cdedaedv WC 181 VCnidcSgtn finplCasdgk Sydnacqike aSCCkCekie VmslgrCCCn tttittkSedd 241 hyartdyaen anklees are hipCpehyn gfcInhgkCeh SinmepSCr CdacytgohC 301 ekkdysvily v VpgpvrfdyV liaavigtig iavicvvvlic itrkcprSnr ihrd kontgh 361 ySSdnttras trili (SEO TD NO: 1) FIG. 8 Patent Application Publication Jun. 30, 2011 Sheet 9 of 15 US 2011/O159588A1 2 s' -o- PDGF-AA s -- PDGF-AB S 1 -a- PDGF-BB -- PDGF-CC --- PDGFDD 0.5 10 100 1000 TECD-FC (ng/ml) FIG. 9A s -o- PDGF-AA S. -- PDGF-AB -a- PDGF-BB Ok-O-R s 0.1 f 10 100 1000 sRo-bio ng/ml FIG. 9B 0.8 0.6 . S s s .. .. 0.4 : O s: s: 3. 3. 3. 0.00 0.10 020 039 0 78 1.56 3.13 625 12.5025.00 5000 SRO. Lig/ml FIG. 9C Patent Application Publication Jun. 30, 2011 Sheet 10 of 15 US 2011/O159588A1 FIG 10 100 gD-TMEFF2-AFS I/II 10 10 FL2 Patent Application Publication Jun. 30, 2011 Sheet 11 of 15 US 2011/O159588A1 gD-TMEFF2-FL gD-TMEFF2-AFS I Patent Application Publication Jun. 30, 2011 Sheet 12 of 15 US 2011/O159588A1 AdipOSe Adrenal BIOOd VeSSel BOne & (3 BOne marrOW Brain - & BreaSt Colorectal g Endometrium ESOphaguS Head & neCK Heart Kidney Liver Lung & LympOid MuSCle s Ovary six s PanCreaS so c 838 & C. C. XX c PrOState :six&ger gotic coaxco o { {: C { { Skin Small intestine SOft tiSSue i c Stomach IeStiS C Urinary WBC -T - I 0e--00 2e --04 4e–04 6e--04 8e-04 1e--05 Affymetrix signal intensity FIG. 11 Patent Application Publication Jun. 30, 2011 Sheet 13 of 15 US 2011/O159588A1 4000 250 3000 200 150 2000 100 1000 50 ICI 300 250 200 200 150 100 100 50 IC) 25000 2000 20000 1500 15000 1000 10000 5000 500 Prostate All Tissues -u-1 FIG. 12A Number of tissues of each category evaluated TISSue IN) ICI IM) Brain 2333 28 3 Colorectal 336 195 56 ESOphagus 28 25 6 PrOState 71 121 3 Stomach 72 71 12 All tiSSUeS 5177 24. 11 393 FIG. 12B Patent Application Publication Jun. 30, 2011 Sheet 14 of 15 US 2011/O159588A1 ---------------- F. 3 ligi i 3. fit PCS Pros tate Cancer F. 3C F. 3 Patent Application Publication Jun. 30, 2011 Sheet 15 of 15 US 2011/O159588A1 O COad E. luSC gbm El OV a kip Z read luad FIG. 14A 0 0 20 30 40 A 23 P 125382 A 23 P 125387 20 10 O 20 40 60 80 100 O 10 20 30 40 FIG. 14B US 2011/O 159588 A1 Jun. 30, 2011 METHODS FOR MODULATING A PDGF-AA of a variety of growth factors including members of the trans MEDIATED BIOLOGICAL RESPONSE forming growth factor beta (TGF-B family, PDGFs, and vas cular endothelial growth factor (VEGF)) (Kupprion et al., CROSS-REFERENCE TO RELATED 1998: Patel, 1998: Patthy and Nikolics, 1993; Raines et al., APPLICATION 1992) (Chang et al., 2003; Harms and Chang, 2003). 0001. This application claims priority under 35 U.S.C. Section 119(e) and the benefit of U.S. Provisional Applica BRIEF DESCRIPTION OF THE DRAWINGS tion Ser. No. 61/291,030 filed Dec. 30, 2009, the contents of 0006 FIG. 1 Expression and purification of recombinant which is incorporated herein by reference in its entirety. ECD of TMEFF2. (A) 0007 Hydropathy plot of TMEFF2 protein based on the FIELD OF THE INVENTION algorithm of Kyte and Doolittle (Kyte and Doolittle, 1982) and the predicted domain structure based on NT sequencing 0002 The present invention concerns methods and means of the recombinant TECD in this study and Horie et al., 2000 for modulating PDGF-AA mediated biological responses. (Horie et al., 2000). SP signal peptide; FS I, follistatin-like domain I; FS II, follistatin-like domain II: EGF, epidermal BACKGROUND OF THE INVENTION growth factor-like domain; TM, transmembrane domain; 0003 Platelet-derived growth factors (PDGFs) not only N-Gly, potential sites for N-linked glycosylation; GAG, play an important role in developmental and physiological potential site of glycosaminoglycan attachment.