`
`
`o
`
`2
`
`4
`
`6
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`
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`
`~§~ Ba/F3—gp130-myogpt’30
`+ Hypervltve
`
`+ 83!F3~gp1 30~myc~gpt 3OAYY
`
`1 80
`'l 60
`140
`120
`100
`80
`60
`40
`20
`
`120
`
`100
`
`
`
`normalizedproliferationPM>
`
`
`normatizedproliferation{‘70}m
`
`normalizedproliferationPM0 <3)
`
`Blocking Constitutiveiy Active gp 736’) Signaling
`
`oncostatin M, or ciliary neurtrophic factor (19), As a control,
`34% did not
`inhibit
`the proliferation of BafFEgplSG»
`gpluOAYY cells stimulated with IL—3, indicating that 8431} spe—
`cifically blocked the activity of gpilBOAYY in Ba/nggpllhlr—
`gpl,I§GAYY-(1ells (Fig. 4D").
`
`flifiCUSSlGN
`
`
`
`Constitutive activation of the gp130~dependent transcrip-
`tion factor STATE: has been implicated in many human neo—
`Jlasitic malignancies, including multiple rnyelorna (Al, 22, 23),
`imstzatze cancer, melanoma. ovarian cancer, renal carcinoma
`(24). as well as gastric cancer (25). Artificially dimerized S'l‘; ’l‘i’l
`Dias been shown to exliil) it oncoger ic potential, and STAT3 was
`therefore designated as an oncogene (26). The lL-—6/gpl3{l sig—
`ialfng pathway is a candidate for constitutive STATE activation
`‘n tumors (27): increased STATE phosphorylation was found in
`lHCAs (2). interestingly, gplSO gene mutations were found in
`
`60% of the analyzed iHCAs. it: to ned out that these mutations
`"esulted in ligand«independent; dirnerization f gplBO receptor
`chains and constitutive STATE phosphorylationo This was the
`irst report on somatic mutation of go 130 in tumors (2), and in
`Combination with the potential to induce cytokinevindepend—
`ent cellular proliferation shown in this study, gplBO can be
`defined as an oncogene involved in. benign human tumors that
`contributes to the inflammatory phenotype (12)
`All imitations of gp130 found in lHCAs were deletions
`within the cytokine binding interface of domain '2 {33). Here, we
`analyzed a frequently occurring gpl 38 mutation (gpl EBAY 186--
`1190, gplBOA‘f‘I) found in four of '26 ll'lCA patients. Six more
`patients carried mutations from Ser—l87’—Yl9il {gpll‘iimSY}
`that: were also covered in Tyr— 186 to Tyrrlgil ('2). \We sho‘. ' that
`gplBGAYY leads to ligand-iudependent, long—term prolifera-
`tion of Ber/FE cells and constitutive STATB phosphorylation.
`nterestingly, deletion of domain 1 from gplaOAYY resulted
`
`in a signa ingincompetent receptor chain,
`ititli""ting that
`domain 1 contributes to ligandindependent receptor activa»
`tion. However, dimerization of gplBOA‘r’Y was independent of
`the presence of the D1 domain. The neutralizing anti—gp130
`mAl) BrTZ directed against. D1 did not. inhibit receptor activa—
`tion of gp l 15(thYr indicating that the up 130: hornodirnerization
`induced by ILo/lLJGR is fundanientflly different from the
`homodimerization of gplSOAYY. Horriodimerizatioi’i of the
`wild-type gplgil receptor is facilitated by contacts of $3130
`(IBM: ( do main 1?. and 3} to the binding site ll (3flL"6 an d of gpl 30
`D1 to the binding site ill of lL—é, whereas the IL~6R contacts
`lL—6 Via
`the binding site
`I
`(16).
`‘We
`speculate
`that
`ltornodirnerizatiou of gr) 1.3(3AYY is facilitated by the interaction
`of the mutated D2 (CHM) of one receptor with the DB of the
`
`’m Proliferation was measured as
` arriouritso p130 (0,0. 1,5,and 10
`
`
`indicated under ”Experimental Procedure ’As a control, Ba/FB-gpl 30»myc»-
`
`gpl30 cells were treated with l rig/ml Hyper-list) plus sgplBM. Cequal nunt»
`
`bers of Ba/F3rgpl30 cells stably transduced with gplBUA‘Wemyc were all
`
`tureo’ for 3 days in the absence of Hype " 6. and increasing amounts of
`sgpl 3OFC (0, O.l, ‘l
`, 5,and 10 ng/ml). Proliferation was measured as indicated
`under “Experiments
`rocedures.” As a control, Ba/F3—gpl30 W e treated
`
`
`with l rig/ml Hyper-iL—é and 5gp! EGFC, D, after 6 h of serum slant
`on, Eta/F3
`
`
`cells stably transduced with myo-gpl EGAYY or myogplBO welt.
`.irnulated
`for 5 min with Hyperllsfi, Hyper»lL--6
`sgpl 30Fc, or sgpl3OFc or left
`untreated. S'lAl'El phosphorylation was analyzed by Western blot. analysis.
`
`
`curlingmm}popeoIU/noq
`SIOZ‘gzannfnoisonfiAq,Ifim'oql‘mmz
`
`\
`\
`
`89
`60
`
`40
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`20
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`
`~£~ Ba/F3-gp130~myc~gp130
`+ Hyper-it~6
`wow Ba/FB—gplSngtBOctYY—myc
`
`‘~‘**—.
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`
`599130 {uglml}
`
`”AasO{\3OC:
`
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`-‘-~ Ba/Fa—gpi 30*gp'l30AVva3/C
`
`
`“i“- Ba/Fng'lBO + Hyper~it.~6
`
`
`
`4
`
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`
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`mycvgpi 30
`
`Ball???»
`myc-gpi BOAYY
`
`P‘S'i'ATB
`
`STATS
`
`Hypenlbfi
`
`—
`
`++-
`
`- ++—
`
`+
`
`+
`
`sgptBOFc
`-
`-
`+ +
`—
`—
`FEGURE 3. No inhibition of ligand-independent proliferation of Baffi-
`gp'lEO-gp’mtm‘i"! by sngD or sgplBflFc or the anti-gram!) mAb B-—T2.
`
`A, equal numbers of Ba/FB—gp’l30 cells stably transduced with myc—
`gp’l 3OAYY were cultured for 3 days in the absence of Hyper-ll 6 and increas-
`ing amounts of B—TZ (0: 0.1, 0.5,
`i, 5, and it) rig/ml). Proliferation was
`measured as indicated under "Experimental Procedures." A5 a control, Ba/FS—
`
`gpl30»myc—gpl30 cells were treated with l ng/ml Hyper»
`6 plus B»T2,
`8, equal numbers of Ba/FB—gplfio cells stably transduced Witn gol30AYY—
`myc were cultured for 3. days in the absence of Hyperrllfl and increasing
`
`APRll. 20, 2012 women: 287-NLW'BER l7
`
`\wmeyfiylék
`
`JOURN’AL OF BBLOG/CAL CHUx/TI'STRY
`
`137’49
`
`Ex. 2001 - Page414
`Ex. 2001 - Page414
`
`

`

`Blocking Constitutiveiy Active 959131} Signaling
`
`>
`
`:11:
`
`
`
`
`
`normalizedproliferation{0/11} 80
`
`
`
`"-t- BaIF3~gp180 + Hyper—11.6
`--O-- BafF3~gp1 301-9131 SOAYY—myc
`-l- - Baa’F3~gp130—L~gp1 3O
`
`13123456711910
`
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`
`
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`
`140
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`
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`
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`
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`++ +++++
`
`Ba/Fsgmso
`
`E] Ba/F3-gp1so.gp13ni\r¥~myc
`
`a Ba/F3~gp1 30119131 30
`
`13152315 119111111
`
`1L‘3 [1 11911111]
`
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`
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`+-+-+-
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`++++++
`{L53 [1 nglmi]
`
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`
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`
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`F('5UHE 4 Biological activity of $113951“! can be suppressed by the neutralizing anti-991313 mAb 15-1»: but not by 3-4713 A, equal numbers of Ba/ F3—
`gp1'30gpl 3OA‘1’Y--111yr
`
`'alis were 111l1u1'1'=o' f01 3 days.111 the absenre of Hypri r-1.1 6 a1'11l111(reasing 211110111115 of B r13 (0, 0.,1 t)5,12. and if) ,ug/‘ml‘. P1‘Eol1'e1'2'11io1'1
`
`
`wasmeasured as:111101catedunder”Expe11meritai ProceduresKAs acontrol, [323/
`~gp E 30 cei1s were treated with '0! ng/ml'.lyper
`~11 1311.1 3- R3 Eequainumbers
`of Bil/FE
`g-p_13-3_,p13OAYY11in cells were cultured for 3 days11 he presence of ILF3
`(1 1113/1110 and BR31’5 rig/m1) Proliferation was measure.las
`dicated
`
`
`under ”Experimental Procedures." A5 a control, BalFngB-G {ens were treated with i
`ng/ml 11.3 and BR3 (5 fig/m1) C equal numbers of-_"all
`11111307
`grit BOLX‘Y‘r-myc cells were. cultured for 3 days in the absence of Hyper-1L6 and incre351Ing 3111011111
`5 of 8- P4 (C1, C11, OF3, 1,F,and 11’) ,ug/mi) "1ol1’ferat1’on was
`measured as indicated under "Experimental Procedures." A5 a control, Ba/FZ-gpi 30 cells were treated with 10 rig/ml Hyper-1L 6 and BP4 (1 O pug/mi}. 1’), equal
`numbers of BalFS-gp’i3O-gp130131YY-11'1y'c cells were cultured for 3 days in the presence of ll.."3 (i rig/ml) and Bv-Pél ('10 119/111!) Proliferation was measured as
`indimterl under "Experimental Protedures."
`
`other receptor. However, future studies are needed to fully
`explore the mechanism of ligandindepen dent gplBOAYY
`receptor activation.
`Moreover, the wild—type11.113130 receptor formed stable hetl
`erodimerswit‘igal‘SOA‘r’Y,andoverexpessionotthew(«ltype
`g’lp- 30 receptor blorked 10113111111111. a..<::tiv3t1.on of go]3013.831.
`(2). A likely mechanism for this inhibitionis that interaction of
`the extracellular parts of Wtld—t‘ype and mutated gpl30ecep—
`tors resultedin inactive gplfi(i/gnlSOASY heterodirneis. Sur—
`
`prisingly, soluble gngO variants (sgt‘slSO and sgp 1. 310131;) did not
`inhibit gplBOAYY—indnced cellular proliferation. This para~
`doxical situation might be explained by a limited access of
`sgplEG in the sterical correct orientation to the cell surface
`bound gplSl) protein, which might also explain why sgpl3tl
`cannot inhihit gpl I'lliAYY-indncecl cellular proliferation.
`l-lowever, constitutive liganddndependent activation of"
`gpiEOAYY was blocked by the neutralizing anti—golBO mAb
`l’rl’d. The epitope oil’s—P41 is located within the fibronecizin type
`lil domain 4 of 11,111,130 (gpliil)-l)l), Truncation of the fibronec»
`tin/like type ill domains results in gplSO molecules devoid of
`signaling capacity ('28), and it has been speculated that the func ,
`tional role of the flhronectin type ill domains is the assembly of
`
`the transmemhrane domains in close proximi (V to allow '1 ctlvm
`tion of gp130assoriated intracellular IAKS (2 9.) lnterestingly
`BP”; has been shown to blow only gp130 signaling induced by
`1L 1 l but not by ll.6 or the other members of the 1L6 family,
`leukemia inhibitory factor, oncostatin M, and ciliary new
`rotrophic factor (19}. Cardiotrophin ‘1, card. i1) trophin- ike cyto»
`kins, and lL~27 were, however, not investigated so far (1‘9), This
`might indicate that signaling of gp lBOAYY mimics iL— i 1 signal—
`ing. Il..'l_.l was shown to promote gastric cancer via gt) 1.3-3 and
`STAT?) phosphorylation (3C!) This View is supported by the
`finding that lL~il but not 1L6 was overexpressed in ll-iCAs 1:2).
`lL—l l was, however, only overexpressed in IHCAS that did not
`harbor gpif—ifl mutations (f3), sug‘ gesting that lHCAs are,
`to
`some extent, driven hyilrll via‘wildtype go]30 After somatic
`mutation ofgol'éllinto “in “1— . 1—liltecro1stit11t1vely artive gpl 30
`Vawant the neressitv 111114 1 driven31130 signal tiansd1.11tion
`might be abrogated, resulting in (low—nregulation of lL—li
`expression levels.
`No malignant: transformation was found in li-lCAs with
`gpl 30 mutations. but two of 111 analyzed cases of malignant
`transformation of iHCAs into hepatocellul ar ca:clnoma car!
`ried 1r1u1:a1:ions in gp130 andin the ,8 catenin pathway suggest;
`
`13750 .1911\/.1J11<310(—I/K/l/ tlilmllm
`
`VLLVFW 287, NLHv’lBlR l7 APRXL 20, 2012
`
`Ex. 2001 - Page415
`Ex. 2001 - Page415
`
`

`

`ing a rare interplay of these pathways in malignant transforma
`tion (2). ncconclusion, blockade of constitutive activation of
`mutant gp130 by 134% might open a possibility to therapeuti—
`cally block g33130~iriclucecl STATS; plies;)horylation-in hepatic
`acien mas .1 mlin asubclass of hepatocellular carcinoma
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`
`the membrane
`proximal extrac ulartic-mains for activation of the signal
`’11 130. 1'. [mm 'mm‘ 1641, 273 282
`
`:xkiniolis, (.1. 13‘1on
`tub/1.), Gama, K. ...,ant1 \‘4’31'..,'.1 (2005)-.
`(
`Wl‘el' in (‘01P.
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`
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`ll cytoki
`
`1L6 and 11:63 rere
`n‘ch../'1/ml 3.01. 1‘3, 545
`ptor. 19.1: .S.’
`
`Er
`st, M, Najdovska, M, Grail, D, Lundgren-May T, Buchert, M, Tye,
`
` ' 9.15.5011,
`
`1‘. 5.,11ugh '.l\1. R, Ma
`
`
`:
`.20 .,P.1.,and1enkins,l3.1.
`1;. G, Karras,)'(7‘,1
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`7
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`810K‘gzQUnf[10Jean?Aq,Ifim'oql"Jamaa/fidniimm}popeopmioa
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`‘i")11!£.!(1lth‘ 1'95")
` 1.,, and J to
`y, 13 (l99’1)imcl 1)ii1-:l-~
`man, D, 1‘2.
`ing properties of hematopoietic cells tiansfected Mitir low-alunitv recen-
`tors for leuken '
`
`iibitory fact
`oncostatin M. and ciliarv neurotrophic
`
`factor Pmrt, Mad, 11!.(21/1'.
`‘ 11.2.4, 91,
`9 77112.3
`n, K. 1.,
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`K: of human 6
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`John, 15., am. l\<111(11, K 1 (290.3)‘ol'io
`'
`
`inger, 1,1{0
`J
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`118111'011'Op .
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`
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`ir' factor (4 NTE) re
`ed. 11:? lll‘IIPIl..lJl<1i1—f"l r'ei‘eplm'
`serve as an a receptor forCCTNF. /. Biol. Chem. 27S.9528~ 9535
`lb, Rothbauer. U, Zoignadi, K, Muylderman.,S. Schmers. A, Cardoso,
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`Ex. 2001 - Page416
`Ex. 2001 - Page416
`
`

`

`Constitutively Active Mutant gp130 Receptor Protein from Inflammatory
`Hepatocellular Adenoma Is Inhibited by an Anti-gp130 Antibody That Specifically
`Neutralizes Interleukin 11 Signaling
`Jan Sommer, Timo Effenberger, Elena Volpi, Georg H. Waetzig, Marten Bernhardt, Jan
`Suthaus, Christoph Garbers, Stefan Rose—John, Doreen M. Floss and Jiirgen Scheller
`
`J. Biol. Chem. 2012, 287:13743—13751.
`doi: 10. 1074/jbc.M1 12. 349167 originally published online March 6, 2012
`
`Access the most updated version of this article at doi: 10.1674ijbC.Nll 1234916?
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`7
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`This article cites 29 references, 10 of which can be accessed free at
`itttttm’i'www ‘jhcorg/co merit/2873"! 71133743 full htmifiref-Eist- 'E
`
`
`
`SIOZ‘gzannfnotsonfiAq,ifim'oql"trams/firingmm}papeoIU/noa
`
`Ex. 2001 - Page417
`Ex. 2001 - Page417
`
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`Electronic Acknowledgement Receipt
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`Information:
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`Ex. 2001 - Page422
`Ex. 2001 - Page422
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`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
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`DOCKET NO.: M0546.70012USOl
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`First Named Inventor:
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`Stuart Alexander Cook
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`Application No.:
`Confirmation No.2
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`15/988,463
`7597
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`Filed:
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`Art Unit:
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`May 24, 2018
`TREATMENT OF FlBROSlS
`Prema Maria Mertz
`1646
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`Certificate of Electronic Filing under 37 CFR §1.S
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`Signature:
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`lAshley A. Cerrone/
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`MAIL STOP AMENDMENT
`Commissioner for Patents
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`STATEMENT FILED PURSUANT TO THE DUTY OF
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`Ex. 2001 - Page423
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`Application No.: 15/988,463
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`Application No.: 15/988,463
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`lnventor(s)
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`Applicant(s)
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`Stuart Alexander Cook, Singapore, SINGAPORE;
`Sebastian Schaefer, Singapore, SINGAPORE;
`
`Singapore Health Services PTE LTD., Singapore, SINGAPORE;
`National University of Singapore, Singapore, SINGAPORE;
`Assignment For Published Patent Application
`Singapore Health Services PTE LTD., Singapore, SINGAPORE
`National University of Singapore, Singapore, SINGAPORE
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