Method of treating bone metastasis diseases, medicaments therefore, and a method of predicting the clinical outcome of treating bone metastasis diseases

Abstract

A method is used for treating bone metastasis diseases in subjects. The method preferably depends on whether the subject shows certain specific proteins levels in one or more body fluids prior to or during treatment. The treatment includes the administration of at least one pan αv integrin inhibitor to a subject, a medicament for use in said new methods, and a method of predicting the outcome of a treatment with at least one pan αv integrin inhibitor based on the specific protein levels in one or more body fluids of the subject.

Claims

1. A method of treating a bone metastasis disease in a subject, wherein said subject is characterized by: a) a high level of one or more proteins in at least one body fluid of said subject, wherein said one or more proteins are selected from the group consisting of: DCN (UniProt ID: P07585), F5 (UniProt ID: P12259), ICAM3 (UniProt ID: P32942), PIGR (UniProt ID: P01833), STK17B (UniProt ID: 094768), STX1A (UniProt ID: Q16623), and TEK (UniProt ID: Q02763), and/or b) a low level of one or more proteins in at least one body fluid of said subject, wherein said one or more proteins are selected from the group consisting of: ANG (UniProt ID: P03950), IL1B (UniProt ID: P01584), LEPR (UniProt ID: P48357), MAP2K2 (UniProt ID: P36507), MAPK11 (UniProt ID: Q15759), RGMB (UniProt ID: Q6NW40), and TNFRSF17 (UniProt ID: Q02223); said method comprising: administering to said subject at least one pan αv integrin inhibitor; wherein a level of a specific protein of said one or more proteins in at least one body fluid of said subject is a) classified as high, if the level of the specific protein of said one or more proteins in said body fluid is at least 2% higher than a median threshold determined for the specific protein, and/or b) classified as low, if the level of the specific protein of said one or more proteins in said body fluid is at least 2% lower than said median threshold for the specific protein; and wherein a threshold or median threshold for the specific protein is determined from the body fluid of a plurality of subjects being part of a diseased subject population suffering from the bone metastasis disease.

2. The method according to claim 1, wherein said at least one pan αv integrin inhibitor comprises Abituzumab.

3. The method according to claim 1, wherein said at least one pan αv integrin inhibitor is Abituzumab.

4. The method according to claim 1, wherein said subject is characterized by a high level of the protein STX1A (UniProt ID: Q16623) and/or a protein having at least 80% sequence homology to said protein.

5. The method according to claim 1, wherein said subject is characterized by the respective levels of one or more proteins having at least 80% sequence homology to said one or more proteins.

6. The method according to claim 1, wherein said body fluid is selected from the group consisting of blood plasma, blood serum and whole blood.

7. The method according to claim 1, wherein said high levels and/or low levels of one or more of said proteins are present and/or determined prior to administering said at least one pan αv integrin inhibitor.

8. The method according to claim 1, wherein said high levels and/or low levels of one or more of said proteins are present and/or determined during or after administering said at least one pan αv integrin inhibitor.

9. The method according to claim 1, wherein the bone metastasis disease is cancer or is derived from cancer.

10. The method according to claim 1, wherein the bone metastasis disease is derived from prostate cancer, breast cancer and/or lung cancer.

11. The method according to claim 1, wherein said at least one pan αv integrin inhibitor is administered to said subject in an amount of 100 mg to 3000 mg per month.

12. The method according to claim 1, wherein said at least one pan αv integrin inhibitor comprises or is Abituzumab, and wherein the Abituzumab is administered to said subject in an amount of 500 to 2000 mg every week, every second week every or every fourth week.

13. The method according to claim 1, wherein said at least one pan αv integrin inhibitor comprises or is Abituzumab, and wherein the Abituzumab is administered to said subject in an amount of about 500 mg per week, about 750 mg per week, about 1000 mg per week or about 1500 mg per week.

14. The method according to claim 1, wherein said at least one pan αv integrin inhibitor is administered in combination with one or more agents or chemotherapeutic agents, a) selected from the group consisting of Leuproreline, Leuproreline acetate, bicalutamide, nilutamide, triptoreline, gosereline, flutamide, cyproterone, busereline and degarelix, b) selected from the group consisting of Zoledronic acid, Pamidronic acid, Clodronate disodium, Alendronic acid and Ibandronic acid, and/or c) selected from the group consisting of Abiraterone, Abiraterone acetate, Prednisone, Enzalutamide, Radium Ra 223 dichloride, Docetaxel, Sipuleucel-T, Cabazitaxel and Mitoxantrone; and/or the pharmaceutically acceptable derivatives and/or salts thereof.

15. The method according to claim 1, wherein said at least one pan αv integrin inhibitor is administered in combination with, or additionally in combination with, one or more chemotherapeutic agents, selected from the group consisting of cetuximab, Panitumumab, irinotecan, vinorelbine, capecitabine, leucovorine, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil (5-FU), bevacizumab, aflibercept and regorafenib.

16. The method according to claim 1, wherein said at least one pan αv integrin inhibitor is administered in combination with one or more agents or chemotherapeutic agents, selected from the group consisting of a) Leuproreline, Leuproreline acetate, bicalutamid, nilutamide, triptoreline, gosereline, flutamide, cyproterone, busereline and degarelix, and/or the pharmaceutically acceptable derivatives and/or salts thereof; and/or b) Zoledronic acid, Pamidronic acid, Clodronate disodium, Alendronic acid and Ibandronic acid, and/or the pharmaceutically acceptable derivatives and/or salts thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 displays rPFS of patients with high and low expressions of MAPK11 for EMD 525797-treated patients and placebo-treated patients.

(2) FIG. 2 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of MAPK11.

(3) FIG. 3 displays rPFS of patients with high and low expressions of STX1A for EMD 525797-treated patients and placebo-treated patients.

(4) FIG. 4 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of STX1A.

(5) FIG. 5 displays rPFS of patients with high and low expressions of MAPK2K2 for EMD 525797-treated patients and placebo-treated patients.

(6) FIG. 6 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of MAPK2K2.

(7) FIG. 7 displays rPFS of patients with high and low expressions of TNFRSF17 for EMD 525797-treated patients and placebo-treated patients.

(8) FIG. 8 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of TNFRSF17.

(9) FIG. 9 displays rPFS of patients with high and low expressions of RGMB for EMD 525797-treated patients and placebo-treated patients.

(10) FIG. 10 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of RGMB.

(11) FIG. 11 displays rPFS of patients with high and low expressions of LEPR for EMD 525797-treated patients and placebo-treated patients.

(12) FIG. 12 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of LEPR.

(13) FIG. 13 displays rPFS of patients with high and low expressions of IL1B for EMD 525797-treated patients and placebo-treated patients.

(14) FIG. 14 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of IL1B.

(15) FIG. 15 displays rPFS of patients with high and low expressions of ICAM3 for EMD 525797-treated patients and placebo-treated patients.

(16) FIG. 16 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of ICAM3.

(17) FIG. 17 displays rPFS of patients with high and low expressions of F5 for EMD 525797-treated patients and placebo-treated patients.

(18) FIG. 18 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of F5.

(19) FIG. 19 displays rPFS of patients with high and low expressions of ANG for EMD 525797-treated patients and placebo-treated patients.

(20) FIG. 20 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of ANG.

(21) FIG. 21 displays rPFS of patients with high and low expressions of PIGR for EMD 525797-treated patients and placebo-treated patients.

(22) FIG. 22 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of PIGR.

(23) FIG. 23 displays rPFS of patients with high and low expressions of TEK for EMD 525797-treated patients and placebo-treated patients.

(24) FIG. 24 displays rPFS of EMD 525797-treated patients and placebo-treated patients for high and low expressions of TEK.

DETAILED DESCRIPTION OF THE INVENTION

(25) Said specific proteins are preferably characterised by the following sequences and/or sequence IDs (Amino acid sequences of protein listed in Table 1 as identified by UniProt IDs in FASTA format):

(26) TABLE-US-00001 ANG: >sp|P03950|ANGI_HUMAN Angiogenin OS = Homo sapiens GN = ANG PE = 1 SV = 1 MVMGLGVLLLVGVLGLGLTPPTAQDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAI CENKNGNPHRENLRISKSSFQVTTVKLHGGSPWPPCQYRATAGFRNVVVACENGLPVHLDQSIFRRP DCN: >sp|P07585|PGS2_HUMAN Decorin OS = Homo sapiens GN = DCN PE = 1 SV = 1 MKATIILLLLAQVSWAGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGPVCPFRCQCHLRVVQCSDLGLDKVPKDL PPDTTLLDLQNNKITEIKDGDFKNLKNLHALILVNNKISKVSPGAFTPLVKLERLYLSKNQLKELPEKMPKTLQELRAH ENEITKVRKVTFNGLNQMIVIELGTNPLKSSGIENGAFQGMKKLSYIRIADTNITSIPQGLPPSLTELHLDGNKISRVD AASLKGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKLTRVPGGLAEHKYIQVVYLHNNNISVVGSSDFCPPG HNTKKASYSGVSLFSNPVQYWEIQPSTFRCVYVRSAIQLGNYK F5: >sp|P12259|FA5_HUMAN Coagulation factor V OS = Homo sapiens GN = F5 PE = 1 SV = 4 MFPGCPRLWVLVVLGTSWVGWGSQGTEAAQLRQFYVAAQGISWSYRPEPTNSSLNLSVTSFKKIVYREYEPYFKKEKPQ STISGLLGPTLYAEVGDIIKVHFKNKADKPLSIHPQGIRYSKLSEGASYLDHTFPAEKMDDAVAPGREYTYEWSISEDS GPTHDDPPCLTHIYYSHENLIEDFNSGLIGPLLICKKGTLTEGGTQKTFDKQIVLLFAVFDESKSWSQSSSLMYTVNGY VNGTMPDITVCAHDHISWHLLGMSSGPELFSIHFNGQVLEQNHHKSAITLVSATSTTANMTVGPEGKWIISSLTPKHLQ AGMQAYIDIKNCPKKTRNLKKITREQRRHMKRWEYFIAAEEVIWDYAPVIPANMDKKYRSQHLDNFSNQIGKHYKKVMY TQYEDESFTKHTVNPNMKEDGILGPIIRAQVRDTLKIVFKNMASRPYSIYPHGVTFSYEDEVNSSFTSGRNNTMIRAVQ PGETYTYKWNILEFDEPTENDAQCLTRPYYSDVDIMRDIASGLIGLLLICKSRSLDRRGIQRAADIEQQAVFAVFDENK SWYLEDNINKFCENPDEVKRDDPKFYESNIMSTINGYVPESITTLGFCFDDTVQWHFCSVGTQNEILTIHFTGHSFIYG KRHEDTLTLFPMRGESVTVTMDNVGTWMLTSMNSSPRSKKLRLKFRDVKCIPDDDEDSYEIFEPPESTVMATRKMHDRL EPEDEESDADYDYQNRLAAALGIRSFRNSSLNQEEEEFNLTALALENGTEFVSSNTDIIVGSNYSSPSNISKFTVNNLA EPQKAPSHQQATTAGSPLRHLIGKNSVLNSSTAEHSSPYSEDPIEDPLQPDVTGIRLLSLGAGEFKSQEHAKHKGPKVE RDQAAKHRFSWMKLLAHKVGRHLSQDTGSPSGMRPWEDLPSQDTGSPSRMRPWKDPPSDLLLLKQSNSSKILVGRHHLA SEKGSYEIIQDTDEDTAVNNWLISPQNASRAWGESTPLANKPGKQSGHPKFPRVRHKSLQVRQDGGKSRLKKSQFLIKT RKKKKEKHTHHAPLSPRTFHLPRSEAYNTFSERRLKHSLVLHKSNETSLPTDLNQTLPSMDFGWIASLPDHNQNSSNDT GQASCPPGLYQTVPPEEHYQTFPIQDPDQMHSTSDPSHRSSSPELSEMLEYDRSHKSFPTDISQMSPSSEHEVWQTVIS PDLSQVTLSPELSQTNLSPDLSHTTLSPELIQRNLSPALGQMPISPDLSHTTLSPDLSHTTLSLDLSQTNLSPELSQTN LSPALGQMPLSPDLSHTTLSLDFSQTNLSPELSHMTLSPELSQTNLSPALGQMPISPDLSHTTLSLDFSQTNLSPELSQ TNLSPALGQMPLSPDPSHTTLSLDLSQTNLSPELSQTNLSPDLSEMPLFADLSQIPLTPDLDQMTLSPDLGETDLSPNF GQMSLSPDLSQVTLSPDISDTTLLPDLSQISPPPDLDQIFYPSESSQSLLLQEFNESFPYPDLGQMPSPSSPTLNDTFL SKEFNLPLVIVGLSKDGTDYIEIIPKEEVQSSEDDYAEIDYVPYDDPYKTDVRTNINSSRDPDNIAAWYLRSNNGNRRN YYIAAEEISWDYSEFVQRETDIEDSDDIPEDTTYKKVVFRKYLDSTFTKRDPRGEYEEHLGILGPIIRAEVDDVIQVRF KNLASRPYSLHAHGLSYEKSSEGKTYEDDSPEWFKEDNAVQPNSSYTYVWHATERSGPESPGSACRAWAYYSAVNPEKD IHSGLIGPLLICQKGILHKDSNMPMDMREFVLLFMTFDEKKSWYYEKKSRSSWRLTSSEMKKSHEFHAINGMIYSLPGL KMYEQEWVRLHLLNIGGSQDIHVVHFHGQTLLENGNKQHQLGVWPLLPGSFKTLEMKASKPGWWLLNTEVGENQRAGMQ TPFLIMDRDCRMPMGLSTGIISDSQIKASEFLGYWEPRLARLNNGGSYNAWSVEKLAAEFASKPWIQVDMQKEVIITGI QTQGAKHYLKSCYTTEFYAYSSNQINWQIFKGNSTRNVMYFNGNSDASTIKENQFDPPIVARYIRISPTRAYNRPTLRL ELQGCEVNGCSTPLGMENGKIENKQITASSFKKSWWGDYWEPFRARLNAQGRVNAWQAKANNNKQWLEIDLLKIKKITA IITQGCKSLSSEMYVKSYTIHYSEQGVEWKPYRLKSSMVDKIFEGNTNTKGHVKNFFNPPIISRFIRVIPKTWNQSIAL RLELFGCDIY ICAM3: >sp|P32942|ICAM3_HUMAN Intercellular adhesion molecule 3 OS = Homo sapiens GN = ICAM3 PE = 1 SV = 2 MATMVPSVLWPRACWTLLVCCLLTPGVQGQEFLLRVEPQNPVLSAGGSLFVNCSTDCPSSEKIALETSLSKELVASGMG WAAFNLSNVTGNSRILCSVYCNGSQITGSSNITVYRLPERVELAPLPPWQPVGQNFTLRCQVEDGSPRTSLTVVLLRWE EELSRQPAVEEPAEVTATVLASRDDHGAPFSCRTELDMQPQGLGLFVNTSAPRQLRTFVLPVTPPRLVAPRFLEVETSW PVDCTLDGLFPASEAQVYLALGDQMLNATVMNHGDTLTATATATARADQEGAREIVCNVTLGGERREARENLTVFSFLG PIVNLSEPTAHEGSTVTVSCMAGARVQVTLDGVPAAAPGQPAQLQLNATESDDGRSFFCSATLEVDGEFLHRNSSVQLR VLYGPKIDRATCPQHLKWKDKTRHVLQCQARGNPYPELRCLKEGSSREVPVGIPFFVNVTHNGTYQCQASSSRGKYTLV VVMDIEAGSSHFVPVFVAVLLTLGVVTIVLALMYVFREHQRSGSYHVREESTYLPLTSMQPTEAMGEEPSRAE IL1B: >sp|P01584|IL1B_HUMAN Interleukin-1 beta OS = Homo sapiens GN = IL1B PE = 1 SV = 2 MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVVVAMDKLRKMLV PCPQTFQENDLSTFFPFIFEEEPIFFDTWDNEAYVHDAPVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVF SMSFVQGEESNDKIPVALGLKEKNLYLSVFLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWY ISTSQAENMPVFLGGTKGGQDITDFTMQFVSS LEPR: >sp|P48357|LEPR_HUMAN Leptin receptor OS = Homo sapiens GN = LEPR PE = 1 SV = 2 MICQKFCVVLLHWEIFYVITAFNLSYPITPWRFKLSCMPPNSTYDYFLLPAGLSKNTSNSNGHYETAVEPKFNSSGTHF SNLSKTTFHCCFRSEQDRNCSLCADNIEGKTFVSTVNSLVFQQIDANWNIQCWLKGDLKLFICYVESLFKNLFRNYNYK VHLLYVLPEVLEDSPLVPQKGSFQMVHCNCSVHECCECLVPVPTAKLNDTLLMCLKITSGGIFQSPLMSVQPINMVKPD PPLGLHMEITDDGNLKISWSSPPLVPFPLQYQVKYSENSTTVIREADKINSATSLLVDSILPGSSYEVQVRGKRLDGPG IWSDWSTPRVFTTQDVIYFPPKILTSVGSNVSFHCIYKKENKIVPSKEIVWWMNLAEKIPQSQYDVVSDHVSKVTFFNL NETKPRGKFTYDAVYCCNEHECHHRYAELYVIDVNINISCETDGYLTKMTCRWSTSTIQSLAESTLQLRYHRSSLYCSD IPSIHPISEPKDCYLQSDGFYECIFQPIFLLSGYTMWIRINHSLGSLDSPPTCVLPDSVVKPLPPSSVKAEITINIGLL KISWEKPVFPENNLQFQIRYGLSGKEVQWKMYEVYDAKSKSVSLPVPDLCAVYAVQVRCKRLDGLGYWSNWSNPAYTVV MDIKVPMRGPEFWRIINGDTMKKEKNVTLLWKPLMKNDSLCSCQRYVINHHTSCNGTWSEDVGNHTKFTFLWTEQAHTV TVLAINSIGASVANFNLTFSWPMSKVNIVQSLSAYPLNSSCVIVSWILSPSDYKLMYFIIEWKNLNEDGEIKWLRISSS VKKYYIHDHFIPIEKYQFSLYPIFMEGVGKPKIINSFTQDDIEKHQSDAGLYVIVPVIISSSILLGTLLISHQRMKKLF WEDVPNPKNCSWAQGLNFQKPETFEHLFIKHTASVTCGPLLLEPETISEDISVDTSWKNKDEMMPTTVVSLLSTTDLEK GSVCISDQFNSVNFSEAEGTEVTYEDESQRQPFVKYATLISNSKPSETGEEQGLINSSVTKCFSSKNSPLKDSFSNSSW EIEAQAFFILSDQHPNIISPHLTFSEGLDELLKLEGNFPEENNDKKSIYYLGVTSIKKRESGVLLTDKSRVSCPFPAPC LFTDIRVLQDSCSHFVENNINLGTSSKKTFASYMPQFQTCSTQTHKIMENKMCDLTV MAP2K2 >sp|P36507|MP2K2_HUMAN Dual specificity mitogen-activated protein kinase 2 OS = Homo sapiens GN = MAP2K2 PE = 1 SV = 1 MLARRKPVLPALTINPTIAEGPSPTSEGASEANLVDLQKKLEELELDEQQKKRLEAFLTQKAKVGELKDDDFERISELG AGNGGVVTKVQHRPSGLIMARKHIHLEIKPAIRNQIIRELQVLHECNSPYIVGFYGAFYSDGEISICMEHMDGGSLDQV LKEAKRIPEEILGKVSIAVLRGLAYLREKHQIMHRDVKPSNILVNSRGEIKLCDFGVSGQLIDSMANSFVGTRSYMAPE RLQGTHYSVQSDIWSMGLSLVELAVGRYPIPPPDAKELEAIFGRPVVDGEEGEPHSISPRPRPPGRPVSGHGMDSRPAM AIFELLDYIVNEPPPKLPNGVFTPDFQEFVNKCLIKNPAERADLKMLTNHTFIKRSEVEEVDFAGWLCKTLRLNQPGTP TRTAV MAPK11 >sp|Q15759|MK11_HUMAN Mitogen-activated protein kinase 11 OS = Homo sapiens GN = MAPK11 PE = 1 SV = 2 MSGPRAGFYRQELNKTVWEVPQRLQGLRPVGSGAYGSVCSAYDARLRQKVAVKKLSRPFQSLIHARRTYRELRLLKHLK HENVIGLLDVFTPATSIEDFSEVYLVTTLMGADLNNIVKCQALSDEHVQFLVYQLLRGLKYIHSAGIIHRDLKPSNVAV NEDCELRILDFGLARQADEEMTGYVATRWYRAPEIMLNWMHYNQTVDIWSVGCIMAELLQGKALFPGSDYIDQLKRIME VVGTPSPEVLAKISSEHARTYIQSLPPMPQKDLSSIFRGANPLAIDLLGRMLVLDSDQRVSAAEALAHAYFSQYHDPED EPEAEPYDESVEAKERTLEEWKELTYQEVLSFKPPEPPKPPGSLEIEQ PIGR: >sp|P01833|PIGR_HUMAN Polymeric immunoglobulin receptor OS = Homo sapiens GN =  PIGR PE = 1 SV = 4 MLLFVLTCLLAVFPAISTKSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSSKYA GRANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDLGRTVTINCPFKTEN AQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQV LKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCRQSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLR KEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRC PLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVPGNVTAVL GETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYG ETAAVYVAVEERKAAGSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSEE QGGSSRALVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISMSDFENSREFGANDNMGASSITQETSLGG KEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVAAEAQDGPQEA RGMG: >sp|Q6NW40|RGMB_HUMAN RGM domain family member B OS = Homo sapiens GN = RGMB PE = 1 SV = 3 MGLRAAPSSAAAAAAEVEQRRSPGLCPPPLELLLLLLFSLGLLHAGDCQQPAQCRIQKCTTDFVSLTSHLNSAVDGFDS EFCKALRAYAGCTQRTSKACRGNLVYHSAVLGISDLMSQRNCSKDGPTSSTNPEVTHDPCNYHSHAGAREHRRGDQNPP SYLFCGLFGDPHLRTFKDNFQTCKVEGAWPLIDNNYLSVQVTNVPVVPGSSATATNKITIIFKAHHECTDQKVYQAVTD DLPAAFVDGTTSGGDSDAKSLRIVERESGHYVEMHARYIGTTVFVRQVGRYLTLAIRMPEDLAMSYEESQDLQLCVNGC PLSERIDDGQGQVSAILGHSLPRTSLVQAWPGYTLETANTQCHEKMPVKDIYFQSCVFDLLTTGDANFTAAAHSALEDV EALHPRKERWHIFPSSGNGTPRGGSDLSVSLGLTCLILIVFL STK17B: >sp|O94768|ST17B_HUMAN Serine/threonine-protein kinase 17B OS = Homo sapiens GN = STK17B PE = 1 SV = 1 MSRRRFDCRSISGLLTTTPQIPIKMENFNNFYILTSKELGRGKFAVVRQCISKSTGQEYAAKFLKKRRRGQDCRAEILH EIAVLELAKSCPRVINLHEVYENTSEIILILEYAAGGEIFSLCLPELAEMVSENDVIRLIKQILEGVYYLHQNNIVHLD LKPQNILLSSIYPLGDIKIVDFGMSRKIGHACELREIMGTPEYLAPEILNYDPITTATDMWNIGIIAYMLLTHTSPFVG EDNQETYLNISQVNVDYSEETFSSVSQLATDFIQSLLVKNPEKRPTAEICLSHSWLQQWDFENLFHPEETSSSSQTQDH SVRSSEDKTSKSSCNGTCGDREDKENIPEDSSMVSKRFRFDDSLPNPHELVSDLLC STX1A: >sp|Q16623|STX1A_HUMAN Syntaxin-1A OS = Homo sapiens GN = STX1A PE = 1 SV = 1 MKDRTQELRTAKDSDDDDDVAVTVDRDRFMDEFFEQVEEIRGFIDKIAENVEENKRKHSAILASPNPDEKTKEELEELM SDIKKTANKVRSKLKSIEQSIEQEEGLNRSSADLRIRKTQHSTLSRKFVEVMSEYNATQSDYRERCKGRIQRQLEITGR TTTSEELEDMLESGNPAIFASGIIMDSSISKQALSEIETRHSEIIKLENSIRELHDMFMDMAMLVESQGEMIDRIEYNV EHAVDYVERAVSDTKKAVKYQSKARRKKIMIIICCVILGIVIASTVGGIFA TEK: >sp|Q02736|TIE2_HUMAN Angioprotein-1 receptor OS = Homo sapiens GN = TEK PE = 1 SV = 2 MDSLASLVLCGVSLLLSGTVEGAMDLILINSLPLVSDAETSLTCIASGWRPHEPITIGRDFEALMNQHQDPLEVTQDVT REWAKKVVWKREKASKINGAYFCEGRVRGEAIRIRTMKMRQQASFLPATLTMTVDKGDNVNISFKKLIKEEDAVIYKNG SFIHSVPRHEVPDILEVHLPHAQPQDAGVYSARYIGGNLFTSAFTRLIVRRCEAQKWGPECNHLCTACMNNGVCHEDTG ECICPPGFMGRTCEKACELHTFGRTCKERCSGQEGCKSYVFCLPDPYGCSCATGWKGLQCNEACHPGFYGPDCKLRCSC NNGEMCDRFQGCLCSPGWQGLQCEREGIQRMTPKIVDLPDHIEVNSGKFNPICKASGWPLPTNEEMTLVKPDGTVLHPK DFNHTDHFSVAIFTIHRILPPDSGVWVCSVNTVAGMVEKPFNISVKVLPKPLNAPNVIDTGHNFAVINISSEPYFGDGP IKSKKLLYKPVNHYEAWQHIQVTNEIVTLNYEPRTEYELCVQLVRRGEGGEGHPGPVRRFTTASIGLPPPRGLNLLPKS QTTLNLTWQPIFPSSEDDFYVEVERRSVQKSDQQNIKVPGNLTSVLLNNLHPREQYVVRARVNTKAQGEWSEDLTAWTL SDILPPQPENIKISNITHSSAVISWTILDGYSISSITIRYKVQGKNEDQHVDVKIKNATITQYQLKGLEPETAYQVDIF AENNIGSSNPAFSHELVTLPESQAPADLGGGKMLLIAILGSAGMTCLTVLLAFLIILQLKRANVQRRMAQAFQNVREEP AVQFNSGTLALNRKVKNNPDPTIYPVLDWNDIKFQDVIGEGNFGQVLKARIKKDGLRMDAAIKRMKEYASKDDHRDFAG ELEVLCKLGHHPNIINLLGACEHRGYLYLAIEYAPHGNLLDFLRKSRVLETDPAFAIANSTASTLSSQQLLHFAADVAR GMDYLSQKQFIHRDLAARNILVGENYVAKIADFGLSRGQEVYVKKTMGRLPVRWMAIESLNYSVYTTNSDVWSYGVLLW EIVSLGGTPYCGMTCAELYEKLPQGYRLEKPLNCDDEVYDLMRQCWREKPYERPSFAQILVSLNRMLEERKTYVNTTLY EKFTYAGIDCSAEEAA TNFRSF17: <sp|Q02223|TNR17_HUMAN Tumor necrosis factor receptor superfamily member 17 OS = Homo sapiens GN = TNFRSF17 PE = 1 SV = 2 MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRK INSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVT TKTNDYCKSLPAALSATEIEKSISAR

(27) Specific proteins according to the invention are preferably also proteins having at least 80%, more preferably at least 90%, even more preferably at least 95% and especially at least 99% sequence homology to the afore described sequences.

(28) As further described herein, a high level of one or more proteins of a first group of said specific proteins and/or a low level of one or more proteins from a second group of specific proteins is predictive for improved clinical benefit, preferably clinical benefit as described herein, under treatment with at least one pan αv integrin inhibitor, preferably including or consisting of Abituzumab, for subjects suffering from a bone metastasis disease, including but not limited to metastatic prostate cancer, and metastatic castration-resistant prostate cancer (mCRPC). Preferably, a high level of one or more proteins of a first group of said specific proteins and/or a low level of one or more proteins from a second group of specific proteins is predictive for improved overall survival and/or improved progression free survival, under treatment with at least one pan αv integrin inhibitor, preferably including or consisting of Abituzumab, for subjects suffering from a bone metastasis disease, including but not limited to metastatic prostate cancer, and metastatic castration-resistant prostate cancer (mCRPC).

(29) In an alternatively preferred embodiment, Intetumumab (CNTO-95) can be employed as the at least one pan αv integrin inhibitor in the method according to the invention, instead of Abituzumab.

(30) Said protein levels for said specific proteins are preferably at the same time negative prognostic indicating that the biologically addressed by the markers plays a role both for disease prognosis (summarized in Table 2).

(31) TABLE-US-00002 TABLE 1 Clinical outcome dependent on the respective specific protein level under Abituzumab treatment: Patients with benefit have Hazard Ratio High(er) or (HR) of Low(er) progression- plasma levels free survival Logrank Gene symbol UniProt compared to (PFS) test (Somamer ID) ID median [Cl 95%] p-value ANG P03950 Low 0.500 0.03 (SL000003) [0.272-0.916] DCN P07585 High 0.443 0.015 (SL004081) [0.235-0.832] F5 P12259 High 0.416 0.01 (SL000622) [0.219-0.790] ICAM3 P32942 High 0.427 0.0059 (SL003178) [0.239-0.766] IL1B P01584 Low 0.498 0.022 (SL001795) [0.279-0.891] LEPR P48357 Low 0.389 0.0033 (SL003184) [0.211-0.717] MAP2K2 P36507 Low 0.397 0.0023 (SL010501) [0.224-0.702] MAPK11 Q15759 Low 0.321 0.00058 (SL007453) [0.171-0.603] PIGR P01833 High 0.311 0.00046 (SL005797) [0.166-0.582] RGMB Q6NW40 Low 0.457 0.0093 (SL010468) [0.256-0.813] STK17B O94768 High 0.380 0.0078 (SL016566) [0.193-0.747] STX1A Q16623 High 0.250 0.000032 (SL004304) [0.131-0.476] TEK Q02763 High 0.508 0.03 (SL003200) [0.280-0.920] TNFRSF17 Q02223 Low 0.471 0.012 (SL004672) [0.265-0.836 

(32) TABLE-US-00003 TABLE 2 Clinical outcome, preferably determined by radiologic PFS (rPFS) dependent on the respective specific protein level under SoC treatment: High levels indicate (g)ood, or (p)oor Gene symbol UniProt prognosis under SOC (Somamer ID) ID [HR] ANG P03950 Good [0.698] (SL000003) DCN P07585 Poor [1.5] (SL004081) F5 P12259 Poor [2.11] (SL000622) ICAM3 P32942 Poor [2.60] (SL003178) IL1B P01584 Good [0.43] (SL001795) LEPR P48357 Good [0.419] (SL003184) MAP2K2 P36507 Good [0.346] (SL010501) MAPK11 Q15759 Good [0.274] (SL007453) PIGR P01833 Poor [3.44] (SL005797) RGMB Q6NW40 Good [0.393] (SL010468) STK17B O94768 Poor [4.40] (SL016566) STX1A Q16623 Poor [3.77] (SL004304) TEK Q02763 Poor [1.95] (SL003200) TNFRSF17 Q02223 Good [0.425] (SL004672)

(33) The clinical outcome of patients having tumors and/or metastases (both preferably also referred to as tumour lesions or lesions) is preferably analysed according to response (complete and partial), benefit (response and stable disease), and progressive disease. Lesions are preferably evaluated using Response Evaluation Criteria in Solid Tumors (i.e. RECIST criteria) whereby “complete response” (CR) is preferably defined as the disappearance of the target lesions; “partial response” (PR) is preferably defined as at least a 30% decrease in the sum of the longest iron metre of target lesions, preferably taking as reference the baseline sum longest diameter; “progressive disease” (PD) is preferably defined as at least a 20% increase in the sum of the longest diameter of target lesions, preferably taking as reference the smallest sum longest diameter recorded since the treatment started or the appearance of one or more new lesions; and “stable disease” (SD) is preferably defined as neither sufficient shrinkage to qualify for partial response nor sufficient increased to qualify for progressive disease, preferably taking as reference the smallest sum longest diameter since the treatment started.

(34) Preferably, the at least one pan αv integrin inhibitor, preferably Abituzumab or Intetumumab (CNTO-95), is administered to said subject in combination with one or more chemotherapeutic agents.

(35) Treatment of prostate cancer and/or metastases thereof may involve surgery (e.g. radical prostatectomy), radiation therapy including brachytherapy (prostate brachytherapy) and external beam radiation therapy, high-intensity focused ultrasound (HIFU), chemotherapy, oral chemotherapeutic drugs (Temozolomide/TMZ), cryosurgery, hormonal therapy, or combinations thereof.

(36) Most hormone dependent cancers become refractory after one to three years and resume growth despite hormone therapy. Previously considered “hormone-refractory prostate cancer” or “androgen-independent prostate cancer”, the term castration-resistant has replaced “hormone refractory” because while they are no longer responsive to castration treatment (reduction of available androgen/testosterone/DHT by chemical or surgical means), these cancers still show reliance upon hormones for androgen receptor activation. However, there are now several chemotherapeutic treatments available to treat CRPC that improve survival.

(37) Chemotherapeutics in this respect preferably include, but are not limited to docetaxel, cabazitaxel, bevacizumab, docetaxel, thalidomide and prednisone, and combinations thereof. E.g., a combination of bevacizumab, docetaxel, thalidomide and prednisone has shown clinical benefits.

(38) Chemotherapeutics in this respect preferably also include, but are not limited to, cetuximab, Panitumumab, irinotecan, vinorelbine, capecitabine, leucovorine, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil (5-FU), bevacizumab, aflibercept and regorafenib.

(39) More preferably, one or more chemotherapeutic agents, even more preferably two or more and especially one, two or three chemotherapeutic agents

(40) a) selected from the group consisting of leuproreline acetate, bicalutamide, nilutamide, triptoreline, gosereline, flutamide, cyproterone, busereline and degarelix,

(41) b) selected from the group consisting of Zoledronic acid, Pamidronic acid, Clodronate disodium, Alendronic acid and Ibandronic acid, and/or

(42) c) selected from the group consisting of Abiraterone, Abiraterone acetate, Prednisone, Enzalutamide, Radium Ra 223 dichloride, Docetaxel, Sipuleucel-T, Cabazitaxel and Mitoxantrone,

(43) are employed. This is preferred for subjects suffering from a bone metastasis disease, more preferred for subjects suffering from metastatic prostate cancer, and especially for subjects suffering from metastatic castration-resistant prostate cancer (mCRPC).

(44) A subset of subjects appears to respond to androgen signaling blocking drugs, including, but not limited to Luteinizing hormone-releasing hormone (LH-RH) agonists and/or antagonists as well as gonadotropin-releasing hormone (GnRH) agonists and/or antagonists. Luteinizing hormone-releasing hormone (LH-RH) as well as gonadotropin-releasing hormone (GnRH) are hormone therapy drugs that lower the production of testosterone in a man's body. This drop in testosterone usually slows or stops the growth of prostate cancer for a period of time. Thus, it is in many cases preferred to administer this class of compounds in connection with treatment with Abituzumab or Intetumumab (CNTO-95).

(45) Further agents that are preferably regarded as chemotherapeutics in the context of the instant invention include sipuleucel-T, abiraterone and Enzalutamide.

(46) Pain is common in metastatic cancers and especially in case of bone metastases thereof. This is also true with prostate cancer, and cancer pain related to bone metastases can be treated with bisphosphonates, medications such as opioids, and palliative radiation therapy to known metastases. Spinal cord compression can occur with metastases to the spine, and can be treated with steroids, surgery, or radiation therapy.

(47) The traditional treatments for cancer are Radiotherapy and chemotherapy, usually in combination with one another. Scientists and pharmaceutical companies are researching drugs to target different types of cancer, including metastatic bone disease.

(48) High-intensity focused ultrasound (HIFU) has CE approval for palliative care for bone metastasis. As an entirely side-effect free and non-invasive treatment, HIFU has been successfully applied in the treatment of cancer to destroy tumours of the bone, brain, breast, liver, pancreas, rectum, kidney, testes, and prostate.

(49) One treatment option for bone metastases that has to be considered is treatment with bisphosphonates, often in combination of other chemotherapeutics and/or (anti-)hormonal treatment. Bisphosphonates have shown great promise in reducing bone cancer pain, bone destruction, and tumor growth.

(50) Monthly injections of radium-223 chloride (as Xofigo, formerly called Alpharadin) have been approved by the FDA in May 2013 for castration-resistant prostate cancer (CRPC) with bone metastases.

(51) Especially preferably, the at least one pan αv integrin inhibitor, preferably Abituzumab or Intetumumab (CNTO-95), more preferably Abituzumab, is administered to said subject in combination with two or more chemotherapeutic agents, preferably referred to as standards of care (SoC).

(52) Preferred standards of care (SoC) include, but are not limited to:

(53) a) at least one LHRH agonist/antagonist, preferably selected from the group consisting of Leuproreline, Leuproreline acetate, bicalutamid, nilutamide, triptoreline, gosereline, flutamide, cyproterone, busereline and degarelix, and/or

(54) b) at least one bisphosphonate, preferably selected from the group consisting of Zoledronic acid, Pamidronic acid, Clodronate disodium, Alendronic acid and Ibandronic acid.

(55) More preferred standards of care (SoC) include, but are not limited to: a) at least one LHRH agonist/antagonist, preferably selected from the group consisting of Leuproreline, Leuproreline acetate, bicalutamid, nilutamide, triptoreline, gosereline, flutamide, cyproterone, busereline and degarelix, and/or the pharmaceutically acceptable derivatives and/or salts thereof; in combination with
b) at least one bisphosphonate, preferably selected from the group consisting of Zoledronic acid, Pamidronic acid, Clodronate disodium, Alendronic acid and Ibandronic acid,
and/or the pharmaceutically acceptable derivatives and/or salts thereof.

(56) The most preferred standard of care (SoC) includes: a) Leuproreline, Leuproreline acetate and/or pharmaceutically acceptable derivatives and/or salts thereof, in combination with b) Zoledronic acid and/or pharmaceutically acceptable derivatives and/or salts thereof.

(57) αv integrins are cell adhesion molecules involved in cell survival, proliferation, migration, and angiogenesis; they are deregulated in various cancer types, including prostate cancer (Legate K R, et al. Nat Rev Mol Cell Biol 2006; 7:20-31; Guise T A, et al. Clin Cancer Res 2006; 12:6213s-16s). Abituzumab, a humanized monoclonal IgG2 antibody, inhibits αv-integrins expressed on castrate-resistant prostate cancer (CRPC) cells, tumor vessels, and osteoclasts involved in bone metastasis (Mitjans F, et al. J Cell Sci 1995; 108:2825-38; Monnier Y, et al. Cancer Res 2008; 68:7323-31). Abituzumab demonstrated antitumor activity in in vivo CRPC models and was well tolerated in a phase I study in mCRPC patients previously treated with docetaxel (Wirth M, et al. Eur Urol 2014; 65:897-904).

(58) In an randomized, double-blind, placebo-controlled, phase II trial, a total of 180 patients were randomized 1:1:1 to receive a) standard of care (SoC), e.g. continuous treatment with a luteinizing hormone-releasing hormone agonist and bisphosphonate treatment, e.g. with Leuproreline or Leuproreline acetate and Zoledronic acid (and/or pharmaceutically acceptable derivatives and/or salts thereof) plus placebo, b) SoC as described under a) plus abituzumab 750 mg, or c) SoC as described under a) plus abituzumab 1,500 mg.

(59) Patients were treated until rPD in bone or soft tissue lesions, skeletal event, death, or unacceptable toxicity; Patients in the placebo arm who had asymptomatic or mildly symptomatic rPD on treatment could crossover to abituzumab 1,500 mg (open-label).

(60) Median PFS with abituzumab 1,500 mg was modestly longer than with abituzumab 750 mg or placebo: 4.3 (95% Cl: 2.8-6.6) vs 3.4 (95% Cl: 2.8-5.6) and 3.3 (95% Cl: 2.8-4.8) months; HR abituzumab 1,500 mg vs placebo: 0.81 (95% Cl: 0.52-1.26). Patients receiving abituzumab experienced bone progression less frequently than those receiving placebo (23% of patients receiving abituzumab had bone progression, vs 42% of those receiving SoC).

(61) Blood sampling for plasma protein analyses was scheduled pre-treatment. Plasma protein analyses (based on highly protein-specific aptamers [SomaLogic system]) were performed on samples taken from 150 patients prior to treatment in cycle 1.

(62) The original set of simultaneously determined 1,129 plasma protein levels was restricted to 888 proteins on the data level to αvoid potential bias due to cell lysis or platelet activation during plasma preparation. Nine global biomarker search analyses were carried out using different normalization procedures, data sets and biomarker dichotomization thresholds, with the aim of filtering specific proteins that are predictive biomarkers for Abituzumab therapy success. The judgement whether a distinct protein is a predictive biomarker was based on an assessment of outcome (OS or PFS) in dependence of treatment (SoC or Abituzumab) and biomarker levels (continuous levels, and dichotomized categories “high” and “low” using the median of the investigated patient population as a threshold). Statistical tests were carried out per protein to identify those proteins that can be considered as predictive. The statistical tests are prior art and comprised. Among other criteria, logrank tests on selected populations, as for example the biomarker “high” and biomarker “low” populations, for detection of differences in outcome (here OS and/or PFS) for different treatment groups (Abituzumab and SOC; threshold p<=0.05), and Cox regression models investigating dependence of outcome on the interaction effect between treatment and continuous marker levels (interaction term p<=0.05). Further, the prognosticity of the marker levels was assessed on the basis of the patient group receiving SOC therapy using logrank tests (threshold p<=0.05) for the “high” and “low” subgroups.

(63) Said specific proteins include decorin (DCN), a protein known to have a role in TGF-β biology, as do some of the αv integrins inhibited by abituzumab (Munger J S, Sheppard D. Cold Spring Harb Perspect Biol 2011; 3:a005017).

(64) Furthermore, analysis of the biological context of other markers indicated that markers related to known molecular interactions of abituzumab (bone metabolism modulation and angiogenesis) appear to predict OS and/or PFS with abituzumab therapy.

(65) Thus, plasma levels of each of the identified biomarker plasma proteins were surprisingly found to be prognostic of poor survival and predicted increased survival and/or progression free survival with abituzumab compared to SoC alone.

(66) Thus, the clinical study delivered data on the pharmacokinetics and immunogenicity of abituzumab, as well as enabled analyses in search of predictive biomarkers, and surprisingly provided specific predictive protein levels in body fluids, especially specific plasma protein levels that allow predicting the therapy outcome under treatment with at least one pan αv integrin inhibitor, preferably including the pan αv integrin inhibitor abituzumab.

(67) Abituzumab is a monoclonal anti-alpha v antibody also designated herein as DI-17E6, DI117E6, EMR62242 and/or EMD 525797). DI17E6 is an engineered specifically tailored IgG2 hybrid monoclonal antibody directed to alpha-v integrin (receptor). Cancer therapy by means of this antibody reduces side effects associated with this type of therapy, above all immune reactions, thereby reducing immunogenicity. The antibody is described in detail in WO 2009/010290, the disclosure of which is encorporated herein in its entirety.

(68) Its hypervariable regions (CDRs) derive from murine mAb 17E6 (EMD 73034). This parent mouse IgG1 antibody is described, for example by Mitjans et al. (1995; J. Cell Sci. 108, 2825) and U.S. Pat. No. 5,985,278 and EP 719 859. Mouse mAb 17E6 is produced by hybridoma cell line 272-17E6 and deposited under accession number DSM ACC2160.

(69) Its light chain domains derive from humanized monoclaonal anti-EGFR antibody 425 (matuzumab). This antibody is described in detail for example in EP 0 531 472B1, and derives from its murine counterpart 425 (mouse MAb 425, ATCC HB9629), The antibody was raised against the human A431 carcinoma cell line and found to bind to a polypeptide epitope on the external domain of the human epidermal growth factor receptor (EGFR). Matuzumab has shown in clinical trials high efficacy.

(70) Generally DI17E6 as used according to the invention comprises: (i) a CDR light and a heavy chain region deriving from mouse monoclonal anti-αv integrin antibody 17E6 (ii) a light chain framework region which is taken from humanized monoclonal anti-EGFR antibody 425, (iii) a heavy chain framework region deriving from mouse monoclonal anti-αv integrin antibody 17E6, optionally comprising one or more mutations of amino acids at specific positions, and (iv) a heavy chain constant region deriving from human IgG2 and a human constant kappa light chain region, wherein in said IgG2 domain the IgG2 hinge region was replaced by the human IgG1 hinge domain, and; wherein optionally one or more mutations within the IgG2 has been carried out.

(71) Specifically, DI17E6 (designated as “DI-17E6γ2h(N297Q)” or “EMD 525797”) as used for the treatment as claimed and in the clinical trials as described above and below, has the following amino acid sequence:

(72) TABLE-US-00004 (i) variable and constant light chain sequences (SEQ ID No. 1): DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWYQQKPGKAPKLLIY YTSKIHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTFPYTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC and (ii) variable and constant heavy chain sequences (SEQ ID No. 2): QVQLQQSGGELAKPGASVKVSCKASGYTFSSFWMHWVRQAPGQGLEWIG YINPRSGYTEYNEIFRDKATMTTDTSTSTAYMELSSLRSEDTAVYYCAS FLGRGAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT QTYTCNVDHKPSNTKVDKTVEPKSSDKTHTCPPCPAPPVAGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QAQSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK,
wherein the underlined sequences represent the variable regions with the CDRs (in bold, identical with the parent mouse antibody). The modified IgG1 hinge region is represented by EPKSSDKTHTCPPCP (SEQ ID No. 3), and AQ is a substitution within the IgG2 domain.

(73) However, as it was shown in WO 2009/010290, also variants of DI17E6 can be used according to the teaching of this invention. Thus, DI17E6 variants comprising one or more modifications within the heavy chain framework regions

(74) TABLE-US-00005 FR1: (SEQ ID No. ) QVQLQQSGAELAEPSGASVKMSCKASGYTFS FR2: (SEQ ID No. 17) WVKQRPGQGLEWIG FR3: (SEQ ID No. ) KATMTADTSSSTAYMQLSGLTSEDSAVYYCAS FR4: (SEQ ID No. 19) WGQGTSVTVSS,
wherein one or more of the bold and underlined positions are mutated, can be used in the treatment of prostate cancer patients as described. In more detail, the following position heavy chain framework region is mutated at one, more or all of the following positions can be mutated: A9, E13, M20, K38, R40, A72, S76, Q82, G85, T87, S91 and S113. These variants show the same or very similar biological activity and efficacy as compared to DI17E6 defined by its sequences above.

(75) In general, the invention as described includes also modifications and variants of the DI17E6 antibody that are functionally and/or pharmaceutically identical or similar to unmodified DI17E6, and wherein the CDR regions and heavy and light chain variable regions are at least 80%, or at least 85%, or at least 90%, or at least 95% identical in their amino acid sequence compared to the respective variable regions of DI17E6. In addition, the invention also includes modifications and variants of the DI17E6 antibody that are functionally and/or pharmaceutically identical or similar to unmodified DI17E6, and wherein the constant regions are at least 80%, or at least 85%, or at least 90%, or at least 98% identical in their amino acid sequence compared to the respective constant regions of DI17E6. Changes is the constant regions of the IgG chains of the antibody may improve specific properties like immunogenicity, ADCC, and so on.

(76) Thus, for use according the invention, also functional derivatives, biologically active variants or modifications of DI17E6 can be employed. Accordingly, in the context of the present invention, the terms “Abituzumab” and/or “DI17E6” preferably also comprise:

(77) a biologically active variant or modification thereof that comprises the CDR regions and heavy and light chain variable regions, which are 80%-95% identical in amino acid sequence compared to the variable regions of Abituzumab;

(78) a biologically active variant or modification that comprises a constant region, which is at least 80%-98% identical with the amino acid sequence compared to the constant region of Abituzumab;

(79) an antibody that comprises one or more modifications within the heavy chain framework regions

(80) TABLE-US-00006 FR1: (SEQ ID No. 16) QVQLQQSGAELAEPGASVKMSCKASGYTFS FR2: (SEQ ID No. 17) WVKQRPGQGLEWIG FR3: (SEQ ID No. 18) KATMTADTSSSTAYMQLSGLTSEDSAVYYCAS FR4: (SEQ ID No. 19) WGQGTSVTVSS,
wherein one or more of the bold and underlined positions are mutated and are different compared to the original respective sequence of abituzumab;
and/or
a modified DI17E6 antibody comprising a human IgG1 constant region instead of human IgG2, or a human IgG2 hinge region instead of the human IgG1 hinge.

(81) Intetumumab or CNTO-95 is a human monoclonal antibody, preferably used in the treatment of solid tumors. It is also an anti-αv integrin antibody, which is preferably comprising human heavy chain and human light chain variable regions comprising the amino acid sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, as shown below:

(82) TABLE-US-00007 <210> SEQ ID NO 7 <211> LENGTH: 119 <212> TYPE: PRY <213> ORGANISM: Homo sapiens <400> SEQUENCE: 7 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1               5                   10                  15 Ser Arg Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr             20                  25                  30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35                  40                  45 Ala Val Ile Ser Phe Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val     50                  55                  60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Thr Leu Tyr 65                  70                  75                  80 Leu Gln Val Asn Ile Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85                  90                  95 Ala Arg Glu Ala Arg Gly Ser Tyr Ala Phe Asp Ile Trp Gly Gln Gly             100                 105                 110 Thr Met Val Thr Val Ser Ser         115 <210> SEQ ID NO 8 <211> LENGTH: 103 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 8 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1               5                   10                  15 Glu Arg Ale Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr             20                  25                  30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Len Ile         35                  40                  45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50                  55                  60 Ser Gly Ser Gly Thr Asp Thr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65                  70                  75                  80 Gln Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro                 85              90                      95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys             100                 105
and/or

(83) TABLE-US-00008 LOCUS ABN29020 119 aa linear PAT 07-FEB-2007 DEFINITION Sequence 7 from patent US 7163681. ACCESSION ABN29020 VERSION ABN29020.1  GI:125142205 DBSOURCE accession ABN29020.1 KEYWORDS . SOURCE Unknown. ORGANISM Unknown. Unclassified. REFERENCE 1 (residues 1 to 119) AUTHORS Giles-Komar,J., Snyder,L., Trikha,M. and Nakada,M.T. TITLE Anti-integrin antibodies, compositions, methods and uses JOURNAL Patent: US 7163681-A 7 16-JAN-2007; Centocor, Inc.; Malvern, PA; US; REMARK CAMBIA Patent Lens: US 7163681 FEATURES Location/Qualifiers source 1..119 /organism=“unknown” ORIGIN  1 qvqlvesggg vvqpgrsrrl scaasgftfs rytmhwvrqa pgkglewvav isfdgsnkyy 61 vdsvkgrfti srdnsently lqvnilraed tavyycarea rgsyafdiwg qgtmvtvss // LOCUS ABN29021 108 aa linear PAT 07-FEB-2007 DEFINITION Sequence 8 from patent US 7163681. ACCESSION ABN29021 VERSION ABN29021.1 GI:125142207 DBSOURCE accession ABN29021.1 KEYWORDS . SOURCE Unknown. ORGANISM Unknown. Unclassified. REFERENCE 1  (residues 1 to 108) AUTHORS Giles-Komar,J., Snyder,L., Trikha,M. and Nakada,M.T. TITLE Anti-integrin antibodies, compositions, methods and uses JOURNAL Patent: US 7163681-A 8 16-JAN-2007; Centocor, Inc.; Malvern, PA; US; REMARK CAMBIA Patent Lens: US 7163681 FEATURES Location/Qualifiers source 1..108 /organism=“unknown” Region 2..107 /region_name=“IgV_L_kappa” /note=“Immunoglobulin (Ig) light chain, kappa type, variable (V) domain; cd04980” /db_xref=“CDD:143181” Region 8..100 /region_name=“IG_like” /note=“Immunoglobulin like; smart00410” /db_xref=“CDD:214653” Site order(12,104,106..107) /site_type=“other” /note=“intrachain domain interface” /db_xref=“CDD:143181” Site 25..27 /site_type=“other” /note=“L1 hypervariable region” /db_xref=“CDD:143181” Site order(32,49,93) /site_type=“other” /note=“antigen binding site” /db_xref=“CDD:143181” Site order (34,36,38,43,46,50,87) /site_type=“other” /note=“heterodimer interface [polypeptide binding]” /db_xref=“CDD:143181” Site 66..70 /site_type=“other” /note=“L2 hypervariable region” /db_xref=“CDD:143181” Site order(92..94,96..98) /site_type=“other” /note=“L3 hypervariable region” /db_xref=“CDD:143181” ORIGIN  1 eivltqspat lslspgerat lscrasqsvs sylawyqqkp gqaprlliyd asnratgipa 61 rfsgsgsgtd ftltisslep edfavyycqq rsnwppftfg pgtkvdik //

(84) Intetumumab is further characterised in WO02/12501 and U.S. Pat. No. 7,163,681, the disclosure of which is incorporated in their entirety into this application by reference.

(85) Preferably, also functional derivatives, biologically active variants or modifications of Intetumumab can be employed in the instant invention.

(86) For ease of use, the one or more proteins that are preferably active as biomarkers in the context of the present invention, i.e.

(87) DCN (Somamer ID: SL004081; UniProt ID: P07585),

(88) F5 (Somamer ID: SL000622; UniProt ID: P12259),

(89) ICAM3 (Somamer ID: SL003178; UniProt ID: P32942),

(90) PIGR (Somamer ID: SL005797; UniProt ID: P01833),

(91) STK17B (Somamer ID: SL016566; UniProt ID: 094768),

(92) STX1A (Somamer ID: SL004304; UniProt ID: Q16623), and

(93) TEK (Somamer ID: SL003200; UniProt ID: Q02763),

(94) and/or

(95) b) one or more proteins, selected from the group consisting of

(96) ANG (Somamer ID: SL000003; UniProt ID: P03950),

(97) IL1B (Somamer ID: SL001795; UniProt ID: P01584),

(98) LEPR (Somamer ID: SL003184; UniProt ID: P48357),

(99) MAP2K2 (Somamer ID: SL010501; UniProt ID: P36507),

(100) MAPK11 (Somamer ID: SL007453; UniProt ID: Q15759),

(101) RGMB (Somamer ID: SL010468; UniProt ID: Q6NW40), and

(102) TNFRSF17 (Somamer ID: SL004672; UniProt ID: Q02223),

(103) are preferably also referred to collectively as “specific proteins” or “said specific proteins” of the present invention,

(104) and preferably also referred to individuality as “the specific protein” or “said specific protein”.

(105) As used herein, the term “sequence homology” is understood by the ones skilled in the art, and methods for determining sequence homology are also known in the art.

(106) As used herein, sequence homology is preferably determined using the BLAST algorithm. BLAST preferably stands for Basic Local Alignment Search Tool and is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences. A BLAST search enables a researcher to compare a query sequence with a library or database of sequences, and identify library sequences that resemble the query sequence above a certain threshold. The BLAST algorithm and the computer program that implements it were developed by Stephen Altschul, Warren Gish, and David Lipman at the U.S. National Center for Biotechnology Information (NCBI), Webb Miller at the Pennsylvania State University, and Gene Myers at the University of Arizona. It is available on the web on the NCBI website. Alternative implementations include AB-BLAST (formerly known as WU-BLAST), FSA-BLAST (last updated in 2006), and ScalaBLAST.

(107) Different types of BLASTs are available according to the query sequences. For example, following the discovery of a previously unknown gene in the mouse, a scientist will typically perform a BLAST search of the human genome to see if humans carry a similar gene; BLAST will identify sequences in the human genome that resemble the mouse gene based on similarity of sequence. The BLAST algorithm and program were designed by Stephen Altschul, Warren Gish, Webb Miller, Eugene Myers, and David J. Lipman at the NIH and was published in the Journal of Molecular Biology in 1990.

(108) In the context of the present invention, the sequence homology of the proteins described herein is preferably determined on the basis of the longest local alignments generated using BLASTp.

(109) In the context of the present invention, subjects and especially human subjects are preferably also referred to as patients.

(110) As used herein, the term “about” with respect to numbers, amounts, dosings, hours, times, timings, durations, and the like, is preferably understood to mean “approximately” with respect to said numbers, amounts, dosings, hours, times, timings, durations, and the like. More Preferably, the term “about” means+/−10%, more preferably +/−5% of the given specific value with respect to numbers, amounts, dosings, hours, times, timings, durations, and the like.

(111) If not specified otherwise, amounts administered to a subject, human subject or patient given in “mg”, such as in 500 mg, 1000 mg, or the like, are preferably intended to mean the respective amounts to be administered “flat”, i.e. as a fixed dose that is not adjusted to the bodyweight and/or body surface of the respective subject, human subject or patient.

(112) If not explicitly indicated otherwise, the term “one or more” as used herein, e.g. with respect to the number of compounds, agents, cancer cotherapeutic agents, cancer chemotherapeutic agents and the like, preferably means “one or more than one” and thus preferably includes “two or more” (or “two or more than two”), “three or more” (or “three or more than three”) and/or “four more” (or “more or more than four”). Accordingly, the term “one or more” as used herein preferably includes the numbers one, two, three, four, five, six and/or higher numbers. With respect to the number of agents, cancer cotherapeutic agents, cancer chemotherapeutic agents, it especially preferably includes the numbers one, two, three, four and/or five, even more preferably the numbers one, two, three and/or four and especially the numbers one, two and/or three.

(113) Preferably, especially preferred subjects of the instant invention relate to aspects, subjects, uses, methods and/or embodiments, wherein one or more features of two or more of the herein described aspects, subjects, uses, methods and/or embodiments are combined in one subject.

(114) The invention is explained in greater detail below by means of examples. The invention can be carried out throughout the range claimed and is not restricted to the examples given here.

(115) The following examples are given in order to assist the skilled artisan to better understand the present invention by way of exemplification. The examples are not intended to limit the scope of protection conferred by the claims. The features, properties and advantages exemplified for the compounds and uses defined in the examples may be assigned to other compounds and uses not specifically described and/or defined in the examples, but falling under the scope of what is defined in the claims.

EXPERIMENTAL SECTION

Example 1

(116) PERSEUS Phase II Clinical Study

(117) c) Leuproreline, Leuproreline acetate and/or pharmaceutically acceptable derivatives and/or salts thereof,

(118) in combination with

(119) Zoledronic acid and/or pharmaceutically acceptable derivatives and/or salts thereof

(120) PERSEUS Phase II Clinical Trial

(121) In this randomized, double-blind, placebo-controlled, international phase II trial, a total of 180 patients were randomized 1:1:1 to receive

(122) a) Standard of Care (SoC), e.g. continuous treatment with a luteinizing hormone-releasing hormone agonist, preferably Leuproreline, Leuproreline acetate and/or pharmaceutically acceptable derivatives and/or salts thereof, and bisphosphonate treatment, preferably Zoledronic acid and/or pharmaceutically acceptable derivatives and/or salts thereof, plus placebo,
b) abituzumab 750 mg plus SoC, or
c) abituzumab 1,500 mg plus SoC.
Pharmacokinetic Analysis Equal numbers of patients per arm were included in the pharmacokinetic analysis subgroup. Blood sampling for pharmacokinetic assessments was scheduled at various timepoints during cycles 1, 3, 4, 5, 6, and 7 of therapy. Pharmacokinetic parameters were calculated according to standard non-compartmental methods using the program KINETICATM v4.1.1 (Innaphase).
Immunogenicity Blood sampling for immunogenicity was scheduled pre-dose in cycles 1, 3, 5 and 6, and at the end-of-treatment visit and safety follow-up visits. Generation of antibodies directed against abituzumab was evaluated centrally using a validated ELISA method.
Biomarker Analyses Archived tumor blocks or punch biopsy materials were collected to explore tumor expression of integrins and their ligands as well as proteins related to angiogenesis and the underlying disease, and their potential relationship to clinical outcomes. Availability of samples had to be confirmed at patient screening Analyses were performed using immunohistochemistry. Blood sampling for plasma protein analyses was scheduled pre-treatment. Plasma protein analyses (based on highly protein-specific aptamers [SomaLogic system]) were performed on samples taken from 150 patients prior to treatment in cycle 1 The original set of simultaneously determined 1,129 plasma protein levels was restricted to 888 proteins on the data level to αvoid potential bias due to cell lysis or platelet activation during plasma preparation Nine global biomarker search analyses were carried out using different normalization procedure, data sets and biomarker dichotomization thresholds, with the aim of filtering biomarker proteins based on data robustness independent of biological annotations. The search process comprised a set of criteria ensuring that identified proteins are significantly (p<0.05) associated with outcome (here exemplary radiologic PFS) for either the patients with low or high levels. These tests comprise, among others, logrank tests for differences in survival (here PFS) for Abituzumab-treated/untreated patients in the biomarker-low and biomarker-high groups according to the median threshold, tests for an interaction effect on outcome (here PFS) between continuous marker levels and treatment based on Cox regression models. This process identified 15 biomarker plasma proteins: DCN (Somamer ID: SL004081; UniProt ID: P07585),
F5 (Somamer ID: SL000622; UniProt ID: P12259),
ICAM3 (Somamer ID: SL003178; UniProt ID: P32942),
PIGR (Somamer ID: SL005797; UniProt ID: P01833),
STK17B (Somamer ID: SL016566; UniProt ID: 094768),
STX1A (Somamer ID: SL004304; UniProt ID: Q16623),
TEK (Somamer ID: SL003200; UniProt ID: Q02763),
ANG (Somamer ID: SL000003; UniProt ID: P03950),
IL1B (Somamer ID: SL001795; UniProt ID: P01584),
LEPR (Somamer ID: SL003184; UniProt ID: P48357),
MAP2K2 (Somamer ID: SL010501; UniProt ID: P36507),
MAPK11 (Somamer ID: SL007453; UniProt ID: Q15759),
RGMB (Somamer ID: SL010468; UniProt ID: Q6NW40), and
TNFRSF17 (Somamer ID: SL004672; UniProt ID: 002223)
Results
Biomarker Analyses IHC analysis of tumor samples has not identified any relevant biomarkers to date. The details documenting why the 14 biomarker plasma proteins identified are judged as active and whether levels above or below the median are judged as predictive are shown in Table 1, Table 2 and/or one or more of FIGS. 1 to 24. The biomarker proteins include decorin (DCN), a protein known to have a role in TGF-β biology, as do some of the αv integrins inhibited by abituzumab Furthermore, analysis of the biological context of other markers indicated that markers related to known molecular interactions of abituzumab (bone metabolism modulation and angiogenesis) appeared to predict OS with abituzumab therapy. Plasma levels of some of the identified 14 biomarker plasma proteins were prognostic under SoC of either good or poor survival and all 14 predicted increased survival with abituzumab compared to SoC alone. Table 1, Table 2 and/or one or more of FIGS. 1 to 24 show the prognostic and predictive value of the identified 14 predictive marker proteins, as for example TEK, for PFS.

Example 2

(123) Proteomic Affinity Assay Method

(124) All steps of the proteomic affinity assay are performed at room temperature unless otherwise indicated.

(125) Sample Thawing and Plating.

(126) Aliquots of 100% serum or EDTA-plasma, stored at −80° C., are thawed by incubating in a 25° C. water bath for ten minutes. After thawing the samples are stored on ice during mixing and prior to sample dilution. Samples are mixed by gentle vortexing (setting #4 on Vortex Genie, Scientific Industries) for 8 seconds. A 20% sample solution is prepared by transferring 16 μL of thawed sample into 96-well plates (Hybaid Omnitube 0.3 mL, ThermoFisher Scientific) containing 64 μL per well of the appropriate sample diluent at 4° C. Sample diluent for serum is 0.8×SB17 with 0.6 mM MgCl.sub.2, 2 mM EGTA, 2 μM Z-Block_2, 0.05% Tween and for EDTA-plasma is 0.8×SB18 with 0.8 mM MgCl.sub.2, 2 mM EGTA, 2 μM Z-Block_2, 0.05% Tween. This plate is stored on ice until the next sample dilution steps are initiated.

(127) Preparation of 10%, 1% and 0.03% SOMAmer Solutions.

(128) SOMAmers are grouped into three unique mixes. The placing of a SOMAmer within a mix is empirically determined by assaying a dilution series of serum or plasma with each SOMAmer and identifying the sample dilution that gave the largest linear range of signal. The segregation of SOMAmers and mixing with different dilutions of sample (10%, 1% or 0.03%) allow the assay to span a 10.sup.7-fold range of protein concentration. The composition of the custom SOMAmer mixes is slightly different between plasma and serum as expected due to variation in protein composition of these two media. The custom stock SOMAmer solutions for 10%, 1% and 0.03% serum and plasma are prepared and stored at 8× concentration in SB17T. For each assay run, the three 8× SOMAmer solutions are diluted separately 1:4 into SB17T to achieve 2× concentration. Each diluted SOMAmer master mix is heated to 95° C. for five minutes and then to 37° C. for 15 minutes. 55 μL of each 2× SOMAmer mix is manually pipetted into a 96-well plate resulting in three plates with 10%, 1% or 0.03% SOMAmer mixes. After mixing with sample, the final individual SOMAmer concentration ranged from 0.25-4 nM for serum, 0.5 nM for plasma.

(129) Equilibration.

(130) A 2% sample plate is prepared by diluting the 20% sample 1:10 into SB17T using the Beckman Coulter Biomek Fx.sup.P (Beckman Coulter). A 0.06% sample plate is prepared by diluting the 2% sample plate 1:31 into SB17T. The three sample dilutions are then transferred to their respective SOMAmer solutions by adding 55 μL of the sample to 55 μL of the appropriate 2× SOMAmer mix. The plates are sealed with a foil seal (Microseal ‘F’ Foil, Bio-Rad) and incubated at 37° C. for 3.5 hours.

(131) Preparation of Catch-1 Bead Plates.

(132) 133.3 μL of a 7.5% Streptavidin-agarose bead slurry in SB17T is added to each well of three pre-washed 0.45 um filter plates. Each well of beads is washed once with 200 μL SB17T using vacuum filtration to remove the wash and then resuspended in 200 μL SB17T.

(133) Catch-1 Bead Capture.

(134) All subsequent steps are performed by the Beckman Coulter Biomek Fx.sup.P robot unless otherwise noted. After the 3.5 hour equilibration, 100 μL of the 10%, 1% and 0.03% equilibration binding reactions is transferred to their respective Catch-1 Streptavidin agarose filter plates and incubated with shaking for ten minutes. Unbound solution is removed via vacuum filtration. Each set of Catch-1 beads is washed with 190 μL of 100 μM biotin in SB17T and then 190 mL of SB17T using vacuum filtration to remove the wash. 190 μL SB17T is added to each well in the Catch-1 plates and incubated with shaking for ten minutes at 25° C. The wash is removed via vacuum filtration and the bottom of the filter plates blotted to remove droplets using the on-deck blot station.

(135) Biotinylation of Proteins.

(136) An aliquot of 100 mM NHS-PEO4-biotin in DMSO is thawed at 37° C. for six minutes and diluted to 1 mM with SB17T at pH 7.25. 100 μL of the NHSPEO4-biotin is added to each well of each Catch-1 filter plate and incubated with shaking for five minutes. Each biotinylation reaction is quenched by adding 150 μL of 20 mM glycine in SB17T to the Catch-1 plates with the NHS-PEO4-biotin. Plates are incubated for one minute with shaking, vacuum filtrated, and 190 μL 20 mM glycine SB17T is added to each well in the plate. The plates are incubated for one minute, shaking before removal by vacuum filtration. 190 μL of SB17T is added to each well and removed by vacuum filtration. The wells of the Catch-1 plates are subsequently washed three times by adding 190 μL SB17T, incubating for one minute with shaking followed by vacuum filtration. After the last wash the plates are centrifuged at 1000 rpm for one minute over a 1 mL deep-well plate to remove extraneous volume before elution. Centrifugation is performed off deck.

(137) Kinetic Challenge and Photo-Cleavage.

(138) 85 μL of 10 mM dextran sulfate in SB17T is added to each well of the filter plates. The filter plates are placed onto a Thermal Shaker (Eppendorf) under a BlackRay light source and irradiated for ten minutes with shaking. The photo-cleaved solutions are sequentially eluted from each Catch-1 plate into a common deep well plate by centrifugation at 1000 rpm for one minute each.

(139) Catch-2 Bead Capture.

(140) In bulk, MyOne-Streptavidin C1 beads are washed two times for 5 minutes each with equal volume of 20 mM NaOH and three times with an equal volume of SB17T. Beads are resuspended in SB17T to a concentration of 10 mg/mL. After resuspension, 50 μL of this solution is manually pipetted into each well of a 96-well plate and stored at 4° C. until Catch-2. During Catch-2, the wash supernatant is removed via magnetic separation. All of the photo-cleaved eluate is pipetted onto the MyOne magnetic beads and incubated with shaking at 25° C. for five minutes. The supernatant is removed from the MyOne beads via magnetic separation and 75 μL of SB17T is transferred to each well. The plate is mixed for one minute at 37° C. with shaking and then 75 μL of 60% glycerol (in SB17T) at 37° C. is transferred to each well. The plate is mixed for another minute at 37° C. with shaking. The wash is removed via magnetic separation. These washes are repeated two more times. After removal of the third glycerol wash from the MyOne beads, 150 μL of SB17T is added to each well and the plates incubated at 37° C. with shaking for one minute before removal by magnetic separation. The MyOne beads are washed a final time using 150 μL SB19T with incubation for one minute, prior to magnetic separation.

(141) Catch-2 Bead Elution and Neutralization.

(142) SOMAmers are eluted from MyOne beads by incubating each well of beads with 105 μL of 100 mM CAPSO pH 10, 1 M NaCl, 0.05% Tween with shaking for five minutes. 90 μL of each eluate is transferred during magnetic separation to a new 96-well plate containing 10 μL of 500 mM HCl, 500 mM HEPES, 0.05% Tween-20, pH 7.5.

(143) Hybridization.

(144) 20 μL of each neutralized Catch-2 eluate is transferred to a new 96-well plate and 5 μL of 10× Agilent Block (Oligo aCGH/ChIP-on-chip Hybridization Kit, Large Volume, Agilent Technologies 5188-5380), containing a 10× spike of hybridization controls (10 Cy3 SOMAmers) is added to each well. After removing the plate from the robot, 25 μL of 2× Agilent Hybridization buffer (Oligo aCGH/ChIP-on-chip Hybridization Kit, Agilent Technologies) is manually pipetted to the each well of the plate containing the neutralized samples and blocking buffer. 40 μL of this solution is manually pipetted into each “well” of the hybridization gasket slide (Hybridization Gasket Slide—8 microarrays per slide format, Agilent Technologies). Custom Agilent microarray slides containing 10 probes per array complementary to 40 nucleotide selected region of each SOMAmer with a 20× dT linker are placed onto the gasket slides according to the manufacturer's protocol. Each assembly (Hybridization Chamber Kit—SureHyb enabled, Agilent Technologies) is tightly clamped and loaded into a hybridization oven for 19 hours at 60° C. rotating at 20 rpm.

(145) Post-Hybridization Washing.

(146) Approximately 400 mL Wash Buffer 1 (Oligo aCGH/ChIP-on-chip Wash Buffer 1, Agilent Technologies) is placed into each of two separate glass staining dishes. Six of the twelve slide/gasket assemblies are sequentially disassembled into the first staining dish containing Wash Buffer 1.

(147) Once disassembled, the slide is quickly transferred into a slide rack in a second staining dish containing Wash Buffer 1. The slides are incubated for five minutes in Wash Buffer 1 with mixing via magnetic stir bar. The slide rack is then transferred to the 37° C. Wash Buffer 2 (Oligo aCGH/ChIP-onchip Wash Buffer 2, Agilent Technologies) and allowed to incubate for five minutes with stirring. The slide rack is transferred to a fourth staining dish containing acetonitrile and incubated for five minutes with stirring.

(148) Microarray Imaging.

(149) The microarray slides are imaged with a microarray scanner (Agilent G2565CA Microarray Scanner System, Agilent Technologies) in the Cy3-channel at 5 μm resolution at 100% PMT setting and the XRD option enabled at 0.05. The resulting tiff images are processed using Agilent feature extraction software version 10.5.1.1 with the GE1_105_Dec08 protocol.