IPR2016-00458, No. 1041 Exhibit - Ex 1041 Provisional App 60076788 (P.T.A.B. Jan. 14, 2016) (2024)

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`DOCKET NO. : UPN-3106
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`PATENT
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`~ A Provisional Patent Application under 37 C.F.R. 1.53(c).
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`LILLY EX. 1041 - 3/44
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`LILLY EX. 1041 - 5/44
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`undersigned.
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`Date: ~ c-rcL ~ l q ~£
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`Mark DeLuca
`Registration No. 33,229
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`Woodco*ck Washburn Kurtz
`Mackiewicz & Norris LLP
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`Philadelphia PA 19103
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`LILLY EX. 1041 - 6/44
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`PROVISIONAL APPLICATION COVER SHEET
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`This is a request for filing a PROVISIONAL APPLICATION under 37 CFR 1.53(b) (2).
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`3106
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`INVENTOR(S)/APPLICANT(S)
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`(CITY AND EITHER
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`STATE OR FOREIGN COUNTRY)
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`CITIZENSHIP
`(if known)
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`O'Rourke
`
`Greene
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`Donald
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`Mark
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`M.
`
`I.
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`Philadelphia, Pennsylvania
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`Penn Valley, Pennsylvania
`
`United
`States
`United
`States
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`INHIBITION OF A NATURALLY OCCURRING EGFR ONCOPROTEIN BY THE P185 NEU ECTODOMAIN:
`FOR SUBDOMAIN CONTRIBUTIONS TO RECEPTOR ASSEMBLY
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`IMPLICATIONS
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`TITLE OF THE INVENTION (280 characters max)
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`CORRESPONDENCE ADDRESS
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`:fr!ark DeLuca, Esq.
`~~OODco*ck WASHBURN KURTZ
`'~CKIEWICZ & NORRIS LLP
`~ne Liberty Place
`46th Floor
`o,j'hiladelphia
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`19103
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`ENCLOSED APPLICATION PARTS
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`Respectfully submitted,
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`SIGNATURE ( L.:::-::~ l ~ y ___
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`TYPED or PRINTED NAME M~a~r~k~D~e~L~u~c~a~---------------
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`Date: March 4, 1998
`REGISTRATION NO. 33,229
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`PROVISIONAL APPLICATION FILING ONLY
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`LILLY EX. 1041 - 7/44
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`INHIBITION OF A NATURALLY
`
`INHIBITION OF A NATURALLY
`OCCURRING EGFR ONCOPROTEIN BY
`OCCURRING EGFR ONCOPROTEIN BY
`THE P185 NEU ECTODOMAIN:
`THE P185 NEU ECTODOMAIN:
`IMPLICATIONS FOR SUBDOMAIN
`IMPLICATIONS FOR SUBDOMAIN
`CONTRIBUTIONS TO RECEPTOR
`CONTRIBUTIONS TO RECEPTOR
`ASSEMBLY
`
`ASSEMBLY
`
`INVENTORS: DONALD M. O'ROURKE
`INVENTORS: DONALD M. O’ROURKE
`AND MARK I. GREENE
`
`AND MARK I. GREENE
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`1
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`LILLY EX. 1041 - 8/44
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`LILLY EX. 1041 - 8/44
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`

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`Abstract
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`Mutant Epidermal Growth Factor Receptor (EGFR) oncoproteins lacking most of subdomains I and II of
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`the extracellular region, a deletion which includes most of the first of two cysteine-rich sequences,
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`have been observed in multiple human epithelial tumors, including malignant gliomas. These EGFR
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`oncoproteins, designated 6.EGFR or EGFRvlll, confer increased tumorigenicity in v1vo and are often
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`coexpressed with full-length EGFR in human tumors. We have expressed an ectodomain-derived,
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`carboxyl-terminal deletion mutant of the p185neu oncogene (T691stop) in human glioblastoma cells
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`coexpressing endogenous EGFR and activated 6.EG FR oncoproteins. The p 185neu ectodomam-derived
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`mutant forms heterodimers with 6.EGFR proteins and reduces the phosphotyrosine content and kinase
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`activity of 6.EG FR monomers. As a consequence of T691 stop neu expression and surface localization,
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`cell proliferation in conditions of full growth and reduced serum and anchorage-mdependent growth in
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`soft agar was reduced in glioblastoma cells expressing either endogenous EGFR alone or coexpressing
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`EGFR and elevated levels of 6.EGFRs. T691 stop neu mutant receptors abrogate the dramatic growth
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`advantage conferred by 6.EGFR in vivo, suggesting that physical associations primanly between
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`subdomains Ill and IV of the p185neu and EGFR ectodomains are sufficient to modulate signaling from
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`activated EGFR complexes. Receptor-based inhibitory strategies exploit the thermodynamic preference
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`for erbB ectodomains to heterodimerize, thereby creating erbB receptor assemblies which are
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`defective in signaling and do not internalize. Pharmaceuticals which mimic the p185neu ectodomain
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`may therefore have important therapeutic applications in advanced human malignancies expressing
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`erbB receptors.
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`2
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`LILLY EX. 1041 - 9/44
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`Introduction
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`Ligand-induced receptor dimerization is thought to account for the activation of eukaryot1c signaling
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`through transmembrane receptor tyrosine kinases (RTKs) (reviewed in Heldin, 1995). Alterations of
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`receptor subunits as a result of structural changes may be coupled to receptor oligomerization
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`resulting in amplification of signaling (Gadella and Jovin, 1995).
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`In addition to the binding of ligand,
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`the erbB family of eukaryotic RTKs is regulated by interdomain interactions in cis (Bertics and G1ll,
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`1985; Honegger et al., 1987; Weiner et al., 1989; Honegger et al., 1990; Wells et al., 1990; Wiley et
`
`al., 1991; Helin and Beguinot, 1991; Sorkin et al., 1991; Guy et al., 1992; Myers et al., 1992; LeVea
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`et al., 1993; Opresko et al., 1995) and trans (Wada et al., 1990; Samanta et al., 1994; Carraway and
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`Cantley, 1994; Karunagaran et al., 1995; Karunagaran et al., 1996; Pinkas et al., 1996; Worthylake
`
`and Wiley, 1997).
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`Receptor activation of the erbB family involves both hom*odimer and heterodimer assembly formation
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`(Ullrich and Schlessinger, 1990; Wada et al., 1990; Carraway and Cantley, 1994; O'Rourke et al.,
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`1997b).
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`In many cases, the formation of heterodimers between erbB family members increases
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`ligand-binding affinity and results in the formation of a more active signaling complex which
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`influences cell phenotype (Kokai et al., 1989; Wada et al., 1990; Alimandi et al., 1995; Beerli et al.,
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`1995; Pinkas et al., 1996; Pinkas et al., 1996). Using p185neu and EGF receptor mutants, we have
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`previously shown that the ectodomain alone of these erbB receptors is sufficient to allow for a
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`thermodynamically preferred heteromeric physical association and that cytoplasmic contacts in the
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`resultant dimer affect ligand affinity, signaling and phenotype (Qian et al., 1994a,b; 1995; 1996;
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`O'Rourke et al., 1997a,b). Biochemical analysis of p185neu and EGFR suggests that the consequences
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`of dimer formation between extracellular domains alone is different from the signaling resultant from
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`endodomain dimer formation (Qian et al., 1994a; 1995). p185neu ectodomain-derived mutants are
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`capable of specific trans-inhibition of EGF receptor signaling in both murine fibroblasts and primarily
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`transformed EGFR-overexpressing human cells (Qian et al., 1994a; O'Rourke et al., 1997a). The
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`active receptor complex for Neu Differentiation Factor (NDF/heregulin) appears to be either an
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`3
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`LILLY EX. 1041 - 10/44
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`erb82-erb83 or erbB2-erbB4 heterodimer, suggesting that p185neu/erbB2 funct1ons, in part, as a
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`trans-regulator of other erbB family receptor kinases (Beerli et al., 1995; Karunagaran et al., 1996;
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`Pinkas et al., 1996).
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`To further examine receptor subdomains responsible for trans regulatory interactions mediated by the
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`extracellular domain in the erbB family, we analyzed the interaction between the EGFR and
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`p 185neu/c-erbB2 in transformed cells. An EGFR oncoprotein commonly observed 1n human glial
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`neoplasms and other human epithelial malignancies (~EGFR or EGFRvlll) results from an in-frame
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`truncation involving exons 2 through 7 (amino acids 6 to 273) in the gene encoding the extracellular
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`region of the molecule resulting in the expression of truncated, constitutively phosphorylated ~EGFRs
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`of 140-155kDa (Nishikawa et al., 1994; Moscatello et al., 1995; Reist et al., 1995).
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`t..EGF
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`receptors have been observed to exist spontaneously in a dimeric form and mediate constitutive
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`signaling and oncogenic transformation of rodent fibroblasts in a ligand-independent manner, while
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`overexpressed p 170 holo-EGFRs are only weakly transforming in the presence of EGF (Haley et al.,
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`1989; Carter and Kung, 1994; Qian et al., 1994a; Moscatello et al., 1996).
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`Importantly, ~EGFR
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`oncoproteins confer a dramatic growth advantage in vivo in human glioblastoma cells (Nishikawa et a!.,
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`1994) and in murine fibroblasts (Moscatello et al., 1996).
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`Recent reports indicate that ~EGF receptors are present on the cell surface and internalize more slowly
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`than ligand-stimulated holo-EGFRs {Moscatello et al., 1 996; Huang et al., 1 997), which may increase
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`transforming efficiency of .6.EGFR oncoproteins (Chen et al., 1989; Wells et al., 1990; Masui et al.,
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`1991; Huang et al., 1997). Other mutations which functionally separate the extracellular domain
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`from the transmembrane and cytoplasmic region of RTK polypeptides have also been observed to lead to
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`spontaneous dimerization {Sorokin, 1995) and to the acquisition of transforming potential (Haley et
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`al., 1989; Sorokin, 1995), suggesting that a portion of the extracellular domain imposes a structural
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`constraint on dimer formation which is presumably removed by ligand-binding or mass action.
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`Extracellular deletions observed in .6.EGFRs or avian v-erbB oncogenes presumably facilitate dimer
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`4
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`LILLY EX. 1041 - 11/44
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`formation by mimicking the conformational changes resulting
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`from
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`llgand-bindi"'g, Soluble
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`extracellular domalna of the EGFR have been observed to oligomerlze (Kohda et al., 1 993) and
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`structural alteration In the ectodomaln can Induce spontaneous oligomerization of extracellular
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`domains, cytoplasmic domains, or both (Hurwitz at al., 1991; Mohammad! et ai .• 1993; LaVes et al.,
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`1993; Samanta et al., 1994; Sorokln, 1995; Cochran and Kim, 1996).
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`The extracellular deletion In .o.EGFR removes the majority of amino acids comprising subdomalns I and
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`II of the EGFR (see Kohda et at., 1993; Carter and Kung, 1994}, which lneludes a large portion of the
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`first (mQ\"e amino·termlnal) of two cysteine-rich sequences in the extracellular r'glon of the
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`reQeptor fSugawa at a!., 1990; Ekstrand et al., 1992; Wong et al., 1992). Subdomaln IIJ, which has
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`been repr,rtecl to confer ligand-binding properties to the EGFR (Kohcla at al., 1993), is preserved In
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`the 6EGF1R oncoprotein, although AEGFRa clo not appear to bind ligand in NIH3T3 cella (Moecetello et al.,
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`1996). Ooexpression of holo-EGFRa and .O.EGFFis has been observed In human glioblastoma and other
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`tumor sa~ples, suggesting that EGFR/6EGFR co-expressing cells may be a close correlate of human
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`disease (~ugawa at al., 1890; Nishikawa at al., 1994; Moacatelto et al., 1995}. We have expressed an
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`actodomain-darlved, carboxyl-terminal deletion mutant of the p18Sneu oncogene (TS91stop neu),
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`Jacking ~he entire kinase domain and carboxyl-terminal autophosphorylatlon altai, in human
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`glioblastQma calla coexpresslng full·length EGFR and 4EGFRs to examine whether the p185neu
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`actodomaln could associate with truncated, actodomaln-cleleted 6EGFRs and modulate 4\EGFR-medlatec:l
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`signaling
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`s
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`LILLY EX. 1041 - 12/44
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`Results
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`Expression of EGFR and p185neu mutant forms In human glioblastoma cells
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`U87MG human glioblastoma cells express elevated levels (=1 as receptors/cell) of endogenous, wtld(cid:173)
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`type EGFR (Nishikawa et al., 1994; O'Rourke et al., 1997a). Three clonal derivatives of parental
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`U87MG human glioblastoma cells were utilized for these studies: U87!T691-1 cells contain T691stop
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`neu in the U87MG background; U87MG.D.EGFR cells express elevated levels (=1 o6 receptors/cell) of
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`human D.EGFR proteins in parental U87MG cells (Nishikawa et al., 1994; Huang et al., 1997); and
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`doubly transfected U87MG.D.EGFRIT691s cells contam endogenous EGFR, D.EGFRs, and T691 stop mutant
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`neu proteins. Subclones derived from parental U87MG human glioblastoma cells notable for the
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`expression of D.EGFR and/or T691 stop neu mutant receptors are shown
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`in Fig. 1 A.
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`lmmunoprecipttating with mAb 528 (Oncogene Science) reacttve with EGFR and ll.EGFR demonstrated
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`all EGFR forms expressed in the U87MG-derived cell lines (lanes 1 ,3,5,7). D.EGFRs were identified in
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`U87MG.D.EGFR and U87MG . .1EGFR/T691 s cells only (lanes 5,7). Metabolic
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`labeling and
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`immunoprecipitating with mAb 7.16.4 reactive with the p185neu ectodomain (O'Rourke et al., 1997a)
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`allowed for the identification of T691stop mutant neu receptors of =115kDa in U87/T691-1 cells
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`(Fig. 1A, lane 4) and in U87MG.D.EGFR!T691s cells (Fig. 1A, lane 8). Flow cytometric analysis of
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`U87fT691-1 and U87MG.D.EGFR!T691s subclones with mAb 7.16.4 confirmed surface localization of
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`T691stop neu proteins (data not shown). Flow cytometric analysis also confirmed cell surface
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`localization of D.EGFRs on both U87MG.D.EGFR and U87MG.D.EGFR!T691s subclones (data not shown).
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`We have previously reported that U87MG glioblastoma cells contain negligible levels of erbB-2 or
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`erbB-3 (O'Rourke et al., 1997a).
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`lmmunoprecipitation and immunoblotting of EGFRs in U87MG.D.EGFR cells revealed the presence of
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`endogenous EGFR (170kDa) and transfected .1EGFRs running as a doublet species of Mr=140kDa and
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`::::155kDa (Nishikawa et al., 1994; Han et al., 1996; Huang et al., 1997) (Fig. 1 B). This pattern was
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`also demonstrated for U87MG.D.EGFR/T691s double transfectants. We used scanning densitometry to
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`confirm a stoichiometric ratio of .1EGFR:EGFR of 10:1 by immunoprecipitation of cell lysates using
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`6
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`LILLY EX. 1041 - 13/44
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`mAb 528 reactive with the extracellular domains of EGFR and D.EGFR, followed by immunoblotting wtth
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`a polyclonal antisera reactive with EGFR
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`in both U87MG . .:lEGFR cells
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`(Fig. 1 B) and
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`U87MG.D.EGFRfT691s cells (data not shown).
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`We demonstrated that T691stop neu ectodomain efficiently forms heterodimers with full-length, Wild(cid:173)
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`type EGFR on the surface of parental U87MG cells (Fig. 2, lane 6} and in rodent fibroblasts (D.M.
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`O'Rourke, M.l. Greene, unpublished observations), using the membrane-impermeable cross-linker
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`DTSSP (3,3'-dithiobis(sulfosuccinimidylpropionate). The p185neu ectodomain inhibited the EGF(cid:173)
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`induced downregulation of endogenous EGFR in U87MG-derived cells as determined by immunoblotting
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`(Fig. 2, lanes 1-2, 4-5). Flow cytometric analysis indicated that receptor association occurring at
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`the cell-surface mediates inhibition of EGFR, rather than endocytosis and degradation (data not shown).
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`These data are in agreement with prior studies performed in rodent fibroblasts (Qian et al., 1994a).
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`The T691stop neu ectodomain inhibited the EGF-induced phosphorylation of wild-type endogenous EGFR
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`in U87MG-derived cells (Fig. 3,
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`lanes 1-4). D.EGFR
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`is constitutively phosphorylated
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`in
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`U87MG . .:lEGFR cells, while p170 EGFR is phosphorylated in U87MG parental cells and
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`in
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`U87MG . .1EGFR cells only upon the addition of EGF (Fig. 3, lanes 1-2,5-6). Blotting with an
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`antiphosphotyrosine antibody revealed that the lower molecular weight species (p 140) of LlEGFR is
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`underphosphorylated relative to the p155 species in U87MG . .1EGFR cells (Fig. 3, lanes 5-6).
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`Modulation of 6EGFR-medlated cell growth and transformation by the T691 stop neu
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`mutant
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`Cell proliferation and transforming efficiency of U87MG-derived human glioblastoma cell lines was
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`assessed in vitro and in vivo in order to determine modulation of LlEGF receptor signaling by
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`ectodomain-derived p185neu mutant proteins. 6EGFR expression (U87MG . .:lEGFR cells) increased cell
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`proliferation in reduced serum conditions (Fig. 4A) over parental U87MG cells, which is consistent
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`with ligand-independent activation of 6EGFRs. Expression of T691 stop mutant neu proteins inhibited
`
`cell growth in reduced serum and, notably, in full-growth media in both EGFR/.1EGFR-coexpressing
`
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`glioblastoma cells and in parental U87MG cells containing endogenous EGF'R only (~ig. 4A). Of note,
`
`U87MG.~eGFR/TS91a subclones exhibited decreased cell proliferation than parental U87MG cells
`
`lacking AEGFRs In both full·grcwth media and, to a greater degree, in reduced serum conditions.
`
`AEGFR did not Increase transforming efficiency In vitro in anchorage·lndependent growth assays over
`
`parental UB7MG cells (Fig. 49). U87MG cells are primary transformed human cells containing
`
`multiple aomatic genetic alterations, including deletions of p16 (He et al., 1 995; Nishikawa at al.,
`
`1895) arjd In the putative protein tyrosine phosphatase gene, PTeN (LI at al., 1997). Soft agar
`
`growth of U87MG.AEGFR/r891a cells was reduced 41.~% and 45% compared to parents U87MG and
`
`U87MG.ll!EGFR cells, respectively.
`
`Inhibition of anchorage·lndependent growth achieved by T69, stop
`
`neu proteins was more •lgnlflcant in U87MG parental cella lacking AEGFR (mean 75~% Inhibition
`
`relative tc parental U87MG cells In three independent experiments) (Fig. 49).
`
`The AEGF receptor confers a selective growth advantage In vivo in the. U87MG cell background (Fig. 4C)
`
`(Nishikawa at al., 1994), while many studies have shown that hclo-EGFRs are nontr$nsformlng in
`
`vivo, exe1ept under defined conditions In which p1BSneu receptors are co-expressed (Clan at al ..
`
`1 992). Expression of the TEi91 stop mutant neu ectodomaln In UB7MG cells was prevlolJisly shown to
`
`preferenttally inhibit the U87MG oncogenic phenotype when compared to a form of truncated p185neu
`
`(N891 stop) which differs from T681stop by containing the protooncogenlc: transmembrane region
`
`(O'Rourke et al., 1997a). TS91atop neu protein expression abrogated the selective In vivo growth
`
`advantage mediated by 4EGFR In U87MG cella (Fig. 4C). This result was confirmed by an analysis of
`
`three adi:Uttonal UB7MG.4EGFRIT681a aubclones (data not shown}. The UB7MG • .:!!.EGFRIT691s
`
`aubclone shown exhibited growth kinetics whic;h are similar to parental U87MG calla, thc'-'gh appeared
`
`to be mere
`
`Inhibited
`
`than U87MG cells
`
`In vivo.
`
`Inhibition obaerved
`
`In vivo
`
`for all
`
`US7MG • .&EGFRIT891s aubclones was directly related 10 the stoichiometry of T691 stop mutant neu
`
`exprassic:ln (data not shown).
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`The Tl91atop ntu mutant forme hat.rcdimera with AEGFR In vivo
`
`Because of the genetic complexity of U87MG.AEGFR transfactanta and U87MG.AEGFRIT891a doubly
`
`transfected aubclonea, and the multiple hom*odlmeric and hetarodlmerlc complexes migrating at similar
`
`molaculat weights In gradient SDS-PAGE analyale. we elected to employ the thioclaavable, membrana(cid:173)
`
`Impermeable croaa·linker DTSSP to examine individual components of putative surface-localized
`
`heteromaric complexes. The mAb 528 was utilized to lmmunopreclpitate all EGFRa (WIId·ty~e and
`
`6EGFRs} which might form heteromers with mutant neu proteins. We detected eo-precipitated 6E:GFR
`
`monome{s from antl-neu lmmunocomplaxas by In vivo c:roaa-linking experiments using DTSSP.
`
`Crosa-lln~lng of U87MG.6EGFRIT691s cells and separating immunocomplexes by SOS/6-8% PAGE
`
`under reducing conditions revealed evidence of heterodlmer formation between T691stop neu proteins
`
`and the p140AE:GFR, p1554EGFR, and p170EGFA forms (Fig. SA). Moat cf the T691stao neu mutant
`
`receptors associated with the p140~EGFR form using these methode, although faint bands identifying
`
`heterodlmerlzed p1554EGFR and p170EGFR proteins ware repeatedly observed. Identification of
`
`AEGFR proteins on the cell surface of U87MG.45GFR and U87MG.AEGFR.ITf5B1s double tranafectants by
`
`flow CY\~metry supports the observations made with the membrane-Impermeable cross-linker (Fig.
`
`SA). T691atop nau mutant receptors form heterodimera with AEGF receptors on the c:all surface. The
`
`cell·surface localization of AEGFR mutant receptors has been confirmed by several independent
`
`Investigators using different methoda (Nishikawa et al., 1894; Moscatello at al., 199S, Wlkstrand et
`
`al., 1991).
`
`Blotting with an antlphosphotyroslne antibody confirmed that both &EGFR apeclea associated with
`
`T891atop nau are underphosphorylatad (Fig. SB, lane e). We ccnaiatantly demonstrated negligible
`
`phospho~roaina content for .AEGFR monomers lmmunocomplexad to T891 stop neu receptors in all
`
`exparlmanta. EGF has been observed to Increase the efficiency cf hetercdlmer formation between
`
`pHOEGFfR and the n•u ectodomaln, although this association Ia ligand-Independent (O.M. O'Rourke and
`
`M.l. Greer.e, unpublished data). EGF minimally increased the formation of AEGFR·p18Snt~u ,ctodomain
`
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`immunocomplexes, suggesting that EGF may stabilize heteromeric formation,
`
`if not ll.EGFR
`
`hom*odimeric formation (Nishikawa et al., 1994; Oian et al., 1994a; Moscatello et al., 1996).
`
`Reduction of phosphotyrosine content of .:lEGFR monomers
`
`in vivo by T691 stop neu
`
`coexpression
`
`The phosphotyrosine content for .:lEGFR monomers immunocomplexed to T691 stop neu receptors was
`
`negligible in all experiments (Fig. 5A,B; lane 6). Our results also revealed that the lower molecular
`
`weight form of .:lEGFR (140kDa) was relatively underphosphorylated as compared to the p155D.EGFR
`
`form in both U87MG . .:lEGFR cells and U87MG . .:lEGFR/T691 s double transfectants (Figs. 3, 58), as
`
`reported previously (Moscatello et al., 1995).
`
`Differences in the phosphotyrosine content of .:lEGFR monomers between U87MG . .:lEGFR cells and
`
`U87MG . .:lEGFRIT691 s cells were not observed in immunoprecipitations of larger cell lysates using
`
`mAb 528, which reacts with all EGFR forms '(Fig. 18), for in vivo cross-linking experiments (Fig.
`
`5A,B; lanes 3,5). Therefore, in order to specifically examine the phosphotyrosine content in vivo of
`
`.:lEGFRs in T691 stop neu-expressing cells, an antibody reactive with the D.EGFR only was used to
`
`precipitate .:lEGFRs from cell lysates containing reduced protein concentrations from those required to
`
`detect heterodimeric complexes. Blotting with PY-20 after immunoprecipitating with mAb tJ.124
`
`reactive with
`
`.:lEGFR only
`
`revealed several phosphoproteins
`
`in U87MG . .:lEGFR and
`
`U87MG.ll.EGFR/T691 s cells (Fig. 5C, lanes 1 ,2). The slower migrating form of .:lEGFR (155kDa) is
`
`indicated by the arrow, while the identities of the lW.Q. other .D.EGFR-associated phosphoproteins are
`
`unknown. Using these smaller protein lysates, the faster migrating form of AEGFR (140kDa) was not
`
`detected by the PY -20 antibody, indicating relatively lower phosphotyrosine content than p 155kDa.
`
`After stripping the membrane and reprobing with Ab-4 reactive with all EGFRs, both AEGFR forms are
`
`visualized in both U87MG.t.~.EGFR and U87MG.ll.EGFR/T691s double transfectants. Scanning
`
`densitometric analysis of the phosphorylation content of immunoprecipitated p 155.:lEGFR monomers in
`
`these cell lines revealed a decrease of 33.7% in U87MG.ll.EGFR/T691 s cells when compared to
`
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`
`U87MG.~EGFR cells, under conditions of full-growth (Fig. 5C). Observed constitutive differences 1n
`
`~EGFR phosphotyrosine content could therefore not be overcome by serum-containing factors. The
`
`ratio of PTyr/~EGFRs in U87MG.~EGFR cells was 1.57; this ratio in U87MG.~EGFR/T691 s cells was
`
`found to be 1.04, as determined by scanning densitometry. This difference was observed in two
`
`additional experiments.
`
`We analyzed the phosphotyrosine content in living cells of total immunoprecipitated ~EGFR monomers,
`
`not only ~EGFR monomers immunocomplexed to T691 stop neu prote1ns. Thus, 1n addition to the finding
`
`that ~EGFRs immunocomplexed to T691 stop neu mutant receptors have negligible phosphotyrosme
`
`content (Fig. 58, lane 6), these data indicate that T691 stop neu surface expression alone is sufficient
`
`to reduce ~EGFR monomeric phosphotyrosine content in trans.
`
`In T691stop neu-containing cells, the
`
`observed 33.7% reduction in phosphotyrosine content of immunoprecipitated ~EGFR monomers may
`
`diminish signaling from the activated ~EGF receptor complex, since the signaling complex may be a
`
`higher order multimer (O'Rourke et at., 1997a) and ~EGFR has been reported to have a lower
`
`stoichiometry of phosphotyrosine content than EGF-stimulated wild-type EGFR (Huang et al., 1997).
`
`Substrate binding and/or catalytic activity of the ~EGFR receptor kinase could be altered by a reduction
`
`of monomeric ~EGFR phosphotyrosine content. The lower level of constitutive phosphotyrosine content
`
`of ~EGFR relative to ligand-stimulated wild-type EGFR may account for the disabling effect on in vivo
`
`growth behavior of individual point mutations in terminal autophosphorylation sites of ~EGFR (Huang
`
`et al., 1997}. Hetero-oligomers formed by the association between ~EGFR dimers and T691stop neu
`
`dimers may be one mechanism for the reduction of ~EGFR phosphotyrosine content and the phenotypic
`
`inhibition resulting from T691 stop neu expression and surface localization.
`
`Reduction of In vitro kinase activity of ~EGFR by T691 stop neu expression
`
`Since we observed a reduction of monomeric ~EGFR phosphotyrosine content in cells expressing
`
`T691 stop neu mutant receptors (Fig. SC), we investigated whether the catalytic activity of the ~EGFR
`
`receptor kinase could be altered by T691stop neu protein expression. Using conditions identical to
`
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`LILLY EX. 1041 - 18/44
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`

`
`those which confirmed the presence of ~EGFRs in cross-linked T691stop neu-associated heterodimers
`
`(Fig. SA), we showed that anti-~EGFR immune complexes had increased in vitro kinase