US 20100129929A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0129929 A1 Polakewicz et al. (43) Pub. Date: May 27, 2010
(54) TYROSINE PHOSPHORYLATION SITES (86). PCT No.: PCT/US2007/O16927 (76) Inventors: Roberto Polakewicz, Lexington, S371 (c)(1), MA (US); Charles Farnsworth, (2), (4) Date: Feb. 13, 2010 Concord, MA (US); Ailan Guo, Related U.S. Application Data Burlington, MA (US); Klarisa Rikova, Reading, MA (US); (60) Provisional application No. 60/833,826, filed on Jul. Albrecht Moritz, Salem, MA (US); 27, 2006. Kimberly Lee, Seattle, WA (US); O O Erik Spek, Cambridge, MA (US) Publication Classification (51) Int. Cl. Correspondence Address: GOIN 33/53 (2006.01) Nancy Chiu Wilker, Ph.D. C07K 6/8 (2006.01) Chief Intellectual Property Counsel (52) U.S. Cl...... 436/501; 530/387.9 CELL SIGNALING TECHNOLOGY, INC., 3 Trask Lane (57) ABSTRACT Danvers, MA 01923 (US) The invention discloses 349 novel phosphorylation sites iden tified in carcinoma, peptides (including AQUA peptides) (21) Appl. No.: 12/309,727 comprising a phosphorylation site of the invention, antibod ies specifically bind to a novel phosphorylation site of the (22) PCT Filed: Jul. 27, 2007 invention, and diagnostic and therapeutic uses of the above. Patent Application Publication May 27, 2010 Sheet 1 of 22 US 2010/0129929 A1
FIGURE 1
Crude protein (e.g., cell extractor cellular organelle) or in vitrokinase reaction or purified phosphoprotein
obtain protein fraction (e.g., lysis, organic extraction, detergent treatment, etc.) immobilize general protein modification-specific antibody to resin denature proteins (e.g., heat treatment)
digest crude protein mixture to peptides with immobilized trypsin or other protease contact digest with antibody-resin and incubate (e.g., 4?, 1-16 hours)
remove unbound peptides by centrifugation, filtration, or column washing
w wash antibody-resin extensively t elute bound peptides (e.g., 30% acetic acid, 0.1 M glycine, pH 2.3, or 0.1%trifluoroacetic acid)
desalt and concetrate peptides with microcolumn (e.g., reversed-phase ZipTip) (optional)
analyze, e.g., MS for mass, MS after phosphatase treatement to confirm phosphorylation, or tandem MS (MS/MS or MS) for partial sequence Patent Application Publication May 27, 2010 Sheet 2 of 22 US 2010/0129929 A1
Figure 2
Protein . Accessie Phosphe. a lirassis isie No. Protein ype Residue Phosphorylation Site Sequence Disease airs S
Aiii. NP_001611.1 Adaptofiscaffold Y8S vKGEyDWTMPK isfiasts 33 S. iii. is: fix P 3503.2 Adap::iscaffod 33 FOCTYPSGGEEACCR go aire SW620 SE. O. 2. Åir N 0403.2 Adagirisatil i. CEC-IEVO Gwysey K ui rice it: a bifas. Si 3. casessi: 3: Cixi NP 55227. Adapteriscified s KyEDGEODMKR cio &ce vis (a-i. NP C06394.1 Adapteriscaffei: y: TGHGYWYEPSR S. 8S (Eastors Squaits ce. G-GS as- NPOS334. Acaptiscarcic f Swhiy Ywi (KEEFER 8::: 33; S.C. far breast Caice, 3. NP {5,343.2 Adaptofiscaffold y MTSDAvy K &as case fee s NSCC Cat-3 cks NF 060589.2 Adaptoriscaffold Y23 GIEiySYPC PT, MPR Cogs 3-cef
ES N-03855, Atiatoriscaicis Y148 ALEvy VPPAR - reast casses HCC423 enopaki: N 979, Asia:Criscaffold Y7s2 WOCAA, OFK tung cancer 5.
iS3 siycipiiri NP 10:379, Adapteriscaicii Yt703 is EP KDMSPYEAyKR S. s ps3 PO4.433.3 Adaptatisaig 232 Cy-FQEETPE, AAR S -82 S. 323s 3.298 a 75767.2 Adaptofiscaicii y SOESGWEyFSK gastric cancer Cis
ischest of exigea : f 574332 matrix protei; Y844 yYPMPFFDELK glioblastefa M059. SEQ) NO. 4 GAG iS X 3. 38 i. isis, KSE2 is C-87 ..? C. liSC s: gastric 3 icef s: giliastina scs oria ga4
Agasif exaces: iting carcer 3. AA POS 438.2 raisix picts: 83 YPyPPPLPDEDKK saia case; is i? 2. S. 15 Achest r exaceria: six MPC592.2 matrix proteir 3. YSyEVHVSGER NSC is8S SE is 16
Aches: extrace.3 aia iOS 2.2 at ti Y;i SEGEy. STONGSPESPOPNAEYSEPK coor cancer 8,82 Si. 17 Asif : 3:3:3: 3 hip 07842 at rotein: y DNVFYyGEEGGGEEDCOYD, OR 333 as: iC3S SiO, 18
Aires: , 8xacif giadii. 3 EP (, 531 atrix riter y: Kiy DGGARTEDEVGSYPSKHY, horia iuri S3 SEQE: NO. 49 Aisic; exactiis C. N 0895. Patrix protein Yi YiyNGASWA SSR irriga; Cer 3. SEO NO2 Aasif acaf Caf2 N° 0183.2 matrix protein Y3. CyPGESGEKGSGCKSF gicia: A172 Sii 2. Adhesis; taxatieta -25s Chis *: O(85.1 matrix proteir 3. AAVSHWQQQSy DSG HSGATTAPSLSGK St. CC82 S. O ki is a cer sts Aciesis fatalia: Siasis : iS; sis NP 08:3, matrix protein 8 HyGPWvK 3::::::::: sji: S.
Aless crexasia: is a reas case Patent Application Publication May 27, 2010 Sheet 3 of 22 US 2010/0129929 A1
Figure 2
f 3. s
Prote Access Phospho Celtic isstei liaise is, Protein ype Reside Phosphoryliatic Site Segence sease arist SEO Aches of extraceliar i?ian reas cance: : CXA NOC329 patrix fairs SYGSNHSSGSMSPSNAEGySK if is acs: Cirie SEO iO2S: fides of extraces: 8. s C-S PO3728.1 matrix ceil LDREERDAyNLR east are: i. S. iii. Acision 3 extrasiliar f:38 cas: CC8 8 sissistakin if CG44.08.2 matrix protein SVEORFDOCKhiy OQLOK coincarcif Si62 SEQDK.C. 27 ises axiacetitisf esota Fe O2 atrix rosei SykDANRSAVEDITG R S. six SEQD NO. 28. Aces of f extracelia 3. S. N 777 AC. matrix proteir YS WYLCNQDENHKHAQDywi YNYEGR CC8 SE 29 Assis a sex acetia S. N 07774.1 matrix protein SCC 32? SEQD No. 30 3. CASP8AF2 NF 3634 Assissis NCNSDyCGSEGMEMK SC Cat-3 SEC E iO. 3 gastric cancer 3. t axia NPOC43.1 i-dig Rite: Yi PAQy DASEK gic tastora C-87 SEOD NO 32 ski, NP C04030, F-ixir; ifosi YS AFLNVOCONKPYFADRy Ski K gastric career C-87 SEO. O. 33 C2 NO38259.2 Cecycle regulation Y8. EvKGSES-AYGwww.GYCR Sea SEQD NO. 34 C NP 036259.2 Ceil cycle regulation Y 38 EWKGSETYSPNTayGWOFWAASYCR N. S. Cel cycle regiation y EWKGSEYSPAYGVFAP GyCR - - SEOSENO NC 3.39
-8 C isit S. . ico carcer Siii.5 gastic catcar XG . 3. class ;P 71394.2 Cast cycle regain ESA. Ly-Eik See X2 SEQID NO. 37 3A 3. Cl2 NP35823 Cai cycle regula-Gi My SJSALNNK tung carcef s: SEQ O NO. 38 3. arets 3 ip633483. Chapeities FyASARFKPFS-K gas,Caitei --8 SEQD NO 39 Chromain, DNA-binding DNA repair of NA : 3. NP 00:48.3 regication rosei y rostate acer SEC DNO. 40 Croatin, Dia-bindirig, A regar of NA Bright NC 525 replication protein CVLDLFMywVTEK is six SEC D. No. 4 3ctii, alia NP 009. Cytoskeietal protei TGIVESGGV NWYEGALP-AMR SCC 5X. SEQDNO. 42,
al 328 s ;SC. at a...ces fe: iC; NPO1C33, Cytoskeleia protein X-Phili IAFC AEKypix ce. A iOS SEQ to No. 43. S A. NPO1631. Cytoskeletal proteir SLAACDEGGPELEPDyGTATR 3. 8s: 2. SEOD NO. 44 is capoir 3 NF (33). Cytoskeieia protein CYCySDQG: OY seats as: .A.A-SS SEC DNC is C a feast career sis east 3.8F NC-8
O42.2 Cytoskelia ite LASy. KiR gastric sée: 22 DNC 46 0413, Cytoskelstartei EGEDAF-LTOK gast 10 NO.47 iP25. Cytoskeletaliotein STFSTNyR ci. is 3. S. iii. 8 kiPOCO25, Cytoskeletal fier Y255 AOy AR gastric aficer C. S C ; reas:cancer CC86 AAA33A, Cytoskeleta iteir YS ySSSSSGGGGGYGGAES GAGNEK assassi Ci-- S. NSCC (als-3 Cix: AAA35044. Cytoskeletal protein SS FVSSSSSGGYGGGyGGv.IASDG. AGNEK gastric carcer is SE x-E CK13 NFCO228.2 Cytoskeletarie DYSHYYTIGER. . . . . gastric cance... gzi St. i. Patent Application Publication May 27, 2010 Sheet 4 of 22 US 2010/0129929 A1
Figure 2
ri Accessic Phospho Celine fissisi is: . Proteiype Reside rosphorylation Site Sequence Disease aii SEC SCL feast acci S. gastric carcer iC8 status ce. iCC827 3. NP (2267.2. Cytoskeleta frien Y256 SCEVRAEONRK acic C-8 SECD No. 53 C3 NP C02237,2:Cytoskeleiai ser Y8. SRLEQEATyr asic cances C-87 SEC D. No 54 CC cs: treas:(3.8; g22 C Pi3.2 Cytoskel:aise wSLAGACGGGYGSRSyNGGSKR Colo? cance? s4. SEC his C. P.O.S, Cytoskeletal protein 283 CVCAA vitive AK ling cancer ga SE is | gait is is is 0452 Cytoskeiseia its Y3A AOyEEANR as 3. iting tumor 26 S. N. S. 8:8 carce; 22 NPO452 Cytoskeleta protein Y. SRTEAESWyCTKYEELCQTAGR coccaces sit SEC - 8 C38
NP (45.2 Cytoskeletarifier 3. SRTEAESNY OTKOOTAR east case: gE S. O. SS kia, is case: ca
NP775103, Cytoskeisa protein 32 Sly GGGSKR 3s cate gii SE : . . trait seast carcer at is NP 77 109.1 Cytoskeietal protein Y3. ISIGGGSCAISGSyGSR feast acer ge: S NSCO NPOS473 Cytoskeleta protein YSS SAyGGPVGAGR gastric cance ii. 3 SEQ DNQ 62 888 34
its CC13, i-CC S. *:C-87 gastric arcer s if OOSSAT 3 Cytoskeleta protein y (DVOAA SK prostate cancer assi SE 3 8. iPO06543 Cytoskeleta prote: 283 AEAEAWyCIKFECACAGK ESCC CC8 SEC x; }; NP 002264. 1 Cytoskeletal protein 2S SySGPGSR i? cance Ciá3. Six, ; iO236, Cytoskeleta prote: ix.EAELGNOGLEDFKNKyCENKR gastic career NC-N87 Sii, S. is & seascace 23 C: Cicer C3 s
gas, it cance: Ci-- Safius ce. via 3 NP_0022641. Cytoskeletal protein YS SRAEAESMyCKYFLOSLAGK carcino: Sé8. ice ii. is N22EA, Cytoskeleta prote: SS SRAEAESMYCKyEES AGK gastic carcer AC32 Cat-3
S.C. C-87 is NPS5:17.1 Cytoskeleta protein YS SNYyCAYOAOPATR gastric C3: Ce: s:
C-3 Caci, 2 (Cytoskeleta rotein Y: SNYYOAyOAQPLATR astric carcer ga ciatic Cytoskeleta Ectein Y. SENSSTOYi cs: cacsi CC:33 is catar 2.
Scarious ce. id at 3 NP_001297. 1 Cytoskeletal protein y wwySAR Cicilia 43 SEO NO2 s
gastricances A 3 (Otacts NPO3222.2 Cytoskeleta role Yig SAG-Dy(GKEK 3 :: C.E. SEQD No. 73. Ca P (1894.2 Cytoskeleta rotein Y2C, OCA, GSAAAASOASOHOGGGGGENSCC SEQD NO 74 Cia iP834. Cytosieleasotei y NGREEKEW&CyåOVFR NSCLC 5 x SEQDNC 75 SCC Cat-3 s Cixi NP (323. Cytoskeleta, ?iteir Y2 GGSAPEGAy APR SC S. S. C. reas casei CO2: 78 desmogiaking NFSU2221. Cytoskeletal protein ... Y480 ... NyGIPAVK gastricance C-3 SEQDNC 77 Patent Application Publication May 27, 2010 Sheet 5 of 22 US 2010/0129929 A1
Prote: Accessi: Phospho Cities issue ! as: hi, Protein pe resis: Posphoryliatic Site Secuence issass: Pier SEC NO Cas s 3. 3.
i. KS i.js Sii C. ga SC 247 gascace ge ing arcs: isis: s dismoriaks, 3° 2321. Cytoskeleta frie : HvAAGQQPyDGVR {{state cases Stig of 23 SEC C. 78. 388&vist. **- : *i; & i fast ceiline $5. 3. is i $33, 3. 4.38 i. Jikai iSC calateria KA tiss Case: is as carag giastia ce isig carcinoia t N5343 Cytoskeleta protei Y77 EFSSYOFVyjiyG risiae carices a s NP 573.4031 Cytoskeleta protein y? SSYDF/YiyG rostate cacie DJ is g ii (4.4252 Cytoskeleiai grotein 8. MSESKPKEPwFSAEEGyvK SCC 28 h; 5744. Cytoskeletal protein Y2 SSSTMyNEK iSC. CC82 i PCO2O. Cytoskeletai protein 215 LEEKENEEGwyyExOR gtasia R iPG(2007. Cytosksitat protei yigs SGSFLOSTHK glioblastoria i.S.S it is 53 s Jia 3. s -á, x Kargas-108; crg. RC-KS R 32. essee S-5 Ere plasmic reticism of is 38 iSJ-- AGIRA. NP 35083.3 gog 3. SayCESAEAPADPFAPEGR it is yelofia g32 S. E. ASS...N. Erzya, SC, Patent Application Publication May 27, 2010 Sheet 6 of 22 US 2010/0129929 A1
Figure 2 s
reig Accession Phospho- Cie iss; aire ics. Proteiype Rese Phosphoryliation Site Sequence isease Pairs Sc his .
& :33 33 S33 ki. is: i. 3 is: 8. f i. Si. SCC s s: : $3. SC bug i? of 57 i: :00859.2 Enzyfrie, fisc, '3s KPFSEOve APPKAviv SCC a hia in St. i. 37 issi:3x: 3: iii. fice i8 tai-3 is is is i85 XC SE3 SS 3. i8 SCC ... 8 gastric acer St. NP (13.1 Erzyitis, fisc, i y AA, SAA G. 4)State age 8 SEOID No. 88 as: 3:3: . as site a: 3
-: SOX: 8.
355 C8 S.C. i-fi: s gastric carcis Sis is: NP 00118. Enzyme, finisc. y Yvia AMVLSAAGDAGYNGK prostate cance? g58 SEC 89. i i ::::::: xiii. 8 siastice: {Cat is:
3OXG 8
its 5 355 iC3 S.C. CSF astric carcer Sis f N 0.36. Enzyme, is, Yi Air SA&GAGYN 3. is as 3i. g258 is: NP (1619. Enzyme, rise Yi HDCAHvyQNENEVGVAOEK reas: CC335 s: : scia is ce. fix NP (134.42 Erzyne, is: Yg RED-Fy SK 3:iiii. CC8 APE/PR SCC iii.
Patent Application Publication May 27, 2010 Sheet 11 of 22 US 2010/0129929 A1
Figure 2
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risis Accession Phosphe. C3 is iss; e i. Proteiyge Reside Piosphorylation Site Secueice isease { ai S iO 33: {3xi A8: 393 CC ii. is
ki s it acreatic C. to: g SC s
& Protein kinase, Tyr is 3: s NF. 1945.3 receio Y55 HyAGOYYROGR acreatic case s2 SE nose entryo if its ce. feasis 53. giassia iss Frotein kinase, yi :::::::::::::: Sa-i-AS is 2 P32 rece; or Y48 FPLRFDyoEPSR etice isota SK--- i. Potein inase, y, St. NP_005223.3 (receptor, Y A DVDREDKLW KFyVOLCAYEDPAGGALOPTFNSCLC 3. Protei kinase, ty i. KPOGE233 receior YES AIDVDREDKWKFYVOCAyEOPAC3AFF. NSCC 3. SEC No. 180; Protein kinase, ly 18 (A2 is 00-422.2 recestor YS28 VIGAGEFGEvy K NSC 32 Si; i. 18 Protest kinase, yi NS, Cat 32 ii. 2, teetor Yi YKPM. TyMENGAEK posta's caca s SE: i. 182 Protein kinase, y 33 A2 NP 4.222 receptor s AySt. GKOWNygii; &:::::::::s: 88: St. iii. 183 Protein kinase, y 36:A NCO:224.2 receiter, 3. yiSEMGYWHRI. AAR SCC f SEC OMO. 84 s Pritain kinase, yi is sp33 NC 434.2 receptor Y3 GSDSy KCOYAPGK to acci S8. SI, 185 Protein itase, yi 8:8 iP (4.433.3 receptor 3S F.C. At 8 isiasis S. S: , , 18 Protein kinase, it 8:38 if CA438, tor y (SIGYEO (CySSPGG, K ::::::::: S; SEC .. 8 Protein kinase, tyr ississ NP CC-438, receiter YS yYiDPSTYEOPCOAR co, cace S2 S iProtein kirtase, yi ; s: i. NCC2002.3 receptor GV-1DyYKK re3st carcer iii.3-3 S. Protein kinase, yr Ri 2002.3 receptor A3 GVH-Yyk: treast Caice, ilf-i-3 SE * : Receptor, cane, ansporter ceis race is fix NP C01597.2 p. Y27 yADKPAGTYSGGNKRK (C$33 carice: SE: Receptos, chairie, transporte a ce's face Cé NPOS372 protein Y2487 YADKPAGySGSNKRK cit are 3 SEO DNC 192; Faceptor, channel, SC. C88 : 330tef tre, striac: 83s 8:8; 83 &SC PO4987, protei YS C.SSSSSySGDSR gastria 223 SC 38 193 Receptor, i3rine, transporter or ceisurface : ACC8 if (343.2 protei Y3 ykhiv. ACSLCD, DLF GOTO SER tug carcer 3i SEG 8, 34. Receptor channel, % 3.Serrar cesiace -Cl28 133 fx3. Yi y}{}i (E. D. SFFY., KCYPK feast cate X S. v. 135 arsefief of Cesiac -Ca A32 09:20, protein Y-32 YQ-LOCQLD DISPLEy. MKCYPEK 38 cases SEQ DNC 196 Receptor, chartei, transporter if eistriace
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Figure 2
reir accessic fisphe (eiline iisse iaise i. Protein ype Reside Phosphorylation Site Sequeries isease Piet SEC
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is sea: acci als: astry is: is Fascace isis Receit, case, cc carcer S. it arsporter arcsi, Siirface is tasis 273 Alix EA 39856. groteir API/ASy. GGR bias is 28 Receito, arse, SEQD No. 198i transpicist or ce. Strace apos hi G375.2 protein 38 FLKNIPvy K glicist:3 S{ } NC 99; Recept, iaiti,
transporte? of ce: striac: six NPC 84.1 protein 783 SySEPEKNEVGTR gastricance i. S . Receptor, late,
transporter of cesiriace S.C. Casis, POSS2 protein s yiLYKNik ficia Receptor, care,
transporter or ce. S. A.C., NP {00532 proteir Yis Yy'NEWS. ilia Receptor, caffei, transpo?e of cistrace
NPCS92, protes 3 y Avvavy Aik iotasta regist, :38, transporter 3, ca: Surface Senis ce: Ci NP C05$90,4 protein s PyLFSFC carcia recast, fate, C
iasporte of estace treas: 3; ca. s R NP 06477, groteir 33; wSETEySAEECSTK 33 :63 Receptor chara, transportes of Cesurface CR NPOSS25, protein Y: SGFySAK GAMG GiG 33 3 3. CCS: 323
t ;SCC jit aeria:s astrictoria Six f ;3f Isai reas EE se: & his fiss iiia feast 8:8 girlastoria a: it groia Rece, care, rostate carce 8: traise; e Siacs: ifica: eit g27A i N 5525, rotein Y:3 ON CAD-Fy. ecies: sia: SEO D No. 207 Receps), are transpore of cesisce is feast Eace C P 6589.2 protein Y52 yFIESNSMLKEAKK gi:38 ass: fee SEOD NO. 208 Recept, carriei
as?if f castiface 3. it 3i: prot 3. SRMSSINSEK ;3: . SEC DNC. 209 Receptor, chartiei if a.scies of ge C3 it is
Patent Application Publication May 27, 2010 Sheet 17 of 22 US 2010/0129929 A1
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TYROSINE PHOSPHORYLATION SITES Some type of carcinoma during 2005. (Source: American Cancer Society (2005)). The worldwide incidence of carci RELATED APPLICATIONS noma is much higher. 0001 Pursuant to 35 U.S.C. S119(e) this application 0007 As with many cancers, deregulation of receptor claims the benefit of, and priority to, provisional application tyrosine kinases (RTKs) appears to be a central theme in the U.S. Ser. No. 607833,826, filed Jul. 27, 2006, the disclosure of etiology of carcinomas. Constitutively active RTKs can con which is incorporated herein, in its entirety, by reference. tribute not only to unrestricted cell proliferation, but also to other important features of malignant tumors, such as evading FIELD OF THE INVENTION apoptosis, the ability to promote blood vessel growth, the 0002 The invention relates generally to novel tyrosine ability to invade other tissues and build metastases at distant phosphorylation sites, methods and compositions for detect sites (see Blume-Jensen et al., Nature 411: 355-365 (2001)). ing, quantitating and modulating same. These effects are mediated not only through aberrant activity of RTKs themselves, but, in turn, by aberrant activity of their BACKGROUND OF THE INVENTION downstream signaling molecules and Substrates. 0008. The importance of RTKs in carcinoma progression 0003. The activation of proteins by post-translational has led to a very active search for pharmacological com modification is an important cellular mechanism for regulat pounds that can inhibit RTKactivity in tumor cells, and more ing most aspects of biological organization and control, recently to significant efforts aimed at identifying genetic including growth, development, homeostasis, and cellular mutations in RTKs that may occur in, and affect progression communication. Protein phosphorylation, for example, plays of different types of carcinomas (see, e.g., Bardell et al., a critical role in the etiology of many pathological conditions Science 300: 949 (2003); Lynch et al., N. Eng. J. Med. 350: and diseases, including to mention but a few: cancer, devel 2129-2139 (2004)). For example, non-small cell lung carci opmental disorders, autoimmune diseases, and diabetes. Yet, noma patients carrying activating mutations in the epidermal in spite of the importance of protein modification, it is not yet growth factor receptor (EGFR), an RTK, appear to respond well understood at the molecular level, due to the extraordi better to specific EGFR inhibitors than do patients without nary complexity of signaling pathways, and the slow devel opment of technology necessary to unravel it. Such mutations (Lynch et al., Supra.; Paez et al., Science 304: 0004 Protein phosphorylation on a proteome-wide scale 1497-1500 (2004)). is extremely complex as a result of three factors: the large 0009 Clearly, identifying activated RTKs and down number of modifying proteins, e.g., kinases, encoded in the stream signaling molecules driving the oncogenic phenotype genome, the much larger number of sites on Substrate proteins of carcinomas would be highly beneficial for understanding that are modified by these enzymes, and the dynamic nature the underlying mechanisms of this prevalent form of cancer, of protein expression during growth, development, disease identifying novel drug targets for the treatment of Such dis states, and aging. The human genome, for example, encodes ease, and for assessing appropriate patient treatment with over 520 different protein kinases, making them the most selective kinase inhibitors of relevant targets when and if they abundant class of enzymes known. (Hunter, Nature 411: 355 become available. The identification of key signaling mecha 65 (2001)). Most kinases phosphorylate many different sub nisms is highly desirable in many contexts in addition to strate proteins, at distinct tyrosine, serine, and/or threonine CaCC. residues. Indeed, it is estimated that one-third of all proteins 0010. However, although a few key RTKs involved in car encoded by the human genome are phosphorylated, and many cinoma progression are known, there is relatively scarce are phosphorylated at multiple sites by different kinases. information about kinase-driven signaling pathways and 0005. Many of these phosphorylation sites regulate criti phosphorylation sites that underlie the different types of car cal biological processes and may prove to be important diag cinoma. Therefore there is presently an incomplete and inac nostic or therapeutic targets for molecular medicine. For curate understanding of how protein activation within signal example, of the more than 100 dominant oncogenes identified ing pathways is driving these complex cancers. Accordingly, to date, 46 are protein kinases. See Hunter, Supra. Under there is a continuing and pressing need to unravel the molecu standing which proteins are modified by these kinases will lar mechanisms of kinase-driven ontogenesis incarcinoma by greatly expand our understanding of the molecular mecha identifying the downstream signaling proteins mediating cel nisms underlying oncogenic transformation. Therefore, the lular transformation in these cancers. identification of and ability to detect, phosphorylation sites on 0011 Presently, diagnosis of carcinoma is made by tissue a wide variety of cellular proteins is crucially important to biopsy and detection of different cell surface markers. How understanding the key signaling proteins and pathways impli ever, misdiagnosis can occur since some carcinoma cases can cated in the progression of disease states like cancer. be negative for certain markers and because these markers 0006 Carcinoma is one of the two main categories of may not indicate which genes or protein kinases may be cancer, and is generally characterized by the formation of deregulated. Although the genetic translocations and/or malignant tumors or cells of epithelial tissue original, such as mutations characteristic of a particular form of carcinoma can skin, digestive tract, glands, etc. Carcinomas are malignant by be sometimes detected, it is clear that other downstream definition, and tend to metastasize to other areas of the body. effectors of constitutively active kinases having potential The most common forms of carcinoma are skin cancer, lung diagnostic, predictive, or therapeutic value, remain to be elu cancer, breast cancer, and colon cancer, as well as other cidated. numerous but less prevalent carcinomas. Current estimates 0012. Accordingly, identification of downstream signal show that, collectively, various carcinomas will account for ing molecules and phosphorylation sites involved in different approximately 1.65 million cancer diagnoses in the United types of diseases including for example, carcinoma and States alone, and more than 300,000 people will die from development of new reagents to detect and quantify these US 2010/0129929 A1 May 27, 2010 sites and proteins may lead to improved diagnostic/prognos effective amount of an antibody or antigen-binding fragment tic markers, as well as novel drug targets, for the detection and thereof that specifically binds at a novel phosphorylation site treatment of many diseases. of the invention. 0021. In a further aspect, the invention provides methods SUMMARY OF THE INVENTION for detecting and quantitating phosphorylation at a novel tyrosine phosphorylation site of the invention. 0013 The present invention provides in one aspect novel 0022. In another aspect, the invention provides a method tyrosine phosphorylation sites (Table 1) identified in carci for identifying an agent that modulates tyrosine phosphory noma. The novel sites occur in proteins such as: protein lation at a novel phosphorylation site of the invention, com kinases (such as serine/threonine dual specificity kinases or prising: contacting a peptide or protein comprising a novel tyrosine kinases), adaptor/scaffold proteins, transcription phosphorylation site of the invention with a candidate agent, factors, phosphatases, tumor Suppressors, ubiquitin conjugat and determining the phosphorylation state or level at the ing system proteins, translation initiation complex proteins, novel phosphorylation site. A change in the phosphorylation RNA binding proteins, apoptosis proteins, adhesion proteins, state or level at the specified tyrosine in the presence of the G protein regulators/GTPase activating protein/Guanine test agent, as compared to a control, indicates that the candi nucleotide exchange factor proteins, and DNA binding/rep date agent potentially modulates tyrosine phosphorylation at lication/repair proteins. a novel phosphorylation site of the invention. 0023. In another aspect, the invention discloses immu 0014. In another aspect, the invention provides peptides noassays for binding, purifying, quantifying and otherwise comprising the novel phosphorylation sites of the invention, generally detecting the phosphorylation of a protein or pep and proteins and peptides that are mutated to eliminate the tide at a novel phosphorylation site of the invention. novel phosphorylation sites. 0024. Also provided are pharmaceutical compositions and 0015. In another aspect, the invention provides modula tors that modulate tyrosine phosphorylation at a novel phos kits comprising one or more antibodies or peptides of the phorylation site of the invention, including Small molecules, invention and methods of using them. peptides comprising a novel phosphorylation site, and bind BRIEF DESCRIPTION OF THE DRAWINGS ing molecules that specifically bind at a novel phosphoryla tion site, including but not limited to antibodies or antigen 0025 FIG. 1 is a diagram depicting the immuno-affinity binding fragments thereof. isolation and mass-spectrometric characterization methodol 0016. In another aspect, the invention provides composi ogy (IAP) used in the Examples to identify the novel phos tions for detecting, quantitating or modulating a novel phos phorylation sites disclosed herein. phorylation site of the invention, including peptides compris 0026 FIG. 2 is a table (corresponding to Table 1) summa ing a novel phosphorylation site and antibodies or antigen rizing the 349 novel phosphorylation sites of the invention: binding fragments thereof that specifically bind at a novel Column A-the parent proteins from which the phosphoryla phosphorylation site. In certain embodiments, the composi tion sites are derived; Column B=the SwissProt accession tions for detecting, quantitating or modulating a novel phos number for the human homologue of the identified parent phorylation site of the invention are Heavy-Isotype Labeled proteins; Column C=the protein type/classification; Column Peptides (AQUA peptides) comprising a novel phosphoryla D=the tyrosine residues at which phosphorylation occurs tion site. (each number refers to the amino acid residue position of the tyrosine in the parent human protein, according to the pub 0017. In another aspect, the invention discloses phospho lished sequence retrieved by the SwissProt accession num rylation site specific antibodies orantigen-binding fragments ber); Column E-flanking sequences of the phosphorylatable thereof. In one embodiment, the antibodies specifically bind tyrosine residues; sequences (SEQID NOs: 1-169, 171-269, to an amino acid sequence comprising a phosphorylation site 271-347) were identified using Trypsin digestion of the par identified in Table 1 when the tyrosine identified in Column D ent proteins; in each sequence, the tyrosine (see correspond is phosphorylated, and do not significantly bind when the ing rows in Column D) appears in lowercase; Column F-the tyrosine is not phosphorylated. In another embodiment, the type of carcinoma in which each of the phosphorylation site antibodies specifically bind to an amino acid sequence com was discovered; Column G=the cell type(s)/Tissue/Patient prising a phosphorylation site when the tyrosine is not phos Sample in which each of the phosphorylation site was dis phorylated, and do not significantly bind when the tyrosine is covered; and Column H=the SEQ ID NOs of the trypsin phosphorylated. digested peptides identified in Column E. 0018. In another aspect, the invention provides a method 0027 FIG. 3 is an exemplary mass spectrograph depicting for making phosphorylation site-specific antibodies. the detection of the phosphorylation of tyrosine 367 in 0019. In another aspect, the invention provides composi DYRK3, as further described in Example 1 (red and blue tions comprising a peptide, protein, or antibody of the inven indicate ions detected in MS/MS spectrum); Y (and pY) tion, including pharmaceutical compositions. indicates the phosphorylated tyrosine (corresponds to lower 0020. In a further aspect, the invention provides methods case “y” in Column E of Table 1: SEQID NO: 155). of treating or preventing carcinoma in a subject, wherein the 0028 FIG. 4 is an exemplary mass spectrograph depicting carcinoma is associated with the phosphorylation state of a the detection of the phosphorylation of tyrosine 693 in Axl, as novel phosphorylation site in Table 1, whether phosphory further described in Example 1 (red and blue indicate ions lated or dephosphorylated. In certain embodiments, the meth detected in MS/MS spectrum); Y (and pY) indicates the ods comprise administering to a subject a therapeutically phosphorylated tyrosine (corresponds to lowercase “y” in effective amount of a peptide comprising a novel phosphory Column E of Table 1: SEQID NO: 175). lation site of the invention. In certain embodiments, the meth 0029 FIG. 5 is an exemplary mass spectrograph depicting ods comprise administering to a subject a therapeutically the detection of the phosphorylation of tyrosine 755 in DDR1. US 2010/0129929 A1 May 27, 2010
as further described in Example 1 (red and blue indicate ions rived cell lines and tissue samples: 293T, 293T TAT, 293T detected in MS/MS spectrum); Y (and pY) indicates the ZNF198/FGFR, 3T3-EGFR(L858R), 3T3-EGFR(del), 3T3 phosphorylated tyrosine (corresponds to lowercase “y” in EGFRwt, A 431, A172, A549, A549 tumor, AML-4833, Column E of Table 1: SEQID NO: 177). AML-6246, AML-6735, AML-7592, BaF3-FLT3(WT), 0030 FIG. 6 is an exemplary mass spectrograph depicting BxPC-3, CCF-STTG1 CHRF, CI-1, CTV-1, Calu-3, the detection of the phosphorylation of tyrosine 5 in Aldolase DBTRG-05MG, DMS 153, DMS 53, DMS 79, DU-528, A, as further described in Example 1 (red and blue indicate DU145, GAMG, GMS-10, H1299, H1373, H1437, H1563, ions detected in MS/MS spectrum);Y* (and pY) indicates the H1568, H1648, H1650, H1650 XG, H1666, H1693, H1703, phosphorylated tyrosine (corresponds to lowercase “y” in H1734, H1793, H1869, H1944, H1975, H1993, H2023, Column E of Table 1: SEQID NO: 98). 0031 FIG. 7 is an exemplary mass spectrograph depicting H2030, H2170, H2172, H2286, H2347, H3255, H358, H460, the detection of the phosphorylation of tyrosine 456 in H520, H524, H526, H661, H810, H82, H838, HCC1395, cPLA2, as further described in Example 1 (red and blue HCC1428, HCC1435, HCC1806, HCC1937, HCC366, indicate ions detected in MS/MS spectrum); Y (and pY) HCC44, HCC78, HCC827, HCT 116, HCT116, HER4-JMb, indicates the phosphorylated tyrosine (corresponds to lower HL107B, HL116B, HL117A, HL117B, HL129A, HL130A, case “y” in Column E of Table 1: SEQID NO: 111). HL131A, HL131B, HL132A, HL132B, HL133A, HL1881, HL25A, HL41A, HL53A, HL53B, HL55B, HL59A, HL59b, DETAILED DESCRIPTION OF THE INVENTION HL61a, HL61b, HL66A, HL66B, HL75A, HL79B, HL83A, HL84A, HL84B, HL87A, HL87B, HL92B, HL97A, HL98A, 0032. The inventors have discovered and disclosed herein HT29, HUVEC, HeLa, Human lung tumor, Jurkat, K562, novel tyrosine phosphorylation sites in signaling proteins KG-1, KG1-A, KMS18, KY821, Karpas 299, Karpas-1 106p. extracted from carcinoma cells. The newly discovered phos LN18, LN229, LOU-NH91, M-07e, MO59.J, MO59K, phorylation sites significantly extend our knowledge of MC-116, MCF-10A (Y561F), MCF-10A(Y969F), MCF7, kinase substrates and of the proteins in which the novel sites MDA-MB-435S, MDA-MB-453, MDA-MB-468, MDS occur. The disclosure herein of the novel phosphorylation 851, MKPL-1, ML-1, MO-91, MOLT15, MV4-11, Marimo, sites and reagents including peptides and antibodies specific Me-F2, Molm 14, NCI-H196, NCI-N87, Nomo-1, OCI-M1, for the sites add important new tools for the elucidation of OCI/AML3, OPM-1, PT7-pancreatic tumor, Pfeiffer, RC signaling pathways that are associate with a host of biological K8, RI-1, RKO, RPMI8266, SCLC T1, SCLC T2, SEM, processes including cell division, growth, differentiation, SH-SY5Y, SK-N-AS, SK-N-MC, SK-N-SH, SNB-19, SU develomental changes and disease. Their discovery in carci DHL1, SW1088, SW1783, SW620, Su.86.86, SuDHL5, T17, noma cells provides and focuses further elucidation of the T98G, TS, U118 MG, UT-7, VACO432, VAL, Verona 2, disease process. And, the novel sites provide additional diag WSU-NHL, XG 1, XG2, XG5, cs001, cs012, cs015, cs019, nostic and therapeutic targets. cs024, cs025, cs026, cs029, cs037, cs041, cs042, cs048, cs057, cs068, cs069, cs070, gz21, gz33, gz42, gz47, gziš8, 1. Novel Phosphorylation Sites in Carcinoma gz70, gz74, gz75, gzB1, h2228, hl 144a, hl 144b, hl145a, 0033. In one aspect, the invention provides 347 novel h1145b, hl146b, hl 148a, hl148b, hl152a, hl 152b, lung tumor tyrosine phosphorylation sites in signaling proteins from cel T26, lung tumor T57, normal human lung, pancreatic lular extracts from a variety of human carcinoma-derived cell xenograft, Swa80. In addition to the newly discovered phos lines and tissue samples (such as H3255, lung tumor T26, etc., phorylation sites (all having a phosphorylatable tyrosine), as further described below in Examples), identified using the many known phosphorylation sites were also identified. techniques described in “Immunoaffinity Isolation of Modi 0036. The immunoaffinity/mass spectrometric technique fied Peptides From Complex Mixtures.” U.S. Patent Publica described in Rush etal, i.e., the “IAP method, is described in tion No. 20030044848, Rush et al., using Table 1 summarizes detail in the Examples and briefly summarized below. the identified novel phosphorylation sites. 0037. The IAP method generally comprises the following 0034. These phosphorylation sites thus occur in proteins steps: (a) a proteinaceous preparation (e.g., a digested cell found incarcinoma. The sequences of the human homologues extract) comprising phosphopeptides from two or more dif are publicly available in SwissProt database and their Acces ferent proteins is obtained from an organism; (b) the prepa sion numbers listed in Column B of Table 1. The novel sites ration is contacted with at least one immobilized general occur in proteins such as: protein kinases (such as serine/ phosphotyrosine-specific antibody; (c) at least one phospho threonine dual specificity kinases or tyrosine kinases), adap peptide specifically bound by the immobilized antibody in tor/scaffold proteins, transcription factors, phosphatases, step (b) is isolated; and (d) the modified peptide isolated in tumor Suppressors, ubiquitin conjugating system proteins, step (c) is characterized by mass spectrometry (MS) and/or translation initiation complex proteins, RNA binding pro tandem mass spectrometry (MS-MS). Subsequently, (e) a teins, apoptosis proteins, adhesion proteins, G protein regu search program (e.g., Sequest) may be utilized to Substan lators/GTPase activating protein/Guanine nucleotide tially match the spectra obtained for the isolated, modified exchange factor proteins, and DNA binding/replication/re peptide during the characterization of step (d) with the spectra pair proteins (see Column C of Table 1). for a known peptide sequence. A quantification step, e.g., 0035. The novel phosphorylation sites of the invention using SILAC or AQUA, may also be used to quantify isolated were identified according to the methods described by Rush peptides in order to compare peptide levels in a sample to a et al., U.S. Patent Publication No. 20030044848, which are baseline. herein incorporated by reference in its entirety. Briefly, phos 0038. In the IAP method as disclosed herein, a general phorylation sites were isolated and characterized by immu phosphotyrosine-specific monoclonal antibody (commer noaffinity isolation and mass-spectrometric characterization cially available from Cell Signaling Technology, Inc., Bev (IAP) (FIG. 1), using the following human carcinoma-de erly, Mass., Cat #9411 (p-Tyr-100)) may be used in the immu US 2010/0129929 A1 May 27, 2010 noaffinity step to isolate the widest possible number of using a ThermoFinnigan LCQ Deca XP Plus ion trap mass phospho-tyrosine containing peptides from the cell extracts. spectrometer or LTQ). MS/MS spectra can be evaluated using, e.g., the program Sequest with the NCBI human pro 0039. As described in more detail in the Examples, lysates tein database. may be prepared from various carcinoma cell lines or tissue 0040. The novel phosphorylation sites identified are sum samples and digested with trypsin after treatment with DTT marized in Table1/FIG. 2. Column A lists the parent (signal and iodoacetamide to alkylate cysteine residues. Before the ing) protein in which the phosphorylation site occurs. Col immunoaffinity step, peptides may be pre-fractionated (e.g., umn D identifies the tyrosine residue at which by reversed-phase solid phase extraction using Sep-Pak Cs phosphorylation occurs (each number refers to the amino acid columns) to separate peptides from other cellular compo residue position of the tyrosine in the parent human protein, nents. The Solid phase extraction cartridges may then be according to the published sequence retrieved by the Swis eluted (e.g., with acetonitrile). Each lyophilized peptide frac sProt accession number). Column E shows flanking tion can be redissolved and treated with phosphotyrosine sequences of the identified tyrosine residues (which are the specific antibody (e.g., P-Tyr-100, CST #9411) immobilized sequences of trypsin-digested peptides). FIG. 2 also shows on protein Agarose. Immunoaffinity-purified peptides can be the particular type of carcinoma (see Column G) and cell eluted and a portion of this fraction may be concentrated (e.g., line(s) (see Column F) in which a particular phosphorylation with Stage or Zip tips) and analyzed by LC-MS/MS (e.g., site was discovered.
TABL 1. Novel Phosphorylation Sites in Carcinoma.
B D A. Accession C Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 2 AHNAK NPOO1611.1 Adaptor / scaffold Y836 VKGEyDVTMPK SEO ID NO: 1
3 Alix NPO37506.2 Adaptor / scaffold Y39 FIQQTYPSGGEEQAQyCR SEO ID NO: 2
4 ARRB1 NPOO4. O32.2 Adaptor/scaffold Y47 DFVDHIDLVDPVDGVVLVDPEyLK SEO ID NO: 3
5 CACYBP NP O55227.1 Adaptor / scaffold Y199 KIyEDGDDDMKR SEO ID NO: 4
6 Cas-I NPOO 6394.1 Adaptor / scaffold Y168 TGHGYVYEyPSR SEO ID NO: 5
7 Cas-L NP OO6394.1 Adaptor / scaffo c Y629 SWMDDyDYVHLQGKEEFER SEO ID NO: 6
8 DLG3 NPO 66943.2 Adaptor / scaffold Y3 O6 NTSDMVyLK SEO ID NO: 7
9 Dok4 NP O6058O.2 Adaptor / scaffold Y255 GTEHySYPCTPTTMLPR SEO ID NO: 8
O EFS NPOO5855.1 Adaptor / scaffo c Y148 DALEVyDVPPTALR SEO ID NO: 9
1 envoplakin NP OO1979.1 Adaptor / scaffold Y752 VVQDAALTyQQFK SEO ID NO: 10
2 envoplakin NP OO1979.1 Adaptor / scaffold Y17O3 ISILEPETGKDMSPYEAyKR SEO ID NO: 11
3 Eps8 NP 004 438.3 Adaptor / scaffold Y252 QyHEQEETPEMMAAR SEO ID NO: 12
4 FLJ32798 NP 775767.2 Adaptor / scaffold Y417 SQESDGVEyIFISK SEO ID NO: 13
5 ADAM22 NPO57 435.2 Adhesion or Y844 yPYPMPPLPDEDK SEO ID NO: 14 extracellular matrix protein
6 ADAM22 NPO57 435.2 Adhesion or Y846 YPyPMPPLPDEDKK SEO ID NO: 15 extracellular matrix protein
7 afadin NPOO5927.2 Adhesion or Y94 YSLyEVHVSGER SEO ID NO: 16 extracellular matrix protein
8 afadin NPOO5927.2 Adhesion or Y1116 SEGFELyNNSTONGSPESPOLPWAEYSEPK SEQ ID NO: 17 extracellular matrix protein
9 CDH3 NP OO1784.2 Adhesion or Y7 O1 DNVFYyGEEGGGEEDODYDITOLHR SEO ID NO: 18 extracellular matrix protein US 2010/0129929 A1 May 27, 2010
TABLE 1 - continued Novel Phosphorylation Sites in Carcinoma.
B D A. Accession C Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO
20 claudin 18 NP O57453.1 Adhesion or Y241 KIyDGGARTEDEVOSYPSKHDYV SEQ ID NO : 19
21 CLDN10 NPOO8915.1 Adhesion or Y194 YTyNGATSVMSSR SEQ ID NO: 2O
22 COL14A2 NPOO1837.2 Adhesion or Y3 O6 GyPGLSGEKGSPGQKGSR SEQ ID NO: 21
23 CTNNB NPOO1895. Adhesion or Y3 O AAVSHWQQQSyLDSGIHSGATTTAPSLSGK SEQ ID NO: 22
24 CTNNB NPOO1895. Adhesion or Y489 LHyGLPVVVK SEQ ID NO: 23
25 CXADR NP OO1329. Adhesion or Y294 SyIGSNHSSLGSMSPSNMEGYSK SEQ ID NO: 24
26 CXADR NP OO1329. Adhesion or Y313 SYIGSNHSSLGSMSPSNMEGySK SEO ID NO : 25 extrace la matrix protein
27 DCHS1 NPOO3728. Adhesion or Y438 LDREERDAyNLR SEQ ID NO: 26
28 desmoplakin NP OO44O6.2 Adhesion or Y1139 SVEDRFDQQKNDyDQLQK SEO ID NO : 27
29 desmoplakin NP OO44O6.2 Adhesion or Y278s ISyKDAINRSMVEDITGLR SEQ ID NO: 28 extracellular matrix protein
3 O DSC2 NP of 774O1 Adhesion or Y853 VYLCNODENHKHAQDyVLTYNYEGR SEQ ID NO : 29
31 DSC2 NP O7774O1 Adhesion or Y859 HAQDYVLTYNyEGR SEQ ID NO : 30 extracellular matrix protein
32 CASP8AP2 NPO3 6247.1 Apoptosis Y739 NDNSDyCGISEGMEMK SEQ ID NO : 31
33 ANXA2 NPOO4 O3 O. 1 Calcium-binding Y109 TPAQyDASELK SEQ ID NO: 32 protein
34 ANXA2 NPOO4 O3 O. 1 Calcium-binding Y27s GDLENAFLNLVOCIQNKPLYFADRLyDSMK SEQ ID NO: 33 protein
35 CIZ1. NPO36259.2 Cell cycle Y782 EEWKGSETySPNTAYGVDFLVPVMGYICR SEQ ID NO: 34 regulation
36 CIZ1. NPO36259.2 Cell cycle Y788 EEWKGSETYSPNTAyGVDFLVPVMGYICR SEO ID NO : 35 regulation
37 CIZ1. NPO36259.2 Cell cycle Y799 EEWKGSETYSPNTAYGVDFLVPVMGyICR SEQ ID NO: 36 regulation
38 claspin NPO71394.2 Cell cycle Y3 O6 ESALNLPyHMPENK SEO ID NO : 37 regulation US 2010/0129929 A1 May 27, 2010
TABLE 1 - continued Novel Phosphorylation Sites in Carcinoma.
B D A. Accession C Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO
39 CUL2 NPOO3582.2 Cell cycle Y477 MyTDMSVSADLNNK SEQ ID NO: 38 regulation
40 calreticulin NP 659.483.1 Chaperone Y75 FyAISARFKPFSNK SEO ID NO : 39 3
41 BLM NPOOOO 48.1 Chromatin, DNA- Y894 HHPYDSGIIyCLSR SEQ ID NO: 40 binding, DNA repair or DNA replication protein
42 Bright NPOO5215.1 Chromatin, DNA- Y276 QVLDLFMLyVLVTEK SEQ ID NO: 41 binding, DNA repair or DNA replication protein 43 actin, NP OO1 O91.1 Cytoskeleta Y171 TTGIVLDSGDGVTHNVPIYEGyALPHAIMR SEQ ID NO : 42 alpha 1. protein
44 ACTN1 NP OO1 O93.1 Cytoskeleta Y215 KDDPLTNLNTAFDVAEKyLDIPK SEQ ID NO : 43 protein
45 ARWCF NP OO1661.1 Cytoskeleta Y282 SLAADDEGGPELEPDyGTATR SEQ ID NO: 44 protein 46 callponin 3 NP OO1830.1 Cytoskeleta Y322 DYQySDQGIDY SEQ ID NO: 45 protein
47 CK1 O NPOOO412.2 Cytoskeleta Y16 O LASyLDKVR SEQ ID NO: 46 protein
48 CK17 NPOOO413.1 Cytoskeleta Y398 LLEGEDAHLTQyK SEO ID NO : 47 protein
49 CK1.8 NPOOO215.1 Cytoskeleta Y13 STFSTNyR SEQ ID NO : 48 protein
50 CK1.8 NPOOO215.1 Cytoskeleta Y256 AQyDELAR SEQ ID NO: 49 protein
51 CK19 AAA36O44.1 Cytoskeleta Y61 FVSSSSSGGyGGGYGGVLTASDGLLAGNEK SEQ ID NO: 50 protein
52 CK19 AAA36O44.1 Cytoskeleta Y65 FVSSSSSGGYGGGyGGVLTASDGLLAGNEK SEQ ID NO: 51 protein
53 CK19 NPOO2267.2 Cytoskeleta Y13 O DYSHyYTTIODLR SEO ID NO : 52 protein
54. CK19 NPOO2267.2 Cytoskeleta Y256 SQyEVMAEQNRK SEO ID NO : 53 protein
55 CK19 NPOO2267.2 Cytoskeleta Y38O SRLEQEIATyR SEQ ID NO: 54 protein
56 CK5 NPOOO415.2 Cytoskeleta Y66 VSLAGACGVGGYGSRSLyNLGGSKR SEO ID NO : 55 protein
st CKs NPOOO 415.2 Cytoskeleta Y283 DVDAAyMNKVELEAK SEO ID NO : 56 protein
58 CK5 NPOOO 415.2 Cytoskeleta Y346 AQyEEIANR SEO ID NO : 57 protein
59 CK5 NPOOO 415.2 Cytoskeleta Y361 SRTEAESWyoTKYEELOQTAGR SEO ID NO : 58 protein US 2010/0129929 A1 May 27, 2010
TABLE 1 - continued Novel Phosphorylation Sites in Carcinoma.
B D A. Accession C Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 60 CK5 NPOOO 415.2 Cytoskeleta Y365 SRTEAESWYOTKyEELOQTAGR SEO ID NO : 59 protein
61 CK6 NP 775109.1 Cytoskeleta Y62 SLyGLGGSKR SEQ ID NO: 60 protein
62 CK6 NP 775109.1 Cytoskeleta Y83 ISIGGGSCAISGGyGSR SEQ ID NO : 61 protein
63 CK7 NPOO5547.3 Cytoskeleta Yss SAyCGPVGAGIR SEQ ID NO: 62 protein
64. CK7 NPOO5547.3 Cytoskeleta Y2O5 DVDAAyMSK SEQ ID NO: 63 protein
6s CK7 NPOO5547.3 Cytoskeleta Y283 AEAEAWyOTKFETLQAQAGK SEQ ID NO: 64 protein
66 CK8 NPOO2264. Cytoskeleta Y25 SyTSGPGSR SEO ID NO : 65 protein
67 CK8 NPOO2264. Cytoskeleta Y179 LKLEAELGNMOGLVEDFKNKyEDEINKR SEQ ID NO: 66 protein
68 CK8 NPOO2264. Cytoskeleta Y282 SRAEAESMyOIKYEELQSLAGK SEO ID NO : 67 protein
69 CK8 NPOO2264. Cytoskeleta Y286 SRAEAESMYQIKyEELQSLAGK SEQ ID NO: 68 protein
70 claudin 2 NPO65117. Cytoskeleta Y195 SNYyDAYQAQPLATR SEQ ID NO: 69 protein
71 claudin 2 NPO65117. Cytoskeleta Y198 SNYYDAyOAQPLATR SEO ID NO : 7 O protein
72 claudin 2 NPO65117. Cytoskeleta Y224 SEFNSySLTGYV SEO ID NO : 71 protein
73 claudin 3 NPOO1297. Cytoskeleta Y198 VVySAPR SEO ID NO : 72 protein
74 cortactin NPOO5222.2 Cytoskeleta Y178 SAVGFDyOGKTEK SEO ID NO : 73 protein
7s CTNNA1 NP OO1894.2 Cytoskeleta Y28 O QLOOAVTGISNAAQATASDDASOHOGGGG SEO ID NO : 74 protein GELAyALNNFDK
76 CTNNA1 NP OO1894.2 Cytoskeleta Y419 NGNEKEVKEyAQVFR SEO ID NO : 75 protein
77 CTNND2 NP OO1323.1 Cytoskeleta Y292 GGSAPEGATyAAPR SEO ID NO : 76 protein
78 desmoplakin 3 NP OO2221.1 Cytoskeleta Y48O LNyGIPAIVK SEO ID NO : 77 protein
79 desmoplakin 3 NP OO2221.1 Cytoskeleta YSSO HVAAGTQQPyTDGVR SEO ID NO : 78 protein
80 ELMO2 NP 5734 O3.1 Cytoskeleta Y717 EPSSYDFVyHYG SEO ID NO : 79 protein
81 ELMO2 NP 5734 O3.1 Cytoskeleta Y719 EPSSYDFVYHyG SEQ ID NO: 80 protein
82 EMAP NPOO 4425.2 Cytoskeleta Y186 NSESKPKEPVFSAEEGyVK SEQ ID NO: 81 protein
US 2010/0129929 A1 May 27, 2010
TABLE 1 - continued Novel Phosphorylation Sites in Carcinoma.
B D A. Accession C Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO
15DHX15 NP OO1349.2 Enzyme, misc. Y13 HRLDLGEDyPSGK SEQ ID NO: 114
16DNTT NPOO4O79.3 Enzyme, misc. Y477 MILDNHALyDKTK SEQ ID NO: 115
17 EHHADH AAA53289.1 Enzyme, misc. Y58O TGKGWyOYDKPLGRIHKPDPWLSTFLSRYRK SEQ ID NO : 116 18EHHADH AAA53289.1 Enzyme, misc. Y582 TGKGWYOyDKPLGRIHKPDPWLSTFLSRYRK SEQ ID NO : 117
19 ENO1 NP OO1419.1 Enzyme, misc. Y189 IGAEVyHNLK SEQ ID NO: 118
2 OFBPase NPOO3828.2 Enzyme, misc. Y216 IYSLNEGyAK SEQ ID NO: 119 21ARFIP1 NP_00102O766. 1 G protein or Y3O4 EKyDKMRNDVSVKLK SEQ ID NO: 12 O regulator 22ARHGAP12 NP O6O757.4 G protein or Y355 GHTLyTSDYTNEK SEQ ID NO: 121 regulator 23ARHGAP12 NPO6O757.4 G protein or Y359 GHTLYTSDyTNEK SEQ ID NO: 122 regulator
24ARHGAPs NP OO1164.2 G protein or Y1104 QKGySDEIYVVPDDSQNR SEQ ID NO: 123 regulator 25ARHGEF5 NPOO5426.2 G protein or Y641 TEQTPDLVGMLLSYSHSELPQRPPKPAIySS SEQ ID NO: 124 regulator WTPR
26ARHGEF5 NPOO5426.2 G protein or Y137O RTEELIyLSQK SEQ ID NO: 125 regulator
27ARHGEFf NPOO3 890.1 G protein or Y595 SLVDTVyALKDEVOELR SEQ ID NO: 126 regulator
28 BCAR3 NPOO3558.1 G protein or Y42 SPLAEHRPDAyODVSIHGTLPR SEO ID NO : 127 regulator
29 BCAR3 NPOO3558.1 G protein or Y341 AHOSESyLPIGCK SEQ ID NO: 128 regulator
3 O Coca-2EP3 NPOO 64 4 O.2 G protein or Y8 MPAKTPIyLKAANNK SEQ ID NO: 129 regulator
31DOCK4 NP O5552O.3 G protein or Y821 LQCIGKTVESQLyTNPDSR SEQ ID NO : 13 O regulator 32DOCK4 NPO5552O3 G protein or Y1727 ERPCSAIyPTPVEPSQR SEQ ID NO : 131 regulator 33 DOCK4 NPO5552O3 G protein or Y1831 GSVOSFTPSPVEyHSPGLISNSPVLSGSYS SEQ ID NO : 132 regulator SGISSISR
34 FARP2 NPO55623.1 G protein or Y365 DSGMKRIPyER SEQ ID NO : 133 regulator
35 FARP2 NP O55623.1 G protein or Y4O7 TPASPSSANAFySLSPSTLVPSGLPEFK SEQ ID NO : 134 regulator 36A2M NPOOOOO5.2 Inhibitor protein Y497 LSFYyLIMAKGGIVRTGTHGLLVK SEQ ID NO : 135
37 CIAS1 NPOO 4886.3 Inhibitor protein Y13 yLEDLEDVDLKK SEQ ID NO : 136 38 CSTB NPOOOO 91.1 Inhibitor protein Y53 SQVVAGTNyFIK SEO ID NO : 137
39AFG3L2 NP OO6787.1 Mitochondrial Y179 DFVNNyLSKGVVDR SEQ ID NO : 138 protein
4 OATPSI NPOO9 O31.1 Mitochondrial Y3 O yNYLKPRAEEER SEQ ID NO : 139 protein US 2010/0129929 A1 May 27, 2010 10
TABLE 1- Continued Novel Phosphorylation Sites in Carcinoma.
A. Accession C Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 41 ATPSI POO 9 O31. itochondrial Y32 YNyLKPRAEEER SEQ ID NO: 14 O protein
42 CHCHD3 PO 6 O282. itochondrial Y141 QDAFyKEQLAR SEQ ID NO: 141 protein
43DNAH7 PO6172O. cotto or Y1272 yYWQENHLETK SEQ ID NO: 142 contractile protein
44DNAH8 POO13 62. cotto or Y251O RIGSTyGPPGGR SEQ ID NO: 143 contractile protein
45DNAI2 PO75462. cotto or Y4 OS yHMAYLTDAAWSPVR SEQ ID NO: 144 contractile protein
PO75462. cotto or Y4O9 YHMAyLTDAAWSPVR SEQ ID NO: 145 contractile protein
47DUSP11 POO3575. Phosphatase Y313 RWyPYNYSR SEQ ID NO: 146
48 CPA6 P 065094. Protease Y326 KHIRAYLSFHAyAQMLLYPYSYK SEO ID NO : 147
49 CPA6 PO65094. Protease Y334 KHIRAYLSFHAYAQMLLYPySYK SEQ ID NO : 148
50 CPA6 P 065094. Protease Y336 KHIRAYLSFHAYAOMLLYPYSyK SEQ ID NO: 149
51 CPA6 PO 65094. Protease Y368 yGPASTTLYVSSGSSMDWAYK SEQ ID NO: 150
52 CPA6 PO 65094. Protease Y376 YGPASTTLyVSSGSSMDWAYK SEQ ID NO: 151
53 CPA6 PO 65094. Protease Y387 YGPASTTLYVSSGSSMDWAyK SEQ ID NO: 152
54 ECEL1 POO 4817. Protease Y512 LOYMMVMVGyPDFLLK SEQ ID NO: 153
55 DYRK2 POO3574. Protein kinase, Y3. Of VyTYIQSR SEQ ID NO: 154 dual-specificity
5 6 DYRK3 POO3573. Protein kinase, Y367 LyTYIQSR SEO ID NO : 155 dual-specificity
57AMPKB POO 6244. Protein kinase, Y24 O MLNHLyALSIK SEQ ID NO: 156 regulatory subunit
POO5456. Protein kinase, Y173 yYAMKILKKEVIIAK SEO ID NO : 157 Ser/Thr (non receptor)
POO5456. Protein kinase, Y174 YyAMKILKKEVIIAK SEO ID NO : 158 Ser/Thr (non receptor)
6 OASK1 POO5914. Protein kinase, Yst O IyOPSYLSINNEVEEKTISIWHVLPDDK SEO ID NO : 159 Ser/Thr (non receptor)
61ASK1 POO 5914 . Protein kinase, Ysf4 IYOPSyLSINNEVEEKTISIWHVLPDDK SEQ ID NO: 16 O Ser/Thr (non receptor)
62 BRDT P OO1717. Protein kinase, Y291 HFSyAWPFYNPVDVNALGLHNYYDVVK SEQ ID NO: 161 Ser/Thr (non receptor)
63 BRDT P OO1717. Protein kinase, Y296 HFSYAWPFyNPVDVNALGLHNYYDVVK SEQ ID NO: 162 Ser/Thr (non receptor) US 2010/0129929 A1 May 27, 2010 11
TABLE 1 - continued Novel Phosphorylation Sites in Carcinoma.
B D A. Accession C Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO
64 BRDT NP OO1717.2 Protein kinase, Y309 HFSYAWPFYNPVDVNALGLHNyYDVVK SEQ ID NO : 63 Ser/Thr (non receptor)
65BRDT NPOO1717.2 Protein kinase, Y31 O HFSYAWPFYNPVDVNALGLHNYyDVVK SEQ ID NO : 64 Ser/Thr (non receptor)
66 CAMKK2 NP OO654. O.3 Protein kinase, Y183 LAyNENDNTYYAMKVLSKKKLIR SEQ ID NO : 65 Ser/Thr (non receptor)
67DCAMKL3 XPO47355.. 6 Protein kinase, Y68O HRETROAyAMK SEQ ID NO : 66 Ser/Thr (non receptor)
68 ALK1. NPOOOO11.2 Protein kinase, Y375 RyMAPEVLDEQIR SEQ ID NO : 67 Ser/Thr (receptor)
69 Ack NPOO5772.3 Protein kinase, Y868 KVSSTHYYLLPERPSyLERYOR SEQ ID NO : 68 Tyr (non receptor)
7 OAck NPOO5772.3 Protein kinase, Y872 VSSTHYYLLPERPSYLERyOR SEQ ID NO : 69 Tyr (non receptor)
71 FAK NPOO5598.3 Protein kinase, Y463 CIGEGQFGDVHQGIyMSPENPALAVAIK SEQ ID NO : 71. Tyr (non receptor)
72 FASTK NP OO6703.1 Protein kinase, Y357 yLSLLDTAVELELPGYR SEQ ID NO : 72 Tyr (non receptor)
73ALK NPOO 42.95.2 Protein kinase, Y1586 HFPCGNVNYGyOQQGLPLEAATAPGAGHY SEQ ID NO : 73 Tyr (receptor) EDTILK
74 Axl NP OO169 O.2 Protein kinase, Y689 KIyNGDYYR SEQ ID NO : 74 Cyr (receptor)
75AX. NP OO169 O.2 Protein kinase, Y693 KIYNGDyYR SEQ ID NO : 7s Tyr (receptor)
76Axl NP OO169 O.2 Protein kinase, Y7 O GQTPYPGVENSEIyDYLR SEQ ID NO : 76 Tyr (receptor)
77 DDR1 NPOO1945.3 Protein kinase, Y7ss NLyAGDYYRVQGR SEQ ID NO : 77 Tyr (receptor)
78DDR2 NPOO6173.2 Protein kinase, Y481 IFPLRPDyQEPSR SEQ ID NO : 78 Tyr (receptor)
79 EphA1 NPOO5223.3 Protein kinase, Y599 ATDVDREDKLWLKPyVDLQAYEDPAQGAL SEQ ID NO : 79 Tyr (receptor) DFTR
80 EphA1 NPOO5223.3 Protein kinase, Y605 ATDVDREDKLWLKPYVDLQAyEDPAQGAL SEQ ID NO : 8O Tyr (receptor) DFTR
81 EphA2 NPOO 4422.2 Protein kinase, Y628 VIGAGEFGEVyK SEQ ID NO : 81 Cyr (receptor)
82 EphA2 NPOO 4422.2 Protein kinase, Y694 YKPMMIITEyMENGALDK SEQ ID NO : 82 Tyr (receptor)
83 EphA2 NPOO 4422.2 Protein kinase, Y96 O IAySLLGLKDQVNTVGIPI SEQ ID NO : 83 Tyr (receptor) US 2010/0129929 A1 May 27, 2010 12
TABLE 1 - continued Novel Phosphorylation Sites in Carcinoma.
B D A. Accession C Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO
84 EphA3 NPOO5224.2 Protein kinase, Y736 yLSDMGYVHRDLAAR SEQ ID NO: 184 Cyr (receptor)
85 EphB3 NPOO4434.2 Protein kinase, Y593 HGSDSEyTEKLQQYIAPGMK SEQ ID NO : 85 Tyr (receptor)
86 EphB4 NPOO 4435.3 Protein kinase, Y357 EDLTyALR SEQ ID NO : 86 Tyr (receptor)
87 EphB6 NPOO 443 6.1 Protein kinase, Y62O GTGYTEQLQQySSPGLGVK SEQ ID NO : 87 Tyr (receptor)
88 EphB6 NPOO 443 6.1 Protein kinase, Y629 yYIDPSTYEDPCQAIR SEQ ID NO : 88 Tyr (receptor)
89 FGFR4 NPOO2OO2.3 Protein kinase, Y642 GVHHIDyYKK SEQ ID NO : 89 Tyr (receptor)
9 OFGFR4 NPOO2 OO2.3 Protein kinase, Y643 GVHHIDYyKK SEQ ID NO : 9 O Tyr (receptor)
91ABCA2 NP OO1597.2 Receptor, Y21.79 yADKPAGTYSGGNKRK SEQ ID NO : 191 channel, transporter or cell surface protein 192ABCA2 NP OO1597.2 Receptor, Y21.87 YADKPAGTySGGNKRK SEQ ID NO : 192 channel, transporter or cell surface protein
193ABCC1 NPOO4987.2 Receptor, Y92 O QLSSSSSySGDISR SEQ ID NO : 193 channel, transporter or cell surface protein
194 ABCC8 NPOOO343.2 Receptor, Y798 yKMVIEACSLQPDIDILPHGDQTQIGER SEQ ID NO : 194 channel, transporter or cell surface protein 195ABCF2 NPOO912 O. 1 Receptor, Y465 yHQHLQEQLDLDLSPLEYMMKCYPEIK SEO ID NO : 195 channel, transporter or cell surface protein
196ABCF2 NPOO912 O. 1 Receptor, Y482 YHQHLQEQLDLDLSPLEyMMKCYPEIK SEQ ID NO : 196 channel, transporter or cell surface protein 197 albumin NPOOO468.1 Receptor, Y108 ETyGEMADCCAK SEO ID NO : 197 channel, transporter or cell surface protein
198ANTXR1 EAW.99856.1 Receptor, Y92 WPTVDASyYGGR SEQ ID NO : 198 channel, transporter or cell surface protein US 2010/0129929 A1 May 27, 2010 13
TABLE 1 - continued Novel Phosphorylation Sites in Carcinoma.
B D A. Accession C Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 199ApoB NPOOO375.2 Receptor, Y368O FLKNIILPVyDK SEO ID NO : 199 channel, transporter or cell surface protein
2OOAPXL NP OO164 O. 1 Receptor, Y763 SySEPEKMNEVGLTR SEQ ID NO: 2OO channel, transporter or cell surface protein
2O1 CACNA1A NPOOOO59.2 Receptor, Y1421 yLLYEKNEVK SEQ ID NO: 2O1 channel, transporter or cell surface protein
2O2 CACNA1A NPOOOO59.2 Receptor, Y1424 YLLyEKNEVK SEQ ID NO: 2O2 channel, transporter or cell surface protein
2O3 CCR8 NPOO51.92.1 Receptor, Y132 yLAVVHAVYALK SEQ ID NO: 2O3 channel, transporter or cell surface protein
2O4 CHRNG NPOO5190 .. 4 Receptor, Y512 PyLPSPD SEQ ID NO: 2O4 channel, transporter or cell surface protein
205 CMKOR1 NPO647 O7.1 Receptor, Y354 VSETEySALEQSTK SEQ ID NO: 2O5 channel, transporter or cell surface protein
2O6 CRIM1 NPO57525.1 Receptor, Y1019 FSGFySMOK SEQ ID NO: 2O6 channel, transporter or cell surface protein
2Of CRIM1 NPO57525.1 Receptor, Y1033 QNHLQADNFyOTV SEO ID NO : 2O7 channel, transporter or cell surface protein
2O8DPP10 NPO65919.2 Receptor, Y512 yFILESNSMLKEAILKK SEQ ID NO: 2O8 channel, transporter or cell surface protein
209EDAR NPO71731.1 Receptor, Y364 TSRMLSSTyNSEK SEQ ID NO: 209 channel, transporter or cell surface protein US 2010/0129929 A1 May 27, 2010 14
TABLE 1- Continued Novel Phosphorylation Sites in Carcinoma.
D A. Accession C Phospho- E 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO
21 O exportin 7 NP O55839.2 Receptor, KIyIGDQVOK SEQ ID NO: 210 channel, transporter or cell surface protein
211 exportin 7 NPO55839.2 Receptor, TLQLLNDLSIGySSVR SEQ ID NO: 211 channel, transporter or cell surface protein
212 ataxin-1 N POOO323.2 RNA binding Y334 yGAPSSADLGLGK SEQ ID NO: 212 protein
213 CPSF1 N PO37423.2 RNA binding Y155 CAAMLVyGTRLVVLPFR SEQ ID NO: 213 protein
214 DDX3 N POO13 47.3 RNA binding Y266 KQyPISLVLAPTR SEQ ID NO: 214 protein
21.5 CHI3L2 N POO3991.2 Secreted protein DKSEVMLyQTINSLK SEQ ID NO: 215
216DEFA1 N POO4O75.1 Secreted protein YGTCIyQGR SEQ ID NO: 216
217ASCL3 N PO65697.1 Transcriptional GCEYSyGPAFTRKRNER SEQ ID NO: 217 regulator
218 CBP N POO 4371.2 Transcriptional Y1391 FVDSGEMSESFPyR SEQ ID NO: 218 regulator
219 COPS2 N POO 4227.1 Transcriptional Y15.9 LyLEREEYGKLQKILR SEQ ID NO: 219 regulator
22 O COPS2 N POO4227.1 Transcriptional Y1.65 LYLEREEyGKLQKILR SEQ ID NO: 220 regulator
221EDF1 N POO3783.1 ranscriptional Y109 INEKPOVIADyESGR SEQ ID NO: 221 regulator
22282 - FIP N PO65823.1 TGyGELNGNAGER SEQ ID NO: 222
223 eBEF1A-1 N POO1393.1 Y177 YEEIVKEVSTyIK SEQ ID NO: 223
224 FAT N POO5236.2 Tumor Y4356 KPLEEKPSQPySAR SEQ ID NO: 224 suppressor
225 CCNB1IP1 N PO67OO1.3 Ubiquitin Y125 QMEKIyTOOIQSKDVELTSMKGEVTSMKK SEQ ID NO: 225 conjugating system
226 Cezanne N PO6459 O.2 Ubiquitin Y218 ALyALMEKGVEKEALK SEQ ID NO: 226 conjugating system
227 FBW1A N POO3930.1 Ubiquitin TyTYISR SEO ID NO : 227 conjugating system
228Fox46 N P OO1073938.1 Ubiquitin Y309 ITCDLyOLISPSR SEQ ID NO: 228 conjugating system
2291810O31KO2RikN PO7357O.1 Unknown function LYEAyLPETFR SEQ ID NO: 229
23 OABHD10 N PO6 O864. 1 Unknown function Y112 FDySGVGSSDGNSEESTLGKWR SEQ ID NO: 23 O US 2010/0129929 A1 May 27, 2010 15
TABLE 1- Continued Novel Phosphorylation Sites in Carcinoma.
B D A. Accession Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 231ADNP NPO56154. Un function Y172 KCTyRDPLYEIVRKHIYR SEQ ID NO: 231
232 ADNP NPO56154. Un function Y185 KCTYRDPLYEIVRKHIyR SEQ ID NO: 232
233AGXT2 NP 1141. O6. Un function Y298 DTLSTSVAKSIAGFFAEPIQGVNGVVoyPK SEQ ID NO: 233
234 ALS2CR11 NP 689738. Un function Y177 RyDDKRNNILLELIQYDNR SEQ ID NO: 234
235 ANKRD25 Un function Y7 O2 QNRAGySPIMLTALATLK SEQ ID NO: 235
236ANKRD26 NPO55730. Un function Y7 O4 WKNOIOSMDDVDDLTOSSETASEDCELPHS SEQ ID NO: 236 Syk
237 ANKRD26 NPO55730. Un function Y12 O9 QYOyENEK SEO ID NO. 237
238APCDD1 NP 694,545. Un function Y98 SGPEFITRSYRFyHNNTFKAYOFYYGSNR SEQ ID NO: 238
239 APCDD1 NP 694,545. Un function Y11 O SGPEFITRSYRFYHNNTFKAYOFYyGSNR SEQ ID NO: 239
24 OARRDC3 NPO65852. Un function Y382 ALQGPLFAyIQEFR SEQ ID NO: 24 O
NP 116274. Un function Y317 LGGETLNPVyVPCVK SEQ ID NO: 241
242 ATP13A1 NP O651.43. Un function SEQ NO: 242
243 BAT2D1 NPO55987. Un function Y1218 GHTRDyPQYR SEQ NO: 243
244 BAT2D1 NPO55987. Un function Y219 O ESVTDyTTPSSSLPNTVATNNTK SEQ NO: 244
245BAT3 NPOO 463 O. Un function Y1116 LQEDPNySPQRFPNAQR SEQ NO: 245
246 BAZ2B NPO38478. Un function Y398 PLSLVNOAKKETyMK SEQ NO: 246
247BAZ2B NPO38478. Un function Y772 LQGEVAyYAPCGKK SEQ NO: 247
248 BAZ2B NPO38478. 2 Un function Y773 LQGEVAYyAPCGKK SEQ NO: 248
249 C10orf78 GI: 73 62 OO88 Un function Y44 MNYKVKLEEISGLLVDALYTNTIyYLIK SEQ NO: 249
25 OC10orf78 GI: 73 62 OO88 Un function Y45 MNYKVKLEEISGLLVDALYTNTIYyLIK SEQ NO: 25 O
251C10orf 81 NPO79165. 3 Un function Y215 QTHLODLSEATODVKEENHyLTPR SEQ NO: 251
252 C10orf 81 NPO79165. 3 Un function Y254 IECHyEPMESYFFK SEQ NO: 252
253 C10orf2 2 Un function Y675 SyOSLKRHMESVEHRR SEQ NO: 253
254 C11orf2 NPO37397. 2 Un function Y41 LYyGLSEGEAAGR SEQ NO: 254
255 C 4 or f8 NP 776245.1 Un function Y155 GIAGREEMTDNTGyVSGYKGSGTYDK SEQ NO: 255
256 C for f6 O NP OO1078892.1 Un function Y313 HSQELQyATPVFQEVAPR SEQ NO: 256
27C 8 or f3 O BACO5412.1 Un function Y103 VyLIINSIKK SEQ NO: 257
258 C. 8 or f3 O BACO5412.1 Un function Y408 YFINYFFyK SEQ NO: 258
259 C. 9 orf21 NP 77575.2.1 Un function Y95 GLHSENREDEGWOVyR SEQ NO: 259
26 OC 9 orf21 NP 77575.2.1 Un function Y124 TyRLDAGDADPR SEQ NO: 26 O
261C 9 orf21 NP 77575.2.1 Un function Y638 KKEQWyAGINPSDGINSEVLEAIR SEQ NO: 261
262C orf116 NPO76427.2 Un function Y4 O SGSSDSSyDFLSTEEK SEQ NO: 262
263 C orf21 O NP 872323.1 Un function Y94 GGRPOVAEDEDDDGFIEDNyIQPGTGELGT SEQ NO: 263 EGSR
264 C2Oorf77 NPO67038.1 Un function Y161 TFQQIQEEEDDDyPGSYSPQDPSAGPLLTE SEQ NO: 264 ELIK
US 2010/0129929 A1 May 27, 2010 17
TABLE 1- Continued Novel Phosphorylation Sites in Carcinoma.
D A. Accession Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ NO
PO57113. 1. Un function Y33 O SGVDADSSyFK SEQ NO. 299
P 1155.05. 1. Un function SCSSGPAGPyLLSK SEQ NO.
P 1155.05. 1. Un function SRDPGyDHLWDETLSSSHOK SEQ NO. 3O1
POO1O 92284.1 Un function Y379 DIVEDPDKFyIFK SEQ NO.
P OO1073 O27.1 Un function FYGRDyEYNR SEQ NO.
3O3DNA2I N POO 107391.8.1 Un function Y928 LTyEGKLECGSDKVANAVINLRHFKDVK SEQ NO.
3O4 DNAJB4. N POO8965. 2 Un function Y296 RIIGyGLPFPKNPDQR SEQ NO. 305
3. OSDZIP1 N P O55749. 1. Un function Y414 LRTSMIDDLNASNVFyK SEQ NO. 3 O 6
3 O 6 ELMO3 N PO78988. Un function VNALTyGEVLR SEQ NO. 3. Of
3. Of EMSY N P 064578. Un function LELEAyAGVISALRAQGDLTKEKK SEQ NO.
3O8EPS8L1 N PO6O199. Un function Y399 VRDPAGQEGyVPYNILTPYPGPR SEQ NO. 309
309EPS8L1 N PO6O199. Un function VRDPAGQEGYVPyNILTPYPGPR SEQ NO.
31 OEPS8L1 N PO6O199. Un function Yssf VySQVTVQR SEQ NO.
311 ERH N POO 44 41. Un function Y36 TYADYESVNECMEGVCKMyEEHLK SEQ NO.
312 ETEA N P O554.28. Un function IySYVVSRPQPR SEQ NO.
313 exophilin 5 N Un function Y4 OS YVyPRGFOENK SEQ NO.
314 FADS6 N P 83.5229. Un function Y16 O yVYMFLAPFLLPIATPLVAVERLR SEQ NO.
315 FADS6 N P 83.5229. Un function Y162 YVyMFLAPFLLPIATPLVAVERLR SEQ NO.
316 FAM48A N PO6 OO39. Un function LVMQETLSCLVVNLYPGNEGySLMLR SEQ NO.
317 FAM62A N PO561 O7. Un function Y588 LLTAPELILDQWFQLSSSGPNSRLyMKLVMR SEQ NO.
318 FAM62A N PO56107. Un function FLGGLVKGKSDPyVK SEQ NO.
319 FAM83D N P 1121,81. Un function Y176 GATRVETHFQPRGAGEGGPygCK SEQ NO.
32 OFAM83F N P 612444. Un function yFLEMCODLQLTDFR SEQ NO. 321
321 FAM83F N P 612444. 1. Un function Y146 VGLHySSTVAR SEQ NO. 322
322 FBX43 N POO1 O25 O31.2 Un function YSO3 KMGIEKLDILTELKyR SEQ NO. 323
323 FCHO2 N P 62O137. 1. Un function Y22 NSGFDVLyHNMK SEQ NO. 324
324 FLEG1 N Un function RHRyKSRMNKTYCK SEQ NO. 3.25
325 FLJ OO128 N P 06 0541. Un function Y242 SPGDGHNAPVEGPEGEyVELLEVTLPVR SEQ NO. 326
326 FLJOO258 N P 689 619. Un function Yssf VyDDVPYEK SEQ NO. 327
327 FLJ11273 N PO6 O844. Un function Y18 SLSHLPLHSSKEDAyDGVTSENMR SEQ NO. 328
328 FLJ11273 N PO6 O844. Un function YSO NGLVNSEVHNEDGRNGDVSQFPyVEFTGR SEQ NO. 329
329 FLJ11305 N PO6 O856. Un function Y258 AIDSSNLKDDySTAQR SEQ NO. 33 O
33 OFLJ12949 N PO75384. Un function AQEEADyIEWLK SEQ NO. 331
331FLJ13941 N PO79.124. Un function Y435 PEGRATEEQAAAAHLGEyVLMIRDVTTPPFL SEQ NO. 332 GR US 2010/0129929 A1 May 27, 2010 18
TABLE 1 - continued Novel Phosphorylation Sites in Carcinoma.
B D A. Accession C Phospho- E H 1 Protein Name No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 332 FLJ14564 NP 115939.1 Unknown function Y54 SSSSDEEyIYMNKVTINK SEQ ID NO. 333
333 FLJ14564 NP 115939.1 Unknown function Y56 SSSSDEEYIyMNKVTINK SEQ ID NO. 334
334 FLJ14564 NP 115939.1 Unknown function Y307 YLSASEyGSSVDGHPEVPETK SEO ID NO. 335
335 FLJ14564 NP 115939.1 Unknown function Y383 DNHLHFyODR SEQ ID NO. 336
336 FLJ2O485 NPO61915.2 Unknown function Y279 ENKDTMDAINVLSKyLRVK SEO ID NO. 337
337FLT21439 NPO7941.3.3 Unknown function Y21.69 yNEMTYIFDLLHKK SEQ ID NO. 338
338 FLJ21 610 NPO73588.1 Unknown function Y700 SASySLESTDVK SEQ ID NO. 339
339FLJ21901 NPO78898.2 Unknown function Y486 QLKLLQKLDHyGR SEQ ID NO. 34 O 340 adaptin, beta NP OO1273.1 Vesicle protein Y276 FLELLPKDSDyYNMLLK SEQ ID NO. 341 341 atlastin NPO56999.2 Vesicle protein Y429 RYLQQLESEIDELyIQYIK SEQ ID NO. 342
342 atlastin NP O56999.2 Vesicle protein Y432 RYLQQLESEIDELYIQyIK SEQ ID NO. 343
343 BET1 NPOO5859.1 Vesicle protein Y2O AGLGEGVPPGNYGNYGyANSGYSACEEEN SEQ ID NO. 344 ERLTESLR
344 COP beta NPOO4757.1 Vesicle protein Y29 O GSNNVALGyDEGSIIVK SEQ ID NO. 345 prime
345 CPLX2 NP OO6641.1 Vesicle protein Y70 DKyGLKKKEEK SEQ ID NO. 346
346 EHD2 NP O55416.2 Vesicle protein Y309 ARLVRVHAyIISYLK SEO ID NO. 347
3.47 EHD2 NP O55416.2 Vesicle protein Y313 ARLVRVHAYIISyLK SEQ ID NO. 348 348 epsin2 NPO55779.1 Vesicle protein Y17 NIVNNySEAEIK SEQ ID NO. 349
0041. One of skill in the art will appreciate that, in many stabilization, may play a role in cell shape control and endot instances the utility of the instant invention is best understood helial barrier function. (PhosphoSiteREGISTERED, Cell in conjunction with an appreciation of the many biological Signaling Technology (Danvers, Mass.), Human roles and significance of the various target signaling proteins/ PSDTRADEMARK, Biobase Corporation, (Beverly, polypeptides of the invention. The foregoing is illustrated in Mass.)). the following paragraphs summarizing the knowledge in the 0044 ALK, phosphorylated at Y1586, is among the pro art relevant to a few non-limiting representative peptides con teins listed in this patent. ALK, Anaplastic lymphoma kinase, taining selected phosphorylation sites according to the inven receptor protein tyrosine kinase, regulates cell growth, cell tion. differentiation and neurite outgrowth; gene fusions are asso 0042 ABCF2, phosphorylated at Y465, is among the pro ciated with anaplastic large cell non-Hodgkin's lymphomas teins listed in this patent. ABCF2, ATP-binding cassette sub and inflammatory myofibroblastic tumors. This protein has family F (GCN20) member 2, a putative component of ABC potential diagnostic and/or therapeutic implications based on transporter complex that may act in mitochondrial transport; the following findings. Induced inhibition of the protein bind gene expression is upregulated in chemotherapy resistance ing of ALK may prevent increased transmembrane receptor ovarian cancer and clear cell ovarian adenocarcinomas. This protein tyrosine kinase signaling pathway associated with protein has potential diagnostic and/or therapeutic implica Ki-1 large-cell lymphoma (Cancer Res 62: 1559-66 (2002)). tions based on the following findings. Amplification of the Increased receptor signaling protein tyrosine kinase activity ABCF2 gene may correlate with drug-resistant form of neo of ALK may cause Ki-1 large-cell lymphoma (Blood 94: plasms (Cancer Res 64: 1403-10 (2004)). (PhosphoSiteREG 3265-8 (1999)). Translocation of the ALK gene may cause ISTERED, Cell Signaling Technology (Danvers, Mass.), increased cell Surface receptor linked signal transduction Human PSDTRADEMARK, Biobase Corporation, (Beverly, associated with Ki-1 large-cell lymphoma (Mol Cell Biol. 18: Mass.)). 6951-61 (1998)). Translocation of the ALK gene may cause 0043 ACTN1, phosphorylated at Y215, is among the pro increased cell Surface receptor linked signal transduction teins listed in this patent. ACTN1, Alpha-actinin isoform 1 a associated with Ki-1 large-cell lymphoma (Mol. Cell Biol 18: non-muscle cell actin-binding protein that interacts with col 6951-61 (1998)). Translocation mutation in the Protein lagen (human COL17A1) and functions in actin filament kinase domain of ALK may cause Ki-1 large-cell lymphoma US 2010/0129929 A1 May 27, 2010
(Science 263: 1281-4 (1994)). Translocation of the ALK gene tyrosine kinases (Axl, Mer and Tyro3) have been shown to may cause increased tyrosine phosphorylation of STAT pro express identical flanking sequences at the homologous sites. tein associated with T-cell lymphoma (JImmunol 168: 466 The conservation of this site (FGLSKRKIpYNGDYYRO) 74 (2002)). Increased phosphorylation of ALK may correlate Suggests that it may play an important role in the regulating with Ki-1 large-cell lymphoma (Blood 95: 2144-9 (2000)). the biological function of members of the Axl family of Translocation of the ALK gene may cause lymphoma (Blood tyrosine kinases. 90: 2901-10 (1997)). Translocation of the ALK gene corre 0047 Accordingly, the elucidation of the phosphorylation lates with non-Hodgkin's lymphoma (Blood 85: 3416-22 sites reported herewith along with the concomitant tools (1995)). Translocation of the ALK gene may cause increased enabled by the instant discoveries provide much needed diag cell Surface receptor linked signal transduction associated nostic and/or therapeutic modalities useful in light of the with Ki-1 large-cell lymphoma (Mol Cell Biol 18: 6951-61 many events in which Axl has been implicated to date. For (1998)). Translocation of the ALK gene may cause increased example, increased expression of AXL protein has been anti-apoptosis associated with Ki-1 large-cell lymphoma shown to correlate with neoplasm metastasis associated with (Blood 96:4319-27 (2000)). Translocation of the ALK gene colonic neoplasms (IntJ Cancer 60:791-7 (1995)); increased causes hematologic neoplasms (Blood 98: 1209-16 (2001)). severity of malignant form of colonic neoplasms (IntJCancer Translocation of the ALK gene may cause increased cell 60: 791-7 (1995)). Similarly, increased expression of AXL Surface receptor linked signal transduction associated with mRNA has been shown to correlate with acute myelomono Ki-1 large-cell lymphoma (Mol. Cell. Biol. 18: 6951-61 cytic leukemia (Blood 84: 1931-41 (1994); hepatocellular (1998)). Amplification of the ALK gene may correlate with carcinoma (Genomics 50:331-40 (1998)). Increased expres neuroblastoma (Oncogene 21: 5823-34 (2002)). Transloca sion of AXL protein has been postulated to correlate with tion of the ALK gene may cause increased tyrosine phospho more severe form of stomach neoplasms (Anticancer Res 22: rylation of STAT protein associated with Ki-1 large-cell lym 1071-8. (2002)). Lack of expression of AXL protein is sus phoma (Cancer Res 61: 6517-23 (2001)). Translocation of the pected to correlate with abnormal cell-matrix adhesion asso ALK gene may cause increased cell Surface receptor linked ciated with lung neoplasms (Eur J Cancer 37: 2264-74. signal transduction associated with Ki-1 large-cell lymphoma (2001)). Increased expression of AXL protein has been cor (MCB 18: 6951-61 (1998)). Increased expression of ALK in related with disease progression associated with colonic neo T-lymphocytes may cause plasma cell granuloma (Blood plasms (IntJ Cancer 60:791-7 (1995)). Lack of expression of 101: 1919-27 (2003)). Translocation of the ALK gene causes AXL protein may also correlate with abnormal cell-matrix increased transmembrane receptor protein tyrosine kinase adhesion associated with small cell carcinoma (EurJ Cancer signaling pathway associated with Ki-1 large-cell lymphoma 37: 2264-74. (2001)). Increased expression of AXL mRNA (Blood 94: 3265-8 (1999)). Increased receptor signaling pro has been associated with more severe form of myeloid leu tein tyrosine kinase activity of ALK may cause neuroblas kemia (Leukemia 13: 1352-8. (1999)). Increased expression toma (Oncogene 21: 5823-34 (2002)). Increased expression of AXL mRNA has also been correlated with chronic myeloid of ALK in T-lymphocytes may cause T-cell lymphoma leukemia (Blood 84: 1931-41 (1994)). (PhosphoSiteREGIS (Blood 101: 1919-27 (2003)). (PhosphoSiteREGISTERED, TERED, Cell Signaling Technology (Danvers, Mass.), Cell Signaling Technology (Danvers, Mass.), Human Human PSDTRADEMARK, Biobase Corporation, (Beverly, PSDTRADEMARK, Biobase Corporation, (Beverly, Mass.)). Mass.)). 0048 CAMKK2, phosphorylated at Y183, is among the 0045 ARRB1, phosphorylated at Y47, is among the pro proteins listed in this patent. CAMKK2, Calcium/calmodu teins listed in this patent. ARRB1, Arrestin beta1, an adaptor lin-dependent protein kinase kinase 2 beta, a protein kinase protein regulating desensitization and internalization of G that selectively phosphorylates and activates Ca2+-calmodu protein-coupled receptors, interacts with phosphorylated lin (CaM)-dependent protein kinases I and IV in a Ca2+- receptors to disrupt G protein coupling and induce endocyto CaM-dependent manner, acts in calcium mediated cellular sis. This protein has potential diagnostic and/or therapeutic responses. (PhosphoSiteREGISTERED, Cell Signaling implications based on the following findings. Decreased Technology (Danvers, Mass.), Human PSDTRADEMARK, expression of ARRB1 protein may cause decreased ubiquitin Biobase Corporation, (Beverly, Mass.)). dependent protein catabolic process associated with mela 0049 Cas-L (NEDD9), phosphorylated atY629, is among noma (JBC 280: 24412-9 (2005)). Decreased expression of the proteins listed in this patent. Cas-L is a widely expressed ARRB1 protein may cause decreased ubiquitin-dependent docking protein which is believed to play a central coordinat protein catabolic process associated with melanoma (J Biol ing role for tyrosine-kinase-based signaling in cell adhesion. Chem 280: 24412-9 (2005)). Increased expression of ARRB1 May function in transmitting growth control signals between in thyroid correlates with thyroid nodule (FEBS Lett 486: focal adhesions at the cell periphery and the mitotic spindle in 208-212 (2000)). (PhosphoSiteREGISTERED, Cell Signal response to adhesion or growth factor signals initiating cell ing Technology (Danvers, Mass.), Human PSDTRADE proliferation. Integrin beta-1 stimulation leads to recruitment MARK, Biobase Corporation, (Beverly, Mass.)). of various proteins including CRK, NCK and SHPTP2 to the 0046 Axl, phosphorylated at Y689, Y693 and Y750, is tyrosine phosphorylated form. Phosphorylated following among the proteins elucidated herein. Axl is an oncogenic integrin beta-1, antigen receptor, or Cla calcitonin receptor receptor tyrosine kinase that induces neoplastic growth when signaling. Transformation of fibroblasts with V-ABL results overexpressed. It has been implicated in the regulation of cell in an increase in its tyrosine phosphorylation. Phosphorylated proliferation and the immune response. Axl binds to and is by focal adhesion kinase. Highly expressed in kidney, lung, activated by the growth and survival factor Gasó. The binding and placenta. Also detected in T-cells, B-cells and diverse cell of Gasó to Axl is tought to induce cellular proliferation and to lines. A series of functional, biochemical, and clinical studies inhibit apoptosis in target cells (Gastroenterology 129:1633 established CasL as a bona fide melanoma metastasis gene in 42 (2005)). All members of the Axl family of receptor the mouse. CasL enhanced invasion in vitro and metastasis in US 2010/0129929 A1 May 27, 2010 20
Vivo of both normal and transformed melanocytes, function 94: 8732-7 (1997)). Decreased transcription factor complex ally interacted with focal adhesion kinase and modulated localization of CREBBP may cause abnormal regulation of focal contact formation, and exhibited frequent robust over transcription, DNA-dependent associated with Huntington expression in human metastatic melanoma relative to primary disease (Science 291: 2423-8 (2001)). Increased PML body melanoma. Thus, comparative oncogenomics has identified localization of CREBBP may cause abnormal regulation of CasL as a highly relevant cancer gene governing metastatic transcription, DNA-dependent associated with acute promy potential in murine and human melanoma (Cell 125:1230-3 elocytic leukemia (Proc Natl Acad Sci USA 96: 2627-32 (2006)). Cas-L is involved in integrin-induced T cell migra (1999)). Mutation in the CREBBP gene may cause autosomal tion, and binds adhesion and adaptor proteins to coordinate dominant form of Rubinstein-Taybi syndrome (Nature 376: cell cycle with migration signals. PhosphoSiteREGIS 348-51 (1995)). Translocation mutation in the Bromodomain TERED, Cell Signaling Technology (Danvers, Mass.), of CREBBP may cause drug-induced form of myeloid leu Human PSDTRADEMARK, Biobase Corporation, (Beverly, kemia (Proc Natl Acad Sci USA 94: 8732-7 (1997)). Mis Mass.)). sense mutation in the CREBBP gene may cause myelodys 0050 CBP phosphorylated at Y1391, is among the pro plastic syndromes (Cancer Lett 213: 11-20 (2004)). teins listed in this patent. CBP, CREB binding protein, a Translocation of the CREBBP gene may cause drug-induced transcriptional coactivator that has histone acetyltransferase form of myeloid leukemia (PNAS 94: 8732-7 (1997)). Dele activity; translocation of the corresponding gene is associated tion mutation in the CREBBP gene causes Rubinstein-Taybi with various leukemias; mutation of the corresponding gene syndrome (Hum Mol Genet 10: 1071-6 (2001)). Nonsense causes Rubinstein-Taybi syndrome. This protein has poten mutation in the CREBBP gene may correlate with colonic tial diagnostic and/or therapeutic implications based on the neoplasms (Proc Natl Acad Sci USA 101: 1273-8 (2004)). following findings. Mutation in the CREBBP gene may cause Point mutation in the CREBBP gene causes abnormal multi leukemia (Cancer Lett 213: 11-20 (2004)). Increased PML cellular organismal development associated with Rubinstein body localization of CREBBP may cause abnormal regula Taybi syndrome (Nature 376:348-51 (1995)). Translocation tion of transcription, DNA-dependent associated with acute of the CREBBP gene may cause myelodysplastic syndromes promyelocytic leukemia (Proc Natl AcadSci USA96: 2627 (Blood 89: 3945-50 (1997)). Translocation of the CREBBP 32 (1999)). Nonsense mutation in the CREBBP gene may gene may cause myeloid leukemia (EMBO J. 19: 4655-64 correlate with colonic neoplasms (Proc Natl Acad Sci USA (2000)). Translocation of the CREBBP gene may cause acute 101: 1273-8 (2004)). Translocation of the CREBBP gene monocytic leukemia (Hum Mol Genet 10: 395-404 (2001)). may cause myeloid leukemia (EMBOJ 19:4655-64 (2000)). (PhosphoSiteREGISTERED, Cell Signaling Technology Induced stimulation of the protein binding of CREBBP may (Danvers, Mass.), Human PSDTRADEMARK, Biobase Cor prevent increased cell proliferation associated with breast poration, (Beverly, Mass.)). neoplasms (Biochemistry 39: 4863-8 (2000)). Translocation 0051 CDH3, phosphorylated at Y701, is among the pro mutation in the Bromodomain of CREBBP may cause drug teins listed in this patent. CDH3, P-cadherin, a calcium-de induced form of myeloid leukemia (PNAS 94: 8732-7 pendent cell Surface adhesion molecule that mediates cell (1997)). Translocation of the CREBBP gene may cause drug cell interactions, involved in epidermal stratification and induced form of myeloid leukemia (Proc Natl AcadSci USA morphogenesis; aberrant expression is associated with breast 94: 8732-7 (1997)). Missense mutation in the CREBBP gene and stomach cancer and melanoma. This protein has potential causes Rubinstein-Taybi syndrome (Hum Mol Genet 10: diagnostic and/or therapeutic implications based on the fol 1071-6 (2001)). Increased PML body localization of lowing findings. Increased expression of CDH3 protein cor CREBBP may cause abnormal regulation of transcription, relates with disease progression associated with ovarian neo DNA-dependent associated with acute promyelocytic leuke plasms (Int J Cancer 106: 172-7 (2003)). Increased mia (PNAS 96: 2627-32 (1999)). Translocation of the expression of CDH3 mRNA correlates with invasive form of CREBBP gene may cause leukemia (Blood 90: 535-41 cervix neoplasms (Cancer 89: 2053-8 (2000)). Increased (1997)). Induced stimulation of the protein binding of expression of CDH3 protein correlates with esophageal neo CREBBP may prevent increased cell proliferation associated plasms associated with squamous cell carcinoma (Int J Can with breast neoplasms (Biochemistry Usa 39: 4863-8 cer 79:573-9 (1998)). Increased expression of CDH3 protein (2000)). Translocation of the CREBBP gene may cause acute correlates with increased occurrence of death associated with monocytic leukemia (Nat Genet 14:33-41 (1996)). Nonsense breast neoplasms (Cancer 86: 1263-72 (1999)). Increased mutation in the CREBBP gene causes Rubinstein-Taybi syn expression of CDH3 mRNA correlates with glandular and drome (Hum Mol Genet 10: 1071-6 (2001)). Absence of the epithelial neoplasms associated with cervix neoplasms (Can histone acetyltransferase activity of CREBBP may cause cer 89: 2053-8 (2000)). Increased expression of CDH3 pro Rubinstein-Taybi syndrome (Hum Mol Genet 10: 1071-6 tein correlates with advanced Stage or high grade form of (2001)). Translocation of the CREBBP gene may cause ovarian neoplasms (Int J Cancer 106: 172-7 (2003)). myeloid leukemia (EMBO 19:4655-64 (2000)). Transloca Increased expression of CDH3 mRNA correlates with adeno tion mutation in the Bromodomain of CREBBP may cause carcinoma associated with cervix neoplasms (Cancer 89: drug-induced form of myeloid leukemia (Proc Natl Acad Sci 2053-8 (2000)). Increased expression of CDH3 mRNA may USA94: 8732-7 (1997)). Nonsense mutation in the CREBBP correlate with abnormal cytokine and chemokine mediated gene may correlate with colonic neoplasms (PNAS 101: signaling pathway associated with prostatic neoplasms (Can 1273-8 (2004)). Translocation of the CREBBP gene may cer 77: 1862-72 (1996)). Alternative form of CDH3 protein cause myelodysplastic syndromes (Blood 90: 535-41 correlates with melanoma (Exp Cell Res 305:418-26 (2005)). (1997)). Frameshift mutation in the CREBBP gene causes Increased expression of CDH3 mRNA correlates with Rubinstein-Taybi syndrome (Hum Mol Genet 10: 1071-6 advanced stage or high grade form of ovarian neoplasms (Int (2001)). Translocation of the CREBBP gene may cause drug JCancer 106: 172-7 (2003)). Increased expression of CDH3 induced form of myeloid leukemia (Proc Natl AcadSci USA mRNA correlates with disease progression associated with US 2010/0129929 A1 May 27, 2010
ovarian neoplasms (Int J Cancer 106: 172-7 (2003)). ated with ovarian neoplasms (IntJ Cancer 82: 625-9 (1999)). Increased expression of CDH3 protein may cause invasive Increased tyrosine phosphorylation of CTNNB1 correlates form of breast neoplasms (Cancer Res 64: 8309-17 (2004)). with hepatocarcinoma tumors associated with hepatitis B Decreased expression of CDH3 mRNA may correlate with (Oncogene 20:3323-31 (2001)). Decreased membrane local decreased response to radiation associated with esophageal ization of CTNNB1 correlates with decreased cell differen neoplasms (Br J Cancer 91: 1543-50 (2004)). Increased tiation associated with esophageal neoplasms (Int J Cancer expression of CDH3 protein correlates with squamous cell 79: 573-9 (1998)). Increased stability of CTNNB1 may cor carcinoma associated with esophageal neoplasms (IntJ Can relate with malignant form of melanoma (Science 275: cer 79:573-9 (1998)). Abnormal expression of CDH3 protein 1790-2 (1997)). Point mutation in the CTNNB1 gene corre correlates with disease progression associated with stomach lates with carcinoma tumors associated with endometrial neoplasms (Int J Cancer 54: 49-52 (1993)). Hypermethyla neoplasms (Cancer Res 59: 3346-51 (1999)). Increased tion of the CDH3 gene correlates with pancreatic neoplasms nucleus localization of CTNNB1 correlates with squamous (Cancer Res 63: 3735-42 (2003)). (PhosphoSiteREGIS cell carcinoma associated with esophageal neoplasms (Int J TERED, Cell Signaling Technology (Danvers, Mass.), Cancer 84: 174-8 (1999)). Decreased nucleus localization of Human PSDTRADEMARK, Biobase Corporation, (Beverly, CTNNB1 may prevent increased cell proliferation associated Mass.)). with colonic neoplasms (J Cell Biol 154: 369-87 (2001)). 0052 COPS2, phosphorylated at Y159 and Y165, is Increased expression of CTNNB1 protein correlates with among the proteins listed in this patent. COPS2, COP9 (con increased occurrence of death associated with breast neo stitutive photomorphogenic) homolog subunit 2, transcrip plasms (PNAS 97: 4262-6 (2000)). Increased cleavage of tion corepressor, COP9 signalosome complex subunit, may CTNNB1 may correlate with increased apoptosis associated mediate repression by recruiting histone deacetylases, bind with colonic neoplasms (Oncogene 21: 8414-27 (2002)). ing to DAX1 (NROB1) may be impaired in adrenal hypopla Mutation in the CTNNB1 gene correlates with defective sia congenita. (PhosphoSiteREGISTERED, Cell Signaling DNA-dependent DNA replication associated with hepatitis C Technology (Danvers, Mass.), Human PSDTRADEMARK, (Proc Natl Acad Sci USA 101: 4262-7 (2004)). Increased Biobase Corporation, (Beverly, Mass.)). expression of CTNNB1 protein correlates with increased 0053 CTNNB, phosphorylated at Y30 and Y489, is occurrence of death associated with breast neoplasms (Proc among the proteins listed in this patent. CTNNB, catenin beta Natl AcadSci USA97: 4262-6 (2000)). Abnormal cytoplasm 1 (beta catenin), is a regulator of cell adhesion and a key localization of CTNNB1 correlates with neoplasm invasive downstream effector in the Wnt signaling pathway. CTNNB ness associated with melanoma (Exp Cell Res 245: 79-90 links adhesion receptors, the cytoskeleton, and nuclear tran (1998)). Decreased membrane localization of CTNNB1 may scriptional regulation. CTNNB is implicated both in early correlate with osteosarcoma tumors associated with bone embryonic development and tumorigenesis. Under normal neoplasms (Cancer Res 64: 2734-9 (2004)). Abnormal physiological conditions, CTNNB is phosphorylated and nucleus localization of CTNNB1 correlates with increased destabilized by CK1 and GSK-3beta. The stabilization of Wnt receptor signaling pathway associated with melanoma cytoplasmic beta-catenin, a hallmark of a variety of cancers, (Biochem Biophys Res Commun 288: 8-15 (2001)). translocates to the nucleus, where it acts as a transcriptional Decreased membrane localization of CTNNB1 correlates activator of transcription factors including T-cell factor with increased occurrence of disease progression associated (TCF). CTNNB Y30 lies 3 residues N-terminal to the S33, with colonic neoplasms (Cancer Res 61: 8085-8 (2001)). which is an important determinant of CTNNB stability. The Increased nucleus localization of CTNNB1 may correlate phosphorylation of S33 by GSK-3? destabilizes -catenin by with carcinoma tumors associated with prostatic neoplasms (Genes Dev. 10, 1443-1454 (1996)). Mutations in these phos (J Biol Chem 277:30935-41 (2002)). Increased expression of phorylation sites, which result in the stabilization of B-catenin CTNNB1 protein may correlate with increased cell motility protein levels, have been found in many tumor cell lines associated with breast neoplasms (JCell Sci: 425-37 (2000)). (Science 275, 1787-1790 (1997)). It is possible that phospho Increased expression of CTNNB1 protein may correlate with rylation of Y30 may inhibit the ability of GSK-3 ?tophospho increased occurrence of death associated with breast neo rylate S33, thus leading to the stabilization of CTNNB, plasms (Cancer 100: 2084-92 (2004)). Abnormal nucleus increasing the oncogenic potential of CTNNB. This protein localization of CTNNB1 correlates with endometrial neo has potential diagnostic and/or therapeutic implications plasms (Oncogene 21: 7981-90 (2002)). Increased androgen based on the following findings. Increased nucleus localiza receptor binding of CTNNB1 may cause increased cell cycle tion of CTNNB1 correlates with increased severity of prog arrest associated with prostatic neoplasms (Oncogene 22: nosis associated with melanoma (Int J Cancer 103: 652-6 5602-13 (2003)). Gain of function mutation in the CTNNB1 (2003)). Increased transcriptional activator activity of gene may cause invasive form of breast neoplasms (J Biol CTNNB1 may cause increased cell motility associated with Chem 276: 28443-50 (2001)). Increased nucleus localization melanoma (Cancer Res 63: 6626-34 (2003)). Decreased of CTNNB1 may cause increased cell proliferation associ DNA binding of CTNNB1 may correlate with increased ated with melanoma (JCB 158: 1079-87 (2002)). Increased response to drug associated with colonic neoplasms (FASEB expression of CTNNB1 protein may cause decreased apop J 19: 1353-5 (2005)). Point mutation in the CTNNB1 gene tosis associated with leukemia (Blood 100: 982-90 (2002)). causes biliary tract neoplasms (Cancer Res 61: 3406-9 Deletion mutation in the CTNNB1 gene correlates with (2001)). Decreased expression of CTNNB1 mRNA may cor adenoma tumors associated with colorectal neoplasms (Can relate with increased response to drug associated with mela cer Lett 159: 73-8 (2000)). Decreased expression of noma (Oncogene 21: 4060-4 (2002)). Abnormal nucleus CTNNB1 protein may prevent increased cell proliferation localization of CTNNB1 correlates with melanoma (Int J associated with colonic neoplasms (Cancer Res 61: 6563-8 Cancer 92: 839-42 (2001)). Increased cytoplasm localization (2001)). Decreased expression of CTNNB1 protein corre of CTNNB1 correlates with endometrioid carcinoma associ lates with increased occurrence of death associated with non US 2010/0129929 A1 May 27, 2010 22 small-cell lung carcinoma (Cancer 94: 752-8 (2002)). nonpolyposis colorectal neoplasms (Cancer Res 59: 4506-9 Increased membrane localization of CTNNB1 correlates with (1999)). Abnormal cytoplasm localization of CTNNB1 may increased occurrence of inflammation associated with breast cause increased cell migration associated with melanoma neoplasms (Cancer Res 61: 5231-41 (2001)). Increased sta (Biochem Biophys Res Commun 288: 8-15 (2001)). bility of CTNNB1 may cause increased protein import into Increased nucleus localization of CTNNB1 may cause nucleus associated with ovarian neoplasms (Int J Cancer 82: increased Wnt receptor signaling pathway associated with 625-9 (1999)). Increased cytoplasm localization of CTNNB1 melanoma (JCell Biol 158: 1079-87 (2002)). Increased pro correlates with osteosarcoma associated with bone neo tein binding of CTNNB1 correlates with melanoma (Bio plasms (Int J Cancer 102: 338-42 (2002)). Mutation in the chem Biophy's Res Commun 288: 8-15 (2001)). Decreased CTNNB1 gene correlates with defective DNA-dependent expression of CTNNB1 mRNA may correlate with Alzhe DNA replication associated with hepatitis C (PNAS 101: imer disease (Nature 395: 698-702 (1998)). Decreased 4262-7 (2004)). Alternative form of CTNNB1 protein corre expression of CTNNB1 protein may cause increased apopto lates with increased occurrence of neoplasm metastasis asso sis associated with colorectal neoplasms (Carcinogenesis 23: ciated with colorectal neoplasms (Int J Cancer 82: 504-11 107-14 (2002)). Decreased expression of CTNNB1 protein (1999)). Abnormal nucleus localization of CTNNB1 may correlates with increased occurrence of lymphatic metastasis cause malignant form of melanoma (Biochem BiophyS Res associated with breast neoplasms (Anticancer Res 17:561-7 Commun 288: 8-15 (2001)). Decreased cadherin binding of (1997)). Increased tyrosine phosphorylation of CTNNB1 CTNNB1 correlates with adenocarcinoma tumors associated correlates with colorectal neoplasms (BrJCancer 77: 605-13 with pancreatic neoplasms (Int J Cancer 95: 194-7 (2001)). (1998)). Decreased stability of CTNNB1 may prevent abnor Increased transcriptional activator activity of CTNNB1 may mal I-kappaB kinase/NF-kappaB cascade associated with cause decreased cell cycle arrest associated with melanoma prostatic neoplasms (Anticancer Res 23: 2077-83 (2003)). (Cancer Res 63: 6626-34 (2003)). Decreased expression of Increased regulation of transcription, DNA-dependent asso CTNNB1 protein may cause increased cell cycle arrest asso ciated with CTNNB1 may correlate with non-small-cell lung ciated with colorectal neoplasms (Carcinogenesis 23: 107-14 carcinoma (Oncogene 21:7497-506 (2002)). Mutation in the (2002)). Increased cytoplasm localization of CTNNB1 cor CTNNB1 gene may correlate with papillary carcinoma (Can relates with squamous cell carcinoma associated with esoph cer Res 61: 8401-4 (2001)). Increased expression of ageal neoplasms (IntJ Cancer 84: 174-8 (1999)). Increased CTNNB1 protein correlates with decreased apoptosis asso nucleus localization of CTNNB1 may correlate with invasive ciated with melanoma (Cancer Res 61: 3819-25 (2001)). form of colorectal neoplasms (Proc Natl Acad Sci USA 98: Decreased expression of CTNNB1 protein correlates with 10356-61 (2001)). Increased expression of CTNNB1 protein increased occurrence of lymphatic metastasis associated with may correlate with increased signal transduction associated esophageal neoplasms (Anticancer Res 23: 4435-42 (2003)). with myeloid leukemia (Oncogene 24: 2410-20 (2005)). Decreased nucleus localization of CTNNB1 may prevent Decreased membrane localization of CTNNB1 may correlate increased cell proliferation associated with colonic neo with increased response to drug associated with colonic neo plasms (JBC 276: 401 13-9 (2001)). Missense mutation in the plasms (FASEB J 19: 1353-5 (2005)). Decreased membrane CTNNB1 gene may cause increased protein import into localization of CTNNB1 correlates with increased occur nucleus associated with ovarian neoplasms (Int J Cancer 82: rence of non-familial form of colonic neoplasms (Cancer 89: 625-9 (1999)). Gene instability of CTNNB1 may cause car 733-40 (2000)). Increased nucleus localization of CTNNB1 cinoma tumors associated with cervix neoplasms (BrJ Can correlates with adenocarcinoma associated with esophageal cer 77: 192-200 (1998)). Increased expression of CTNNB1 neoplasms (Anticancer Res 24: 1369-75 (2004)). Decreased protein correlates with advanced stage or high grade form of stability of CTNNB1 may prevent increased cell proliferation melanoma (Cancer Res 61: 7318-24 (2001)). Decreased associated with prostatic neoplasms (Anticancer Res 23: expression of CTNNB1 protein may prevent disease progres 2077-83 (2003)). Loss of heterozygosity at the CTNNB1 sion associated with melanoma (Cancer Res 64: 5385-9 gene may cause carcinoma tumors associated with cervix (2004)). Gain of function mutation in the CTNNB1 gene may neoplasms (Br J Cancer 77: 192-200 (1998)). Decreased cause invasive form of breast neoplasms (JBC 276: 28443-50 expression of CTNNB1 protein correlates with increased (2001)). Increased nucleus localization of CTNNB1 may cor occurrence of disease progression associated with colorectal relate with invasive form of colorectal neoplasms (PNAS 98: neoplasms (Anticancer Res 17: 2241-7 (1997)). Decreased 10356-61 (2001)). Point mutation in the CTNNB1 gene cor expression of CTNNB1 protein correlates with breast ductal relates with hepatocellular carcinoma (Oncogene 21: 4863 carcinoma (IntJCancer 106: 208-15 (2003)). Missense muta 71 (2002)). Abnormal cytoplasm localization of CTNNB1 tion in the CTNNB1 gene correlates with hepatocellular car correlates with neoplastic cell transformation associated with cinoma (IntJ Cancer 104: 745-51 (2003)). Increased expres melanoma (Exp Cell Res 245: 79-90 (1998)). Increased sion of CTNNB1 mutant protein correlates with adenoma nucleus localization of CTNNB1 correlates with carcinoid tumors associated with colorectal neoplasms (Cancer Lett tumor associated with gastrointestinal neoplasms (Cancer 159: 73-8 (2000)). Increased cytoplasm localization of Res 61: 6656-9 (2001)). Increased expression of CTNNB1 CTNNB1 may cause increased protein import into nucleus protein correlates with hepatocellular carcinoma (Cancer Lett associated with ovarian neoplasms (Int J Cancer 82: 625-9 199: 201-8 (2003)). Decreased membrane localization of (1999)). Abnormal cytoplasm localization of CTNNB1 cor CTNNB1 may correlate with invasive form of bone neo relates with increased Wnt receptor signaling pathway asso plasms (Cancer Res 64: 2734-9 (2004)). Abnormal mRNA ciated with melanoma (Biochem Biophys Res Commun288: splicing of CTNNB1 may correlate with malignant form of 8-15 (2001)). Missense mutation in the CTNNB1 gene may melanoma (Science 275: 1790-2 (1997)). Increased expres correlate with malignant form of melanoma (Science 275: sion of CTNNB1 protein may correlate with increased cell 1790-2 (1997)). Point mutation in the CTNNB1 gene corre motility associated with breast neoplasms (JCell Sci: 425-37 lates with increased occurrence of invasive form of hereditary (2000)). Decreased expression of CTNNB1 protein corre US 2010/0129929 A1 May 27, 2010 lates with increased severity of pancreatic ductal carcinoma protein has potential diagnostic and/or therapeutic implica associated with pancreatic neoplasms (Anticancer Res 23: tions based on the following findings. Polymorphism in the 5043-7 (2003)). Increased cytoplasm localization of CUL2 gene correlates with pheochromocytoma (JClin Endo CTNNB1 correlates with Barrett esophagus associated with crinol Metab 84: 3207-11 (1999)). (PhosphoSiteREGIS esophageal neoplasms (Oncogene 21: 6071-81 (2002)). TERED, Cell Signaling Technology (Danvers, Mass.), Increased cytoplasm localization of CTNNB1 correlates with Human PSDTRADEMARK, Biobase Corporation, (Beverly, carcinoid tumor associated with gastrointestinal neoplasms Mass.)). (Cancer Res 61: 6656-9 (2001)). Increased protein binding of 0055 CXADR, phosphorylated at Y294 and Y313, is CTNNB1 may cause malignant form of melanoma (Biochem among the proteins listed in this patent. CXADR, Coxsack Biophy's Res Commun 288: 8-15 (2001)). Increased expres ievirus and adenovirus receptor, acts in cell adhesion, aber sion of CTNNB1 protein may cause increased cell prolifera rant protein expression is associated with multiple neoplasms tion associated with leukemia (Blood 100: 982-90 (2002)). and viral infections; presence ofrat Cxadrprotein in the heart Increased nucleus localization of CTNNB1 may correlate of adult rat model is associated with autoimmune myocardi with increased cell proliferation associated with colonic neo tis. This protein has potential diagnostic and/or therapeutic plasms (PNAS 102: 6027-32 (2005)). Mutation in the CTNNB1 gene correlates with endometrial neoplasms (On implications based on the following findings. Decreased cogene 21: 7981-90 (2002)). Decreased membrane localiza expression of CXADR protein correlates with squamous cell tion of CTNNB1 correlates with adenocarcinoma tumors carcinoma (Anticancer Res 22: 2629-34 (2002)). Alternative associated with pancreatic neoplasms (IntJ Cancer 95: 194-7 form of CXADR protein may prevent increased initiation of (2001)). Decreased proteolysis of CTNNB1 may correlate viral infection associated with coxsackievirus infections with increased cell adhesion associated with breast neo (JBC 279: 18497-503 (2004)). Decreased expression of plasms (Endocrinology 137: 3265-73 (1996)). Mutation in CXADR protein may cause increased cell proliferation asso the CTNNB1 gene may cause carcinoid tumor associated ciated with glioma (Br J Cancer 88: 1411-6 (2003)). with gastrointestinal neoplasms (Cancer Res 61: 6656-9 Decreased expression of CXADR protein may correlate with (2001)). Abnormal cytoplasm localization of CTNNB1 cor glioma (Cancer Res 58: 5738-48 (1998)). Decreased expres relates with melanoma (Int J Cancer 92: 839-42 (2001)). sion of CXADR protein may correlate with increased cell Increased nucleus localization of CTNNB1 correlates with proliferation associated with glioma (IntJ Cancer 103: 723-9 increased occurrence of invasive form of hereditary nonpoly (2003)). Increased expression of CXADR protein correlates posis colorectal neoplasms (Cancer Res 59: 4506-9 (1999)). with invasive form of prostatic neoplasms (Cancer Res 62: Decreased membrane localization of CTNNB1 may correlate 3812-8 (2002)). Decreased expression of CXADR in placenta with decreased cell-cell adhesion associated with bone neo may preventadenoviridae infections (Biol Reprod 64: 1001-9 plasms (Cancer Res 64; 2734-9 (2004)). Increased androgen (2001)). Decreased expression of CXADR protein correlates receptor binding of CTNNB1 may cause hormone-dependent with squamous cell carcinoma tumors associated with head neoplasms associated with prostatic neoplasms (Oncogene and neck neoplasms (Anticancer Res 22: 2629-34 (2002)). 22: 5602-13 (2003)). Increased nucleus localization of Viral exploitation of the receptor activity of CXADR may CTNNB1 may cause increased Wnt receptor signaling path cause increased interferon-alphabiosynthetic process associ way associated with melanoma (JCB 158: 1079-87 (2002)). ated with coxsackievirus infections (JGenVirol 82: 1899-907 Mutation in the CTNNB1 gene correlates with malignant (2001)). Decreased expression of CXADR protein correlates form of melanoma (Int J Cancer 100: 549-56 (2002)). with more severe form of prostatic neoplasms (Cancer Res Increased nucleus localization of CTNNB1 correlates with 62:3812-8 (2002)). Decreased expression of CXADR protein adenocarcinoma tumors associated with esophageal neo correlates with prostatic neoplasms (Cancer Res 60: 5031-6 plasms (Int J Cancer 86: 533-7 (2000)). Increased nucleus (2000)). Increased expression of CXADR protein may pre localization of CTNNB1 may cause increased mRNA tran vent increased cell proliferation associated with prostatic Scription associated with colorectal neoplasms (Int J Cancer neoplasms (Cancer Res 60: 5031-6 (2000)). Decreased 108: 321-6 (2004)). Deletion mutation in the CTNNB1 gene expression of CXADR protein may correlate with bladder correlates with carcinoma tumors associated with colorectal neoplasms (Cancer Res 59: 325-30 (1999)). Alternative form neoplasms (Cancer Res 58: 1021-6 (1998)). Missense muta of CXADR protein may prevent increased initiation of viral tion in the CTNNB1 gene causes abnormal Wnt receptor infection associated with coxsackievirus infections (J Biol signaling pathway associated with ovarian neoplasms (Can Chem 279: 18497-503 (2004)). Decreased expression of cer Res 61: 8247-55 (2001)). Increased nucleus localization CXADR protein correlates with more severe form of brain of CTNNB1 may cause increased transcription from RNA neoplasms (Int J Cancer 103: 723-9 (2003)). Decreased polymerase II promoter associated with esophageal neo expression of CXADR protein may cause decreased cell-cell plasms (Br J Cancer 90: 892-9 (2004)). Decreased DNA adhesion associated with bladder neoplasms (Cancer Res 61: binding of CTNNB1 may correlate with increased response 6592-600 (2001)). Decreased expression of CXADR protein to drug associated with colonic neoplasms (FASEB 19: may cause abnormal regulation of progression through cell 1353-5 (2005)). Deletion mutation in the CTNNB1 gene cycle associated with bladder neoplasms (Cancer Res 61: correlates with malignant form of mesothelioma (Oncogene 6592-600 (2001)). Decreased expression of CXADR protein 20: 4249-57. correlates with more severe form of astrocytoma (IntJCancer 0054 CUL2, phosphorylated at Y477, is among the pro 103: 723-9 (2003)). Decreased expression of CXADR protein teins listed in this patent. CUL2, Cullin 2, member of the E3 may correlate with increased cell proliferation associated ubiquitin ligase complex that contains VHL, TCEB1 and with brain neoplasms (IntJ Cancer 103: 723-9 (2003)) (Phos TCEB2, conjugation by NEDD8 may be important for VHL phoSiteREGISTERED, Cell Signaling Technology (Dan tumor Suppressor function, associated with uveal melanoma; vers, Mass.), Human PSDTRADEMARK, Biobase Corpora gene mutation is associated with pheochromocytoma. This tion, (Beverly, Mass.)). US 2010/0129929 A1 May 27, 2010 24
0056 desmoplakin 3, phosphorylated at Y480 and Y550, ISTERED, Cell Signaling Technology (Danvers, Mass.), is among the proteins listed in this patent. desmoplakin 3. Human PSDTRADEMARK, Biobase Corporation, (Beverly, Junction plakoglobin, interacts with cadherins and mediates Mass.)). linkage to the cytoskeleton, also binds desmoplakin (DSP) 0057 DNTT, phosphorylated at Y477, is among the pro and PECAM1, altered expression or localization is linked to teins listed in this patent. DNTT, Terminal deoxynucleotidyl various cancers; gene mutations are linked to breast and ova transferase, participates in the generation of diversity in rian tumors. This protein has potential diagnostic and/or immunoglobulins and T cell receptors, aberrantly expressed therapeutic implications based on the following findings. in myeloid and acute myelocytic leukemias. This protein has Increased expression of JUP mRNA may correlate with potential diagnostic and/or therapeutic implications based on malignant form of colonic neoplasms (GenesDev 16: 2058 the following findings. Abnormal expression of DNTT pro 72 (2002)). Increased expression of JUP mRNA may cause tein correlates with acute myelocytic leukemia (Cancer 68: less severe form of non-small-cell lung carcinoma (Oncogene 2161-8 (1991)). Increased expression of DNTT protein may correlate with leukemia (Leukemia 9: 583-7 (1995)). Abnor 21:7497-506 (2002)). Decreased expression of JUP protein mal expression of DNTT protein may correlate with lym correlates with lobular carcinoma associated with breast neo phoma associated with skin neoplasms (Cancer 94: 2401-8 plasms (IntJ Cancer 106: 208-15 (2003)). Decreased expres (2002)). Abnormal expression of DNTT protein correlates sion of JUP protein correlates with breast ductal carcinoma with acute myelocytic leukemia (Blood 87: 1162-9 (1996)). associated with breast neoplasms (Int J Cancer 106: 208-15 Abnormal expression of DNTT protein may correlate with (2003)). Increased expression of JUP mRNA may correlate acute B-cell leukemia (Cancer 68: 2161-8 (1991)). Abnormal with malignant form of colonic neoplasms (Genes Dev 16: expression of DNTT protein may correlate with acute B-cell 2058-72 (2002)). Polymorphism in the JUP gene correlates leukemia (Leukemia 10: 1159-63 (1996)) (PhosphoSiteREG with familial form of breast neoplasms (Proc Natl Acad Sci ISTERED, Cell Signaling Technology (Danvers, Mass.), USA 92: 6384-8 (1995)). Increased expression of JUP Human PSDTRADEMARK, Biobase Corporation, (Beverly, mRNA may correlate with malignant form of melanoma Mass.)). (Genes Dev 16: 2058-72 (2002)). Loss of heterozygosity at 0058 DYRK2, phosphorylated at Y307, is among the pro the JUP gene correlates with breast neoplasms (Proc Natl teins listed in this patent. DYRK2, Dual-specificity tyrosine AcadSci USA92: 6384-8 (1995)). Loss of heterozygosity at (Y)-phosphorylation regulated kinase 2, may play a role in the JUP gene correlates with breast neoplasms (PNAS 92: spermatogenesis; gene amplification causes gastric cancer 6384-8 (1995)). Loss of heterozygosity at the JUP gene cor and Barrett adenocarcinoma and mRNA overexpression is relates with ovarian neoplasms (PNAS 92: 6384-8 (1995)). associated with esophageal and lung adenocarcinomas. This Increased expression of JUP mRNA may correlate with protein has potential diagnostic and/or therapeutic implica malignant form of colonic neoplasms (Genes Dev. 16: 2058 tions based on the following findings. Increased expression of 72 (2002)). Decreased expression of JUP mRNA correlates DYRK2 mRNA correlates with esophageal neoplasms (Can with non-small-cell lung carcinoma (Oncogene 21:7497-506 cer Res 63:4136-43 (2003)). Increased expression of DYRK2 (2002)). Polymorphism in the JUP gene correlates with famil mRNA correlates with increased incidence of malignant form ial form of breast neoplasms (Proc Natl Acad Sci USA 92: of gastrointestinal neoplasms (Gut 53: 235-40 (2004)) (Phos 6384-8 (1995)). Loss of heterozygosity at the JUP gene cor phoSiteREGISTERED, Cell Signaling Technology (Dan relates with ovarian neoplasms (Proc Natl AcadSci USA 92: vers, Mass.), Human PSDTRADEMARK, Biobase Corpora 6384-8 (1995)). Loss of heterozygosity at the JUP gene cor tion, (Beverly, Mass.)). relates with breast neoplasms (Proc Natl Acad Sci USA 92: 0059 eEF1A-1, phosphorylated at Y177, is among the 6384-8 (1995)). Decreased expression of JUP protein corre proteins listed in this patent, eEF1A-1, CC chemokine recep lates with carcinoma tumors associated with colorectal neo tor 5, a G protein-coupled receptor that binds chemokines and plasms (Anticancer Res 17: 2241-7 (1997)). Polymorphism is a coreceptor for HIV-1 glycoprotein 120, may modulate in the JUP gene correlates with familial form of ovarian immune and inflammatory responses, inhibition may be neoplasms (Proc Natl Acad Sci USA 92: 6384-8 (1995)). therapeutic for HIV infections and multiple sclerosis. This Increased expression of JUP mRNA may correlate with protein has potential diagnostic and/or therapeutic implica malignant form of melanoma (Gene Develop 16: 2058-72 tions based on the following findings. Induced inhibition of (2002)). Polymorphism in the JUP gene correlates with famil the coreceptor activity of CCR5 may prevent HIV infections ial form of ovarian neoplasms (Proc Natl Acad Sci USA 92: (JVirol 74: 9328-32 (2000)). Increased expression of CCR5 6384-8 (1995)). Polymorphism in the JUP gene correlates in T-lymphocytes correlates with more severe form of HIV with familial form of ovarian neoplasms (PNAS 92: 6384-8 infections (J Infect Dis 181:927-32 (2000)). Induced inhibi (1995)). Decreased expression of JUP protein correlates with tion of the viral receptor activity of CCR5 may prevent abnor adenoma tumors associated with colorectal neoplasms (Anti mal initiation of viral infection associated with HIV infec cancer Res 17: 2241-7 (1997)). Loss of heterozygosity at the tions (Cell 86: 367-77 (1996)). Induced inhibition of the JUP gene correlates with ovarian neoplasms (Proc Natl Acad coreceptor activity of CCR5 may prevent HIV infections (J Sci USA 92: 6384-8 (1995)). Increased expression of JUP Virol 73: 3443-8 (1999)). Increased expression of CCR5 in mRNA may correlate with malignant form of colonic neo leukocytes correlates with pulmonary tuberculosis associated plasms (Gene Develop 16: 2058-72 (2002)). Increased with AIDS-related opportunistic infections (J Infect Dis 183: expression of JUP mRNA may correlate with malignant form 1801-4 (2001)). Decreased expression of CCR5 in T-lympho of melanoma (GenesDev 16: 2058-72 (2002)). Polymor cytes correlates with abnormal T-lymphocytes migration phism in the JUP gene correlates with familial form of breast associated with chronic hepatitis C (J Infect Dis 185: 1803-7 neoplasms (PNAS 92: 6384-8 (1995)). Increased expression (2002)). Decreased expression of CCR5 in leukocytes corre of JUP mRNA may correlate with malignant form of mela lates with type I diabetes mellitus (Diabetes 51: 2474-80 noma (Genes Dev. 16: 2058-72 (2002)) (PhosphoSiteREG (2002)). Increased expression of CCR5 in T-lymphocytes US 2010/0129929 A1 May 27, 2010
correlates with Schistosomiasis mansoni (Infect Immun 71: associated with HIV infections (JNeuroimmunol 110: 230-9 6668-71 (2003)). Increased expression of CCR5 in T-lym (2000)). Antibody to CCR5 may prevent increased initiation phocytes correlates with advanced stage or high grade form of of viral infection associated with HIV infections (PNAS 97: HIV infections (J Immunol 163: 4597-603 (1999)). Viral 805-10 (2000)). Increased presence of CCR5 antibody may exploitation of the coreceptor activity of CCR5 may cause prevent HIV infections (Clin Exp Immunol 129: 493-501 HIV infections (JVirol 79: 1686-700 (2005)). Increased viral (2002)). Antibody to CCR5 may prevent increased initiation receptor activity of CCR5 correlates with advanced stage or of viral infection associated with HIV infections (Proc Natl high grade form of acquired immunodeficiency syndrome (J AcadSci USA 97: 805-10 (2000)). Viral exploitation of the Virol 73: 9741-55 (1999)). Increased expression of CCR5 in coreceptor activity of CCR5 causes increased initiation of dendritic cells correlates with optic neuritis associated with viral infection associated with HIV infections (J Exp Med multiple sclerosis (Clin Exp Immunol 127: 519-26 (2002)). 185: 621-8 (1997)). Increased expression of CCR5 in lym Monoclonal antibody to CCR5 may prevent abnormal initia phocytes correlates with autoimmune diseases associated tion of viral infection associated with HIV infections (Proc with thyroid diseases (J Clin Endocrinol Metab 86: 5008-16 Natl AcadSci USA 97: 3388-93 (2000)). Decreased plasma (2001)). Loss of function mutation in the CCR5 gene corre membrane localization of CCR5 may prevent HIV infections lates with decreased severity of disease progression associ (PNAS 94: 11567-72 (1997)). Viral exploitation of the core ated with HIV infections (Mol Med 3: 23-36 (1997)). ceptor activity of CCR5 may cause defective initiation of viral Absence of the viral receptor activity of CCR5 causes infection associated with HIV infections (JVirol 71: 7478-87 decreased initiation of viral infection associated with HIV (1997)). Increased expression of CCR5 in T-lymphocytes infections (Nature 382: 722-5 (1996)). Increased expression correlates with more severe form of HIV infections (Blood of CCR5 in leukocytes correlates with AIDS-related oppor 96: 2649-54 (2000)). Increased expression of CCR5 protein tunistic infections associated with HIV infections (J Infect correlates with kidney diseases (Kidney Int 56: 52-64 Dis 183: 1801-4 (2001)). Deletion mutation in the CCR5 gene (1999)). Single nucleotide polymorphism in the CCR5 pro correlates with decreased occurrence of recurrence associ moter correlates with diabetic nephropathies (Diabetes 51: ated with multiple sclerosis (J Neuroimmunol 102: 98-106 238-42 (2002)). Polymorphism in the CCR5 gene correlates (2000)). Induced inhibition of the coreceptor activity of with decreased occurrence of AIDS-related lymphoma asso CCR5 may prevent HIV infections (Proc Natl AcadSci USA ciated with acquired immunodeficiency syndrome (Blood 93: 98: 12718-23 (2001)). Absence of plasma membrane local 1838-42 (1999)). Decreased plasma membrane localization ization of CCR5 causes decreased initiation of viral infection of CCR5 may prevent HIV infections (Proc Natl Acad Sci associated with HIV infections (Nature 382: 722-5 (1996)). USA 94: 11567-72 (1997)). Absence of plasma membrane Increased expression of CCR5 in T-lymphocytes may corre localization of CCR5 causes decreased initiation of viral late with pulmonary tuberculosis associated with AIDS-re infection associated with HIV infections (Cell 86: 367-77 lated opportunistic infections (J Infect Dis 183: 1801-4 (1996)). Abnormal expression of CCR5 in T-lymphocytes (2001)). Viral exploitation of the chemokine receptor activity correlates with rheumatoid arthritis (Clip Exp Immunol 132: of CCR5 may cause increased induction by virus of cell-cell 371-8 (2003)). Viral exploitation of the coreceptor activity of fusion in host associated with HIV infections (J Virol 71: CCR5 causes increased initiation of viral infection associated 8405-15 (1997)). Deletion mutation in the CCR5 gene may with HIV infections (Cell 85: 1135-48 (1996)). Deletion prevent HIV infections (Science 273: 1856-62 (1996)). mutation in the CCR5 gene correlates with abnormal immune Increased expression of CCR5 in monocytes correlates with response associated with HIV infections (Mol Med 6: 28-36 more severe form of HIV infections (J Exp Med 187: 439-44 (2000)). Single nucleotide polymorphism in the CCR5 pro (1998)). Increased expression of CCR5 in B-lymphocytes moter correlates with increased incidence of diabetic neph correlates with relapsing-remitting multiple Sclerosis (JNeu ropathies associated with type II diabetes mellitus (Diabetes roimmunol 122: 125-31 (2002)). Deletion mutation in the 51: 238-42 (2002)). Deletion mutation in the CCR5 gene CCR5 gene causes decreased initiation of viral infection correlates with decreased occurrence of non-Hodgkin’s lym associated with HIV infections (Cell 86: 367-77 (1996)). phoma associated with acquired immunodeficiency syn Increased expression of CCR5 in B-lymphocytes correlates drome (Blood 93: 1838-42 (1999)). Antibody to CCR5 may with Hodgkin's disease (Blood 97: 1543-8 (2001)). Abnor prevent increased initiation of viral infection associated with mal expression of CCR5 in NK cells may correlate with HIV infections (Proc Natl AcadSci USA97: 805-10 (2000)). increased severity of leukemia associated with lymphoprolif Viral exploitation of the coreceptor activity of CCR5 corre erative disorders (Leukemia 19: 1169-74 (2005)). Mono lates with acute form of HIV infections (Blood 98: 3169-71 clonal antibody to CCR5 may prevent abnormal initiation of (2001)). Increased expression of CCR5 in fibroblasts corre viral infection associated with HIV infections (Proc Natl lates with rheumatoid arthritis (J Immunol 167: 5381-5 AcadSci USA 97.3388-93 (2000)). Induced inhibition of the (2001)). Increased expression of CCR5 in T-lymphocytes coreceptor activity of CCR5 may prevent HIV infections may correlate with AIDS-related opportunistic infections (Proc Natl Acad Sci USA 98: 12718-23 (2001)). Increased associated with HIV infections (J Infect Dis 183: 1801-4 expression of CCR5 in T-lymphocytes correlates with (2001)). Mutation in the CCR5 gene correlates with Hodgkin's disease (Blood97: 1543-8 (2001)). Polymorphism decreased occurrence of acquired immunodeficiency syn in the CCR5 gene correlates with increased initiation of viral drome associated with HIV infections (Science 277: 959-65 infection associated with HIV infections (J Infect Dis 183: (1997)). Loss of function mutation in the CCR5 gene causes 1574-85 (2001)). Decreased expression of CCR5 protein cor decreased initiation of viral infection associated with HIV relates with chronic myeloid leukemia (J Immunol 162: infections (Mol Med 3: 23-36 (1997)). Decreased expression 6191-9 (1999)). Decreased plasma membrane localization of of CCR5 in T-lymphocytes correlates with Crohn disease CCR5 may prevent HIV infections (Proc Natl AcadSci USA (Clin Exp Immunol 132: 332-8 (2003)). Viral exploitation of 94: 11567-72 (1997)). Decreased expression of CCR5 in the CCR5 protein causes increased entry of virus into host cell T-lymphocytes may prevent HIV infections (Proc Natl Acad US 2010/0129929 A1 May 27, 2010 26
Sci USA 100: 183-8 (2003)). Increased expression of CCR5 viral infection associated with acquired immunodeficiency in NK cells correlates with inflammation associated with syndrome (PNAS96:7496-501 (1999)). Deletion mutationin chronic hepatitis C (J Infect Dis 190: 989-97 (2004)). Poly the CCR5 gene correlates with decreased occurrence of morphism in the CCR5 gene correlates with increased inci AIDS-related lymphoma associated with acquired immuno dence of death associated with breast neoplasms (J Exp Med deficiency syndrome (Blood 93: 1838-42 (1999)). Increased 198: 1381-9 (2003)). Deletion mutation in the CCR5 gene expression of CCR5 in lymphocytes correlates with increased correlates with late onset form of HIV infections (Mol Med 6: T-helper 1 type immune response associated with Behcet 28-36 (2000)). Polymorphism in the CCR5 promoter corre Syndrome (Clin Exp Immunol 139: 371-8 (2005)). Viral lates with increased occurrence of acquired immunodefi exploitation of the coreceptor activity of CCR5 correlates ciency syndrome associated with HIV infections (Science with AIDS dementia complex (Virology 279:509-26 (2001)). 282: 1907-11 (1998)). Deletion mutation in the CCR5 gene Induced inhibition of the chemokine receptor activity of correlates with decreased occurrence of disease susceptibility CCR5 may prevent recurrence associated with multiple scle associated with asthma (Lancet 354: 1264-5 (1999)). rosis (J Neuroimmunol 102: 98-106 (2000)). Viral exploita Increased expression of CCR5 mRNA correlates with inflam tion of the chemokine receptor activity of CCR5 may cause mation (J Clin Invest 101: 746-54 (1998)). Monoclonal anti increased initiation of viral infection associated with acquired body to CCR5 may prevent abnormal initiation of viral infec immunodeficiency syndrome (Proc Natl Acad Sci USA 96: tion associated with HIV infections (PNAS 97: 3388-93 7496-501 (1999)). Polymorphism in the CCR5 promoter cor (2000)). Polymorphism in the CCR5 gene correlates with relates with more severe form of HIV infections (J Virol 73: increased occurrence of disease Susceptibility associated with 10264-71 (1999)). Deletion mutation in the CCR5 gene may diabetic nephropathies (Diabetes 54:3331-5 (2005)). Abnor prevent disease progression associated with acquired immu mal expression of CCR5 protein correlates with Graves dis nodeficiency syndrome (Science 273: 1856-62 (1996)). Dele ease (Clin Exp Immunol 127: 479-85 (2002)). Increased tion mutation in the CCR5 gene causes decreased initiation of expression of CCR5 in T-lymphocytes correlates with inflam viral infection associated with HIV infections (Nature 382: mation associated with chronic hepatitis C (J Infect Dis 190: 722-5 (1996)). Polymorphism in the CCR5 gene correlates 989-97 (2004)). Increased expression of CCR5 in monocytes with decreased (delayed) early viral mRNA transcription correlates with Schistosomiasis mansoni (Infect Immun 71: associated with HIV seropositivity (J Virol 76: 662-72 6668-71 (2003)). Polymorphism in the CCR5 gene correlates (2002)). Absence of the viral receptor activity of CCR5 with diabetic angiopathies associated with type I diabetes causes decreased initiation of viral infection associated with mellitus (Cytokine 26: 114-21 (2004)). Polymorphism in the HIV infections (Cell 86: 367-77 (1996)). Increased expres CCR5 promoter correlates with increased occurrence of dis sion of CCR5 in leukocytes correlates with pulmonary tuber ease progression associated with acquired immunodeficiency culosis associated with HIV infections (J Infect Dis 183: syndrome (Science 282: 1907-11 (1998)). Induced inhibition 1801-4 (2001)). Increased expression of CCR5 mRNA cor of the viral receptor activity of CCR5 may prevent abnormal relates with periapical granuloma (Cytokine 16: 62-6 initiation of viral infection associated with HIV infections (2001)). Increased expression of CCR5 in T-lymphocytes (Nature 382: 722-5 (1996)). Polymorphism in the CCR5 pro may cause increased T-helper 1 type immune response asso moter correlates with more severe form of HIV infections (J ciated with relapsing-remitting multiple Sclerosis (J Neu Infect Dis 183: 814-8 (2001)). Increased expression of CCR5 roimmunol 114: 207-12 (2001)). Viral exploitation of the in B-lymphocytes correlates with inflammation associated CCR5 protein may cause increased induction by virus of with chronic hepatitis C (J Infect Dis 190: 989-97 (2004)). cell-cell fusion in host associated with HIV infections (Blood Induced inhibition of the coreceptor activity of CCR5 may 103: 1211-7 (2004)). Polymorphism in the CCR5 promoter prevent HIV infections (PNAS 98: 12718-23 (2001)). correlates with decreased occurrence of acquired immunode Decreased expression of CCR5 in T-lymphocytes may pre ficiency syndrome associated with HIV infections (Lancet vent HIV infections (PNAS 100: 183-8 (2003)). Viral exploi 352: 866-70 (1998)). Viral exploitation of the chemokine tation of the chemokine receptor activity of CCR5 may cause receptor activity of CCR5 may cause increased induction by increased initiation of viral infection associated with acquired virus of cell-cell fusion in host associated with acquired immunodeficiency syndrome (Proc Natl Acad Sci USA 96: immunodeficiency syndrome (J Virol 71: 8405-15 (1997)) 7496-501 (1999)). Increased expression of CCR5 in T-lym (PhosphoSiteREGISTERED, Cell Signaling Technology phocytes correlates with rheumatoid arthritis (J Immunol (Danvers, Mass.), Human PSDTRADEMARK, Biobase Cor 174: 1693-700 (2005)). Increased expression of CCR5 in poration, (Beverly, Mass.)). T-lymphocytes may correlate with pulmonary tuberculosis 0060 ENO1, phosphorylated at Y189, is among the pro associated with HIV infections (J Infect Dis 183: 1801-4 teins listed in this patent. ENO1, Enolase 1 (alpha enolase), (2001)). Increased expression of CCR5 in lymphocytes cor converts 2-phospho-D-glycerate to phosphoenolpyruvate in relates with chronic hepatitis C Immunol 163: 6236-43 glycolysis, shorter alternative is a transcriptional repressor, (1999)). Decreased expression of CCR5 in T-lymphocytes expression in increased in various cancers and serves as an may prevent HIV infections (Proc Natl Acad Sci USA 100: autoantigen in multiple autoimmune diseases. This protein 183-8 (2003)). Increased expression of CCR5 protein corre has potential diagnostic and/or therapeutic implications lates with inflammation associated with periodontitis (Cytok based on the following findings. Increased expression of ine 20: 70-7 (2002)). Polymorphism in the CCR5 promoter ENO1 protein may prevent increased positive regulation of correlates with more severe form of HIV infections (J Infect protein biosynthetic process associated with prostatic neo Dis184: 89-92 (2001)). Decreased chemokine receptor activ plasms (JBC 280: 14325-30 (2005)). Increased presence of ity of CCR5 correlates with decreased occurrence of recur ENO1 autoimmune antibody correlates with systemic lupus rence associated with multiple sclerosis (J Neuroimmunol erythematosus (Biochem Biophy's Res Commun 298: 169-77 102: 98-106 (2000)). Viral exploitation of the chemokine (2002)). Increased expression of ENO1 mRNA may correlate receptor activity of CCR5 may cause increased initiation of with mouth neoplasms (Oncogene 18: 827-31 (1999)). US 2010/0129929 A1 May 27, 2010 27
Increased expression of ENO1 protein correlates with glio 90 (1996)). Increased presence of ENO1 autoimmune anti blastoma (J Neurochem 66: 2484-90 (1996)). Increased body correlates with chronic brain damage associated with expression of ENO1 protein correlates with adenocarcinoma autoimmune thyroiditis (FEBS Lett 528: 197-202 (2002)). associated with pancreatic neoplasms (Cancer Res 64: 9018 Autoimmune antibody to ENO1 correlates with inflamma 26 (2004)). Increased expression of ENO1 protein correlates tion associated with pituitary diseases (J Clin Endocrinol with astrocytoma associated with brain neoplasms (JNeuro Metab 87: 752-7 (2002)) (PhosphoSiteREGISTERED, Cell chem 66: 2484-90 (1996)). Increased expression of ENO1 Signaling Technology (Danvers, Mass.), Human protein may prevent increased cell proliferation associated PSDTRADEMARK, Biobase Corporation, (Beverly, with prostatic neoplasms (J Biol Chem 280: 14325-30 Mass.)). (2005)). Increased expression of ENO1 protein may prevent 0061 EphA2, phosphorylated at Y628 and Y694, is increased cell proliferation associated with prostatic neo among the proteins listed in this patent. EphA2, Eph receptor plasms (JBC 280: 14325-30 (2005)). Increased expression of A2, ephrin receptor, inhibits cell-matrix adhesion and prolif ENO1 protein may prevent increased activation of MAPK eration, induces apoptosis, regulates tumor angiogenesis, activity associated with prostatic neoplasms (J Biol Chem overexpressed in several cancers, expression is prognostic of 280: 14325-30 (2005)). Decreased phosphopyruvate poor Survival in cancer patients. This protein has potential hydratase activity of ENO1 correlates with astrocytoma (J diagnostic and/or therapeutic implications based on the fol Neurochem 66: 2484-90 (1996)). Increased expression of ENO1 protein may prevent increased positive regulation of lowing findings. Increased phosphorylation of EPHA2 may protein biosynthetic process associated with prostatic neo correlate with increased induction of apoptosis associated plasms (J Biol Chem 280: 14325-30 (2005)). Increased with non-small-cell lung carcinoma (Oncogene 20: 6503-15 expression of ENO1 protein may prevent invasive form of (2001)). Induced inhibition of the GPI-linked ephrin receptor breast neoplasms (Cancer Res 55: 3747-51 (1995)). activity of EPHA2 may prevent increased angiogenesis asso Increased presence of ENO1 autoimmune antibody correlates ciated with breast neoplasms (Oncogene 19: 6043–52 with drug-sensitive form of autoimmune thyroiditis (FEBS (2000)). Increased GPI-linked ephrin receptor activity of Lett 528: 197-202 (2002)). Increased expression of ENO1 EPHA2 may prevent increased cell proliferation associated protein may prevent increased activation of NF-kappaB tran with breast neoplasms (Cancer Res 61: 2301-6 (2001)). Scription factor associated with prostatic neoplasms (JBC Induced inhibition of the GPI-linked ephrin receptor activity 280: 14325-30 (2005)). Decreased phosphopyruvate of EPHA2 may prevent increased angiogenesis associated hydratase activity of ENO1 correlates with astrocytoma asso with lung neoplasms (Oncogene 19: 6043-52 (2000)). ciated with brain neoplasms (J Neurochem 66: 2484-90 Induced inhibition of the GPI-linked ephrin receptor activity (1996)). Increased expression of ENO1 protein correlates of EPHA2 may prevent increased angiogenesis associated with meningioma (J Neurochem 66: 2484-90 (1996)). with colonic neoplasms (Oncogene 19: 6043-52 (2000)). Autoimmune antibody to ENO1 may correlate with discoid Induced inhibition of the GPI-linked ephrin receptor activity lupus erythematosus (Immunology 92: 362-8 (1997)). of EPHA2 may prevent increased angiogenesis associated Decreased phosphopyruvate hydratase activity of ENO1 cor with fibroadenoma (Oncogene 19: 6043-52 (2000)). relates with glioblastoma (JNeurochem 66: 2484-90 (1996)). Increased expression of EPHA2 in epithelium/epithelial cells Autoimmune antibody to ENO1 correlates with connective may cause decreased cell-cell adhesion associated with breast tissue diseases (Eur J. Immunol 30: 3575-3584 (2000)). neoplasms (Cancer Res 61: 2301-6 (2001)). Monoclonal anti Increased expression of ENOL protein correlates with astro body to EPHA2 may prevent increased cell proliferation cytoma (JNeurochem 66: 2484-90 (1996)). Increased expres associated with breast neoplasms (Cancer Res 62: 2840-7 sion of ENO1 protein may prevent increased activation of (2002)). Increased expression of EPHA2 in epithelium/epi NF-kappaB transcription factor associated with prostatic thelial cells may cause increased cell proliferation associated neoplasms (J Biol Chem 280: 14325-30 (2005)). Decreased with breast neoplasms (Cancer Res 61: 2301-6 (2001)). phosphopyruvate hydratase activity of ENO1 correlates with Induced inhibition of the GPI-linked ephrin receptor activity glioblastoma associated with brain neoplasms (JNeurochem of EPHA2 may prevent increased angiogenesis associated 66: 2484-90 (1996)). Increased expression of ENO1 protein with gynecomastia (Oncogene 19: 6043-52 (2000)). Mono correlates with glioblastoma associated with brain neoplasms clonal antibody to EPHA2 may prevent malignant form of (J Neurochem 66: 2484-90 (1996)). Increased expression of breast neoplasms (Cancer Res 62: 2840-7 (2002)). Induced ENO1 protein correlates with meningioma associated with inhibition of the GPI-linked ephrin receptor activity of brain neoplasms (J Neurochem 66: 2484-90 (1996)). EPHA2 may prevent increased angiogenesis associated with Decreased phosphopyruvate hydratase activity of ENO1 cor stomach neoplasms (Oncogene 19: 6043-52 (2000)). relates with meningioma associated with brain neoplasms (J Increased phosphorylation of EPHA2 may correlate with Neurochem 66: 2484-90 (1996)). Increased expression of increased induction of apoptosis associated with breast neo ENO1 protein may cause decreased viral genome replication plasms (Oncogene 20: 6503-15 (2001)). Induced inhibition associated with HIV infections (J Cell Biochem 64: 565-72 of the GPI-linked ephrin receptor activity of EPHA2 may (1997)). Increased expression of ENO1 protein may prevent prevent increased angiogenesis associated with kidney neo increased activation of MAPK activity associated with pros plasms (Oncogene 19: 6043-52 (2000)). Increased expres tatic neoplasms (JBC 280: 14325-30 (2005)). Increased pres sion of EPHA2 protein correlates with malignant form of ence of ENO1 autoimmune antibody correlates with Behcet melanoma (Cancer Res 61: 3250-5 (2001)). Decreased Syndrome (Cancer 101: 2106-15 (2004)). Increased expres expression of EPHA2 protein may prevent increased cell sion of ENO1 in cerebrospinal fluid correlates with early proliferation associated with breast neoplasms (Cancer Res onset form of lymphocytic leukemia (Leukemia 1: 820-1 62: 2840-7 (2002)) (PhosphoSiteREGISTERED, Cell Sig (1987)). Decreased phosphopyruvate hydratase activity of naling Technology (Danvers, Mass.), Human PSDTRADE ENO1 correlates with meningioma (J Neurochem 66: 2484 MARK, Biobase Corporation, (Beverly, Mass.)). US 2010/0129929 A1 May 27, 2010 28
0062 FAK, phosphorylated atY463, is among the proteins associated with prostatic neoplasms (IntJ Cancer 68: 164-71 listed in this patent. FAK, Protein tyrosine kinase 2 (focal (1996)). Increased phosphorylation of PTK2 may correlate adhesion kinase), a non-receptor tyrosine kinase involved in with increased cytokine and chemokine mediated signaling integrin-mediated signaling and cell adhesion, migration, pathway associated with multiple myeloma (Cancer Res 63: chemotaxis, and proliferation, contributes to melanoma 5850-8 (2003)). Increased protein-tyrosine kinase activity of metastasis. This protein has potential diagnostic and/or thera PTK2 may correlate with disease progression associated with peutic implications based on the following findings. Induced prostatic neoplasms (Int J Cancer 68: 164-71 (1996)). inhibition of the protein kinase activity of PTK2 may cause Increased dephosphorylation of PTK2 may cause increased increased apoptosis associated with breast neoplasms (JBC apoptosis associated with breast neoplasms (J Mol Endo 277: 38978-87 (2002)). Increased phosphorylation of PTK2 crinol 22: 141-50 (1999)) (PhosphoSiteREGISTERED, Cell correlates with more severe form of lung neoplasms (Br J Signaling Technology (Danvers, Mass.), Human Cancer 74: 780-7 (1996)). Increased tyrosine phosphoryla PSDTRADEMARK, Biobase Corporation, (Beverly, tion of PTK2 may correlate with increased cell proliferation Mass.)). associated with non-small-cell lung carcinoma (Cancer Lett 0063 FGFR4, phosphorylated at Y642 and Y643, is 162: 87-95 (2001)). Increased protein-tyrosine kinase activ among the proteins listed in this patent. FGFR4, Fibroblast ity of PTK2 may correlate with increased cell migration asso growth factor receptor 4, involved in cholesterol metabolism, ciated with prostatic neoplasms (Oncogene 20: 1152-63 bile acid synthesis, and cell adhesion, elevated protein levels (2001)). Increased expression of PTK2 protein may correlate correlate with arteriosclerosis and several cancers; gene poly with astrocytoma associated with glioma (Cancer Res 61: morphism is associated with prostate cancer. This protein has 5688-91 (2001)). Increased expression of PTK2 protein may potential diagnostic and/or therapeutic implications based on correlate with increased cell migration associated with pros the following findings. Gain of function mutation in the tatic neoplasms (Oncogene 20: 1152-63 (2001)). Increased FGFR4 gene may correlate with breast neoplasms (Biochem tyrosine phosphorylation of PTK2 may correlate with Biophy's Res Commun 287: 60-5 (2001)). Increased expres decreased cell motility associated with breast neoplasms sion of FGFR4 mRNA correlates with increased occurrence (Exp Cell Res 247: 17-28 (1999)). Increased phosphorylation of death associated with prostatic neoplasms (BrJCancer 92: of PTK2 may correlate with increased protein secretion asso 320-7 (2005)). Alternative form of FGFR4 protein may cause ciated with small cell carcinoma (Biochem Biophy's Res abnormal regulation of cell adhesion associated with pituitary Commun290: 1123-7 (2002)). Increased expression of PTK2 neoplasms (JClin Invest 109: 69-78 (2002)). Increased cyto mRNA may correlate with disease progression associated plasm localization of FGFR4 correlates with non-familial with prostatic neoplasms (Int J Cancer 68: 164-71 (1996)). form of pituitary neoplasms (JClin Invest 109: 69-78 (2002)). Increased expression of PTK2 protein may cause increased Increased expression of FGFR4 mRNA correlates with breast apoptosis associated with breast neoplasms (J Biol Chem neoplasms (Int J Cancer 61: 170-6 (1995)). Alternative form 275: 30597-604 (2000)). Increased expression of PTK2 pro of FGFR4 protein may cause non-familial form of pituitary tein may correlate with neoplasm invasiveness associated neoplasms (JClin Invest 109: 69-78 (2002)). Alternative form with colonic neoplasms (Cancer Res 55: 2752-5 (1995)). of FGFR4 protein correlates with non-familial form of pitu Induced inhibition of the protein kinase activity of PTK2 may itary neoplasms (J Clin Invest 109: 69-78 (2002)). Increased cause increased apoptosis associated with breast neoplasms cytoplasm localization of FGFR4 correlates with adenoma (J Biol Chem 277: 38978-87 (2002)). Decreased expression tumors associated with pituitary neoplasms (J Clin Endo of PTK2 protein may correlate with increased response to crinol Metab 89: 1904-11 (2004)). Amplification of the drug associated with prostatic neoplasms (Int J Cancer 98: FGFR4 gene correlates with ovarian neoplasms (IntJ Cancer 167-72 (2002)). Increased expression of PTK2 protein may 54:378-82 (1993)). Alternative form of FGFR4 mRNA cor correlate with neoplasm invasiveness associated with pros relates with pituitary neoplasms (JClin Endocrinol Metab 82: tatic neoplasms (Int J Cancer 68: 164-71 (1996)). Increased 1160-6 (1997)). Increased cytoplasm localization of FGFR4 expression of PTK2 protein may correlate with neoplasm correlates with increased cell proliferation associated with invasiveness associated with breast neoplasms (Cancer Res pituitary neoplasms (J Clin Endocrinol Metab 89: 1904-11 55: 2752-5 (1995)). Increased protein-tyrosine kinase activ (2004)). Polymorphism in the FGFR4 gene correlates with ity of PTK2 may correlate with neoplasm invasiveness asso more severe form of colonic neoplasms (Cancer Res 62: ciated with prostatic neoplasms (Int J Cancer 68: 164-71 840-7 (2002)). Alternative form of FGFR4 protein may cause (1996)). Increased expression of PTK2 protein may correlate abnormal cell proliferation associated with pituitary neo with disease progression associated with glioma (Cancer Res plasms (JClin Invest 109: 69-78 (2002)). Amplification of the 61:5688-91 (2001)). Increased expression of PTK2 mRNA FGFR4 gene correlates with breast neoplasms (Int J Cancer may correlate with neoplasm invasiveness associated with 54:378-82 (1993)). Polymorphism in the FGFR4 gene cor prostatic neoplasms (Int J Cancer 68: 164-71 (1996)). relates with more severe form of breast neoplasms (Cancer Increased tyrosine phosphorylation of PTK2 correlates with Res 62: 840-7 (2002)) (PhosphoSiteREGISTERED, Cell colonic neoplasms (J Histochem Cytochem 51: 1041-8 Signaling Technology (Danvers, Mass.), Human (2003)). Increased expression of PTK2 protein may cause PSDTRADEMARK, Biobase Corporation, (Beverly, increased apoptosis associated with breast neoplasms (JBC Mass.)). 275: 30597-604 (2000)). Increased dephosphorylation of 0064. The invention also provides peptides comprising a PTK2 correlates with increased cell-cell adhesion associated novel phosphorylation site of the invention. In one particular with colonic neoplasms (Oncogene 21: 1450-60 (2002)). embodiment, the peptides comprise any one of the an amino Increased tyrosine phosphorylation of PTK2 may correlate acid sequences as set forth in SEQID NOs: 1-169, 171-269, with increased cell migration associated with prostatic neo 271-347, which are trypsin-digested peptide fragments of the plasms (Oncogene 20: 1152-63 (2001)). Increased expres parent proteins. Alternatively, a parent signaling protein listed sion of PTK2 protein may correlate with disease progression in Table 1 may be digested with another protease, and the US 2010/0129929 A1 May 27, 2010 29 sequence of a peptide fragment comprising a phosphorylation by Screening compound libraries. Alternative appropriate site can be obtained in a similar way. Suitable proteases modulators of this class are natural products, particularly include, but are not limited to, serine proteases (e.g. hepsin), secondary metabolites from organisms such as plants or metallo proteases (e.g. PUMP 1), chymotrypsin, cathepsin, fungi, which can also be identified by screening compound pepsin, thermolysin, carboxypeptidases, etc. libraries. Methods for generating and obtaining compounds 0065. The invention also provides proteins and peptides are well known in the art (Schreiber SL, Science 151: 1964 that are mutated to eliminate a novel phosphorylation site of 1969(2000); Radmann J. and Gunther J., Science 151: 1947 the invention. Such proteins and peptides are particular useful 1948 (2000)). as research tools to understand complex signaling transduc 0071. The modulators also include peptidomimetics, tion pathways of cancer cells, for example, to identify new Small protein-like chains designed to mimic peptides. Pepti upstream kinase(s) or phosphatase(s) or other proteins that domimetics may be analogues of a peptide comprising a regulates the activity of a signaling protein; to identify down phosphorylation site of the invention. Peptidomimetics may stream effector molecules that interact with a signaling pro also be analogues of a modified peptide that are mutated to tein, etc. eliminate a phosphorylation site of the invention. Peptidomi 0.066 Various methods that are well known in the art can metics (both peptide and non-peptidyl analogues) may have be used to eliminate a phosphorylation site. For example, the improved properties (e.g., decreased proteolysis, increased phosphorylatable tyrosine may be mutated into a non-phos retention or increased bioavailability). Peptidomimetics gen phorylatable residue. Such as phenylalanine. A "phosphory erally have improved oral availability, which makes them latable' amino acid refers to an amino acid that is capable of especially Suited to treatment of disorders in a human or being modified by addition of a phosphate group (any animal. includes both phosphorylated form and unphosphorylated 0072. In certain embodiments, the modulators are pep form). Alternatively, the tyrosine may be deleted. Residues tides comprising a novel phosphorylation site of the inven other than the tyrosine may also be modified (e.g., delete or tion. In certain embodiments, the modulators are antibodies mutated) if such modification inhibits the phosphorylation of or antigen-binding fragments thereofthat specifically bind at the tyrosine residue. For example, residues flanking the a novel phosphorylation site of the invention. tyrosine may be deleted or mutated, so that a kinase can not recognize/phosphorylate the mutated protein or the peptide. 3. Heavy-Isotope Labeled Peptides (AQUA Peptides). Standard mutagenesis and molecular cloning techniques can 0073. In another aspect, the invention provides peptides be used to create amino acid Substitutions or deletions. comprising a novel phosphorylation site of the invention. In a particular embodiment, the invention provides Heavy-Iso 2. Modulators of the Phosphorylation Sites type Labeled Peptides (AQUA peptides) comprising a novel 0067. In another aspect, the invention provides a modula phosphorylation site. Such peptides are useful to generate tor that modulates tyrosine phosphorylation at a novel phos phosphorylation site-specific antibodies for a novel phospho phorylation site of the invention, including Small molecules, rylation site. Such peptides are also useful as potential diag peptides comprising a novel phosphorylation site, and bind nostic tools for screening carcinoma, or as potential therapeu ing molecules that specifically bind at a novel phosphoryla tic agents for treating carcinoma. tion site, including but not limited to antibodies or antigen 0074 The peptides may be of any length, typically six to binding fragments thereof. fifteen amino acids. The novel tyrosine phosphorylation site 0068 Modulators of a phosphorylation site include any can occur at any position in the peptide; if the peptide will be molecules that directly or indirectly counteract, reduce, used as an immnogen, it preferably is from seven to twenty antagonize or inhibit tyrosine phosphorylation of the site. The amino acids in length. In some embodiments, the peptide is modulators may compete or block the binding of the phos labeled with a detectable marker. phorylation site to its upstream kinase(s) or phosphatase(s), 0075) “Heavy-isotope labeled peptide' (used interchange or to its downstream signaling transduction molecule(s). ably with AQUA peptide) refers to a peptide comprising at 0069. The modulators may directly interact with a phos least one heavy-isotope label, as described in WO/03016861, phorylation site. The modulator may also be a molecule that “Absolute Quantification of Proteins and Modified Forms does not directly interact with a phosphorylation site. For Thereof by Multistage Mass Spectrometry” (Gygiet al.) (the example, the modulators can be dominant negative mutants, teachings of which are hereby incorporated herein by refer i.e., proteins and peptides that are mutated to eliminate the ence, in their entirety). The amino acid sequence of an AQUA phosphorylation site. Such mutated proteins or peptides peptide is identical to the sequence of a proteolytic fragment could retain the binding ability to a downstream signaling of the parent protein in which the novel phosphorylation site molecule but lose the ability to trigger downstream signaling occurs. AQUA peptides of the invention are highly useful for transduction of the wild type parent signaling protein. detecting, quantitating or modulating a phosphorylation site 0070 The modulators include small molecules that modu of the invention (both in phosphorylated and unphosphory late the tyrosine phosphorylation at a novel phosphorylation lated forms) in a biological sample. site of the invention. Chemical agents, referred to in the art as 0076 A peptide of the invention, including an AQUA pep “Small molecule' compounds are typically organic, non-pep tides comprises any novel phosphorylation site. Preferably, tide molecules, having a molecular weight less than 10,000, the peptide or AQUA peptide comprises a novel phosphory less than 5,000, less than 1,000, or less than 500 daltons. This lation site of a protein in Table 1 that is an adaptor/scaffold class of modulators includes chemically synthesized mol protein, an adhesion or extracellular matrix protein, a cell ecules, for instance, compounds from combinatorial chemi cycle regulation protein, a cytoskeletal protein, an enzyme, a cal libraries. Synthetic compounds may be rationally G protein regulator protein, a protein kinase, a receptor/chan designed or identified based on known or inferred properties nel/transporter/cell Surface protein, a transcriptional regula of a phosphorylation site of the invention or may be identified tor, or a ubiquitin conjugating system protein. US 2010/0129929 A1 May 27, 2010 30
0077 Particularly preferred peptides and AQUA peptides I0083 Generally, to develop a suitable internal standard, a are these comprising a novel tyrosine phosphorylation site particular peptide (or modified peptide) within a target pro (shown as a lower case “y” in a sequence listed in Table 1) tein sequence is chosen based on its amino acid sequence and selected from the group consisting of SEQID NOs: 6 (Cas a particular protease for digestion. The peptide is then gener L): 7 (DLG3), 8 (Dok4), 9 (EFS), 16 (afadin), 19 (claudin 18), ated by Solid-phase peptide synthesis Such that one residue is 22 (CTNNB), 23 (CTNNB), 27 (desmoplakin), 38 (CUL2), replaced with that same residue containing stable isotopes 49 (CK18), 54(CK19), 75 (CTNNA1), 87 (ADH1B), 91 ('C, 'N). The result is a peptide that is chemically identical (AKR1B1), 98 (adolase A), 111 (cPLA2), 121 (AR to its native counterpart formed by proteolysis, but is easily HGAP12), 124 (ARHGEF5), 127 (BCAR3), 129 distinguishable by MS via a mass shift. A newly synthesized (Cdc42EP3), 154 (DYRK2), 156 (AMPKB), 159 (ASK1), 167 (ALK1), 171 (FAK), 177 (DDR1), 193 (ABCC1), 198 AQUA internal standard peptide is then evaluated by LC-MS/ (ANTXR1), 199 (ApoB), 201 (CACNA1A), 217 (ASCL3), MS. This process provides qualitative information about pep 218 (CBP), 219 (COPS2), 221 (EDF1), 226 (Cezanne), 227 tide retention by reverse-phase chromatography, ionization (FBW1A), 309 (Fbx46), and 323 (FBX43). efficiency, and fragmentation via collision-induced dissocia 0078. In some embodiments, the peptide or AQUA peptide tion. Informative and abundant fragment ions for sets of comprises the amino acid sequence shown in any one of the native and internal standard peptides are chosen and then above listed SEQID NOS. In some embodiments, the peptide specifically monitored in rapid succession as a function of or AQUA peptide consists of the amino acid sequence in said chromatographic retention to form a selected reaction moni SEQ ID NOS. In some embodiments, the peptide or AQUA toring (LC-SRM) method based on the unique profile of the peptide comprises a fragment of the amino acid sequence in peptide standard. said SEQ ID NOS., wherein the fragment is six to twenty I0084. The second stage of the AQUA strategy is its imple amino acid long and includes the phosphorylatable tyrosine. mentation to measure the amount of a protein or the modified In some embodiments, the peptide or AQUA peptide consists form of the protein from complex mixtures. Whole cell of a fragment of the amino acid sequence in said SEQ ID lysates are typically fractionated by SDS-PAGE gel electro NOS., wherein the fragment is six to twenty amino acid long phoresis, and regions of the gel consistent with protein migra and includes the phosphorylatable tyrosine. tion are excised. This process is followed by in-gel proteoly 0079. In certain embodiments, the peptide or AQUA pep sis in the presence of the AQUA peptides and LC-SRM tide comprises any one of SEQ ID NOs: 1-169, 171-269, analysis. (See Gerber et al. supra.) AQUA peptides are spiked 271-347, which are trypsin-digested peptide fragments of the into the complex peptide mixture obtained by digestion of the parent proteins. whole cell lysate with a proteolytic enzyme and subjected to 0080. It is understood that parent protein listed in Table 1 immunoaffinity purification as described above. The reten may be digested with any Suitable protease (e.g., serine pro tion time and fragmentation pattern of the native peptide teases (e.g. trypsin, hepsin), metallo proteases (e.g. PUMP1), formed by digestion (e.g., trypsinization) is identical to that of chymotrypsin, cathepsin, pepsin, thermolysin, carboxypepti the AQUA internal standard peptide determined previously; dases, etc), and the resulting peptide sequence comprising a thus, LC-MS/MS analysis using an SRM experiment results phosphorylated site of the invention may differ from that of in the highly specific and sensitive measurement of both trypsin-digested fragments (as set forth in Column E), internal standard and analyte directly from extremely com depending the cleavage site of a particular enzyme. An AOUA plex peptide mixtures. Because an absolute amount of the peptide for a particular a parent protein sequence should be AQUA peptide is added (e.g. 250 fmol), the ratio of the areas chosen based on the amino acid sequence of the parent protein under the curve can be used to determine the precise expres and the particular protease for digestion; that is, the AQUA sion levels of a protein orphosphorylated form of a protein in peptide should match the amino acid sequence of a pro the original cell lysate. In addition, the internal standard is teolytic fragment of the parent protein in which the novel present during in-gel digestion as native peptides are formed, phosphorylation site occurs. Such that peptide extraction efficiency from gel pieces, abso 0081. An AQUA peptide is preferably at least about 6 lute losses during sample handling (including vacuum cen amino acids long. The preferred ranged is about 7 to 15 amino trifugation), and variability during introduction into the LC acids. MS system do not affect the determined ratio of native and 0082. The AQUA method detects and quantifies a target AQUA peptide abundances. protein in a sample by introducing a known quantity of at least I0085. An AQUA peptide standard may be developed for a one heavy-isotope labeled peptide standard (which has a known phosphorylation site previously identified by the IAP unique signature detectable by LC-SRM chromatography) LC-MS/MS method within a target protein. One AQUA pep into a digested biological sample. By comparing to the pep tide incorporating the phosphorylated form of the site, and a tide standard, one may readily determines the quantity of a second AQUA peptide incorporating the unphosphorylated peptide having the same sequence and protein modification form of site may be developed. In this way, the two standards (s) in the biological sample. Briefly, the AQUA methodology may be used to detect and quantify both the phosphorylated has two stages: (1) peptide internal standard selection and and unphosphorylated forms of the site in a biological validation; method development; and (2) implementation sample. using validated peptide internal standards to detect and quan I0086 Peptide internal standards may also be generated by tify a target protein in a sample. The method is a powerful examining the primary amino acid sequence of a protein and technique for detecting and quantifying a given peptide?pro determining the boundaries of peptides produced by protease tein within a complex biological mixture. Such as a cell lysate, cleavage. Alternatively, a protein may actually be digested and may be used, e.g., to quantify change in protein phospho with a protease and a particular peptide fragment produced rylation as a result of drug treatment, or to quantify a protein can then sequenced. Suitable proteases include, but are not in different biological states. limited to, serine proteases (e.g. trypsin, hepsin), metallo US 2010/0129929 A1 May 27, 2010 proteases (e.g. PUMP1), chymotrypsin, cathepsin, pepsin, example by collision-induced dissociation (CID) using, e.g., thermolysin, carboxypeptidases, etc. argon or helium as a collision gas. The fragments are then 0087. A peptide sequence within a target protein is analyzed, for example by multi-stage mass spectrometry selected according to one or more criteria to optimize the use (MS) to obtain a fragmention spectrum, to obtain a peptide of the peptide as an internal standard. Preferably, the size of fragmentation signature. Preferably, peptide fragments have the peptide is selected to minimize the chances that the pep significant differences in m/z ratios to enable peaks corre tide sequence will be repeated elsewhere in other non-target sponding to each fragment to be well separated, and a signa proteins. Thus, a peptide is preferably at least about 6 amino ture that is unique for the target peptide is obtained. If a acids. The size of the peptide is also optimized to maximize Suitable fragment signature is not obtained at the first stage, ionization frequency. Thus, peptides longer than about 20 additional stages of MS are performed until a unique signa amino acids are not preferred. The preferred ranged is about ture is obtained. 7 to 15 amino acids. A peptide sequence is also selected that is not likely to be chemically reactive during mass spectrom 0092. Fragment ions in the MS/MS and MS spectra are etry, thus sequences comprising cysteine, tryptophan, or typically highly specific for the peptide of interest, and, in methionine are avoided. conjunction with LC methods, allow a highly selective means 0088 A peptide sequence that is outside a phosphoryla of detecting and quantifying a target peptide?protein in a tion site may be selected as internal standard to determine the complex protein mixture, such as a cell lysate, containing quantity of all forms of the target protein. Alternatively, a many thousands or tens of thousands of proteins. Any bio peptide encompassing a phosphorylated site may be selected logical sample potentially containing a target protein/peptide as internal standard to detect and quantify only the phospho of interest may be assayed. Crude or partially purified cell rylated form of the target protein. Peptide standards for both extracts are preferably used. Generally, the sample has at least phosphorylated form and unphosphorylated form can be used 0.01 mg of protein, typically a concentration of 0.1-10 together, to determine the extent of phosphorylation in a mg/mL, and may be adjusted to a desired buffer concentration particular sample. and pH. 0089. The peptide is labeled using one or more labeled 0093. A known amount of a labeled peptide internal stan amino acids (i.e. the label is an actual part of the peptide) or dard, preferably about 10 femtomoles, corresponding to a less preferably, labels may be attached after synthesis accord target protein to be detected/quantified is then added to a ing to standard methods. Preferably, the label is a mass biological sample, such as a cell lysate. The spiked sample is altering label selected based on the following considerations: The mass should be unique to shift fragment masses produced then digested with one or more protease(s) for a suitable time by MS analysis to regions of the spectrum with low back period to allow digestion. A separation is then performed ground; the ion mass signature component is the portion of (e.g., by HPLC, reverse-phase HPLC, capillary electrophore the labeling moiety that preferably exhibits a unique ion mass sis, ion exchange chromatography, etc.) to isolate the labeled signature in MS analysis; the Sum of the masses of the con internal standard and its corresponding target peptide from stituent atoms of the label is preferably uniquely different other peptides in the sample. Microcapillary LC is a preferred than the fragments of all the possible amino acids. As a result, method. the labeled amino acids and peptides are readily distinguished 0094. Each isolated peptide is then examined by monitor from unlabeled ones by the ion/mass pattern in the resulting ing of a selected reaction in the MS. This involves using the mass spectrum. Preferably, the ion mass signature component prior knowledge gained by the characterization of the peptide imparts a mass to a protein fragment that does not match the internal standard and then requiring the MS to continuously residue mass for any of the 20 natural amino acids. monitor a specific ion in the MS/MS or MS" spectrum for both 0090 The label should be robust under the fragmentation the peptide of interest and the internal standard. After elution, conditions of MS and not undergo unfavorable fragmenta the area under the curve (AUC) for both peptide standard and tion. Labeling chemistry should be efficient under a range of target peptide peaks are calculated. The ratio of the two areas conditions, particularly denaturing conditions, and the provides the absolute quantification that can be normalized labeled tag preferably remains soluble in the MS buffer sys for the number of cells used in the analysis and the protein's tem of choice. The label preferably does not suppress the molecular weight, to provide the precise number of copies of ionization efficiency of the protein and is not chemically the protein percell. Further details of the AQUA methodology reactive. The label may contain a mixture of two or more are described in Gygiet al., and Gerber et al. Supra. isotopically distinct species to generate a unique mass spec 0.095 Accordingly, AQUA internal peptide standards trometric pattern at each labeled fragment position. Stable (heavy-isotope labeled peptides) may be produced, as isotopes, such as 'C, N, O, O, or 'S, are among pre described above, for any of the 349 novel phosphorylation ferred labels. Pairs of peptide internal standards that incorpo sites of the invention (see Table 1/FIG. 2). For example, rate a different isotope label may also be prepared. Preferred peptide standards for a given phosphorylation site (e.g., an amino acid residues into which a heavy isotope label may be AQUA peptidehaving the sequence MPAKTPlyLKAANNK incorporated include leucine, proline, Valine, and phenylala (SEQ ID NO: 129), wherein “y” corresponds to phosphory 1C. latable tyrosine 8 of Cdc42EP3) may be produced for both the 0091 Peptide internal standards are characterized accord phosphorylated and unphosphorylated forms of the sequence. ing to their mass-to-charge (m/z) ratio, and preferably, also Such standards may be used to detect and quantify both according to their retention time on a chromatographic col phosphorylated form and unphosphorylated form of the par umn (e.g. an HPLC column). Internal standards that co-elute ent signaling protein (e.g., Cdc42EP3) in a biological sample. with unlabeled peptides of identical sequence are selected as 0096 Heavy-isotope labeled equivalents of a phosphory optimal internal standards. The internal standard is then ana lation site of the invention, both in phosphorylated and lyzed by fragmenting the peptide by any suitable means, for unphosphorylated form, can be readily synthesized and their US 2010/0129929 A1 May 27, 2010 32 unique MS and LC-SRM signature determined, so that the 0104. An antibody or antigen-binding fragment thereof peptides are validated as AQUA peptides and ready for use in specially binds an antigen when the dissociation constant is quantification. s1 mM, preferably s100 nM, and more preferably s 10 nM. 0097. The novel phosphorylation sites of the invention are 0105. In some embodiments, the antibody or antigen particularly well Suited for development of corresponding binding fragment of the invention binds an amino acid AQUA peptides, since the IAP method by which they were sequence that comprises a novel phosphorylation site of a identified (see Part A above and Example 1) inherently con protein in Table 1 that is an adaptor/scaffold protein, an adhe firmed that such peptides are in fact produced by enzymatic sion or extracellular matrix protein, a cell cycle regulation digestion (e.g., trypsinization) and are in fact Suitably frac protein, a cytoskeletal protein, an enzyme, a G protein regu tionated/ionized in MS/MS. Thus, heavy-isotope labeled lator protein, a protein kinase, a receptor/channel/transporter/ equivalents of these peptides (both in phosphorylated and cell Surface protein, a transcriptional regulator, or a ubiquitin unphosphorylated form) can be readily synthesized and their conjugating System protein. unique MS and LC-SRM signature determined, so that the 0106. In particularly preferred embodiments, an antibody peptides are validated as AQUA peptides and ready for use in orantigen-binding fragment thereof of the invention specially quantification experiments. binds an amino acid sequence comprising a novel tyrosine 0098. Accordingly, the invention provides heavy-isotope phosphorylation site shown as a lower case “y” in a sequence labeled peptides (AQUA peptides) that may be used for listed in Table 1 selected from the group consisting of SEQID detecting, quantitating, or modulating any of the phosphory NOS: 6 (Cas-L);7(DLG3), 8 (Dok4), 9 (EFS), 16 (afadin), 19 lation sites of the invention (Table 1). For example, an AQUA (claudin 18), 22 (CTNNB), 23 (CTNNB), 27 (desmoplakin), peptide having the sequence WPTVDASyYGGR (SEQ ID 38 (CUL2), 49 (CK18), 54(CK19), 75 (CTNNA1), 87 NO: 198), whereiny (Tyr382) may be either phosphotyrosine (ADH1B), 91 (AKR1B1), 98 (adolase A), 111 (cPLA2), 121 or tyrosine, and wherein V-labeled valine (e.g., ''C)) is pro (ARHGAP12), 124 (ARHGEF5), 127 (BCAR3), 129 vided for the quantification of phosphorylated (or unphos (Cdc42EP3), 154 (DYRK2), 156 (AMPKB), 159 (ASK1), phorylated) form of ANTXR1 (a receptor/channel/trans 167 (ALK1), 171 (FAK), 177 (DDR1), 193 (ABCC1), 198 porter/cell Surface protein) in a biological sample. (ANTXR1), 199 (ApoB), 201 (CACNA1A), 217 (ASCL3), 0099 Example 4 is provided to further illustrate the con 218 (CBP), 219 (COPS2), 221 (EDF1), 226 (Cezanne), 227 struction and use, by standard methods described above, of (FBW1A), 309 (Fbx46), and 323 (FBX43). exemplary AQUA peptides provided by the invention. For 0107. In some embodiments, an antibody or antigen-bind example, AQUA peptides corresponding to both the phospho ing fragment thereof of the invention specifically binds an rylated and unphosphorylated forms of SEQID NO: 198 (a amino acid sequence comprising any one of the above listed trypsin-digested fragment of ANTXR1, with a tyrosine 382 SEQID NOS. In some embodiments, an antibody or antigen phosphorylation site) may be used to quantify the amount of binding fragment thereof of the invention especially binds an phosphorylated ANTXR1 in a biological sample, e.g., a amino acid sequence comprises a fragment of one of said tumor cell sample or a sample before or after treatment with SEQID NOS., wherein the fragment is four to twenty amino a therapeutic agent. acid long and includes the phosphorylatable tyrosine. 0108. In certain embodiments, an antibody or antigen 0100 Peptides and AQUA peptides provided by the inven binding fragment thereof of the invention specially binds an tion will be highly useful in the further study of signal trans amino acid sequence that comprises a peptide produced by duction anomalies underlying cancer, including carcinomas. proteolysis of the parent protein with a protease wherein said Peptides and AQUA peptides of the invention may also be peptide comprises a novel tyrosine phosphorylation site of the used for identifying diagnostic/bio-markers of carcinomas, invention. In some embodiments, the peptides are produced identifying new potential drug targets, and/or monitoring the from trypsin digestion of the parent protein. The parent pro effects of test therapeutic agents on signaling proteins and tein comprising the novel tyrosine phosphorylation site can pathways. be from any species, preferably from a mammal including but 0101 4. Phosphorylation Site-Specific Antibodies not limited to non-human primates, rabbits, mice, rats, goats, 0102. In another aspect, the invention discloses phospho cows, sheep, and guinea pigs. In some embodiments, the rylation site-specific binding molecules that specifically bind parent protein is a human protein and the antibody binds an at a novel tyrosine phosphorylation site of the invention, and epitope comprising the novel tyrosine phosphorylation site that distinguish between the phosphorylated and unphospho shown by a lower case “y” in Column E of Table 1. Such rylated forms. In one embodiment, the binding molecule is an peptides include any one of SEQ ID NOs: 1-169, 171-269, antibody or an antigen-binding fragment thereof. The anti 271-347. body may specifically bind to an amino acid sequence com 0109 An antibody of the invention can be an intact, four prising a phosphorylation site identified in Table 1. immunoglobulin chain antibody comprising two heavy 0103) In some embodiments, the antibody or antigen chains and two light chains. The heavy chain of the antibody binding fragment thereof specifically binds the phosphory can be of any isotype including IgM, IgG, IgE. IgG, IgA or lated site. In other embodiments, the antibody or antigen IgD or Sub-isotype including IgG1, IgG2, IgG3, IgG4, IgE1. binding fragment thereof specially binds the IgE.2, etc. The light chain can be a kappa light chain or a unphosphorylated site. An antibody or antigen-binding frag lambda light chain. ment thereof specially binds an amino acid sequence com 0110. Also within the invention are antibody molecules prising a novel tyrosine phosphorylation site in Table 1 when with fewer than 4 chains, including single chain antibodies, it does not significantly bind any other site in the parent Camelid antibodies and the like and components of the anti protein and does not significantly binda protein other than the body, including a heavy chain or a light chain. The term parent protein. An antibody of the invention is sometimes “antibody’ (or “antibodies') refers to all types of immuno referred to herein as aphospho-specific' antibody. globulins. The term “an antigen-binding fragment of an anti US 2010/0129929 A1 May 27, 2010 body” refers to any portion of an antibody that retains specific region will, in general, be made in order to improve or alter binding of the intact antibody. An exemplary antigen-binding characteristics, such as complement fixation, interaction with fragment of an antibody is the heavy chain and/or light chain membranes and other effector functions. Changes in the vari CDR, or the heavy and/or light chain variable region. The able region will be made in order to improve the antigen term “does not bind.” when appeared in context of an anti binding characteristics. body's binding to one phospho-form (e.g., phosphorylated 0114. The antibodies of the invention include antibodies form) of a sequence, means that the antibody does not Sub of any isotype including IgM, IgG, Ig|D, IgA and IgE, and any stantially react with the other phospho-form (e.g., non-phos Sub-isotype, including IgG1, IgG2a, IgG2b, IgG3 and IgG4. phorylated form) of the same sequence. One of skill in the art IgE.1, IgE2 etc. The light chains of the antibodies can either be will appreciate that the expression may be applicable in those kappa light chains or lambda light chains. instances when (1) a phospho-specific antibody either does 0115 Antibodies disclosed in the invention may be poly not apparently bind to the non-phospho form of the antigenas clonal or monoclonal. As used herein, the term “epitope' ascertained in commonly used experimental detection sys refers to the smallest portion of a protein capable of selec tems (Western blotting. IHC. Immunofluorescence, etc.); (2) tively binding to the antigen binding site of an antibody. It is where there is some reactivity with the Surrounding amino well accepted by those skilled in the art that the minimal size acid sequence, but that the phosphorylated residue is an of a protein epitope capable of selectively binding to the immunodominant feature of the reaction. In cases Such as antigenbinding site of an antibody is about five or six to seven these, there is an apparent difference in affinities for the two amino acids. sequences. Dilutional analyses of Such antibodies indicates 0116. Other antibodies specifically contemplated are oli that the antibodies apparent affinity for the phosphorylated goclonal antibodies. As used herein, the phrase "oligoclonal form is at least 10-100 fold higher than for the non-phospho antibodies’ refers to a predetermined mixture of distinct rylated form; or where (3) the phospho-specific antibody monoclonal antibodies. See, e.g., PCT publication WO reacts no more than an appropriate control antibody would 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163. In one react under identical experimental conditions. A control anti embodiment, oligoclonal antibodies consisting of a predeter body preparation might be, for instance, purified immunoglo mined mixture of antibodies against one or more epitopes are bulin from a pre-immune animal of the same species, an generated in a single cell. In other embodiments, oligoclonal isotype- and species-matched monoclonal antibody. Tests antibodies comprise a plurality of heavy chains capable of using control antibodies to demonstrate specificity are recog pairing with a common light chain to generate antibodies with nized by one of skill in the art as appropriate and definitive. multiple specificities (e.g., PCT publication WO 04/0096.18). 0111. In some embodiments an immunoglobulin chain Oligoclonal antibodies are particularly useful when it is may comprise in order from 5' to 3', a variable region and a desired to target multiple epitopes on a single target molecule. constant region. The variable region may comprise three In view of the assays and epitopes disclosed herein, those complementarity determining regions (CDRS), with inter skilled in the art can generate or selectantibodies or mixtures spersed framework (FR) regions for a structure FR1, CDR1, of antibodies that are applicable for an intended purpose and FR2, CDR2, FR3, CDR3 and FR4. Also within the invention desired need. are heavy or light chain variable regions, framework regions 0117 Recombinant antibodies against the phosphoryla and CDRs. An antibody of the invention may comprise a tion sites identified in the invention are also included in the heavy chain constant region that comprises some or all of a present application. These recombinant antibodies have the CH1 region, hinge, CH2 and CH3 region. same amino acid sequence as the natural antibodies or have 0112 An antibody of the invention may have an binding altered amino acid sequences of the natural antibodies in the affinity (K) of 1x107M or less. In other embodiments, the present application. They can be made in any expression antibody binds with a Kof 1x10M, 1x10M, 1x10'M, systems including both prokaryotic and eukaryotic expres 1x10' M, 1x10' M or less. In certain embodiments, the sion systems or using phage display methods (see, e.g., K, is 1 pM to 500 pM, between 500 pM to 1 uM, between 1 Dower et al., WO91/17271 and McCafferty et al., WO92/ uM to 100 nM, or between 100 mM to 10 nM. 01047; U.S. Pat. No. 5,969,108, which are herein incorpo 0113 Antibodies of the invention can be derived from any rated by reference in their entirety). species of animal, preferably a mammal. Non-limiting exem 0118 Antibodies can be engineered in numerous ways. plary natural antibodies include antibodies derived from They can be made as single-chain antibodies (including Small human, chicken, goats, and rodents (e.g., rats, mice, hamsters modular immunopharmaceuticals or SMIPsTM), Fab and and rabbits), including transgenic rodents genetically engi F(ab')2 fragments, etc. Antibodies can be humanized, chimer neered to produce human antibodies (see, e.g., Lonberg et al., ized, deimmunized, or fully human. Numerous publications WO93/12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et set forth the many types of antibodies and the methods of al., WO91/10741; U.S. Pat. No. 6,150,584, which are herein engineering such antibodies. For example, see U.S. Pat. Nos. incorporated by reference in their entirety). Natural antibod 6,355,245; 6, 180,370; 5,693,762; 6,407,213; 6,548,640; ies are the antibodies produced by a host animal. “Genetically 5,565,332: 5,225,539; 6,103,889; and 5,260,203. altered antibodies' refer to antibodies wherein the amino acid 0119 The genetically altered antibodies should be func sequence has been varied from that of a native antibody. tionally equivalent to the above-mentioned natural antibod Because of the relevance of recombinant DNA techniques to ies. In certain embodiments, modified antibodies provide this application, one need not be confined to the sequences of improved stability or/and therapeutic efficacy. Examples of amino acids found in natural antibodies; antibodies can be modified antibodies include those with conservative substi redesigned to obtain desired characteristics. The possible tutions of amino acid residues, and one or more deletions or variations are many and range from the changing of just one additions of amino acids that do not significantly deleteri or a few amino acids to the complete redesign of for example, ously alter the antigenbinding utility. Substitutions can range the variable or constant region. Changes in the constant from changing or modifying one or more amino acid residues US 2010/0129929 A1 May 27, 2010 34 to complete redesign of a region as long as the therapeutic fragments. Single chain antibodies may be produced by join utility is maintained. Antibodies of this application can be ing VL- and VH-coding regions with a DNA that encodes a modified post-translationally (e.g., acetylation, and/or phos peptide linker connecting the VL and VH protein fragments phorylation) or can be modified synthetically (e.g., the attach 0.126 Papain digestion of antibodies produces two identi ment of a labeling group). cal antigen-binding fragments, called “Fab' fragments, each 0120 Antibodies with engineered or variant constant or with a single antigen-binding site, and a residual "Fo' frag Fc regions can be useful in modulating effector functions, ment, whose name reflects its ability to crystallize readily. Such as, for example, antigen-dependent cytotoxicity Pepsin treatment of an antibody yields an F(ab'), fragment (ADCC) and complement-dependent cytotoxicity (CDC). that has two antigen-combining sites and is still capable of Such antibodies with engineered or variant constant or Fc cross-linking antigen. regions may be useful in instances where a parent singling I0127 “Fv usually refers to the minimum antibody frag protein (Table 1) is expressed in normal tissue; variant anti ment that contains a complete antigen-recognition and -bind bodies without effector function in these instances may elicit ing site. This region consists of a dimer of one heavy- and one the desired therapeutic response while not damaging normal light-chain variable domain in tight, non-covalent associa tissue. Accordingly, certain aspects and methods of the tion. It is in this configuration that the three CDRs of each present disclosure relate to antibodies with altered effector variable domain interact to define an antigen-binding site on functions that comprise one or more amino acid Substitutions, the surface of the V-V, dimer. Collectively, the CDRs confer insertions, and/or deletions. antigen-binding specificity to the antibody. However, even a 0121. In certain embodiments, genetically altered anti single variable domain (or half of an Fv comprising three bodies are chimeric antibodies and humanized antibodies. CDRS specific for an antigen) has the ability to recognize and 0122) The chimeric antibody is an antibody having por bind antigen, although likely at a lower affinity than the entire tions derived from different antibodies. For example, a chi binding site. meric antibody may have a variable region and a constant region derived from two different antibodies. The donoranti 0128. Thus, in certain embodiments, the antibodies of the bodies may be from different species. In certain embodi application may comprise 1, 2, 3, 4, 5, 6, or more CDRS that ments, the variable region of a chimeric antibody is non recognize the phosphorylation sites identified in Column E of human, e.g., murine, and the constant region is human. Table 1. 0123. The genetically altered antibodies used in the inven I0129. The Fab fragment also contains the constant domain tion include CDR grafted humanized antibodies. In one of the light chain and the first constant domain (CH1) of the embodiment, the humanized antibody comprises heavy and/ heavy chain. Fab' fragments differ from Fab fragments by the or light chain CDRS of a non-human donor immunoglobulin addition of a few residues at the carboxy terminus of the and heavy chain and light chain frameworks and constant heavy chain CH1 domain including one or more cysteines regions of a human acceptor immunoglobulin. The method of from the antibody hinge region. Fab'-SH is the designation making humanized antibody is disclosed in U.S. Pat. Nos: herein for Fab' in which the cysteine residue(s) of the constant 5,530,101: 5,585,089; 5,693,761; 5,693,762; and 6,180.370 domains bear a free thiol group. F(ab') antibody fragments each of which is incorporated herein by reference in its originally were produced as pairs of Fab' fragments that have entirety. hinge cysteines between them. Other chemical couplings of 0.124 Antigen-binding fragments of the antibodies of the antibody fragments are also known. invention, which retain the binding specificity of the intact I0130 “Single-chain Fv' or “scFv” antibody fragments antibody, are also included in the invention. Examples of comprise the V, and V, domains of an antibody, wherein these antigen-binding fragments include, but are not limited these domains are present in a single polypeptide chain. In to, partial or full heavy chains or light chains, variable certain embodiments, the Fv polypeptide further comprises a regions, or CDR regions of any phosphorylation site-specific polypeptide linker between the V and V, domains that antibodies described herein. enables the schv to form the desired structure for antigen 0.125. In one embodiment of the application, the antibody binding. For a review of scFv see Pluckthun in The Pharma fragments are truncated chains (truncated at the carboxyl cology of Monoclonal Antibodies, vol. 113, Rosenburg and end). In certain embodiments, these truncated chains possess Moore, eds. (Springer-Verlag: New York, 1994), pp. 269-315. one or more immunoglobulin activities (e.g., complement I0131 SMIPs are a class of single-chain peptides engi fixation activity). Examples of truncated chains include, but neered to include a target binding region and effector domain are not limited to, Fab fragments (consisting of the VL, VH, (CH2 and CH3 domains). See, e.g., U.S. Patent Application CL and CH1 domains); Fd fragments (consisting of the VH Publication No. 20050238646. The target binding region may and CH1 domains): Fv fragments (consisting of VL and VH be derived from the variable region or CDRs of an antibody, domains of a single chain of an antibody); dAb fragments e.g., a phosphorylation site-specific antibody of the applica (consisting of a VH domain); isolated CDR regions: (Fab') tion. Alternatively, the target binding region is derived from a fragments, bivalent fragments (comprising two Fab frag protein that binds a phosphorylation site. ments linked by a disulphide bridge at the hinge region). The I0132 Bispecificantibodies may be monoclonal, human or truncated chains can be produced by conventional biochemi humanized antibodies that have binding specificities for at cal techniques, such as enzyme cleavage, or recombinant least two different antigens. In the present case, one of the DNA techniques, each of which is known in the art. These binding specificities is for the phosphorylation site, the other polypeptide fragments may be produced by proteolytic cleav one is for any other antigen, Such as for example, a cell age of intact antibodies by methods well known in the art, or Surface protein or receptor or receptor Subunit. Alternatively, by inserting stop codons at the desired locations in the vectors atherapeutic agent may be placed on one arm. The therapeu using site-directed mutagenesis, Such as after CH1 to produce tic agent can be a drug, toxin, enzyme, DNA, radionuclide, Fab fragments or after the hinge region to produce (Fab') etc. US 2010/0129929 A1 May 27, 2010
0133. In some embodiments, the antigen-binding frag ers, which bind, in a phospho-specific manner, to an amino ment can be a diabody. The term "diabody' refers to small acid sequence comprising a novel phosphorylation site of the antibody fragments with two antigen-binding sites, which invention. See, e.g., Neuberger et al., Nature 312: 604 (1984). fragments comprise aheavy-chain variable domain (V) con Aptamers are oligonucleic acid or peptide molecules that nected to a light-chain variable domain (V) in the same bind a specific target molecule. DNA or RNA aptamers are polypeptide chain (V-V.). By using a linker that is too short typically short oligonucleotides, engineered through repeated to allow pairing between the two domains on the same chain, rounds of selection to bind to a molecular target. Peptide the domains are forced to pair with the complementary aptamers typically consist of a variable peptide loop attached domains of another chain and create two antigen-binding at both ends to a protein scaffold. This double structural sites. Diabodies are described more fully in, for example, EP constraint generally increases the binding affinity of the pep 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. tide aptamer to levels comparable to an antibody (nanomolar Acad. Sci. USA, 90: 6444-6448 (1993). range). 0134 Camelid antibodies refer to a unique type of anti 0140. The invention also discloses the use of the phospho bodies that are devoid of light chain, initially discovered from rylation site-specific antibodies with immunotoxins. Conju animals of the camelid family. The heavy chains of these gates that are immunotoxins including antibodies have been so-called heavy-chain antibodies bind their antigen by one widely described in the art. The toxins may be coupled to the single domain, the variable domain of the heavy immunoglo antibodies by conventional coupling techniques or immuno bulin chain, referred to as VHH.VHHs show homology with toxins containing protein toxin portions can be produced as the variable domain of heavy chains of the human VHIII fusion proteins. In certain embodiments, antibody conjugates family. The VHHs obtained from an immunized camel, drom may comprise stable linkers and may release cytotoxic agents edary, or llama have a number of advantages, such as effective inside cells (see U.S. Pat. Nos. 6,867,007 and 6,884,869). The production in microorganisms such as Saccharomyces cer conjugates of the present application can be used in a corre evisiae. sponding way to obtain Such immunotoxins. Illustrative of 0135) In certain embodiments, single chain antibodies, such immunotoxins are those described by Byers et al., Semi and chimeric, humanized or primatized (CDR-grafted) anti nars Cell Biol 2:59-70 (1991) and by Fanger et al., Immunol bodies, as well as chimeric or CDR-grafted single chain anti Today 12:51-54 (1991). Exemplary immunotoxins include bodies, comprising portions derived from different species, radiotherapeutic agents, ribosome-inactivating proteins are also encompassed by the present disclosure as antigen (RIPs), chemotherapeutic agents, toxic peptides, or toxic pro binding fragments of an antibody. The various portions of teins. these antibodies can be joined together chemically by con 0.141. The phosphorylation site-specific antibodies dis ventional techniques, or can be prepared as a contiguous closed in the invention may be used singly or in combination. protein using genetic engineering techniques. For example, The antibodies may also be used in an array format for high nucleic acids encoding a chimeric or humanized chain can be throughput uses. An antibody microarray is a collection of expressed to produce a contiguous protein. See, e.g., U.S. Pat. immobolized antibodies, typically spotted and fixed on a Nos. 4,816,567 and 6,331415: U.S. Pat. No. 4,816,397; Solid Surface (such as glass, plastic and silicon chip). European Patent No. 0,120,694; WO 86/01533: European 0142. In another aspect, the antibodies of the invention Patent No. 0,194,276 B1; U.S. Pat. No. 5,225,539; and Euro modulate at least one, or all, biological activities of a parent pean Patent No. 0.239,400 B1. See also, Newman et al., protein identified in Column A of Table 1. The biological BioTechnology, 10: 1455-1460 (1992), regarding primatized activities of a parent protein identified in Column A of Table antibody. See, e.g., Ladner et al., U.S. Pat. No. 4,946,778; and 1 include: 1) ligand binding activities (for instance, these Bird et al., Science, 242: 423-426 (1988)), regarding single neutralizing antibodies may be capable of competing with or chain antibodies. completely blocking the binding of a parent signaling protein 0136. In addition, functional fragments of antibodies, to at least one, or all, of its ligands; 2) signaling transduction including fragments of chimeric, humanized, primatized or activities, such as receptor dimerization, or tyrosine phospho single chain antibodies, can also be produced. Functional rylation; and 3) cellular responses induced by a parent sig fragments of the Subject antibodies retain at least one binding naling protein, such as oncogenic activities (e.g., cancer cell function and/or modulation function of the full-length anti proliferation mediated by a parent signaling protein), and/or body from which they are derived. angiogenic activities. 0.137 Since the immunoglobulin-related genes contain 0143. In certain embodiments, the antibodies of the inven separate functional regions, each having one or more distinct tion may have at least one activity selected from the group biological activities, the genes of the antibody fragments may consisting of: 1) inhibiting cancer cell growth or prolifera be fused to functional regions from other genes (e.g., tion; 2) inhibiting cancer cell Survival; 3) inhibiting angio enzymes, U.S. Pat. No. 5,004,692, which is incorporated by genesis; 4) inhibiting cancer cell metastasis, adhesion, migra reference in its entirety) to produce fusion proteins or conju tion or invasion; 5) inducing apoptosis of cancer cells; 6) gates having novel properties. incorporating a toxic conjugate; and 7) acting as a diagnostic 0.138. Non-immunoglobulin binding polypeptides are also marker. contemplated. For example, CDRs from an antibody dis 0144. In certain embodiments, the phosphorylation site closed herein may be inserted into a suitable non-immuno specific antibodies disclosed in the invention are especially globulin scaffold to create a non-immunoglobulin binding indicated for diagnostic and therapeutic applications as polypeptide. Suitable candidate scaffold structures may be described herein. Accordingly, the antibodies may be used in derived from, for example, members of fibronectin type III therapies, including combination therapies, in the diagnosis and cadherin Superfamilies. and prognosis of disease, as well as in the monitoring of 0.139. Also contemplated are other equivalent non-anti disease progression. The invention, thus, further includes body molecules, such as protein binding domains or aptam compositions comprising one or more embodiments of an US 2010/0129929 A1 May 27, 2010 36 antibody or an antigen binding portion of the invention as four or more amino acids flanking each side of the phospho described herein. The composition may further comprise a rylatable amino acid and encompassing it. Peptide antigens pharmaceutically acceptable carrier. The composition may suitable for producing antibodies of the invention may be comprise two or more antibodies orantigen-binding portions, designed, constructed and employed in accordance with well each with specificity for a different novel tyrosine phospho known techniques. See, e.g., Antibodies: A Laboratory rylation site of the invention or two or more different anti Manual, Chapter 5, p. 75-76, Harlow & Lane Eds. Cold bodies or antigen-binding portions all of which are specific Spring Harbor Laboratory (1988); Czernik, Methods In Enzy for the same novel tyrosine phosphorylation site of the inven mology, 201: 264-283 (1991); Merrifield, J. Am. Chem. Soc. tion. A composition of the invention may comprise one or 85: 21-49 (1962)). more antibodies or antigen-binding portions of the invention 0151. Suitable peptide antigens may comprise all or par and one or more additional reagents, diagnostic agents or tial sequence of a trypsin-digested fragment as set forth in therapeutic agents. Column E of Table 1/FIG. 2. Suitable peptide antigens may 0145 The present application provides for the polynucle also comprise all or partial sequence of a peptide fragment otide molecules encoding the antibodies and antibody frag produced by another protease digestion. ments and their analogs described herein. Because of the 0152 Preferred immunogens are those that comprise a degeneracy of the genetic code, a variety of nucleic acid novel phosphorylation site of a protein in Table 1 that is an sequences encode each antibody amino acid sequence. The adaptor/scaffold protein, an adhesion or extracellular matrix desired nucleic acid sequences can be produced by de novo protein, a cell cycle regulation protein, a cytoskeletal protein, solid-phase DNA synthesis or by PCR mutagenesis of an an enzyme, a G protein regulator protein, a protein kinase, a earlier prepared variant of the desired polynucleotide. In one receptor/channel/transporter/cell Surface protein, a transcrip embodiment, the codons that are used comprise those that are tional regulator, or a ubiquitin conjugating system protein. In typical for human or mouse (see, e.g., Nakamura, Y., Nucleic Some embodiments, the peptide immunogen is an AQUA Acids Res. 28:292 (2000)). peptide, for example, any one of SEQID NOS: 1-169, 171 0146 The invention also provides immortalized cell lines 269, 271-347. that produce an antibody of the invention. For example, hybri 0153. Particularly preferred immunogens are peptides doma clones, constructed as described above, that produce comprising any one of the novel tyrosine phosphorylation site monoclonal antibodies to the targeted signaling protein phos shown as a lower case “y” in a sequence listed in Table 1 phorylation sties disclosed herein are also provided. Simi selected from the group consisting of SEQID NOS: 6 (Cas larly, the invention includes recombinant cells producing an L): 7 (DLG3), 8 (Dok4), 9 (EFS), 16 (afadin), 19 (claudin 18), antibody of the invention, which cells may be constructed by 22 (CTNNB), 23 (CTNNB), 27 (desmoplakin), 38 (CUL2), well known techniques; for example the antigen combining 49 (CK18), 54(CK19), 75 (CTNNA1), 87 (ADH1B), 91 site of the monoclonal antibody can be cloned by PCR and (AKR1B1), 98 (adolase A), 111 (cPLA2), 121 (AR single-chain antibodies produced as phage-displayed recom HGAP12), 124 (ARHGEF5), 127 (BCAR3), 129 binant antibodies or soluble antibodies in E. coli (see, e.g., (Cdc42EP3), 154 (DYRK2), 156 (AMPKB), 159 (ASK1), ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, 167 (ALK1), 171 (FAK), 177 (DDR1), 193 (ABCC1), 198 Sudhir Paul editor.) (ANTXR1), 199 (ApoB), 201 (CACNA1A), 217 (ASCL3), 0147 5. Methods of Making Phosphorylation Site-Spe 218 (CBP), 219 (COPS2), 221 (EDF1), 226 (Cezanne), 227 cific Antibodies (FBW1A), 309 (Fbx46), and 323 (FBX43). 0148. In another aspect, the invention provides a method 0154) In some embodiments the immunogen is adminis for making phosphorylation site-specific antibodies. tered with an adjuvant. Suitable adjuvants will be well known 0149 Polyclonal antibodies of the invention may be pro to those of skill in the art. Exemplary adjuvants include com duced according to standard techniques by immunizing a plete or incomplete Freund's adjuvant, RIBI (muramyl dipep Suitable animal (e.g., rabbit, goat, etc.) with an antigen com tides) or ISCOM (immunostimulating complexes). prising a novel tyrosine phosphorylation site of the invention. 0155 For example, a peptide antigen comprising the novel (i.e. a phosphorylation site shown in Table 1) in either the receptor tyrosine kinase phosphorylation site in SEQID NO: phosphorylated or unphosphorylated State, depending upon 156 shown by the lower case “y” in Table 1 may be used to the desired specificity of the antibody, collecting immune produce antibodies that specifically bind the novel tyrosine serum from the animal, and separating the polyclonal anti phosphorylation site. bodies from the immune serum, in accordance with known 0156 When the above-described methods are used for procedures and screening and isolating a polyclonal antibody producing polyclonal antibodies, following immunization, specific for the novel tyrosine phosphorylation site of interest the polyclonal antibodies which secreted into the blood as further described below. Methods for immunizing non stream can be recovered using known techniques. Purified human animals such as mice, rats, sheep, goats, pigs, cattle forms of these antibodies can, of course, be readily prepared and horses are well known in the art. See, e.g., Harlow and by standard purification techniques, such as for example, Lane, Antibodies. A Laboratory Manual, New York: Cold affinity chromatography with Protein A, anti-immunoglobu Spring Harbor Press, 1990. lin, or the antigen itself. In any case, in order to monitor the 0150. The immunogen may be the full length protein or a Success of immunization, the antibody levels with respect to peptide comprising the novel tyrosine phosphorylation site of the antigen in serum will be monitored using standard tech interest. In some embodiments the immunogen is a peptide of niques such as ELISA, RIA and the like. from 7 to 20 amino acids in length, preferably about 8 to 17 0157 Monoclonal antibodies of the invention may be pro amino acids in length. In some embodiments, the peptide duced by any of a number of means that are well-known in the antigen desirably will comprise about 3 to 8 amino acids on art. In some embodiments, antibody-producing B cells are each side of the phosphorylatable tyrosine. In yet other isolated from an animal immunized with a peptide antigen as embodiments, the peptide antigen desirably will comprise described above. The B cells may be from the spleen, lymph US 2010/0129929 A1 May 27, 2010 37 nodes or peripheral blood. Individual B cells are isolated and encoding the variable region, may be cloned and isolated screened as described below to identify cells producing an from antibody-producing cells using means that are well antibody specific for the novel tyrosine phosphorylation site known in the art. For example, the antigen combining site of of interest. Identified cells are then cultured to produce a the monoclonal antibody can be cloned by PCR and single monoclonal antibody of the invention. chain antibodies produced as phage-displayed recombinant 0158 Alternatively, a monoclonal phosphorylation site antibodies or soluble antibodies in E. coli (see, e.g., Antibody specific antibody of the invention may be produced using Engineering Protocols, 1995, Humana Press, Sudhir Paul standard hybridoma technology, in a hybridoma cell line editor.) according to the well-known technique of Kohler and Mil 0163 Accordingly, in a further aspect, the invention pro stein. See Nature 265: 495-97 (1975); Kohler and Milstein, vides such nucleic acids encoding the heavy chain, the light Eur: J. Immunol. 6: 511 (1976); see also, Current Protocols in chain, a variable region, a framework region or a CDR of an Molecular Biology, Ausubel et al. Eds. (1989). Monoclonal antibody of the invention. In some embodiments, the nucleic antibodies so produced are highly specific, and improve the acids are operably linked to expression control sequences. selectivity and specificity of diagnostic assay methods pro The invention, thus, also provides vectors and expression vided by the invention. For example, a Solution containing the control sequences useful for the recombinant expression of an appropriate antigen may be injected into a mouse or other antibody or antigen-binding portion thereof of the invention. species and, after a Sufficient time (in keeping with conven Those of skill in the art will be able to choose vectors and tional techniques), the animal is sacrificed and spleen cells expression systems that are suitable for the host cell in which obtained. The spleen cells are then immortalized by any of a the antibody or antigen-binding portion is to be expressed. number of Standard means. Methods of immortalizing cells 0164 Monoclonal antibodies of the invention may be pro include, but are not limited to, transfecting them with onco duced recombinantly by expressing the encoding nucleic genes, infecting them with an oncogenic virus and cultivating acids in a suitable host cell under Suitable conditions. Accord them under conditions that select for immortalized cells, sub ingly, the invention further provides host cells comprising the jecting them to carcinogenic or mutating compounds, fusing nucleic acids and vectors described above. them with an immortalized cell, e.g., a myeloma cell, and 0.165 Monoclonal Fab fragments may also be produced in inactivating a tumor Suppressor gene. See, e.g., Harlow and Escherichia coli by recombinant techniques known to those Lane, Supra. If fusion with myeloma cells is used, the skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 myeloma cells preferably do not secrete immunoglobulin (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 polypeptides (a non-secretory cell line). Typically the anti (1990). body producing cell and the immortalized cell (such as but not 0166 If monoclonal antibodies of a single desired isotype limited to myeloma cells) with which it is fused are from the are preferred for a particular application, particular isotypes same species. Rabbit fusion hybridomas, for example, may be can be prepared directly, by selecting from the initial fusion, produced as described in U.S. Pat. No. 5.675,063, C. Knight, or prepared secondarily, from a parental hybridoma Secreting Issued Oct. 7, 1997. The immortalized antibody producing a monoclonal antibody of different isotype by using the sib cells, such as hybridoma cells, are then grown in a Suitable selection technique to isolate class-switch variants selection media, Such as hypoxanthine-aminopterin-thymi (Steplewski, et al., Proc. Natl. Acad. Sci., 82: 8653 (1985); dine (HAT), and the Supernatant Screened for monoclonal Spira et al., J. Immunol. Methods, 74: 307 (1984)). Alterna antibodies having the desired specificity, as described below. tively, the isotype of a monoclonal antibody with desirable The secreted antibody may be recovered from tissue culture propertied can be changed using antibody engineering tech Supernatant by conventional methods such as precipitation, niques that are well-known in the art. ion exchange or affinity chromatography, or the like. 0.167 Phosphorylation site-specific antibodies of the 0159. The invention also encompasses antibody-produc invention, whether polyclonal or monoclonal, may be ing cells and cell lines, such as hybridomas, as described screened for epitope and phospho-specificity according to above. standard techniques. See, e.g., Czernik et al., Methods in 0160 Polyclonal or monoclonal antibodies may also be Enzymology, 201: 264-283 (1991). For example, the antibod obtained through in vitro immunization. For example, phage ies may be screened against the phosphorylated and/or display techniques can be used to provide libraries containing unphosphosphorylated peptide library by ELISA to ensure a repertoire of antibodies with varying affinities for a particu specificity for both the desired antigen (i.e. that epitope lar antigen. Techniques for the identification of high affinity including a phosphorylation site of the invention and for human antibodies from such libraries are described by Grif reactivity only with the phosphorylated (or unphosphory fiths et al., (1994) EMBO.J., 13:3245-3260; Nissim et al., lated) form of the antigen. Peptide competition assays may be ibid, pp. 692-698 and by Griffiths et al., ibid, 12:725-734, carried out to confirm lack of reactivity with other phospho which are incorporated by reference. epitopes on the parent protein. The antibodies may also be 0161 The antibodies may be produced recombinantly tested by Western blotting against cell preparations contain using methods well known in the art for example, according ing the parent signaling protein, e.g., cell lines over-express to the methods disclosed in U.S. Pat. No. 4.349,893 (Reading) ing the parent protein, to confirm reactivity with the desired or U.S. Pat. No. 4,816,567 (Cabilly et al.) The antibodies may phosphorylated epitope/target. also be chemically constructed by specific antibodies made 0168 Specificity against the desired phosphorylated according to the method disclosed in U.S. Pat. No. 4,676.980 epitope may also be examined by constructing mutants lack (Segel et al.) ing phosphorylatable residues at positions outside the desired 0162. Once a desired phosphorylation site-specific anti epitope that are known to be phosphorylated, or by mutating body is identified, polynucleotides encoding the antibody, the desired phospho-epitope and confirming lack of reactiv Such as heavy, light chains or both (or single chains in the case ity. Phosphorylation site-specific antibodies of the invention of a single chain antibody) or portions thereof Such as those may exhibit some limited cross-reactivity to related epitopes US 2010/0129929 A1 May 27, 2010 in non-target proteins. This is not unexpected as most anti specified tyrosine residue, but are not limited only to binding bodies exhibit some degree of cross-reactivity, and anti-pep to the listed signaling proteins of human species, perse. The tide antibodies will often cross-react with epitopes having invention includes antibodies that also bind conserved and high homology to the immunizing peptide. See, e.g., Czernik, highly homologous or identical phosphorylation sites in Supra. Cross-reactivity with non-target proteins is readily respective signaling proteins from other species (e.g., mouse, characterized by Western blotting alongside markers of rat, monkey, yeast), in addition to binding the phosphoryla known molecular weight. Amino acid sequences of cross tion site of the human homologue. The term “homologous' reacting proteins may be examined to identify phosphoryla refers to two or more sequences or Subsequences that have at tion sites with flanking sequences that are highly homologous least about 85%, at least 90%, at least 95%, or higher nucle to that of a phosphorylation site of the invention. otide or amino acid residue identity, when compared and 0169. In certain cases, polyclonal antisera may exhibit Some undesirable general cross-reactivity to phosphotyrosine aligned for maximum correspondence, as measured using itself, which may be removed by further purification of anti sequence comparison method (e.g., BLAST) and/or by visual sera, e.g., over a phosphotyramine column. Antibodies of the inspection. Highly homologous oridentical sites conserved in invention specifically bind their target protein (i.e. a protein other species can readily be identified by standard sequence listed in Column A of Table 1) only when phosphorylated (or comparisons (such as BLAST). only when not phosphorylated, as the case may be) at the site 0.174 Methods for making bispecificantibodies are within disclosed in corresponding Columns D/E, and do not (Sub the purview of those skilled in the art. Traditionally, the stantially) bind to the other form (as compared to the form for recombinant production of bispecific antibodies is based on which the antibody is specific). the co-expression of two immunoglobulin heavy-chain/light 0170 Antibodies may be further characterized via immu chain pairs, where the two heavy chains have different speci nohistochemical (IHC) staining using normal and diseased ficities (Milstein and Cuello, Nature, 305:537-539 (1983)). tissues to examine phosphorylation and activation state and Antibody variable domains with the desired binding speci level of a phosphorylation site in diseased tissue. IHC may be ficities (antibody-antigen combining sites) can be fused to carried out according to well-known techniques. See, e.g., immunoglobulin constant domain sequences. In certain Antibodies: A Laboratory Manual, Chapter 10, Harlow & embodiments, the fusion is with an immunoglobulin heavy Lane Eds. Cold Spring Harbor Laboratory (1988). Briefly, chain constant domain, including at least part of the hinge, paraffin-embedded tissue (e.g., tumor tissue) is prepared for CH2, and CH3 regions. DNAs encoding the immunoglobulin immunohistochemical staining by deparaffinizing tissue sec heavy-chainfusions and, if desired, the immunoglobulin light tions with Xylene followed by ethanol: hydrating in water chain, are inserted into separate expression vectors, and are then PBS, unmasking antigen by heating slide in Sodium co-transfected into a suitable host organism. For further citrate buffer, incubating sections in hydrogen peroxide; details of illustrative currently known methods for generating blocking in blocking solution; incubating slide in primary bispecific antibodies see, for example, Suresh et al., Methods antibody and secondary antibody; and finally detecting using in Enzymology, 121:210 (1986); WO 96/27011: Brennan et ABC avidin/biotin method according to manufacturer's al., Science 229:81 (1985); Shalaby et al., J. Exp. Med. 175: instructions. 217-225 (1992); Kostelny et al., J. Immunol. 148(5):1547 0171 Antibodies may be further characterized by flow 1553 (1992); Hollinger et al., Proc. Natl. Acad. Sci. USA cytometry carried out according to standard methods. See 90:6444-6448 (1993); Gruber et al., J. Immunol. 152:5368 Chow et al., Cytometry (Communications in Clinical Cytom (1994); and Tutt et al., J. Immunol. 147:60 (1991). Bispecific etry) 46: 72-78 (2001). Briefly and by way of example, the antibodies also include cross-linked or heteroconjugate anti following protocol for cytometric analysis may be employed: bodies. Heteroconjugate antibodies may be made using any samples may be centrifuged on Ficoll gradients to remove convenient cross-linking methods. Suitable cross-linking lysed erythrocytes and cell debris. Adherring cells may be agents are well known in the art, and are disclosed in U.S. Pat. scrapped offplates and washed with PBS. Cells may then be No. 4,676.980, along with a number of cross-linking tech fixed with 2% paraformaldehyde for 10 minutes at 37° C. niques. followed by permeabilization in 90% methanol for 30 min 0.175 Various techniques for making and isolating bispe utes on ice. Cells may then be stained with the primary phos cific antibody fragments directly from recombinant cell cul phorylation site-specific antibody of the invention (which ture have also been described. For example, bispecific anti detects a parent signaling protein enumerated in Table 1). bodies have been produced using leucine Zippers. Kostelny et washed and labeled with a fluorescent-labeled secondary al., J. Immunol. 148(5):1547-1553 (1992). The leucine zip antibody. Additional fluorochrome-conjugated marker anti per peptides from the Fos and Jun proteins may be linked to bodies (e.g., CD45, CD34) may also be added at this time to the Fab" portions of two different antibodies by gene fusion. aid in the Subsequent identification of specific hematopoietic The antibody homodimers may be reduced at the hinge region cell types. The cells would then be analyzed on a flow cytom to form monomers and then re-oxidized to form the antibody eter (e.g. a Beckman Coulter FC500) according to the specific heterodimers. This method can also be utilized for the pro protocols of the instrument used. duction of antibody homodimers. A strategy for making 0172 Antibodies of the invention may also be advanta bispecific antibody fragments by the use of single-chain FV geously conjugated to fluorescent dyes (e.g. Alexa488, PE) (scFv) dimers has also been reported. See Gruber et al., J. for use in multi-parametric analyses along with other signal Immunol. 152:5368 (1994). Alternatively, the antibodies can transduction (phospho-CrkL, phospho-Erk 1/2) and/or cell be “linear antibodies' as described in Zapata et al. Protein marker (CD34) antibodies. Eng.8(10): 1057-1062 (1995). Briefly, these antibodies com 0173 Phosphorylation site-specific antibodies of the prise a pair of tandem Fd segments (V-C1-V-Cl) which invention may specifically bind to a signaling protein or form a pair of antigen binding regions. Linear antibodies can polypeptide listed in Table 1 only when phosphorylated at the be bispecific or monospecific. US 2010/0129929 A1 May 27, 2010 39
0176) To produce the chimeric antibodies, the portions and eukaryotic expression systems, such as bacteria, yeast, derived from two different species (e.g., human constant insect cells, plant cells, mammalian cells (for example, NSO region and murine variable or binding region) can be joined cells). together chemically by conventional techniques or can be 0183) Once produced, the whole antibodies, their dimers, prepared as single contiguous proteins using genetic engi individual light and heavy chains, or other immunoglobulin neering techniques. The DNA molecules encoding the pro forms of the present application can be purified according to teins of both the light chain and heavy chain portions of the standard procedures of the art, including ammonium Sulfate chimeric antibody can be expressed as contiguous proteins. precipitation, affinity columns, column chromatography, gel The method of making chimeric antibodies is disclosed in electrophoresis and the like (see, generally, Scopes, R., Pro U.S. Pat. No. 5,677,427; U.S. Pat. No. 6,120,767; and U.S. tein Purification (Springer-Verlag, N.Y., 1982)). Once puri Pat. No. 6,329.508, each of which is incorporated by refer fied, partially or to homogeneity as desired, the polypeptides ence in its entirety. may then be used therapeutically (including extracorpore 0177. Fully human antibodies may be produced by a vari ally) or in developing and performing assay procedures, ety of techniques. One example is trioma methodology. The immunofluorescent staining, and the like. (See, generally, basic approach and an exemplary cell fusion partner, SPAZ-4. Immunological Methods, Vols. I and II (Lefkovits and Pernis, for use in this approach have been described by Oestberg et eds., Academic Press, NY, 1979 and 1981). al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. (0.184 6. Therapeutic Uses 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666 (each 0185. In a further aspect, the invention provides methods of which is incorporated by reference in its entirety). and compositions for therapeutic uses of the peptides or pro 0.178 Human antibodies can also be produced from non teins comprising a phosphorylation site of the invention, and human transgenic animals having transgenes encoding at phosphorylation site-specific antibodies of the invention. least a segment of the human immunoglobulin locus. The 0186. In one embodiment, the invention provides for a production and properties of animals having these properties method of treating or preventing carcinoma in a Subject, are described in detail by, see, e.g., Lonberg et al., WO93/ wherein the carcinoma is associated with the phosphorylation 12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et al., state of a novel phosphorylation site in Table 1, whether WO91/10741; U.S. Pat. No. 6,150,584, which are herein phosphorylated or dephosphorylated, comprising: adminis incorporated by reference in their entirety. tering to a subject in need thereof a therapeutically effective 0179 Various recombinant antibody library technologies amount of a peptide comprising a novel phosphorylation site may also be utilized to produce fully human antibodies. For (Table 1) and/or an antibody or antigen-binding fragment example, one approach is to screen a DNA library from thereof that specifically bind a novel phosphorylation site of human B cells according to the general protocol outlined by the invention (Table 1). The antibodies maybe full-length Huse et al., Science 246:1275-1281 (1989). The protocol antibodies, genetically engineered antibodies, antibody frag described by Huse is rendered more efficient in combination ments, and antibody conjugates of the invention. with phage-display technology. See, e.g., Dower et al., WO 0187. The term “subject” refers to a vertebrate, such as for 91/17271 and McCafferty et al., WO92/01047; U.S. Pat. No. example, a mammal, or a human. Although present applica 5.969,108, (each of which is incorporated by reference in its tion are primarily concerned with the treatment of human entirety). subjects, the disclosed methods may also be used for the 0180 Eukaryotic ribosome can also be used as means to treatment of other mammalian Subjects Such as dogs and cats display a library of antibodies and isolate the binding human for veterinary purposes. antibodies by Screening against the target antigen, as 0188 In one aspect, the disclosure provides a method of described in Coia G, et al., J. Immunol. Methods 1: 254 treating carcinoma in which a peptide or an antibody that (1-2):191-7 (2001); Hanes J. et al., Nat. Biotechnol. 18(12): reduces at least one biological activity of a targeted signaling 1287-92 (2000); Proc. Natl. Acad. Sci. U.S.A. protein is administered to a Subject. For example, the peptide 95(24): 14130-5 (1998); Proc. Natl. Acad. Sci. U.S.A. 94(10): or the antibody administered may disrupt or modulate the 4937-42 (1997), each which is incorporated by reference in interaction of the target signaling protein with its ligand. its entirety. Alternatively, the peptide or the antibody may interfere with, 0181. The yeast system is also suitable for screening mam thereby reducing, the down-stream signal transduction of the malian cell-surface or secreted proteins. Such as antibodies. parent signaling protein. An antibody that specifically binds Antibody libraries may be displayed on the surface of yeast the novel tyrosine phosphorylation site only when the cells for the purpose of obtaining the human antibodies tyrosine is phosphorylated, and that does not substantially against a target antigen. This approach is described by Yeung, bind to the same sequence when the tyrosine is not phospho et al., Biotechnol. Prog. 18(2):212-20 (2002); Boeder, E.T., et rylated, thereby prevents downstream signal transduction al., Nat. Biotechnol. 15(6):553-7 (1997), each of which is triggered by a phospho-tyrosine. Alternatively, an antibody herein incorporated by reference in its entirety. Alternatively, that specifically binds the unphosphorylated target phospho human antibody libraries may be expressed intracellularly rylation site reduces the phosphorylation at that site and thus and screened via the yeast two-hybrid system reduces activation of the protein mediated by phosphoryla (WO0200729A2, which is incorporated by reference in its tion of that site. Similarly, an unphosphorylated peptide may entirety). compete with an endogenous phosphorylation site for same 0182 Recombinant DNA techniques can be used to pro kinases, thereby preventing or reducing the phosphorylation duce the recombinant phosphorylation site-specific antibod of the endogenous target protein. Alternatively, a peptide ies described herein, as well as the chimeric or humanized comprising a phosphorylated novel tyrosine site of the inven phosphorylation site-specific antibodies, or any other geneti tion but lacking the ability to trigger signal transduction may cally-altered antibodies and the fragments or conjugate competitively inhibit interaction of the endogenous protein thereof in any expression systems including both prokaryotic with the same down-stream ligand(s). US 2010/0129929 A1 May 27, 2010 40
0189 The antibodies of the invention may also be used to IgG2/4 fusion domain. Other ways of eliminating effector target cancer cells for effector-mediated cell death. The anti function can be envisioned such as, e.g., mutation of the sites body disclosed herein may be administered as a fusion mol known to interact with FcR or insertion of a peptide in the ecule that includes a phosphorylation site-targeting portion hinge region, thereby eliminating critical sites required for joined to a cytotoxic moiety to directly kill cancer cells. FcR interaction. Variant antibodies with reduced or no effec Alternatively, the antibody may directly kill the cancer cells tor function also include variants as described previously through complement-mediated or antibody-dependent cellu herein. lar cytotoxicity. 0196. The peptides and antibodies of the invention may be 0190. Accordingly in one embodiment, the antibodies of used in combination with other therapies or with other agents. the present disclosure may be used to deliver a variety of Other agents include but are not limited to polypeptides, cytotoxic compounds. Any cytotoxic compound can be fused Small molecules, chemicals, metals, organometallic com to the present antibodies. The fusion can be achieved chemi pounds, inorganic compounds, nucleic acid molecules, oli cally or genetically (e.g., via expression as a single, fused gonucleotides, aptamers, Spiegelmers, antisense nucleic molecule). The cytotoxic compound can be a biological. Such acids, locked nucleic acid (LNA) inhibitors, peptide nucleic as a polypeptide, or a small molecule. As those skilled in the acid (PNA) inhibitors, immunomodulatory agents, antigen art will appreciate, for Small molecules, chemical fusion is binding fragments, prodrugs, and peptidomimetic com used, while for biological compounds, either chemical or pounds. In certain embodiments, the antibodies and peptides genetic fusion can be used. of the invention may be used in combination with cancer 0191 Non-limiting examples of cytotoxic compounds therapies known to one of skill in the art. include therapeutic drugs, radiotherapeutic agents, ribosome 0.197 In certain aspects, the present disclosure relates to inactivating proteins (RIPs), chemotherapeutic agents, toxic combination treatments comprising a phosphorylation site peptides, toxic proteins, and mixtures thereof. The cytotoxic specific antibody described herein and immunomodulatory drugs can be intracellularly acting cytotoxic drugs, such as compounds, vaccines or chemotherapy. Illustrative examples short-range radiation emitters, including, for example, short of Suitable immunomodulatory agents that may be used in range, high-energy C.-emitters. Enzymatically active toxins Such combination therapies include agents that block nega and fragments thereof, including ribosome-inactivating pro tive regulation of T cells or antigen presenting cells (e.g., teins, are exemplified by Saporin, luffin, momordins, ricin, anti-CTLA4 antibodies, anti-PD-L1 antibodies, anti-PDL-2 trichosanthin, gelonin, abrin, etc. Procedures for preparing antibodies, anti-PD-1 antibodies and the like) or agents that enzymatically active polypeptides of the immunotoxins are enhance positive co-stimulation of T cells (e.g., anti-CD40 described in WO84/03508 and WO85/03508, which are antibodies or anti 4-1BB antibodies) or agents that increase hereby incorporated by reference. Certain cytotoxic moieties NK cell number or T-cell activity (e.g., inhibitors such as are derived from adriamycin, chlorambucil, daunomycin, IMiDs, thalidomide, or thalidomide analogs). Furthermore, methotrexate, neocarzinostatin, and platinum, for example. immunomodulatory therapy could include cancer vaccines 0.192 Exemplary chemotherapeutic agents that may be Such as dendritic cells loaded with tumor cells, proteins, attached to an antibody or antigen-binding fragment thereof peptides, RNA, or DNA derived from such cells, patient include taxol, doxorubicin, Verapamil, podophyllotoxin, pro derived heat-shock proteins (hsp’s) or general adjuvants carbazine, mechlorethamine, cyclophosphamide, camptoth stimulating the immune system at various levels such as CpG. ecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitro Luivac(R), BiostimR), Ribomunyl(R), Imudon(R), Broncho Surea, dactinomycin, daunorubicin, doxorubicin, bleomycin, Vaxom R) or any other compound or other adjuvant activating plicomycin, mitomycin, etoposide (VP16), tamoxifen, trans receptors of the innate immune system (e.g., toll like receptor platinum, 5-fluorouracil, Vincristin, vinblastin, or methotrex agonist, anti-CTLA-4 antibodies, etc.). Also, immunomodu ate latory therapy could include treatment with cytokines such as 0193 Procedures for conjugating the antibodies with the IL-2, GM-CSF and IFN-gamma. cytotoxic agents have been previously described and are 0198 Furthermore, combination of antibody therapy with within the purview of one skilled in the art. chemotherapeutics could be particularly useful to reduce 0194 Alternatively, the antibody can be coupled to high overall tumor burden, to limitangiogenesis, to enhance tumor energy radiation emitters, for example, a radioisotope, Such accessibility, to enhance susceptibility to ADCC, to result in as ''I, a Y-emitter, which, when localized at the tumor site, increased immune function by providing more tumor antigen, results in a killing of several cell diameters. See, e.g., S. E. or to increase the expression of the T cell attractant LIGHT. Order, “Analysis, Results, and Future Prospective of the 0199 Pharmaceutical compounds that may be used for Therapeutic Use of Radiolabeled Antibody in Cancer combinatory anti-tumor therapy include, merely to illustrate: Therapy”, Monoclonal Antibodies for Cancer Detection and aminoglutethimide, amsacrine, anastrozole, asparaginase, Therapy, Baldwin et al. (eds.), pp. 303-316 (Academic Press bcg, bicalutamide, bleomycin, buserelin, buSulfan, camptoth 1985), which is hereby incorporated by reference. Other suit ecin, capecitabine, carboplatin, carmustine, chlorambucil, able radioisotopes include C-emitters, such as 'Bi, 'Bi, cisplatin, cladribine, clodronate, colchicine, cyclophospha and ''At, and B-emitters, such as 'Re and 'Y. mide, cyproterone, cytarabine, dacarbazine, dactinomycin, 0.195 Because many of the signaling proteins in which daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxo novel tyrosine phosphorylation sites of the invention occur rubicin, epirubicin, estradiol, estramustine, etoposide, also are expressed in normal cells and tissues, it may also be exemestane, filgrastim, fludarabine, fludrocortisone, fluorou advantageous to administer a phosphorylation site-specific racil, fluoxymesterone, flutamide, gemcitabine, genistein, antibody with a constant region modified to reduce or elimi goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, nate ADCC or CDC to limit damage to normal cells. For interferon, irinotecan, letrozole, leucovorin, leuprolide, example, effector function of an antibodies may be reduced or levamisole, lomustine, mechlorethamine, medroxyprogester eliminated by utilizing an IgG1 constant domain instead of an one, megestrol, melphalan, mercaptopurine, mesna, methotr US 2010/0129929 A1 May 27, 2010 41 exate, mitomycin, mitotane, mitoxantrone, nilutamide, corticosteroids (cortisone, dexamethasone, hydrocortisone, nocodazole, octreotide, Oxaliplatin, paclitaxel, pamidronate, methylprednisolone, prednisone, and prenisolone); growth pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, factor signal transduction kinase inhibitors; mitochondrial rituximab, Streptozocin, Suramin, tamoxifen, temozolomide, dysfunction inducers and caspase activators; and chromatin teniposide, testosterone, thioguanine, thiotepa, titanocene disruptors. dichloride, topotecan, trastuzumab, tretinoin, vinblastine, 0201 In certain embodiments, pharmaceutical com Vincristine, vindesine, and vinorelbine. pounds that may be used for combinatory anti-angiogenesis 0200. These chemotherapeutic anti-tumor compounds therapy include: (1) inhibitors of release of “angiogenic mol may be categorized by their mechanism of action into groups, ecules, such as bFGF (basic fibroblast growth factor); (2) including, for example, the following classes of agents: anti neutralizers of angiogenic molecules, such as anti-BbFGF metabolites/anti-cancer agents, such as pyrimidine analogs antibodies; and (3) inhibitors of endothelial cell response to (5-fluorouracil, floXuridine, capecitabine, gemcitabine and angiogenic stimuli, including collagenase inhibitor, base cytarabine) and purine analogs, folate inhibitors and related ment membrane turnover inhibitors, angiostatic steroids, fun inhibitors (mercaptopurine, thioguanine, pentostatin and gal-derived angiogenesis inhibitors, platelet factor 4, throm 2-chlorodeoxyadenosine (cladribine)); antiproliferative/anti bospondin, arthritis drugs such as D-penicillamine and gold mitotic agents including natural products such as Vinca alka thiomalate, vitamin D analogs, alpha-interferon, and the loids (vinblastine, Vincristine, and vinorelbine), microtubule like. For additional proposed inhibitors of angiogenesis, see disruptors such as taxane (paclitaxel, docetaxel), Vincristine, Blood et al., Biochim. Biophys. Acta, 1032:89-118 (1990), vinblastine, nocodazole, epothilones and navelbine, epidi Moses et al., Science, 248: 1408-1410 (1990), Ingber et al., podophyllotoxins (etoposide, teniposide), DNA damaging Lab. Invest., 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885, agents (actinomycin, amsacrine, anthracyclines, bleomycin, 5,112,946, 5,192,744, 5,202,352, and 6,573.256. In addition, buSulfan, camptothecin, carboplatin, chlorambucil, cisplatin, there are a wide variety of compounds that can be used to cyclophosphamide, cytoxan, dactinomycin, daunorubicin, inhibit angiogenesis, for example, peptides or agents that doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, block the VEGF-mediated angiogenesis pathway, endostatin iphosphamide, melphalan, mechlorethamine, mitomycin, protein or derivatives, lysine binding fragments of angiosta mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol. tin, melanin or melanin-promoting compounds, plasminogen taxotere, teniposide, triethylenethiophosphoramide and eto fragments (e.g., Kringles 1-3 of plasminogen), troponin Sub poside (VP16)); antibiotics Such as dactinomycin (actinomy units, inhibitors of vitronectin CfB, peptides derived from cin D), daunorubicin, doxorubicin (adriamycin), idarubicin, Saposin B, antibiotics or analogs (e.g., tetracycline or neo anthracyclines, mitoxantrone, bleomycins, plicamycin (mith mycin), dienogest-containing compositions, compounds ramycin) and mitomycin; enzymes (L-asparaginase which comprising a MetAP-2 inhibitory core coupled to a peptide, systemically metabolizes L-asparagine and deprives cells the compound EM-138, chalcone and its analogs, and naala which do not have the capacity to synthesize their own aspar dase inhibitors. See, for example, U.S. Pat. Nos. 6,395,718, agine); antiplatelet agents; antiproliferative/antimitotic alky 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810, lating agents such as nitrogen mustards (mechlorethamine, 6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, cyclophosphamide and analogs, melphalan, chlorambucil), 6,538,103, 6,544,758, 6,544,947, 6,548,477, 6,559,126, and ethylenimines and methylmelamines (hexamethylmelamine 6,569,845. and thiotepa), alkyl Sulfonates-buSulfan, nitrosoureas (car 0202 7. Diagnostic Uses mustine (BCNU) and analogs, streptozocin), traZenes-dacar 0203. In a further aspect, the invention provides methods bazinine (DTIC); antiproliferative/antimitotic antimetabo for detecting and quantitating phosphoyrlation at a novel lites Such as folic acid analogs (methotrexate); platinum tyrosine phosphorylation site of the invention. For example, coordination complexes (cisplatin, carboplatin), procarba peptides, including AQUA peptides of the invention, and Zine, hydroxyurea, mitotane, aminoglutethimide; hormones, antibodies of the invention are useful in diagnostic and prog hormone analogs (estrogen, tamoxifen, goserelin, bicaluta nostic evaluation of carcinomas, wherein the carcinoma is mide, nilutamide) and aromatase inhibitors (letrozole, anas associated with the phosphorylation state of a novel phospho trozole); anticoagulants (heparin, synthetic heparin salts and rylation site in Table 1, whether phosphorylated or dephos other inhibitors of thrombin); fibrinolytic agents (such as phorylated. tissue plasminogen activator, Streptokinase and urokinase), 0204 Methods of diagnosis can be performed in vitro aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; using a biological sample (e.g., blood sample, lymph node antimigratory agents; antisecretory agents (breveldin); biopsy or tissue) from a subject, or in vivo. The phosphory immunosuppressives (cyclosporine, tacrolimus (FK-506), lation state or level at the tyrosine residue identified in the Sirolimus (rapamycin), azathioprine, mycophenolate corresponding row in Column D of Table 1 may be assessed. mofetil); immunomodulatory agents (thalidomide and ana A change in the phosphorylation state or level at the phos logs thereof such as lenalidomide (Revlimid, CC-5013) and phorylation site, as compared to a control, indicates that the CC-4047 (Actimid)), cyclophosphamide; anti-angiogenic Subject is Suffering from, or Susceptible to, carcinoma. compounds (TNP-470, genistein) and growth factor inhibi 0205. In one embodiment, the phosphorylation state or tors (vascular endothelial growth factor (VEGF) inhibitors, level at a novel phosphorylation site is determined by an fibroblast growth factor (FGF) inhibitors); angiotensin recep AQUA peptide comprising the phosphorylation site. The tor blocker, nitric oxide donors; anti-sense oligonucleotides; AQUA peptide may be phosphorylated or unphosphorylated antibodies (trastuzumab); cell cycle inhibitors and differen at the specified tyrosine position. tiation inducers (tretinoin); mTOR inhibitors, topoisomerase 0206. In another embodiment, the phosphorylation state inhibitors (doxorubicin (adriamycin), amsacrine, camptoth or levelata phosphorylation site is determined by an antibody ecin, daunorubicin, dactinomycin, eniposide, epirubicin, eto or antigen-binding fragment thereof, wherein the antibody poside, idarubicin and mitoxantrone, topotecan, irinotecan), specifically binds the phosphorylation site. The antibody may US 2010/0129929 A1 May 27, 2010 42 be one that only binds to the phosphorylation site when the analyzed by flow cytometry for target signaling protein phos tyrosine residue is phosphorylated, but does not bind to the phorylation, as well as for markers identifying various same sequence when the tyrosine is not phosphorylated; or hematopoietic cell types. In this manner, activation status of Vice versa. the malignant cells may be specifically characterized. Flow 0207. In particular embodiments, the antibodies of the cytometry may be carried out according to standard methods. present application are attached to labeling moieties, such as See, e.g., Chow et al., Cytometry (Communications in Clini a detectable marker. One or more detectable labels can be cal Cytometry) 46: 72-78 (2001). attached to the antibodies. Exemplary labeling moieties include radiopaque dyes, radiocontrast agents, fluorescent 0213 Alternatively, antibodies of the invention may be molecules, spin-labeled molecules, enzymes, or other label used in immunohistochemical (IHC) staining to detect differ ing moieties of diagnostic value, particularly in radiologic or ences in signal transduction or protein activity using normal magnetic resonance imaging techniques. and diseased tissues. IHC may be carried out according to 0208. A radiolabeled antibody in accordance with this well-known techniques. See, e.g., Antibodies: A Laboratory disclosure can be used for in vitro diagnostic tests. The spe Manual, Supra. cific activity of an antibody, binding portion thereof, probe, or 0214 Peptides and antibodies of the invention may be also ligand, depends upon the half-life, the isotopic purity of the be optimized for use in other clinically-suitable applications, radioactive label, and how the label is incorporated into the for example bead-based multiplex-type assays, Such as biological agent. In immunoassay tests, the higher the spe IGEN, LuminexTM and/or BioplexTMassay formats, or other cific activity, in general, the better the sensitivity. Radioiso wise optimized for antibody arrays formats, such as reversed topes useful as labels, e.g., for use in diagnostics, include phase array applications (see, e.g. Paweletz et al., Oncogene iodine ("'I or 'I), indium ('''In), technetium ('Tc), phos 20(16): 1981-89 (2001)). Accordingly, in another embodi phorus ('P), carbon (''C), and tritium (H), or one of the ment, the invention provides a method for the multiplex therapeutic isotopes listed above. detection of the phosphorylation state or level at two or more 0209 Fluorophore and chromophore labeled biological phosphorylation sites of the invention (Table 1) in a biological agents can be prepared from standard moieties known in the sample, the method comprising utilizing two or more anti art. Since antibodies and other proteins absorb light having bodies or AQUA peptides of the invention. In one preferred wavelengths up to about 310 nm, the fluorescent moieties embodiment, two to five antibodies or AQUA peptides of the may be selected to have substantial absorption at wavelengths invention are used. In another preferred embodiment, six to above 310 nm, such as for example, above 400 nm. A variety ten antibodies or AQUA peptides of the invention are used, of suitable fluorescers and chromophores are described by while in another preferred embodiment eleven to twenty anti Stryer, Science, 162:526 (1968) and Brand et al., Annual bodies or AQUA peptides of the invention are used. Review of Biochemistry, 41:843-868 (1972), which are 0215. In certain embodiments the diagnostic methods of hereby incorporated by reference. The antibodies can be the application may be used in combination with other cancer labeled with fluorescent chromophore groups by conven diagnostic tests. tional procedures such as those disclosed in U.S. Pat. Nos. 0216. The biological sample analyzed may be any sample 3,940,475, 4,289.747, and 4,376,110, which are hereby incor that is Suspected of having abnormal tyrosine phosphoryla porated by reference. tion at a novel phosphorylation site of the invention, Such as a 0210. The control may be parallel samples providing a homogenized neoplastic tissue sample. basis for comparison, for example, biological samples drawn from a healthy Subject, or biological samples drawn from 0217 8. Screening assays healthy tissues of the same subject. Alternatively, the control 0218. In another aspect, the invention provides a method may be a pre-determined reference or threshold amount. If the for identifying an agent that modulates tyrosine phosphory Subject is being treated with a therapeutic agent, and the lation at a novel phosphorylation site of the invention, com progress of the treatment is monitored by detecting the prising: a) contacting a candidate agent with a peptide or tyrosine phosphorylation state level at a phosphorylation site protein comprising a novel phosphorylation site of the inven of the invention, a control may be derived from biological tion; and b) determining the phosphorylation state or level at samples drawn from the Subject prior to, or during the course the novel phosphorylation site. A change in the phosphoryla of the treatment. tion level of the specified tyrosine in the presence of the test 0211. In certain embodiments, antibody conjugates for agent, as compared to a control, indicates that the candidate diagnostic use in the present application are intended for use agent potentially modulates tyrosine phosphorylation at a in vitro, where the antibody is linked to a secondary binding novel phosphorylation site of the invention. ligand or to an enzyme (an enzyme tag) that will generate a 0219. In one embodiment, the phosphorylation state or colored product upon contact with a chromogenic Substrate. level at a novel phosphorylation site is determined by an Examples of Suitable enzymes include urease, alkaline phos AQUA peptide comprising the phosphorylation site. The phatase, (horseradish) hydrogen peroxidase and glucose oxi AQUA peptide may be phosphorylated or unphosphorylated dase. In certain embodiments, secondary binding ligands are at the specified tyrosine position. biotin and avidin or Streptavidin compounds. 0220. In another embodiment, the phosphorylation state 0212 Antibodies of the invention may also be optimized or levelata phosphorylation site is determined by an antibody for use in a flow cytometry (FC) assay to determine the or antigen-binding fragment thereof, wherein the antibody activation/phosphorylation status of a target signaling protein specifically binds the phosphorylation site. The antibody may in Subjects before, during, and after treatment with a thera be one that only binds to the phosphorylation site when the peutic agent targeted at inhibiting tyrosine phosphorylation at tyrosine residue is phosphorylated, but does not bind to the the phosphorylation site disclosed herein. For example, bone same sequence when the tyrosine is not phosphorylated; or marrow cells or peripheral blood cells from patients may be Vice versa. US 2010/0129929 A1 May 27, 2010
0221. In particular embodiments, the antibodies of the 0229. The antibody used in the detection may itself be present application are attached to labeling moieties, such as conjugated to a detectable label, wherein one would then a detectable marker. simply detect this label. The amount of the primary immune 0222. The control may be parallel samples providing a complexes in the composition would, thereby, be determined. basis for comparison, for example, the phosphorylation level 0230. Alternatively, the first antibody that becomes bound of the target protein or peptide in absence of the testing agent. within the primary immune complexes may be detected by Alternatively, the control may be a pre-determined reference means of a second binding ligand that has binding affinity for or threshold amount. the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself 9. Immunoassays often an antibody, which may thus be termed a “secondary' antibody. The primary immune complexes are contacted with 0223. In another aspect, the present application concerns the labeled, secondary binding ligand, or antibody, under immunoassays for binding, purifying, quantifying and other conditions effective and for a period of time sufficient to allow wise generally detecting the phosphorylation state or level at the formation of secondary immune complexes. The second a novel phosphorylation site of the invention. ary immune complexes are washed extensively to remove any 0224 Assays may be homogeneous assays or heteroge non-specifically bound labeled secondary antibodies or neous assays. In a homogeneous assay the immunological ligands, and the remaining label in the secondary immune reaction usually involves a phosphorylation site-specific anti complex is detected. body of the invention, a labeled analyte, and the sample of 0231. An enzyme linked immunoadsorbent assay interest. The signal arising from the label is modified, directly (ELISA) is a type of binding assay. In one type of ELISA, or indirectly, upon the binding of the antibody to the labeled phosphorylation site-specific antibodies disclosed herein are analyte. Both the immunological reaction and detection of the immobilized onto a selected surface exhibiting protein affin extent thereof are carried out in a homogeneous solution. ity, Such as a well in a polystyrene microtiter plate. Then, a Immunochemical labels that may be used include free radi Suspected neoplastic tissue sample is added to the wells. After cals, radioisotopes, fluorescent dyes, enzymes, bacterioph binding and washing to remove non-specifically bound ages, coenzymes, and so forth. immune complexes, the bound target signaling protein may 0225. In a heterogeneous assay approach, the reagents are be detected. usually the specimen, a phosphorylation site-specific anti 0232. In another type of ELISA, the neoplastic tissue body of the invention, and suitable means for producing a samples are immobilized onto the well surface and then con detectable signal. Similar specimens as described above may tacted with the phosphorylation site-specific antibodies dis be used. The antibody is generally immobilized on a Support, closed herein. After binding and washing to remove non Such as a bead, plate or slide, and contacted with the specimen specifically bound immune complexes, the bound Suspected of containing the antigen in a liquid phase. The phosphorylation site-specific antibodies are detected. Support is then separated from the liquid phase and either the 0233. Irrespective of the format used, ELISAs have certain Support phase or the liquid phase is examined for a detectable features in common, such as coating, incubating or binding, signal using means for producing Such signal. The signal is washing to remove non-specifically bound species, and related to the presence of the analyte in the specimen. Means detecting the bound immune complexes. for producing a detectable signal include the use of radioac 0234. The radioimmunoassay (RIA) is an analytical tech tive labels, fluorescent labels, enzyme labels, and so forth. nique which depends on the competition (affinity) of an anti 0226 Phosphorylation site-specific antibodies disclosed gen for antigen-binding sites on antibody molecules. Stan herein may be conjugated to a solid Support Suitable for a dard curves are constructed from data gathered from a series diagnostic assay (e.g., beads, plates, slides or wells formed of samples each containing the same known concentration of from materials such as latex or polystyrene) in accordance labeled antigen, and various, but known, concentrations of with known techniques, such as precipitation. unlabeled antigen. Antigens are labeled with a radioactive 0227. In certain embodiments, immunoassays are the vari isotope tracer. The mixture is incubated in contact with an ous types of enzyme linked immunoadsorbent assays (ELI antibody. Then the free antigen is separated from the antibody SAS) and radioimmunoassays (RIA) known in the art. Immu and the antigen bound thereto. Then, by use of a suitable nohistochemical detection using tissue sections is also detector, such as a gamma or beta radiation detector, the particularly useful. However, it will be readily appreciated percent of either the bound or free labeled antigen or both is that detection is not limited to such techniques, and Western determined. This procedure is repeated for a number of blotting, dot and slot blotting, FACS analyses, and the like samples containing various known concentrations of unla may also be used. The steps of various useful immunoassays beled antigens and the results are plotted as a standard graph. have been described in the scientific literature. Such as, e.g., The percent of boundtracer antigens is plotted as a function of Nakamura et al., in Enzyme Immunoassays: Heterogeneous the antigen concentration. Typically, as the total antigen con and Homogeneous Systems, Chapter 27 (1987), incorporated centration increases the relative amount of the tracer antigen herein by reference. bound to the antibody decreases. After the standard graph is 0228. In general, the detection of immunocomplex forma prepared, it is thereafter used to determine the concentration tion is well known in the art and may beachieved through the of antigen in samples undergoing analysis. application of numerous approaches. These methods are 0235. In an analysis, the sample in which the concentra based upon the detection of radioactive, fluorescent, biologi tion of antigen is to be determined is mixed with a known cal or enzymatic tags. Of course, one may find additional amount of tracer antigen. Tracer antigen is the same antigen advantages through the use of a secondary binding ligand known to be in the sample but which has been labeled with a Such as a second antibody or a biotin/avidin ligand binding suitable radioactive isotope. The sample with tracer is then arrangement, as is known in the art. incubated in contact with the antibody. Then it can be counted US 2010/0129929 A1 May 27, 2010 44 in a suitable detector which counts the free antigen remaining istration, e.g., oral (p.o.), may be used if desired and practi in the sample. The antigen bound to the antibody or immu cable for the particular antibody to be administered. An anti noadsorbent may also be similarly counted. Then, from the body can also be administered in a variety of unit dosage standard curve, the concentration of antigen in the original forms and their dosages will also vary with the size, potency, sample is determined. and in vivo half-life of the particular antibody being admin 0236 10. Pharmaceutical Formulations and Methods of istered. Doses of a phosphorylation site-specific antibody Administration will also vary depending on the manner of administration, the 0237 Methods of administration of therapeutic agents, particular symptoms of the patient being treated, the overall particularly peptide and antibody therapeutics, are well health, condition, size, and age of the patient, and the judg known to those of skill in the art. ment of the prescribing physician. 0238 Peptides of the invention can be administered in the same manner as conventional peptide type pharmaceuticals. 0243 The frequency of administration may also be Preferably, peptides are administered parenterally, for adjusted according to various parameters. These include the example, intravenously, intramuscularly, intraperitoneally, or clinical response, the plasma half-life of the antibody, and the subcutaneously. When administered orally, peptides may be levels of the antibody in a body fluid, such as, blood, plasma, proteolytically hydrolyzed. Therefore, oral application may serum, or synovial fluid. To guide adjustment of the frequency not be usually effective. However, peptides can be adminis of administration, levels of the antibody in the body fluid may tered orally as a formulation wherein peptides are not easily be monitored during the course of treatment. hydrolyzed in a digestive tract, such as liposome-microcap 0244. Formulations particularly useful for antibody-based Sules. Peptides may be also administered in Suppositories, therapeutic agents are also described in U.S. Patent App. Sublingual tablets, or intranasal spray. Publication Nos. 20030202972, 20040091490 and 0239. If administered parenterally, a preferred pharma 20050158316. In certain embodiments, the liquid formula ceutical composition is an aqueous Solution that, in addition tions of the application are substantially free of surfactant to a peptide of the invention as an active ingredient, may and/or inorganic salts. In another specific embodiment, the contain for example, buffers such as phosphate, acetate, etc., liquid formulations have a pH ranging from about 5.0 to about osmotic pressure-adjusting agents such as Sodium chloride, 7.0. In yet another specific embodiment, the liquid formula Sucrose, and Sorbitol, etc., antioxidative or antioxygenic tions comprise histidine at a concentration ranging from agents, such as ascorbic acid or tocopherol and preservatives, about 1 mM to about 100 mM. In still another specific such as antibiotics. The parenterally administered composi embodiment, the liquid formulations comprise histidine at a tion also may be a solution readily usable or in a lyophilized concentration ranging from 1 mM to 100 mM. It is also form which is dissolved in sterile water before administra contemplated that the liquid formulations may further com tion. prise one or more excipients such as a saccharide, an amino 0240. The pharmaceutical formulations, dosage forms, acid (e.g., arginine, lysine, and methionine) and a polyol. and uses described below generally apply to antibody-based Additional descriptions and methods of preparing and ana therapeutic agents, but are also useful and can be modified, lyzing liquid formulations can be found, for example, in PCT where necessary, for making and using therapeutic agents of publications WO 03/106644, WO 04/066957, and WO the disclosure that are not antibodies. O4/09 1658. 0241. To achieve the desired therapeutic effect, the phos 0245 Wetting agents, emulsifiers and lubricants, such as phorylation site-specific antibodies or antigen-binding frag Sodium lauryl Sulfate and magnesium Stearate, as well as ments thereof can be administered in a variety of unit dosage coloring agents, release agents, coating agents, Sweetening, forms. The dose will vary according to the particular anti flavoring and perfuming agents, preservatives and antioxi body. For example, different antibodies may have different dants can also be present in the pharmaceutical compositions masses and/or affinities, and thus require different dosage of the application. levels. Antibodies prepared as Fab or other fragments will 0246. In certain embodiments, formulations of the subject also require differing dosages than the equivalent intact antibodies are pyrogen-free formulations which are substan immunoglobulins, as they are of considerably smaller mass tially free of endotoxins and/or related pyrogenic Substances. than intact immunoglobulins, and thus require lower dosages Endotoxins include toxins that are confined inside microor to reach the same molar levels in the patient's blood. The dose ganisms and are released when the microorganisms are bro will also vary depending on the manner of administration, the ken down or die. Pyrogenic Substances also include fever particular symptoms of the patient being treated, the overall inducing, thermostable Substances (glycoproteins) from the health, condition, size, and age of the patient, and the judg outer membrane of bacteria and other microorganisms. Both ment of the prescribing physician. Dosage levels of the anti of these substances can cause fever, hypotension and shock if bodies for human Subjects are generally between about 1 mg administered to humans. Due to the potential harmful effects, per kg and about 100 mg per kg per patient per treatment, Such it is advantageous to remove even low amounts of endotoxins as for example, between about 5 mg per kg and about 50 mg from intravenously administered pharmaceutical drug solu per kg per patient per treatment. In terms of plasma concen tions. The Food & Drug Administration (“FDA) has set an trations, the antibody concentrations may be in the range from upper limit of 5 endotoxin units (EU) per dose per kilogram about 25 ug/mL to about 500 lug/mL. However, greater body weight in a single one hour period for intravenous drug amounts may be required for extreme cases and Smaller applications (The United States Pharmacopeial Convention, amounts may be sufficient for milder cases. Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic 0242 Administration of an antibody will generally be per proteins are administered in amounts of several hundred or formed by a parenteral route, typically via injection Such as thousand milligrams per kilogram body weight, as can be the intra-articular or intravascular injection (e.g., intravenous case with monoclonal antibodies, it is advantageous to infusion) or intramuscular injection. Other routes of admin remove even trace amounts of endotoxin. US 2010/0129929 A1 May 27, 2010
0247 The amount of the formulation which will be thera invention encompasses modifications and variations of the peutically effective can be determined by standard clinical methods taught herein which would be obvious to one of techniques. In addition, in vitro assays may optionally be ordinary skill in the art. used to help identify optimal dosage ranges. The precise dose to be used in the formulation will also depend on the route of Example 1 administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the prac Isolation of Phosphotyrosine-Containing Peptides titioner and each patient's circumstances. Effective doses from Extracts of Carcinoma Cell Lines and Identifi may be extrapolated from dose-response curves derived from cation of Novel Phosphorylation Sites in vitro or animal model test systems. The dosage of the 0253) In order to discover novel tyrosine phosphorylation compositions to be administered can be determined by the sites in carcinoma, IAP isolation techniques were used to skilled artisan without undue experimentation in conjunction identify phosphotyrosine-containing peptides in cell extracts with Standard dose-response studies. Relevant circumstances from human carcinoma cell lines and patient cell lines iden to be considered in making those determinations include the tified in Column G of Table 1 including 293T, 293T TAT, condition or conditions to be treated, the choice of composi 293T-ZNF198/FGFR, 3T3-EGFR(L858R), 3T3-EGFR(del), tion to be administered, the age, weight, and response of the 3T3-EGFRwt, A 431, A172, A549, A549 tumor, AML-4833, individual patient, and the severity of the patient's symptoms. AML-6246, AML-6735, AML-7592, BaF3-FLT3(WT), For example, the actual patient body weight may be used to BxPC-3, CCF-STTG1 CHRF, CI-1, CTV-1, Calu-3, calculate the dose of the formulations in milliliters (mL) to be DBTRG-05MG, DMS 153, DMS 53, DMS 79, DU-528, administered. There may be no downward adjustment to DU145, GAMG, GMS-10, H1299, H1373, H1437, H1563, “ideal weight. In Such a situation, an appropriate dose may H1568, H1648, H1650, H1650 XG, H1666, H1693, H1703, be calculated by the following formula: H1734, H1793, H1869, H1944, H1975, H1993, H2023, Dose (mL)=patient weight (kg)xdose level (mg/kg). H2030, H2170, H2172, H2286, H2347, H3255, H358, H460, drug concentration (mg/mL) H520, H524, H526, H661, H810, H82, H838, HCC1395, 0248 For the purpose of treatment of disease, the appro HCC1428, HCC1435, HCC1806, HCC1937, HCC366, priate dosage of the compounds (for example, antibodies) HCC44, HCC78, HCC827, HCT 116, HCT116, HER4-JMb, will depend on the severity and course of disease, the patient’s HL107B, HL116B, HL117A, HL117B, HL129A, HL130A, clinical history and response, the toxicity of the antibodies, HL131A, HL131B, HL132A, HL132B, HL133A, HL1881, and the discretion of the attending physician. The initial can HL25A, HL41A, HL53A, HL53B, HL55B, HL59A, HL59b, didate dosage may be administered to a patient. The proper HL61a, HL61b, HL66A, HL66B, HL75A, HL79B, HL83A, dosage and treatment regimen can be established by monitor HL84A, HL84B, HL87A, HL87B, HL92B, HL97A, HL98A, ing the progress of therapy using conventional techniques HT29, HUVEC, HeLa, Human lung tumor, Jurkat, K562, known to those of skill in the art. KG-1, KG1-A, KMS18, KY821, Karpas 299, Karpas-1 106p. 0249. The formulations of the application can be distrib LN18, LN229, LOU-NH91, M-07e, MO59.J, MO59K, uted as articles of manufacture comprising packaging mate MC-116, MCF-10A (Y561F), MCF-10A(Y969F), MCF7, rial and a pharmaceutical agent which comprises, e.g., the MDA-MB-435S, MDA-MB-453, MDA-MB-468, MDS antibody and a pharmaceutically acceptable carrier as appro 851, MKPL-1, ML-1, MO-91, MOLT15, MV4-11, Marimo, priate to the mode of administration. The packaging material Me-F2, Molm 14, NCI-H196, NCI-N87, Nomo-1, OCI-M1, will include a label which indicates that the formulation is for OCI/AML3, OPM-1, PT7-pancreatic tumor, Pfeiffer, RC use in the treatment of prostate cancer. K8, RI-1, RKO, RPMI8266, SCLC T1, SCLC T2, SEM, (0250) 11. Kits SH-SY5Y, SK-N-AS, SK-N-MC, SK-N-SH, SNB-19, SU 0251 Antibodies and peptides (including AQUA pep DHL1, SW1088, SW1783, SW620, Su.86.86, SuDHL5, T17, tides) of the invention may also be used within a kit for T98G, TS, U118 MG, UT-7, VACO432, VAL, Verona 2, detecting the phosphorylation state or level at a novel phos WSU-NHL, XG 1, XG2, XG5, cs001, cs012, cs015, cs019, phorylation site of the invention, comprising at least one of cs024, cs025, cs026, cs029, cs037, cs041, cs042, cs048, the following: an AQUA peptide comprising the phosphory cs057, cs068, cs069, cs070, gz21, gz33, gz42, gz47, gziš8, lation site, or an antibody or an antigen-binding fragment gz70, gz74, gz75, gzB1, h2228, hl 144a, hl 144b, hl145a, thereofthat binds to an amino acid sequence comprising the h1145b, hl146b, hl 148a, hl148b, hl152a, hl 152b, lung tumor phosphorylation site. Such a kit may further comprise a pack T26, lung tumor T57, normal human lung, pancreatic aged combination of reagents in predetermined amounts with Xenograft, SWA80. Tryptic phosphotyrosine-containing pep instructions for performing the diagnostic assay. Where the tides were purified and analyzed from extracts of each of the antibody is labeled with an enzyme, the kit will include sub cell lines mentioned above, as follows. Cells were cultured in strates and co-factors required by the enzyme. In addition, DMEM medium or RPMI 1640 medium supplemented with other additives may be included such as stabilizers, buffers 10% fetal bovine serum and penicillin/streptomycin. and the like. The relative amounts of the various reagents may 0254 Suspension cells were harvested by low speed cen be varied widely to provide for concentrations in solution of trifugation. After complete aspiration of medium, cells were the reagents that substantially optimize the sensitivity of the resuspended in 1 mL lysis buffer per 1.25x10 cells (20 mM assay. Particularly, the reagents may be provided as dry pow HEPES pH 8.0, 9 Murea, 1 mM sodium vanadate, supple ders, usually lyophilized, including excipients that, on disso mented or not with 2.5 mM sodium pyro-phosphate, 1 mM lution, will provide a reagent solution having the appropriate B-glycerol-phosphate) and Sonicated. concentration. (0255. Adherent cells at about 80% confluency were 0252. The following Examples are provided only to fur starved in medium without serum overnight and stimulated, ther illustrate the invention, and are not intended to limit its with ligand depending on the cell type or not stimulated. After Scope, except as provided in the claims appended hereto. The complete aspiration of medium from the plates, cells were US 2010/0129929 A1 May 27, 2010 46 scraped off the plate in 10 ml lysis buffer per 2x10 cells (20 min (eluate 1), followed by a wash of the beads (eluate 2) with mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, 45ul of 0.15% TFA. Both eluates were combined. supplemented with 2.5 mM sodium pyrophosphate, 1 mM B-glycerol-phosphate) and Sonicated. Analysis by LC-MS/MS Mass Spectrometry. 0256 Frozen tissue samples were cut to small pieces, 0261 40 ul or more of IAP eluate were purified by 0.2 ul homogenize in lysis buffer (20 mMHEPES pH 8.0,9 MUrea, StageTips or ZipTips. Peptides were eluted from the micro 1 mN sodium vanadate, supplemented with 2.5 mM sodium columns with 1 ul of 40% MeCN, 0.1%TFA (fractions I and pyrophosphate, 1 mMb-glycerol-phosphate, 1 ml lysis buffer II) or 1 ul of 60% MeCN, 0.1%TFA (fraction III) into 7.6-9.0 for 100 mg of frozen tissue) using a polytron for 2 times of 20 ul of 0.4% acetic acid/0.005% heptafluorobutyric acid. For Sec. each time. Homogenate is then briefly Sonicated. single fraction analysis, 1 ul of 60% MeCN, 0.1% TFA, was 0257 Sonicated cell lysates were cleared by centrifuga used for elution from the microcolumns. This sample was loaded onto a 10 cmx75um PicoFrit capillary column (New tion at 20,000xg, and proteins were reduced with DTT at a Objective) packed with Magic C18 AQ reversed-phase resin final concentration of 4.1 mM and alkylated with iodoaceta (Michrom Bioresources) using a Famos autosampler with an mide at 8.3 mM. For digestion with trypsin, protein extracts inert sample injection valve (Dionex). The column was then were diluted in 20 mMHEPES pH 8.0 to a final concentration developed with a 45-minlinear gradient of acetonitrile deliv of 2 Murea and soluble TLCK-trypsin (Worthington) was ered at 200 ml/min (Ultimate, Dionex), and tandem mass added at 10-20 ug/mL. Digestion was performed for 1-2 days spectra were collected in a data-dependent manner with an at room temperature. LTQ ion trap mass spectrometer essentially as described by 0258 Trifluoroacetic acid (TFA) was added to protein Gygiet al., Supra. digests to a final concentration of 1%, precipitate was removed by centrifugation, and digests were loaded onto Database Analysis & Assignments. Sep-Pak Cs columns (Waters) equilibrated with 0.1% TFA. 0262 MS/MS spectra were evaluated using TurboSequest A column volume of 0.7-1.0 ml was used per 2x10 cells. in the Sequest Browser package (v. 27, rev. 12) Supplied as Columns were washed with 15 volumes of 0.1% TFA, fol part of BioWorks 3.0 (ThermoFinnigan). Individual MS/MS lowed by 4 volumes of 5% acetonitrile (MeCN) in 0.1%TFA. spectra were extracted from the raw data file using the Peptide fraction I was obtained by eluting columns with 2 Sequest Browser program CreateDta, with the following set volumes each of 8, 12, and 15% MeCN in 0.1% TFA and tings: bottom MW, 700; top MW, 4,500; minimum number of combining the eluates. Fractions II and III were a combina ions, 20 (40 for LTQ); minimum TIC, 4x10(2x10 for LTQ): tion of eluates after eluting columns with 18, 22, 25% MeCN and precursor charge state, unspecified. Spectra were in 0.1% TFA and with 30, 35, 40% MeCN in 0.1% TFA, extracted from the beginning of the raw data file before respectively. All peptide fractions were lyophilized. sample injection to the end of the eluting gradient. The Ion 0259 Peptides from each fraction corresponding to 2x10 Quest and Vulota programs were not used to further select cells were dissolved in 1 ml of IAP buffer (20 mM Tris/HClor MS/MS spectra for Sequest analysis. MS/MS spectra were 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM evaluated with the following TurboSequest parameters: pep NaCl) and insoluble matter (mainly in peptide fractions III) tide mass tolerance, 2.5; fragmention tolerance, 0.0 (1.0 for was removed by centrifugation. IAP was performed on each LTQ); maximum number of differential amino acids per peptide fraction separately. The phosphotyrosine monoclonal modification, 4, mass type parent, average; mass type frag antibody P-Tyr-100 (Cell Signaling Technology, Inc., catalog ment, average; maximum number of internal cleavage sites, 10; neutral losses of water and ammonia from b and y ions number 9411) was coupled at 4 mg/ml beads to protein G were considered in the correlation analysis. Proteolytic (Roche), respectively. Immobilized antibody (15 ul, 60 ug) enzyme was specified except for spectra collected from was added as 1:1 slurry in IAP buffer to 1 ml of each peptide elastase digests. fraction, and the mixture was incubated overnight at 4° C. 0263 Searches were performed against the NCBI human with gentle rotation. The immobilized antibody beads were protein database (NCBI RefSeq protein release #11; 8 May washed three times with 1 ml IAP buffer and twice with 1 ml 2005: 1,826,611 proteins, including 47.859 human proteins. water, all at 4°C. Peptides were eluted from beads by incu Peptides that did not match RefSeq were compared to NCBI bation with 75 ul of 0.1% TFA at room temperature for 10 GenPept release #148; 15 Jun. 2005 release date; 2,479,172 minutes. proteins, including 196,054 human proteins.). Cysteine car 0260 Alternatively, one single peptide fraction was boxamidomethylation was specified as a static modification, obtained from Sep-Pak C18 columns by elution with 2 vol and phosphorylation was allowed as a variable modification umes each of 10%, 15%, 20%, 25%, 30%, 35% and 40% on serine, threonine, and tyrosine residues or on tyrosine acetonitirile in 0.1% TFA and combination of all eluates. IAP residues alone. It was determined that restricting phosphory on this peptide fraction was performed as follows: After lyo lation to tyrosine residues had little effect on the number of philization, peptide was dissolved in 1.4 ml IAP buffer phosphorylation sites assigned. (MOPS pH 7.2, 10 mM sodium phosphate, 50 mMNaCl) and 0264. In proteomics research, it is desirable to validate insoluble matter was removed by centrifugation. Immobi protein identifications based solely on the observation of a lized antibody (40 ul, 160 g) was added as 1:1 slurry in IAP single peptide in one experimental result, in order to indicate buffer, and the mixture was incubated overnight at 4°C. with that the protein is, in fact, present in a sample. This has led to gentle shaking. The immobilized antibody beads were the development of statistical methods for validating peptide washed three times with 1 ml IAP buffer and twice with 1 ml assignments, which are not yet universally accepted, and water, all at 4°C. Peptides were eluted from beads by incu guidelines for the publication of protein and peptide identifi bation with 55 ul of 0.15% TFA at room temperature for 10 cation results (see Canet al., Mol. Cell Proteomics 3: 531-533 US 2010/0129929 A1 May 27, 2010 47
(2004)), which were followed in this Example. However, animal to raise antibodies against the antigen, as further because the immunoaffinity strategy separates phosphory described below. Production of exemplary polyclonal anti lated peptides from unphosphorylated peptides, observing bodies is provided below. just one phosphopeptide from a protein is a common result, since many phosphorylated proteins have only one tyrosine A. EFS (Tyrosine 148). phosphorylated site. For this reason, it is appropriate to use additional criteria to validate phosphopeptide assignments. 0269. A 14 amino acid phospho-peptide antigen, Assignments are likely to be correct if any of these additional DALEVy*DVPPTALR (SEQ NO:9; y*=phosphotyrosine), criteria are met: (i) the same phosphopeptide sequence is which comprises the phosphorylation site derived from assigned to co-eluting ions with different charge states, since human EFS (an adaptor/scaffold protein, Tyr 148 being the the MS/MS spectrum changes markedly with charge state; (ii) phosphorylatable residue), plus cysteine on the C-terminal the phosphorylation site is found in more than one peptide for coupling, is constructed according to standard synthesis sequence context due to sequence overlaps from incomplete techniques using, e.g., a Rainin/Protein Technologies, Inc., proteolysis or use of proteases other than trypsin; (iii) the Symphony peptide synthesizer. See ANTIBODIES: A LABORA phosphorylation site is found in more than one peptide TORY MANUAL, supra.; Merrifield, supra. This peptide is then sequence context due to homologous but not identical protein coupled to KLH and used to immunize animals to produce isoforms; (iv) the phosphorylation site is found in more than (and Subsequently screen) phosphorylation site-specific one peptide sequence context due to homologous but not polyclonal antibodies as described in Immunization/Screen identical proteins among species; and (V) phosphorylation ing below. sites validated by MS/MS analysis of synthetic phosphopep tides corresponding to assigned sequences, since the ion trap B. Afadin (Tyrosine 94). mass spectrometer produces highly reproducible MS/MS 0270. A 12 amino acid phospho-peptide antigen, spectra. The last criterion is routinely used to confirm novel YSLy*EVHVSGER (SEQID NO: 16:y*=phosphotyrosine), site assignments of particular interest. which comprises the phosphorylation site derived from 0265 All spectra and all sequence assignments made by human afadin (an adhesion or extracellular matrix protein, Sequest were imported into a relational database. The follow Tyr 94 being the phosphorylatable residue), plus cysteine on ing Sequest scoring thresholds were used to select phospho the C-terminal for coupling, is constructed according to stan peptide assignments that are likely to be correct: RSp-6, dard synthesis techniques using, e.g., a Rainin/Protein Tech XCorre2.2, and DeltaCN>0.099. Further, the sequence nologies, Inc., Symphony peptide synthesizer. See ANTIBOD assignments could be accepted or rejected with respect to IES: A LABORATORY MANUAL, supra.; Merrifield, supra. This accuracy by using the following conservative, two-step pro peptide is then coupled to KLH and used to immunize animals CCSS, to produce (and Subsequently screen) phosphorylation site 0266. In the first step, a Subset of high-scoring sequence specific polyclonal antibodies as described in Immunization/ assignments should be selected by filtering for XCorr values Screening below. of at least 1.5 for a charge state of +1, 2.2 for +2, and 3.3 for +3, allowing a maximum RSp value of 10. Assignments in C. CTNNA1 (Tyrosine 419). this subset should be rejected if any of the following criteria 0271. A 15 amino acid phospho-peptide antigen, are satisfied: (i) the spectrum contains at least one major peak NGNEKEVKEy*AQVFR (SEQ ID NO: 75; (at least 10% as intense as the most intense ion in the spec y*-phosphotyrosine, which comprises the phosphorylation trum) that can not be mapped to the assigned sequence as an site derived from human CTNNA1 (a cytoskeletal protein, a, b, or y ion, as an ion arising from neutral-loss of water or Tyr 419 being the phosphorylatable residue), plus cysteine on ammonia from ab ory ion, or as a multiply protonated ion; (ii) the C-terminal for coupling, is constructed according to stan the spectrum does not contain a series of bory ions equivalent dard synthesis techniques using, e.g., a Rainin/Protein Tech to at least six uninterrupted residues; or (iii) the sequence is nologies, Inc., Symphony peptide synthesizer. See ANTIBOD not observed at least five times in all the studies conducted IES: A LABORATORY MANUAL, supra.; Merrifield, supra. This (except for overlapping sequences due to incomplete pro peptide is then coupled to KLH and used to immunize animals teolysis or use of proteases other than trypsin). to produce (and Subsequently screen) phosphorylation site 0267 In the second step, assignments with below-thresh specific polyclonal antibodies as described in Immunization/ old scores should be accepted if the low-scoring spectrum Screening below. shows a high degree of similarity to a high-scoring spectrum 0272 Immunization/Screening. collected in another study, which simulates a true reference 0273 A synthetic phospho-peptide antigenas described in library-searching strategy. A-C above is coupled to KLH, and rabbits are injected intra dermally (ID) on the back with antigen in complete Freunds Example 2 adjuvant (500 ug antigen per rabbit). The rabbits are boosted Production of Phosphorylation Site-Specific Poly with same antigen in incomplete Freund adjuvant (250 ug clonal Antibodies antigen per rabbit) every three weeks. After the fifth boost, bleeds are collected. The sera are purified by Protein A-affin 0268 Polyclonal antibodies that specifically bind a novel ity chromatography by standard methods (see ANTIBODIES: A phosphorylation site of the invention (Table 1/FIG. 2) only LABORATORY MANUAL, Cold Spring Harbor, supra.). The when the tyrosine residue is phosphorylated (and does not eluted immunoglobulins are further loaded onto an unphos bind to the same sequence when the tyrosine is not phospho phorylated synthetic peptide antigen-resin Knotes column to rylated), and vice versa, are produced according to standard pulloutantibodies that bind the unphosphorylated form of the methods by first constructing a synthetic peptide antigen phosphorylation sites. The flow through fraction is collected comprising the phosphorylation site and then immunizing an and applied onto a phospho-synthetic peptide antigen-resin US 2010/0129929 A1 May 27, 2010 48 column to isolate antibodies that bind the phosphorylated y*-phosphotyrosine), which comprises the phosphorylation form of the phosphorylation sites. After washing the column site derived from human ADH1B (an enzyme protein, Tyr 35 extensively, the bound antibodies (i.e. antibodies that bind the being the phosphorylatable residue), plus cysteine on the phosphorylated peptides described in A-C above, but do not C-terminal for coupling, is constructed according to standard bind the unphosphorylated form of the peptides) are eluted synthesis techniques using, e.g., a Rainin/Protein Technolo and kept in antibody storage buffer. gies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A 0274 The isolated antibody is then tested for phospho LABORATORY MANUAL, supra.; Merrifield, supra. This pep specificity using Western blot assay using an appropriate cell tide is then coupled to KLH and used to immunize animals line that expresses (or overexpresses) target phospho-protein and harvest spleen cells for generation (and Subsequent (i.e. phosphorylated EFS, afadin or CTNNA1), for example, screening) of phosphorylation site-specific monoclonal anti HCC1428, NSCLC or H1650XG. Cells are cultured in bodies as described in Immunization/Fusion/Screening DMEM or RPMI supplemented with 10% FCS. Cell are below. collected, washed with PBS and directly lysed in cell lysis buffer. The protein concentration of cell lysates is then mea B. Adolase A (Tyrosine 5). sured. The loading buffer is added into cell lysate and the mixture is boiled at 100° C. for 5 minutes. 20 ul (10 ug 0279 A 13 amino acid phospho-peptide antigen, protein) of sample is then added onto 7.5% SDS-PAGE gel. PYQy*PALTPEQKK (SEQ ID NO: 98: 0275 A standard Western blot may be performed accord y*-phosphotyrosine), which comprises the phosphorylation ing to the Immunoblotting Protocol set out in the CELL SIG site derived from human adolase A (an enzyme protein, Tyr 5 NALING TECHNOLOGY, INC. 2003-04 Catalogue, p. 390. The being the phosphorylatable residue), plus cysteine on the isolated phosphorylation site-specific antibody is used at C-terminal for coupling, is constructed according to standard dilution 1:1000. Phospho-specificity of the antibody will be synthesis techniques using, e.g., a Rainin/Protein Technolo shown by binding of only the phosphorylated form of the gies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A target amino acid sequence. Isolated phosphorylation site LABORATORY MANUAL, supra.; Merrifield, supra. This pep specific polyclonal antibody does not (Substantially) recog tide is then coupled to KLH and used to immunize animals nize the same target sequence when not phosphorylated at the and harvest spleen cells for generation (and Subsequent specified tyrosine position (e.g., the antibody does not bind to screening) of phosphorylation site-specific monoclonal anti CTNNA1 in the non-stimulated cells, when tyrosine 419 is bodies as described in Immunization/Fusion/Screening not phosphorylated). below. 0276. In order to confirm the specificity of the isolated antibody, different cell lysates containing various phospho C. ARHGAP12 (Tyrosine 355). rylated signaling proteins other than the target protein are 0280 A 13 amino acid phospho-peptide antigen, prepared. The Western blot assay is performed again using GHTLy*TSDYTNEK (SEQ ID NO: 121: these cell lysates. The phosphorylation site-specific poly y-phosphotyrosines), which comprises the phosphorylation clonal antibody isolated as described above is used (1:1000 site derived from human ARHGAP 12 (an enzyme protein, dilution) to test reactivity with the different phosphorylated Tyr355 being the phosphorylatable residue), plus cysteine on non-target proteins. The phosphorylation site-specific anti the C-terminal for coupling, is constructed according to stan body does not significantly cross-react with other phospho dard synthesis techniques using, e.g., a Rainin/Protein Tech rylated signaling proteins that do not have the described phos nologies, Inc., Symphony peptide synthesizer. See ANTIBOD phorylation site, although occasionally slight binding to a IES: A LABORATORY MANUAL, supra.; Merrifield, supra. This highly homologous sequence on another protein may be peptide is then coupled to KLH and used to immunize animals observed. In such case the antibody may be further purified and harvest spleen cells for generation (and Subsequent using affinity chromatography, or the specific immunoreac screening) of phosphorylation site-specific monoclonal anti tivity cloned by rabbit hybridoma technology. bodies as described in Immunization/Fusion/Screening below. Example 3 Immunization/Fusion/Screening. Production of Phosphorylation Site-Specific Mono clonal Antibodies 0281. A synthetic phospho-peptide antigenas described in A-C above is coupled to KLH, and BALB/C mice are injected 0277 Monoclonal antibodies that specifically bind a novel intradermally (ID) on the back with antigen in complete Fre phosphorylation site of the invention (Table 1) only when the unds adjuvant (e.g., 50 ug antigen per mouse). The mice are tyrosine residue is phosphorylated (and does not bind to the boosted with same antigen in incomplete Freund adjuvant same sequence when the tyrosine is not phosphorylated) are (e.g. 25 ug antigen per mouse) every three weeks. After the produced according to standard methods by first constructing fifth boost, the animals are sacrificed and spleens are har a synthetic peptide antigen comprising the phosphorylation vested. site and then immunizing an animal to raise antibodies 0282 Harvested spleen cells are fused to SP2/0 mouse against the antigen, and harvesting spleen cells from Such myeloma fusion partner cells according to the standard pro animals to produce fusion hybridomas, as further described tocol of Kohler and Milstein (1975). Colonies originating below. Production of exemplary monoclonal antibodies is from the fusion are screened by ELISA for reactivity to the provided below. phospho-peptide and non-phospho-peptide forms of the anti gen and by Western blot analysis (as described in Example 1 A. ADH1B (Tyrosine 35). above). Colonies found to be positive by ELISA to the phos 0278 A 19 amino acid phospho-peptide antigen, pho-peptide while negative to the non-phospho-peptide are KPFSIEDVEVAPPKAy*EVA (SEQ ID NO: 87; further characterized by Western blot analysis. Colonies US 2010/0129929 A1 May 27, 2010 49 found to be positive by Western blot analysis are subcloned by peptide is then spiked into a biological sample to quantify the limited dilution. Mouse ascites are produced from a single amount of phosphorylated EDF1 (tyr 109) in the sample, as clone obtained from Subcloning, and tested for phospho further described below in Analysis & Quantification. specificity (against the ADH1B, adolase A or ARHGAP12) phospho-peptide antigen, as the case may be) on ELISA. C. FbX46 (Tyrosine 309). Clones identified as positive on Western blot analysis using cell culture Supernatant as having phospho-specificity, as 0287. An AQUA peptide comprising the sequence indicated by a strong band in the induced lane and a weak ITCDLy*QLISPSR (SEQID NO: 228:y*=phosphotyrosine; band in the uninduced lane of the blot, are isolated and sub Leucine being 'C/N-labeled, as indicated in bold), which cloned as clones producing monoclonal antibodies with the comprises the phosphorylation site derived from human desired specificity. FbX46 (a ubiquitin conjugating system protein, Tyr 309 being 0283 Ascites fluid from isolated clones may be further the phosphorylatable residue), is constructed according to tested by Western blot analysis. The ascites fluid should pro standard synthesis techniques using, e.g., a Rainin/Protein duce similar results on Western blot analysis as observed Technologies, Inc., Symphony peptide synthesizer (see Mer previously with the cell culture Supernatant, indicating phos rifield, supra.) as further described below in Synthesis & pho-specificity against the phosphorylated target. MS/MS Signature. The Fbx46 (tyr 309) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated FbX46 (tyr 309) in the sample, as further Example 4 described below in Analysis & Quantification. Production and Use of AQUA Peptides for Detecting (0288. D. ApoB (Tyrosine 3680). and Quantitating Phosphorylation at a Novel Phos 0289 An AQUA peptide comprising the sequence phorylation Site FLKNIILPVy*DK (SEQID NO: 199; y*=phosphotyrosine; proline being ''C/N-labeled, as indicated in bold), which 0284. Heavy-isotope labeled peptides (AQUA peptides comprises the phosphorylation site derived from human (internal standards)) for the detecting and quantitating a novel ApoB (a receptor/channel/transporter/cell Surface protein, phosphorylation site of the invention (Table 1) only when the Tyr3680 being the phosphorylatable residue), is constructed tyrosine residue is phosphorylated are produced according to according to standard synthesis techniques using, e.g., a Rai the standard AQUA methodology (see Gygi et al., Gerber et nin/Protein Technologies, Inc., Symphony peptide synthe al., Supra.) methods by first constructing a synthetic peptide sizer (see Merrifield, supra.) as further described below in standard corresponding to the phosphorylation site sequence Synthesis & MS/MS Signature. The ApoB (tyr 3680) AQUA and incorporating a heavy-isotope label. Subsequently, the peptide is then spiked into a biological sample to quantify the MS" and LC-SRM signature of the peptide standard is vali amount of phosphorylated ApoB (tyr 3680) in the sample, as dated, and the AQUA peptide is used to quantify native pep tide in a biological sample, Such as a digested cell extract. further described below in Analysis & Quantification. Production and use of exemplary AQUA peptides is provided Synthesis & MS/MS Spectra. below. 0290 Fluorenylmethoxycarbonyl (Fmoc)-derivatized A. ANTXR1 (Tyrosine 92). amino acid monomers may be obtained from AnaSpec (San Jose, Calif.). Fmoc-derivatized stable-isotope monomers 0285 An AQUA peptide comprising the sequence, containing one 'N and five to nine C atoms may be WPTVDASy*YGGR (SEQ ID NO: 198: obtained from Cambridge Isotope Laboratories (Andover, y*-phosphotyrosine; Valine being 'C/N-labeled, as indi Mass.). Preloaded Wang resins may be obtained from Applied cated in bold), which comprises the phosphorylation site Biosystems. Synthesis scales may vary from 5 to 25 umol. derived from human ANTXR1 (a receptor/channel/trans Amino acids are activated in situ with 1-H-benzotriazolium, porter/cell surface protein, Tyr 92 being the phosphorylatable 1-bis(dimethylamino)methylene-hexafluorophosphate (1-), residue), is constructed according to standard synthesis tech 3-oxide: 1-hydroxybenzotriazole hydrate and coupled at a niques using, e.g., a Rainin/Protein Technologies, Inc., Sym 5-fold molar excess over peptide. Each coupling cycle is phony peptide synthesizer (see Merrifield, supra.) as further followed by capping with acetic anhydride to avoid accumu described below in Synthesis & MS/MS Signature. The lation of one-residue deletion peptide by-products. After syn ANTXR1 (tyr 92) AQUA peptide is then spiked into a bio thesis peptide-resins are treated with a standard Scavenger logical sample to quantify the amount of phosphorylated containing trifluoroacetic acid (TFA)-water cleavage ANTXR1 (tyr 92) in the sample, as further described below in Solution, and the peptides are precipitated by addition to cold Analysis & Quantification. ether. Peptides (i.e. a desired AQUA peptide described in A-D above) are purified by reversed-phase C18HPLC using stan B. EDF1 (Tyrosine 109). dard TFA/acetonitrile gradients and characterized by matrix 0286 An AQUA peptide comprising the sequence assisted laser desorption ionization-time of flight (Biflex III, INEKPQVIADy*ESGR (SEQ ID NO: 221 Bruker Daltonics, Billerica, Mass.) and ion-trap (Ther y*-phosphotyrosine; Proline being '"C/N-labeled, as indi moFinnigan, LCQ DecaXP or LTQ) MS. cated in bold), which comprises the phosphorylation site 0291 MS/MS spectra for each AQUA peptide should derived from human EDF1 (a transcriptional regulator, Tyr exhibit a strongy-type ion peak as the most intense fragment 109 being the phosphorylatable residue), is constructed ion that is suitable for use in an SRM monitoring/analysis. according to standard synthesis techniques using, e.g., a Rai Reverse-phase microcapillary columns (0.1 A-150-220mm) nin/Protein Technologies, Inc., Symphony peptide synthe are prepared according to standard methods. An Agilent 1100 sizer (see Merrifield, supra.) as further described below in liquid chromatograph may be used to develop and deliver a Synthesis & MS/MS Signature. The EDF1 (tyr 109) AQUA solvent gradient 0.4% acetic acid/0.005% heptafluorobu US 2010/0129929 A1 May 27, 2010 50 tyric acid (HFBA)/7% methanol and 0.4% acetic acid/0. Jose, Calif.) mass spectrometer (LCQ DecaXP ion trap or 0.05% HFBA/65% methanol/35% acetonitrile to the micro TSQ Quantum triple quadrupole or LTO). On the DecaXP. capillary column by means of a flow splitter. Samples are then parentions are isolated at 1.6 m/z, width, the ion injection time directly loaded onto the microcapillary column by using a being limited to 150 ms per microScan, with two microscans FAMOS inert capillary autosampler (LC Packings, San Fran per peptide averaged, and with an AGC setting of 1x10; on cisco) after the flow split. Peptides are reconstituted in 6% the Quantum, Q1 is kept at 0.4 and Q3 at 0.8 m/z, with a scan acetic acid/0.01% TFA before injection. time of 200 ms per peptide. On both instruments, analyte and Analysis & Quantification. internal standard are analyzed in alternation within a previ 0292 Target protein (e.g. a phosphorylated proteins of ously known reverse-phase retention window; well-resolved A-D above) in a biological sample is quantified using a vali pairs of internal standard and analyte are analyzed in separate dated AQUA peptide (as described above). The IAP method is retention segments to improve duty cycle. Data are processed then applied to the complex mixture of peptides derived from by integrating the appropriate peaks in an extracted ion chro proteolytic cleavage of crude cell extracts to which the AQUA matogram (60.15 m/z from the fragment monitored) for the peptides have been spiked in. native and internal standard, followed by calculation of the 0293 LC-SRM of the entire sample is then carried out. ratio of peak areas multiplied by the absolute amount of MS/MS may be performed by using a ThermoFinnigan (San internal standard (e.g., 500 fmol).
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 352
<21 Os SEQ ID NO 1 &211s LENGTH: 11 212s. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs SEQUENCE: 1 Val Lys Gly Glu Tyr Asp Val Thr Met Pro Llys 1. 5 1O
<21 Os SEQ ID NO 2 &211s LENGTH: 18 212s. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs SEQUENCE: 2 Phe Ile Glin Gln Thr Tyr Pro Ser Gly Gly Glu Glu Glin Ala Glin Tyr 1. 5 1O 15 Cys Arg
<21 Os SEQ ID NO 3 &211s LENGTH: 24 212s. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (22) ... (22) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs SEQUENCE: 3 Asp Phe Val Asp His Ile Asp Lieu Val Asp Pro Val Asp Gly Val Val 1. 5 1O 15
Lieu Val Asp Pro Glu Tyr Lieu Lys 2O US 2010/0129929 A1 May 27, 2010 51
- Continued
<210s, SEQ ID NO 4 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 4 Lys Ile Tyr Glu Asp Gly Asp Asp Asp Met Lys Arg 1. 5 1O
<210s, SEQ ID NO 5 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 5 Thr Gly His Gly Tyr Val Tyr Glu Tyr Pro Ser Arg 1. 5 1O
<210s, SEQ ID NO 6 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 6 Ser Trp Met Asp Asp Tyr Asp Tyr Val His Lieu. Glin Gly Lys Glu Glu 1. 5 1O 15 Phe Glu Arg
<210s, SEQ ID NO 7 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 7 Asn Thr Ser Asp Met Val Tyr Lieu Lys 1. 5
<210s, SEQ ID NO 8 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 8 Gly Thr Glu. His Tyr Ser Tyr Pro Cys Thr Pro Thr Thr Met Leu Pro 1. 5 1O 15 US 2010/0129929 A1 May 27, 2010 52
- Continued
Arg
<210s, SEQ ID NO 9 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 9 Asp Ala Lieu. Glu Val Tyr Asp Val Pro Pro Thr Ala Lieu. Arg 1. 5 1O
<210s, SEQ ID NO 10 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 10 Val Val Glin Asp Ala Ala Lieu. Thir Tyr Glin Glin Phe Llys 1. 5 1O
<210s, SEQ ID NO 11 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 11 Ile Ser Ile Leu Glu Pro Glu Thr Gly Lys Asp Met Ser Pro Tyr Glu 1. 5 1O 15 Ala Tyr Lys Arg 2O
<210s, SEQ ID NO 12 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 12 Gln Tyr His Glu Gln Glu Glu Thr Pro Glu Met Met Ala Ala Arg 1. 5 1O 15
<210s, SEQ ID NO 13 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr US 2010/0129929 A1 May 27, 2010 53
- Continued <4 OOs, SEQUENCE: 13 Ser Glin Glu Ser Asp Gly Val Glu Tyr Ile Phe Ile Ser Lys 1. 5 1O
<210s, SEQ ID NO 14 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 14 Tyr Pro Tyr Pro Met Pro Pro Leu Pro Asp Glu Asp Llys 1. 5 1O
<210s, SEQ ID NO 15 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 15 Tyr Pro Tyr Pro Met Pro Pro Leu Pro Asp Glu Asp Llys Lys 1. 5 1O
<210s, SEQ ID NO 16 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 16 Tyr Ser Leu Tyr Glu Val His Val Ser Gly Glu Arg 1. 5 1O
<210s, SEQ ID NO 17 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 17 Ser Glu Gly Phe Glu Lieu. Tyr Asn Asn Ser Thr Glin Asn Gly Ser Pro 1. 5 1O 15 Glu Ser Pro Gln Leu Pro Trp Ala Glu Tyr Ser Glu Pro Llys 2O 25 3O
<210s, SEQ ID NO 18 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) US 2010/0129929 A1 May 27, 2010 54
- Continued <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 18 Asp Asn Val Phe Tyr Tyr Gly Glu Glu Gly Gly Gly Glu Glu Asp Glin 1. 5 1O 15 Asp Tyr Asp Ile Thr Glin Lieu. His Arg 2O 25
<210s, SEQ ID NO 19 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 19 Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Glin Ser Tyr 1. 5 1O 15 Pro Ser Lys His Asp Tyr Val 2O
<210s, SEQ ID NO 2 O &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 2O Tyr Thr Tyr Asn Gly Ala Thr Ser Val Met Ser Ser Arg 1. 5 1O
<210s, SEQ ID NO 21 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 21 Gly Tyr Pro Gly Lieu Ser Gly Glu Lys Gly Ser Pro Gly Glin Lys Gly 1. 5 1O 15
Ser Arg
<210s, SEQ ID NO 22 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 22 Ala Ala Val Ser His Trp Glin Glin Glin Ser Tyr Lieu. Asp Ser Gly Ile 1. 5 1O 15 His Ser Gly Ala Thr Thr Thr Ala Pro Ser Leu Ser Gly Lys US 2010/0129929 A1 May 27, 2010 55
- Continued
2O 25 3O
<210s, SEQ ID NO 23 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 23 Lieu. His Tyr Gly Lieu Pro Val Val Val Lys 1. 5 1O
<210s, SEQ ID NO 24 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 24 Ser Tyr Ile Gly Ser Asn His Ser Ser Leu Gly Ser Met Ser Pro Ser 1. 5 1O 15 Asn Met Glu Gly Tyr Ser Lys 2O
<210s, SEQ ID NO 25 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (21) ... (21) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 25 Ser Tyr Ile Gly Ser Asn His Ser Ser Leu Gly Ser Met Ser Pro Ser 1. 5 1O 15 Asn Met Glu Gly Tyr Ser Lys 2O
<210s, SEQ ID NO 26 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 26 Lieu. Asp Arg Glu Glu Arg Asp Ala Tyr Asn Lieu. Arg 1. 5 1O
<210s, SEQ ID NO 27 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) US 2010/0129929 A1 May 27, 2010 56
- Continued <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 27 Ser Val Glu Asp Arg Phe Asp Glin Glin Lys Asn Asp Tyr Asp Glin Lieu. 1. 5 1O 15 Glin Lys
<210s, SEQ ID NO 28 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 28 Ile Ser Tyr Lys Asp Ala Ile Asin Arg Ser Met Val Glu Asp Ile Thr 1. 5 1O 15 Gly Lieu. Arg
<210s, SEQ ID NO 29 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 29 Val Tyr Lieu. Cys Asn Glin Asp Glu Asn His Llys His Ala Glin Asp Tyr 1. 5 1O 15 Val Lieu. Thir Tyr Asn Tyr Glu Gly Arg 2O 25
<210s, SEQ ID NO 3 O &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 30 His Ala Glin Asp Tyr Val Lieu. Thir Tyr Asn Tyr Glu Gly Arg 1. 5 1O
<210s, SEQ ID NO 31 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 31 Asn Asp Asn. Ser Asp Tyr Cys Gly Ile Ser Glu Gly Met Glu Met Lys 1. 5 1O 15
<210s, SEQ ID NO 32 US 2010/0129929 A1 May 27, 2010 57
- Continued
&211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 32 Thr Pro Ala Glin Tyr Asp Ala Ser Glu Lieu Lys 1. 5 1O
<210s, SEQ ID NO 33 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (26) ... (26) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 33 Gly Asp Lieu. Glu Asn Ala Phe Lieu. Asn Lieu Val Glin Cys Ile Glin Asn 1. 5 1O 15 Llys Pro Lieu. Tyr Phe Ala Asp Arg Lieu. Tyr Asp Ser Met Lys 2O 25 3O
<210 SEQ ID NO. 34 &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 34 Glu Glu Trp Lys Gly Ser Glu Thr Tyr Ser Pro Asn Thr Ala Tyr Gly 1. 5 1O 15 Val Asp Phe Leu Val Pro Val Met Gly Tyr Ile Cys Arg 2O 25
<210s, SEQ ID NO 35 &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 35 Glu Glu Trp Lys Gly Ser Glu Thr Tyr Ser Pro Asn Thr Ala Tyr Gly 1. 5 1O 15 Val Asp Phe Leu Val Pro Val Met Gly Tyr Ile Cys Arg 2O 25
<210s, SEQ ID NO 36 &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (26) ... (26) <223> OTHER INFORMATION: Phosphorylated Tyr US 2010/0129929 A1 May 27, 2010 58
- Continued
<4 OOs, SEQUENCE: 36 Glu Glu Trp Lys Gly Ser Glu Thr Tyr Ser Pro Asn Thr Ala Tyr Gly 1. 5 1O 15 Val Asp Phe Leu Val Pro Val Met Gly Tyr Ile Cys Arg 2O 25
<210s, SEQ ID NO 37 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 37 Glu Ser Ala Lieu. Asn Lieu Pro Tyr His Met Pro Glu Asn Lys 1. 5 1O
<210s, SEQ ID NO 38 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 38 Met Tyr Thr Asp Met Ser Val Ser Ala Asp Lieu. Asn. Asn Lys 1. 5 1O
<210s, SEQ ID NO 39 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 39 Phe Tyr Ala Ile Ser Ala Arg Phe Llys Pro Phe Ser Asn Lys 1. 5 1O
<210s, SEQ ID NO 4 O &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 4 O His His Pro Tyr Asp Ser Gly Ile Ile Tyr Cys Lieu Ser Arg 1. 5 1O
<210s, SEQ ID NO 41 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 2010/0129929 A1 May 27, 2010 59
- Continued <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 41 Glin Val Lieu. Asp Lieu. Phe Met Lieu. Tyr Val Lieu Val Thr Glu Lys 1. 5 1O 15
<210s, SEQ ID NO 42 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (22) ... (22) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 42 Thir Thr Gly Ile Val Lieu. Asp Ser Gly Asp Gly Val Thr His Asn Val 1. 5 1O 15 Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Met Arg 2O 25 3O
<210s, SEQ ID NO 43 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 43 Lys Asp Asp Pro Lieu. Thir Asn Lieu. Asn. Thir Ala Phe Asp Val Ala Glu 1. 5 1O 15 Llys Tyr Lieu. Asp Ile Pro Llys 2O
<210s, SEQ ID NO 44 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 44 Ser Lieu Ala Ala Asp Asp Glu Gly Gly Pro Glu Lieu. Glu Pro Asp Tyr 1. 5 1O 15 Gly Thr Ala Thr Arg 2O
<210s, SEQ ID NO 45 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 45 Asp Tyr Glin Tyr Ser Asp Glin Gly Ile Asp Tyr 1. 5 1O US 2010/0129929 A1 May 27, 2010 60
- Continued
<210s, SEQ ID NO 46 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 46 Lieu Ala Ser Tyr Lieu. Asp Llys Val Arg 1. 5
<210s, SEQ ID NO 47 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 47 Lieu. Lieu. Glu Gly Glu Asp Ala His Lieu. Thr Glin Tyr Lys 1. 5 1O
<210 SEQ ID NO 48 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 48 Ser Thr Phe Ser Thr Asn Tyr Arg 1. 5
<210s, SEQ ID NO 49 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 49 Ala Glin Tyr Asp Glu Lieu Ala Arg 1. 5
<210s, SEQ ID NO 50 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 50 Phe Val Ser Ser Ser Ser Ser Gly Gly Tyr Gly Gly Gly Tyr Gly Gly 1. 5 1O 15 US 2010/0129929 A1 May 27, 2010 61
- Continued Val Lieu. Thir Ala Ser Asp Gly Lieu. Lieu Ala Gly Asn. Glu Lys 2O 25 3O
<210s, SEQ ID NO 51 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 51 Phe Val Ser Ser Ser Ser Ser Gly Gly Tyr Gly Gly Gly Tyr Gly Gly 1. 5 1O 15 Val Lieu. Thir Ala Ser Asp Gly Lieu. Lieu Ala Gly Asn. Glu Lys 2O 25 3O
<210s, SEQ ID NO 52 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 52 Asp Tyr Ser His Tyr Tyr Thr Thr Ile Glin Asp Lieu. Arg 1. 5 1O
<210s, SEQ ID NO 53 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 53 Ser Glin Tyr Glu Val Met Ala Glu Glin Asn Arg Llys 1. 5 1O
<210s, SEQ ID NO 54 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 54 Ser Arg Lieu. Glu Glin Glu Ile Ala Thr Tyr Arg 1. 5 1O
<210s, SEQ ID NO 55 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: Phosphorylated Tyr US 2010/0129929 A1 May 27, 2010 62
- Continued <4 OO > SEQUENCE: 55 Val Ser Lieu Ala Gly Ala Cys Gly Val Gly Gly Tyr Gly Ser Arg Ser 1. 5 1O 15 Lieu. Tyr Asn Lieu. Gly Gly Ser Lys Arg 2O 25
<210s, SEQ ID NO 56 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 56 Asp Wall Asp Ala Ala Tyr Met Asn Llys Val Glu Lieu. Glu Ala Lys 1. 5 1O 15
<210s, SEQ ID NO 57 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 57 Ala Glin Tyr Glu Glu Ile Ala Asn Arg 1. 5
<210s, SEQ ID NO 58 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 58 Ser Arg Thr Glu Ala Glu Ser Trp Tyr Glin Thr Lys Tyr Glu Glu Lieu. 1. 5 1O 15 Glin Glin Thir Ala Gly Arg 2O
<210s, SEQ ID NO 59 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 59 Ser Arg Thr Glu Ala Glu Ser Trp Tyr Glin Thr Lys Tyr Glu Glu Lieu. 1. 5 1O 15 Glin Glin Thir Ala Gly Arg 2O
<210s, SEQ ID NO 60 US 2010/0129929 A1 May 27, 2010 63
- Continued
&211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 60 Ser Lieu. Tyr Gly Lieu. Gly Gly Ser Lys Arg 1. 5 1O
<210s, SEQ ID NO 61 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 61 Ile Ser Ile Gly Gly Gly Ser Cys Ala Ile Ser Gly Gly Tyr Gly Ser 1. 5 1O 15 Arg
<210s, SEQ ID NO 62 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 62 Ser Ala Tyr Gly Gly Pro Val Gly Ala Gly Ile Arg 1. 5 1O
<210s, SEQ ID NO 63 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 63 Asp Wall Asp Ala Ala Tyr Met Ser Lys 1. 5
<210s, SEQ ID NO 64 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 64 Ala Glu Ala Glu Ala Trp Tyr Glin Thr Llys Phe Glu Thir Lieu. Glin Ala 1. 5 1O 15 Glin Ala Gly Lys US 2010/0129929 A1 May 27, 2010 64
- Continued
<210s, SEQ ID NO 65 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 65 Ser Tyr Thr Ser Gly Pro Gly Ser Arg 1. 5
<210s, SEQ ID NO 66 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (21) ... (21) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 66 Lieu Lys Lieu. Glu Ala Glu Lieu. Gly Asn Met Glin Gly Lieu Val Glu Asp 1. 5 1O 15 Phe Lys Asn Lys Tyr Glu Asp Glu. Ile ASn Lys Arg 2O 25
<210s, SEQ ID NO 67 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 67 Ser Arg Ala Glu Ala Glu Ser Met Tyr Glin Ile Llys Tyr Glu Glu Lieu. 1. 5 1O 15 Glin Ser Lieu Ala Gly Lys 2O
<210s, SEQ ID NO 68 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 68 Ser Arg Ala Glu Ala Glu Ser Met Tyr Glin Ile Llys Tyr Glu Glu Lieu. 1. 5 1O 15 Glin Ser Lieu Ala Gly Lys 2O
<210s, SEQ ID NO 69 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 2010/0129929 A1 May 27, 2010 65
- Continued
22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 69 Ser Asn Tyr Tyr Asp Ala Tyr Glin Ala Glin Pro Lieu Ala Thir Arg 1. 5 1O 15
<210s, SEQ ID NO 70 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 7 O Ser Asn Tyr Tyr Asp Ala Tyr Glin Ala Glin Pro Lieu Ala Thir Arg 1. 5 1O 15
<210s, SEQ ID NO 71 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 71 Ser Glu Phe Asin Ser Tyr Ser Lieu. Thr Gly Tyr Val 1. 5 1O
<210s, SEQ ID NO 72 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 72 Val Val Tyr Ser Ala Pro Arg 1. 5
<210s, SEQ ID NO 73 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 73 Ser Ala Val Gly Phe Asp Tyr Glin Gly Lys Thr Glu Lys 1. 5 1O
<210s, SEQ ID NO 74 &211s LENGTH: 41 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: US 2010/0129929 A1 May 27, 2010 66
- Continued <221s NAME/KEY: MOD RES <222s. LOCATION: (34) . . (34) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 74 Glin Lieu. Glin Glin Ala Val Thr Gly Ile Ser Asn Ala Ala Glin Ala Thr 1. 5 1O 15 Ala Ser Asp Asp Ala Ser Glin His Glin Gly Gly Gly Gly Gly Glu Lieu 2O 25 3O Ala Tyr Ala Lieu. Asn. Asn. Phe Asp Llys 35 4 O
<210s, SEQ ID NO 75 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 75 Asn Gly Asn. Glu Lys Glu Val Lys Glu Tyr Ala Glin Val Phe Arg 1. 5 1O 15
<210s, SEQ ID NO 76 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 76 Gly Gly Ser Ala Pro Glu Gly Ala Thr Tyr Ala Ala Pro Arg 1. 5 1O
<210s, SEQ ID NO 77 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 77 Lieu. Asn Tyr Gly Ile Pro Ala Ile Val Lys 1. 5 1O
<210s, SEQ ID NO 78 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 78 His Val Ala Ala Gly Thr Glin Gln Pro Tyr Thr Asp Gly Val Arg 1. 5 1O 15 US 2010/0129929 A1 May 27, 2010 67
- Continued <210s, SEQ ID NO 79 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 79 Glu Pro Ser Ser Tyr Asp Phe Val Tyr His Tyr Gly 1. 5 1O
<210s, SEQ ID NO 8O &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 80 Glu Pro Ser Ser Tyr Asp Phe Val Tyr His Tyr Gly 1. 5 1O
<210s, SEQ ID NO 81 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 81 Asn Ser Glu Ser Lys Pro Lys Glu Pro Val Phe Ser Ala Glu Glu Gly 1. 5 1O 15 Tyr Val Lys
<210s, SEQ ID NO 82 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 82 Gln Ser Ser Ser Thr Asn Tyr Thr Asn Glu Lieu Lys 1. 5 1O
<210s, SEQ ID NO 83 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 83 Lieu. Glu Glu Lys Glu Asp Asn. Glu Glu Gly Val Tyr Asp Tyr Glu Asn 1. 5 1O 15 US 2010/0129929 A1 May 27, 2010 68
- Continued Thr Gly Arg
<210s, SEQ ID NO 84 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 84 Ser Gly Ser Phe Lieu. Tyr Glin Val Ser Thr His Lys 1. 5 1O
<210s, SEQ ID NO 85 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 85 Ser Ala Tyr Glin Thir Ile Asp Ser Ala Glu Ala Pro Ala Asp Pro Phe 1. 5 1O 15 Ala Val Pro Glu Gly Arg 2O
<210s, SEQ ID NO 86 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 86 Phe Pro Gly Tyr Tyr Val Thr Gly Asp Gly Cys Glin Arg 1. 5 1O
<210s, SEQ ID NO 87 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 87 Llys Pro Phe Ser Ile Glu Asp Val Glu Val Ala Pro Pro Lys Ala Tyr 1. 5 1O 15 Glu Val Arg
<210s, SEQ ID NO 88 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Phosphorylated Tyr US 2010/0129929 A1 May 27, 2010 69
- Continued
<4 OOs, SEQUENCE: 88 Tyr Val Ala Ala Met Val Lieu. Ser Ala Ala Gly Asp Ala Lieu. Gly Tyr 1. 5 1O 15 Tyr Asn Gly Lys 2O
<210s, SEQ ID NO 89 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 89 Tyr Val Ala Ala Met Val Lieu. Ser Ala Ala Gly Asp Ala Lieu. Gly Tyr 1. 5 1O 15 Tyr Asn Gly Lys 2O
<210s, SEQ ID NO 90 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 90 Tyr Val Ala Ala Met Val Lieu. Ser Ala Ala Gly Asp Ala Lieu. Gly Tyr 1. 5 1O 15 Tyr Asn Gly Lys 2O
<210s, SEQ ID NO 91 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 91 His Ile Asp Cys Ala His Val Tyr Glin Asn. Glu Asn. Glu Val Gly Val 1. 5 1O 15 Ala Ile Glin Glu Lys 2O
<210s, SEQ ID NO 92 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 92 Arg Glu Asp Ile Phe Tyr Thr Ser Lys US 2010/0129929 A1 May 27, 2010 70
- Continued
<210s, SEQ ID NO 93 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 93 Lieu. Asn Asp Gly His Phe Met Pro Val Lieu. Gly Phe Gly Thr Tyr Ala 1. 5 1O 15 Pro Pro Glu Val Pro Arg 2O
<210s, SEQ ID NO 94 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 94 His Ile Asp Ser Ala His Lieu. Tyr ASn ASn Glu Glu Gln Val Gly Lieu. 1. 5 1O 15 Ala Ile Arg
<210s, SEQ ID NO 95 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (22) ... (22) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 95 Asn Lieu. His Tyr Phe Asn Ser Asp Ser Phe Ala Ser His Pro Asn Tyr 1. 5 1O 15 Pro Tyr Ser Asp Glu Tyr 2O
<210s, SEQ ID NO 96 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 96 Pro Phe Glin Glu Ser Tyr Ala Lieu. Asp Lieu. Asp Glu Val Ile Llys 1. 5 1O 15
<210s, SEQ ID NO 97 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: US 2010/0129929 A1 May 27, 2010 71
- Continued <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OO > SEQUENCE: 97 Phe Asp His Ile Lieu. Tyr Thr Gly Ser Thr Gly Val Gly Lys 1. 5 1O
<210s, SEQ ID NO 98 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 98 Pro Tyr Glin Tyr Pro Ala Lieu. Thr Pro Glu Gln Lys Lys 1. 5 1O
<210s, SEQ ID NO 99 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 99 His Ile Asp Cys Ala Tyr Val Tyr Glin Asn. Glu. His Glu Val Gly Glu 1. 5 1O 15 Ala Ile Glin Glu Lys 2O
<210s, SEQ ID NO 100 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 1.OO Met Glin Gln His Phe Glu Phe Glu Tyr Glin Thr Lys 1. 5 1O
<210s, SEQ ID NO 101 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 101 Lieu. Ser Lieu. Asp Glu Lieu. His Arg Llys Tyr Gly Thr Asp Lieu. Ser Arg 1. 5 1O 15
<210s, SEQ ID NO 102 &211s LENGTH: 12 212. TYPE: PRT US 2010/0129929 A1 May 27, 2010 72
- Continued <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 102 Llys Lieu. Tyr Cys Ile Tyr Val Ala Ile Gly Glin Lys 1. 5 1O
<210s, SEQ ID NO 103 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 103 Glu Asp Asp Ser Tyr Lieu. Phe Asp Lieu. Asp Asn Llys 1. 5 1O
<210s, SEQ ID NO 104 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 104 Ser Pro Lieu. Asp Glin Arg Ser Tyr Gly Ser Arg 1. 5 1O
<210s, SEQ ID NO 105 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 105 Tyr Glu Asp Lieu Val Gly Asp Pro Val Lys 1. 5 1O
<210s, SEQ ID NO 106 &211s LENGTH: 34 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (24) . . (24) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 106 His Ala Lieu Pro Phe Ala Lys Ile Arg Arg Val Glin Glu Lieu. Cys Ala 1. 5 1O 15 Gly Ala Lieu. Glin Lieu. Lieu. Gly Tyr Arg Pro Val Tyr Ser Glu Asp Glu 2O 25 3O Glin Arg US 2010/0129929 A1 May 27, 2010 73
- Continued
<210s, SEQ ID NO 107 &211s LENGTH: 34 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (28) ... (28) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 107 His Ala Lieu Pro Phe Ala Lys Ile Arg Arg Val Glin Glu Lieu. Cys Ala 1. 5 1O 15 Gly Ala Lieu. Glin Lieu. Lieu. Gly Tyr Arg Pro Val Tyr Ser Glu Asp Glu 2O 25 3O Glin Arg
<210s, SEQ ID NO 108 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 108 Gln Val Glu Ala Ala Cys Ala Pro Ala Met Arg Lieu. Lieu. Ala Tyr Pro 1. 5 1O 15 Arg
<210s, SEQ ID NO 109 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 109 Glu Ile Val Lieu Met Ser Lys Tyr Ser Asn Thr Glu Ile His Lys 1. 5 1O 15
<210s, SEQ ID NO 110 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 110 Glu Lys Ala Ser Trp Ser Ser Lieu. Ser Met Asp Glu Lys Val Glu Lieu. 1. 5 1O 15 Tyr Arg
<210s, SEQ ID NO 111 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 2010/0129929 A1 May 27, 2010 74
- Continued <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 111 Gly Thr Glu Asn. Glu Asp Ala Gly Ser Asp Tyr Glin Ser Asp Asn Glin 1. 5 1O 15 Ala Ser Trp Ile His Arg 2O
<210s, SEQ ID NO 112 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 112 Val His Ala Cys Gly Val Asn Pro Val Glu Thir Tyr Ile Arg 1. 5 1O
<210s, SEQ ID NO 113 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAME/KEY: MOD RES <222s. LOCATION: (19) . . (19) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 113 Phe Ala Leu Pro Ser Pro Gln His Ile Leu Gly Lieu Pro Val Gly Glin 1. 5 1O 15 His Ile Tyr Lieu. Ser Ala Arg 2O
<210s, SEQ ID NO 114 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 114 His Arg Lieu. Asp Lieu. Gly Glu Asp Tyr Pro Ser Gly Lys 1. 5 1O
<210s, SEQ ID NO 115 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 115 Met Ile Lieu. Asp Asn His Ala Lieu. Tyr Asp Llys Thr Lys 1. 5 1O
<210s, SEQ ID NO 116 US 2010/0129929 A1 May 27, 2010 75
- Continued
&211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 116 Thr Gly Lys Gly Trp Tyr Glin Tyr Asp Llys Pro Lieu. Gly Arg Ile His 1. 5 1O 15 Llys Pro Asp Pro Trp Lieu. Ser Thr Phe Lieu. Ser Arg Tyr Arg Llys 2O 25 3O
<210s, SEQ ID NO 117 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 117 Thr Gly Lys Gly Trp Tyr Glin Tyr Asp Llys Pro Lieu. Gly Arg Ile His 1. 5 1O 15 Llys Pro Asp Pro Trp Lieu. Ser Thr Phe Lieu. Ser Arg Tyr Arg Llys 2O 25 3O
<210s, SEQ ID NO 118 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 118 Ile Gly Ala Glu Val Tyr His Asn Lieu Lys 1. 5 1O
<210s, SEQ ID NO 119 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 119 Ile Tyr Ser Lieu. Asn. Glu Gly Tyr Ala Lys 1. 5 1O
<210s, SEQ ID NO 120 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 120 US 2010/0129929 A1 May 27, 2010 76
- Continued Glu Lys Tyr Asp Llys Met Arg Asn Asp Val Ser Val Llys Lieu Lys 1. 5 1O 15
<210s, SEQ ID NO 121 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 121 Gly His Thr Lieu. Tyr Thr Ser Asp Tyr Thr Asn Glu Lys 1. 5 1O
<210s, SEQ ID NO 122 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 122 Gly His Thr Lieu. Tyr Thr Ser Asp Tyr Thr Asn Glu Lys 1. 5 1O
<210s, SEQ ID NO 123 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 123 Glin Lys Gly Tyr Ser Asp Glu Ile Tyr Val Val Pro Asp Asp Ser Glin 1. 5 1O 15 Asn Arg
<210s, SEQ ID NO 124 &211s LENGTH: 35 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 124 Thr Glu Gln Thr Pro Asp Leu Val Gly Met Leu Lleu Ser Tyr Ser His 1. 5 1O 15 Ser Glu Lieu Pro Glin Arg Pro Pro Llys Pro Ala Ile Tyr Ser Ser Val 2O 25 3O Thr Pro Arg 35
<210s, SEQ ID NO 125 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 2010/0129929 A1 May 27, 2010 77
- Continued
22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 125 Arg Thr Glu Glu Lieu. Ile Tyr Lieu. Ser Glin Lys 1. 5 1O
<210s, SEQ ID NO 126 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 126 Ser Lieu Val Asp Thr Val Tyr Ala Lieu Lys Asp Glu Val Glin Glu Lieu. 1. 5 1O 15 Arg
<210s, SEQ ID NO 127 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 127 Ser Pro Lieu Ala Glu. His Arg Pro Asp Ala Tyr Glin Asp Val Ser Ile 1. 5 1O 15 His Gly Thr Lieu Pro Arg 2O
<210s, SEQ ID NO 128 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 128 Ala His Glin Ser Glu Ser Tyr Lieu Pro Ile Gly Cys Lys 1. 5 1O
<210s, SEQ ID NO 129 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyr
<4 OOs, SEQUENCE: 129 Met Pro Ala Lys Thr Pro Ile Tyr Lieu Lys Ala Ala Asn. Asn Lys 1. 5 1O 15