'
`
`_u . DOCKET NO. : UPN-3106
`n
`"'
`~ -.1
`<J
`(..>
`""'O_c:
`~ .
`~ !'l
`
`~()\)
`
`PATENT
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`00
`
`~
`>-i
`0
`
`In Re Application of:
`
`Donald M. O'Rourke and Mark I. Greene
`
`Serial No.:
`
`Filing Date: Herewith
`
`Group Art Unit:
`
`Examiner:
`
`For:
`
`INHIBITION OF A NATURALLY OCCURRING EGFR ONCOPROTEIN
`BY THE P185 NEU ECTODOMAIN: IMPLICATIONS FOR SUBDOMAIN
`CONTRIBUTIONS TO RECEPTOR ASSEMBLY
`
`EXPRESS MAIL LABEL NO: EL065978878US
`DATE OF DEPOSIT: MARCH 4, 1998
`
`Box 0 Patent Application
`~ Provisional 0 Design 0 Sequence
`
`Assistant Commissioner for Patents
`Washington DC 20231
`
`Sir:
`
`0
`
`PATENT APPLICATION TRANSMITTAL LETTER
`
`Transmitted herewith for filing, please find
`
`A Utility Patent Application under 37 C.F.R. 1.53(b).
`
`If this is a continuing application, please check appropriate box:
`
`0 continuation 0 divisional 0 continuation-in-part of prior application number
`_/ ___ _
`
`K \U\FORMS\TRA:-l'SMIT\APPLN 1NS
`
`LILLY EX. 1041 - 1/44
`
`
`
`DOCKET NO. : UPN-3106
`
`-2-
`
`PATENT
`
`~ A Provisional Patent Application under 37 C.F.R. 1.53(c).
`D
`A Design Patent Application (submitted in duplicate).
`
`Including the following:
`
`~ Provisional Application Cover Sheet.
`
`~ New or Revised Specification, including pages 1 to lL containing:
`
`~ Specification
`D
`Claims
`~
`D
`
`Abstract
`
`Substitute Specification, including Claims and Abstract.
`
`D
`
`D
`
`The present application is a continuation application of Application
`No.
`filed
`. The present application includes the
`Specification of the parent application which has been revised in
`accordance with the amendments filed in the parent application. Since
`none of those amendments incorporate new matter into the parent
`application, the present revised Specification also does not include new
`matter.
`
`The present application is a continuation application of Application
`No.
`filed
`, which in turn is a continuation-in-part of
`Application No.
`filed
`. The present application
`includes the Specification of the parent application which has been
`revised in accordance with the amendments filed in the parent
`application. Although the amendments in the parent C-1-P application
`may have incorporated new matter, since those are the only revisions
`included in the present application, the present application includes no
`new matter in relation to the parent application.
`
`D
`
`D
`
`to
`Filed
`A copy of earlier application Serial No.
`which no new matter has been added TOGETHER WITH a copy of the executed oath
`or declaration for such earlier application and all drawings and appendices. Such
`earlier application is hereby incorporated into the present application by reference.
`
`Please enter the following amendment to the Specification under the Cross-Reference
`
`K \U\FORMS\TRANSMIT\APPLN.TNS
`
`LILLY EX. 1041 - 2/44
`
`
`
`DOCKET NO. : UPN-3106
`
`-3-
`
`PATENT
`
`to Related Applications section (or create such a section) :"This Application is a
`D continuation or D divisional of Application Serial No. - - - - - - - - -
`filed ____________________________ _
`
`D
`
`D
`
`181
`
`D
`
`D
`
`D
`
`D
`
`D
`
`Signed Statement attached deleting inventor(s) named in the prior application.
`
`"
`
`A Preliminary Amendment.
`
`Four (4) Sheets of D Formal 181 Informal Drawings.
`
`Petition to Accept Photographic Drawings.
`
`D
`
`Petition Fee
`
`An 0 Executed D Unexecuted Declaration or Oath and Power of Attorney.
`
`An Associate Power of Attorney.
`
`AnD Executed D Copy ofExecuted Assignment of the Invention to ____ _
`
`D A Recordation Form Cover Sheet.
`D Recordation Fee- $40.00.
`Priority is claimed under 35 U.S.C. § 119 of application Serial No. ____ filed
`______ in
`(country).
`D
`
`A Certified Copy of each of the following applications for which priority is
`claimed:
`D is enclosed.
`D has been filed in prior application Serial No.
`
`filed __ _
`
`181 AnD Executed 181 Unexecuted D Statement Claiming Small Entity Status under 37
`C.P.R. 1.9 and 1.27
`181 is enclosed.
`
`K \U\FORMS\TRANSMIT\APPLN TNS
`
`LILLY EX. 1041 - 3/44
`
`
`
`-.
`
`DOCKET NO. : UPN-3106
`
`-4-
`
`PATENT
`
`D has been filed in prior application Serial No.
`said status is still proper and desired in present case.
`
`filed _ _ _
`
`Diskette Containing DNA/ Amino Acid Sequence Information.
`
`Statement to Support Submission ofDNA/Amino Acid Sequence Information.
`
`Letter of Reference to Computer Readable Form.
`
`Information Disclosure Statement.
`D
`D
`
`Attached Form 1449.
`
`Copies of each of the references listed on the attached Form PT0-1449 are
`enclosed herewith.
`
`A copy of Petition for Extension ofTime as filed in the prior case.
`
`Appended Material as follows:
`
`Return Receipt Postcard (should be specifically itemized).
`
`Other as follows: - - - - - - - - - - - - - - - - - - - - - - - - -
`
`of the prior application before
`Cancel in this application original claims
`calculating the filing fee. (At least one original independent claim must be retained
`for filing purposes.)
`
`D
`
`D
`
`D
`
`D
`
`D
`
`D
`
`D
`
`D
`
`K·IU\FORMSITRANSMIT\APPLN.TNS
`
`LILLY EX. 1041 - 4/44
`
`
`
`..
`
`DOCKET NO. : UPN-3106
`
`-5-
`
`PATENT
`
`FEE CALCULATION
`
`SMALL ENTITY
`
`NOT SMALL ENTITY
`
`RATE
`
`FEE
`
`RATE
`
`FEE
`
`PROVISIONAL APPLICATION
`
`DESIGN APPLICATION
`
`UTILITY APPLICATIONS BASE FEE
`
`UTILITY APPLICATION; ALL CLAIMS
`CALCULATED AFTER ENTRY OF ALL
`AMENDMENTS
`
`No. Filed
`
`No. Extra
`
`-20 =
`
`-3 =
`
`$75.00
`
`$165.00
`
`$395.00
`
`$1 I each
`
`$41 each
`
`FIRST PRESENTATION OF MULTIPLE
`DEPENDENT CLAIM
`
`Sl35
`
`ADDITIONAL FILING FEE
`
`TOTAL FILING FEE DUE
`
`$75.00
`
`$
`
`$
`
`s
`
`s
`
`$
`
`$
`
`$75.00
`
`$150.00
`
`$330.00
`
`$790.00
`
`$22 each
`
`$82 each
`
`$270
`
`$
`
`$
`s
`
`$
`
`$
`
`$
`
`$
`
`$
`
`A Check is enclosed in the amount of$75.00
`
`The Commissioner is hereby requested to grant an extension of time for the
`appropriate length of time, should one be necessary, in connection with this filing or
`any future filing submitted to the U.S. Patent and Trademark Office in the above(cid:173)
`identified application during the pendency of this application. The Commissioner is
`further authorized to charge any fees related to any such extension of time to deposit
`account 23-3050. This sheet is provided in duplicate.
`
`The Commissioner is authorized to charge payment of the following fees and to
`refund any overpayment associated with this communication or during the pendency
`of this application to deposit account 23-3050. This sheet is provided in duplicate.
`
`D
`181
`
`181
`
`The foregoing amount due.
`
`Any additional filing fees required, including fees for the presentation of extra
`claims under 37 C.F.R. 1.16.
`
`Any additional patent application processing fees under 37 C.F .R. 1.17 or
`1.20(d).
`
`K \U\FORMS\TRANSMIT\APPLN TNS
`
`LILLY EX. 1041 - 5/44
`
`
`
`D
`
`The issue fee set in 37 C.F.R. 1.18 at the mailing ofthe Notice of Allowance.
`
`SHOULD ANY DEFICIENCIES APPEAR with respect to this application, including
`deficiencies in payment of fees, missing parts of the application or otherwise, the United
`States Patent and Trademark Office is respectfully requested to promptly notify the
`undersigned.
`
`Date: ~ c-rcL ~ l q ~£
`
`Mark DeLuca
`Registration No. 33,229
`
`Woodco*ck Washburn Kurtz
`Mackiewicz & Norris LLP
`One Liberty Place - 46th Floor
`Philadelphia PA 19103
`Telephone: (215) 568-3100
`Facsimile: (215) 568-3439
`
`0 1997 \¥WKI\.1N
`
`LILLY EX. 1041 - 6/44
`
`
`
`PROVISIONAL APPLICATION COVER SHEET
`
`This is a request for filing a PROVISIONAL APPLICATION under 37 CFR 1.53(b) (2).
`
`Docket
`Number
`UPN-
`3106
`
`Type a plus
`sign (+)
`inside this
`box~
`
`+
`
`INVENTOR(S)/APPLICANT(S)
`
`LAST NAME
`
`FIRST NAME
`
`MIDDLE
`INITIAL
`
`(CITY AND EITHER
`RESIDENCE
`STATE OR FOREIGN COUNTRY)
`
`CITIZENSHIP
`(if known)
`
`O'Rourke
`
`Greene
`
`Donald
`
`Mark
`
`M.
`
`I.
`
`Philadelphia, Pennsylvania
`
`Penn Valley, Pennsylvania
`
`United
`States
`United
`States
`
`INHIBITION OF A NATURALLY OCCURRING EGFR ONCOPROTEIN BY THE P185 NEU ECTODOMAIN:
`FOR SUBDOMAIN CONTRIBUTIONS TO RECEPTOR ASSEMBLY
`
`IMPLICATIONS
`
`TITLE OF THE INVENTION (280 characters max)
`
`CORRESPONDENCE ADDRESS
`
`:fr!ark DeLuca, Esq.
`~~OODco*ck WASHBURN KURTZ
`'~CKIEWICZ & NORRIS LLP
`~ne Liberty Place
`46th Floor
`o,j'hiladelphia
`l"'i"!
`
`'*'"~:
`,.
`
`STATE
`
`PA
`
`ZIP
`CODE
`
`19103
`
`COUNTRY
`
`U.S.A.
`
`~-
`
`(check all that apply)
`
`ENCLOSED APPLICATION PARTS
`:
`~ Specification Number of Pages 11
`Number of Sheets .1
`A~ Drawing(s)
`
`XX
`-
`
`Small Entity Statement
`Other (specify)
`
`~~;
`
`,•";::!'
`
`METHOD OF PAYMENT (check one)
`·-·
`:}ex A check or money order is enclosed to cover the Provisional
`~·r filing fee
`
`-
`
`The Commissioner is hereby authorized to charge filing fee
`and credit Deposit Account Number:
`23-3050
`
`PROVISIONAL
`FILING FEE
`AMOUNT
`($)75.00
`
`The invention was made by an agency of the Un1ted States Government or under a contract with a~ age~cy of the Un1ted States
`Government.
`
`XX No.
`
`Yes, the name of the U.S. Government agency and the Government contract number are:
`
`Respectfully submitted,
`
`SIGNATURE ( L.:::-::~ l ~ y ___
`
`TYPED or PRINTED NAME M~a~r~k~D~e~L~u~c~a~---------------
`
`Date: March 4, 1998
`REGISTRATION NO. 33,229
`(if appropriate)
`
`Additional inventors are being named on separately numbered sheets attached hereto.
`
`PROVISIONAL APPLICATION FILING ONLY
`
`LILLY EX. 1041 - 7/44
`
`
`
`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
`
`1
`
`LILLY EX. 1041 - 8/44
`
`LILLY EX. 1041 - 8/44
`
`
`
`Abstract
`
`Mutant Epidermal Growth Factor Receptor (EGFR) oncoproteins lacking most of subdomains I and II of
`
`the extracellular region, a deletion which includes most of the first of two cysteine-rich sequences,
`
`have been observed in multiple human epithelial tumors, including malignant gliomas. These EGFR
`
`oncoproteins, designated 6.EGFR or EGFRvlll, confer increased tumorigenicity in v1vo and are often
`
`coexpressed with full-length EGFR in human tumors. We have expressed an ectodomain-derived,
`
`carboxyl-terminal deletion mutant of the p185neu oncogene (T691stop) in human glioblastoma cells
`
`coexpressing endogenous EGFR and activated 6.EG FR oncoproteins. The p 185neu ectodomam-derived
`
`mutant forms heterodimers with 6.EGFR proteins and reduces the phosphotyrosine content and kinase
`
`activity of 6.EG FR monomers. As a consequence of T691 stop neu expression and surface localization,
`
`cell proliferation in conditions of full growth and reduced serum and anchorage-mdependent growth in
`
`soft agar was reduced in glioblastoma cells expressing either endogenous EGFR alone or coexpressing
`
`EGFR and elevated levels of 6.EGFRs. T691 stop neu mutant receptors abrogate the dramatic growth
`
`advantage conferred by 6.EGFR in vivo, suggesting that physical associations primanly between
`
`subdomains Ill and IV of the p185neu and EGFR ectodomains are sufficient to modulate signaling from
`
`activated EGFR complexes. Receptor-based inhibitory strategies exploit the thermodynamic preference
`
`for erbB ectodomains to heterodimerize, thereby creating erbB receptor assemblies which are
`
`defective in signaling and do not internalize. Pharmaceuticals which mimic the p185neu ectodomain
`
`may therefore have important therapeutic applications in advanced human malignancies expressing
`
`erbB receptors.
`
`2
`
`LILLY EX. 1041 - 9/44
`
`
`
`Introduction
`
`Ligand-induced receptor dimerization is thought to account for the activation of eukaryot1c signaling
`
`through transmembrane receptor tyrosine kinases (RTKs) (reviewed in Heldin, 1995). Alterations of
`
`receptor subunits as a result of structural changes may be coupled to receptor oligomerization
`
`resulting in amplification of signaling (Gadella and Jovin, 1995).
`
`In addition to the binding of ligand,
`
`the erbB family of eukaryotic RTKs is regulated by interdomain interactions in cis (Bertics and G1ll,
`
`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
`
`et al., 1993; Opresko et al., 1995) and trans (Wada et al., 1990; Samanta et al., 1994; Carraway and
`
`Cantley, 1994; Karunagaran et al., 1995; Karunagaran et al., 1996; Pinkas et al., 1996; Worthylake
`
`and Wiley, 1997).
`
`Receptor activation of the erbB family involves both hom*odimer and heterodimer assembly formation
`
`(Ullrich and Schlessinger, 1990; Wada et al., 1990; Carraway and Cantley, 1994; O'Rourke et al.,
`
`1997b).
`
`In many cases, the formation of heterodimers between erbB family members increases
`
`ligand-binding affinity and results in the formation of a more active signaling complex which
`
`influences cell phenotype (Kokai et al., 1989; Wada et al., 1990; Alimandi et al., 1995; Beerli et al.,
`
`1995; Pinkas et al., 1996; Pinkas et al., 1996). Using p185neu and EGF receptor mutants, we have
`
`previously shown that the ectodomain alone of these erbB receptors is sufficient to allow for a
`
`thermodynamically preferred heteromeric physical association and that cytoplasmic contacts in the
`
`resultant dimer affect ligand affinity, signaling and phenotype (Qian et al., 1994a,b; 1995; 1996;
`
`O'Rourke et al., 1997a,b). Biochemical analysis of p185neu and EGFR suggests that the consequences
`
`of dimer formation between extracellular domains alone is different from the signaling resultant from
`
`endodomain dimer formation (Qian et al., 1994a; 1995). p185neu ectodomain-derived mutants are
`
`capable of specific trans-inhibition of EGF receptor signaling in both murine fibroblasts and primarily
`
`transformed EGFR-overexpressing human cells (Qian et al., 1994a; O'Rourke et al., 1997a). The
`
`active receptor complex for Neu Differentiation Factor (NDF/heregulin) appears to be either an
`
`3
`
`LILLY EX. 1041 - 10/44
`
`
`
`erb82-erb83 or erbB2-erbB4 heterodimer, suggesting that p185neu/erbB2 funct1ons, in part, as a
`
`trans-regulator of other erbB family receptor kinases (Beerli et al., 1995; Karunagaran et al., 1996;
`
`Pinkas et al., 1996).
`
`To further examine receptor subdomains responsible for trans regulatory interactions mediated by the
`
`extracellular domain in the erbB family, we analyzed the interaction between the EGFR and
`
`p 185neu/c-erbB2 in transformed cells. An EGFR oncoprotein commonly observed 1n human glial
`
`neoplasms and other human epithelial malignancies (~EGFR or EGFRvlll) results from an in-frame
`
`truncation involving exons 2 through 7 (amino acids 6 to 273) in the gene encoding the extracellular
`
`region of the molecule resulting in the expression of truncated, constitutively phosphorylated ~EGFRs
`
`of 140-155kDa (Nishikawa et al., 1994; Moscatello et al., 1995; Reist et al., 1995).
`
`t..EGF
`
`receptors have been observed to exist spontaneously in a dimeric form and mediate constitutive
`
`signaling and oncogenic transformation of rodent fibroblasts in a ligand-independent manner, while
`
`overexpressed p 170 holo-EGFRs are only weakly transforming in the presence of EGF (Haley et al.,
`
`1989; Carter and Kung, 1994; Qian et al., 1994a; Moscatello et al., 1996).
`
`Importantly, ~EGFR
`
`oncoproteins confer a dramatic growth advantage in vivo in human glioblastoma cells (Nishikawa et a!.,
`
`1994) and in murine fibroblasts (Moscatello et al., 1996).
`
`Recent reports indicate that ~EGF receptors are present on the cell surface and internalize more slowly
`
`than ligand-stimulated holo-EGFRs {Moscatello et al., 1 996; Huang et al., 1 997), which may increase
`
`transforming efficiency of .6.EGFR oncoproteins (Chen et al., 1989; Wells et al., 1990; Masui et al.,
`
`1991; Huang et al., 1997). Other mutations which functionally separate the extracellular domain
`
`from the transmembrane and cytoplasmic region of RTK polypeptides have also been observed to lead to
`
`spontaneous dimerization {Sorokin, 1995) and to the acquisition of transforming potential (Haley et
`
`al., 1989; Sorokin, 1995), suggesting that a portion of the extracellular domain imposes a structural
`
`constraint on dimer formation which is presumably removed by ligand-binding or mass action.
`
`Extracellular deletions observed in .6.EGFRs or avian v-erbB oncogenes presumably facilitate dimer
`
`4
`
`LILLY EX. 1041 - 11/44
`
`
`
`formation by mimicking the conformational changes resulting
`
`from
`
`llgand-bindi"'g, Soluble
`
`extracellular domalna of the EGFR have been observed to oligomerlze (Kohda et al., 1 993) and
`
`structural alteration In the ectodomaln can Induce spontaneous oligomerization of extracellular
`
`domains, cytoplasmic domains, or both (Hurwitz at al., 1991; Mohammad! et ai .• 1993; LaVes et al.,
`
`1993; Samanta et al., 1994; Sorokln, 1995; Cochran and Kim, 1996).
`
`The extracellular deletion In .o.EGFR removes the majority of amino acids comprising subdomalns I and
`
`II of the EGFR (see Kohda et at., 1993; Carter and Kung, 1994}, which lneludes a large portion of the
`
`first (mQ\"e amino·termlnal) of two cysteine-rich sequences in the extracellular r'glon of the
`
`reQeptor fSugawa at a!., 1990; Ekstrand et al., 1992; Wong et al., 1992). Subdomaln IIJ, which has
`
`been repr,rtecl to confer ligand-binding properties to the EGFR (Kohcla at al., 1993), is preserved In
`
`the 6EGF1R oncoprotein, although AEGFRa clo not appear to bind ligand in NIH3T3 cella (Moecetello et al.,
`
`1996). Ooexpression of holo-EGFRa and .O.EGFFis has been observed In human glioblastoma and other
`
`tumor sa~ples, suggesting that EGFR/6EGFR co-expressing cells may be a close correlate of human
`
`disease (~ugawa at al., 1890; Nishikawa at al., 1994; Moacatelto et al., 1995}. We have expressed an
`
`actodomain-darlved, carboxyl-terminal deletion mutant of the p18Sneu oncogene (TS91stop neu),
`
`Jacking ~he entire kinase domain and carboxyl-terminal autophosphorylatlon altai, in human
`
`glioblastQma calla coexpresslng full·length EGFR and 4EGFRs to examine whether the p185neu
`
`actodomaln could associate with truncated, actodomaln-cleleted 6EGFRs and modulate 4\EGFR-medlatec:l
`
`signaling
`
`s
`
`LILLY EX. 1041 - 12/44
`
`
`
`Results
`
`Expression of EGFR and p185neu mutant forms In human glioblastoma cells
`
`U87MG human glioblastoma cells express elevated levels (=1 as receptors/cell) of endogenous, wtld(cid:173)
`
`type EGFR (Nishikawa et al., 1994; O'Rourke et al., 1997a). Three clonal derivatives of parental
`
`U87MG human glioblastoma cells were utilized for these studies: U87!T691-1 cells contain T691stop
`
`neu in the U87MG background; U87MG.D.EGFR cells express elevated levels (=1 o6 receptors/cell) of
`
`human D.EGFR proteins in parental U87MG cells (Nishikawa et al., 1994; Huang et al., 1997); and
`
`doubly transfected U87MG.D.EGFRIT691s cells contam endogenous EGFR, D.EGFRs, and T691 stop mutant
`
`neu proteins. Subclones derived from parental U87MG human glioblastoma cells notable for the
`
`expression of D.EGFR and/or T691 stop neu mutant receptors are shown
`
`in Fig. 1 A.
`
`lmmunoprecipttating with mAb 528 (Oncogene Science) reacttve with EGFR and ll.EGFR demonstrated
`
`all EGFR forms expressed in the U87MG-derived cell lines (lanes 1 ,3,5,7). D.EGFRs were identified in
`
`U87MG.D.EGFR and U87MG . .1EGFR/T691 s cells only (lanes 5,7). Metabolic
`
`labeling and
`
`immunoprecipitating with mAb 7.16.4 reactive with the p185neu ectodomain (O'Rourke et al., 1997a)
`
`allowed for the identification of T691stop mutant neu receptors of =115kDa in U87/T691-1 cells
`
`(Fig. 1A, lane 4) and in U87MG.D.EGFR!T691s cells (Fig. 1A, lane 8). Flow cytometric analysis of
`
`U87fT691-1 and U87MG.D.EGFR!T691s subclones with mAb 7.16.4 confirmed surface localization of
`
`T691stop neu proteins (data not shown). Flow cytometric analysis also confirmed cell surface
`
`localization of D.EGFRs on both U87MG.D.EGFR and U87MG.D.EGFR!T691s subclones (data not shown).
`
`We have previously reported that U87MG glioblastoma cells contain negligible levels of erbB-2 or
`
`erbB-3 (O'Rourke et al., 1997a).
`
`lmmunoprecipitation and immunoblotting of EGFRs in U87MG.D.EGFR cells revealed the presence of
`
`endogenous EGFR (170kDa) and transfected .1EGFRs running as a doublet species of Mr=140kDa and
`
`::::155kDa (Nishikawa et al., 1994; Han et al., 1996; Huang et al., 1997) (Fig. 1 B). This pattern was
`
`also demonstrated for U87MG.D.EGFR/T691s double transfectants. We used scanning densitometry to
`
`confirm a stoichiometric ratio of .1EGFR:EGFR of 10:1 by immunoprecipitation of cell lysates using
`
`6
`
`LILLY EX. 1041 - 13/44
`
`
`
`mAb 528 reactive with the extracellular domains of EGFR and D.EGFR, followed by immunoblotting wtth
`
`a polyclonal antisera reactive with EGFR
`
`in both U87MG . .:lEGFR cells
`
`(Fig. 1 B) and
`
`U87MG.D.EGFRfT691s cells (data not shown).
`
`We demonstrated that T691stop neu ectodomain efficiently forms heterodimers with full-length, Wild(cid:173)
`
`type EGFR on the surface of parental U87MG cells (Fig. 2, lane 6} and in rodent fibroblasts (D.M.
`
`O'Rourke, M.l. Greene, unpublished observations), using the membrane-impermeable cross-linker
`
`DTSSP (3,3'-dithiobis(sulfosuccinimidylpropionate). The p185neu ectodomain inhibited the EGF(cid:173)
`
`induced downregulation of endogenous EGFR in U87MG-derived cells as determined by immunoblotting
`
`(Fig. 2, lanes 1-2, 4-5). Flow cytometric analysis indicated that receptor association occurring at
`
`the cell-surface mediates inhibition of EGFR, rather than endocytosis and degradation (data not shown).
`
`These data are in agreement with prior studies performed in rodent fibroblasts (Qian et al., 1994a).
`
`The T691stop neu ectodomain inhibited the EGF-induced phosphorylation of wild-type endogenous EGFR
`
`in U87MG-derived cells (Fig. 3,
`
`lanes 1-4). D.EGFR
`
`is constitutively phosphorylated
`
`in
`
`U87MG . .:lEGFR cells, while p170 EGFR is phosphorylated in U87MG parental cells and
`
`in
`
`U87MG . .1EGFR cells only upon the addition of EGF (Fig. 3, lanes 1-2,5-6). Blotting with an
`
`antiphosphotyrosine antibody revealed that the lower molecular weight species (p 140) of LlEGFR is
`
`underphosphorylated relative to the p155 species in U87MG . .1EGFR cells (Fig. 3, lanes 5-6).
`
`Modulation of 6EGFR-medlated cell growth and transformation by the T691 stop neu
`
`mutant
`
`Cell proliferation and transforming efficiency of U87MG-derived human glioblastoma cell lines was
`
`assessed in vitro and in vivo in order to determine modulation of LlEGF receptor signaling by
`
`ectodomain-derived p185neu mutant proteins. 6EGFR expression (U87MG . .:lEGFR cells) increased cell
`
`proliferation in reduced serum conditions (Fig. 4A) over parental U87MG cells, which is consistent
`
`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
`
`7
`
`LILLY EX. 1041 - 14/44
`
`
`
`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).
`
`8
`
`LILLY EX. 1041 - 15/44
`
`
`
`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
`
`9
`
`LILLY EX. 1041 - 16/44
`
`
`
`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
`
`10
`
`LILLY EX. 1041 - 17/44
`
`
`
`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
`
`11
`
`LILLY EX. 1041 - 18/44
`
`
`
`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