l
`
`
`
`N4 iiiiil’ioot
`
`American Journal OfObStCtI‘ICS and Gyl’lCCOIOgy
`Founded in 1920
`
`-
`
`(I
`.
`
`r
`\fl
`
`Copyright 53> /993 by ll/Iosbyil’em' Book, Inc.
`
`
`
`January
`Part 1
`1993
`
`CLINICAL SECTION
`
`
`I Clinical Opinion
`
`Reporting the results of cystic fibrosis carrier screening
`David A. Aseh, MD, MBA. James P. t’atton, MD, MBA, John C. Hershey, Phl), and
`Michael T. Mcnnuti, MD
`Philadelphia, Pennsylvania
`
`The 85% detection rate of cystic fibrosis carriers otl‘ers several alternative ways to report the
`results of carrier screening and to act on that information.
`
`Diagnosis of fetal infection in the patient with an ultrasonographically
`detected abnormality but a negative clinical history
`Carl P. Weincr7 MD, Charles F. Gross, MD, and Stanley J. Nairlcs, Ml)
`Iowa City, Iowa
`
`Many ultr'asonographic abnormalities indicate an antenatal evaluation for inl‘cction.
`
`I Clinical Articles
`
`The reliability of ultrasonography in the management of spontaneous
`abortion, clinically thought to be complete: A prospective study
`Marvin C. Rutin, MD, Susan G. Bernstein, MD, and Joseph D. Campbell, M l.)
`I’iltrburg/I,
`l’emu‘ylvmria
`
`Ultrasonngr‘aphic examination is highly reliable in the management of spontaneous abortion,
`clinically thought to be complete.
`
`1
`
`6
`
`12
`
`(Contents continued on page 5A)
`
`I, Part 1, januur} 1993 The American Journal oi Obstetrics and (i'i'nrrr‘nlogy (ISSN “(KB—937M is published monthly,
`I68, No.
`\‘ol
`ext-opt seruirnontlriy in janriru) (thirteen issues per year l. by Aluxln—Ycar Bunk. Inr'.v lIHflO \\’r'.\t|irir- industrial |)ri\(‘, St. Louis, ,\|()
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`‘3‘
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`i513 .
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`IMMUNOGEN 2037, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2037, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`BASIC SCIENCE SECTION
`
`The effect of antibodies and immunotoxins reactive with
`
`HER-Z/neu on growth of ovarian and breast cancer
`
`cellJinesi"
`
`Gustavo C. Rodriguez, MD," Matthew P. Bocnte, MD," Andrew Berchuck, MD,"
`Regina S. Whitaker, MD,u Kathy C. O’Briant, BS," Fengji Xu, MD,h and
`Robert C. Bast, _]r., MD‘" ‘
`Durham, North CrIt‘IIIIIHI
`
`OBJECTIVE: Because HER-2/netl is overexpressed in one third of breast and ovarian cancers, we
`examined the effect of unconjugated monoclonal antibodies (lD-5, PB-S, TA<1) and an immunotoxin
`(TA»1—ricin) reactive with this protooncogene on the growth of breast and ovarian cancer cell lines.
`STUDY DESIGN: The tritiated thymidine incorporation assay was used to examine the effect of
`unconjugated antibodies on proliferation. A limiting dilution clonogenic assay was used to assess the
`effect of immunotoxin on cellular cytotoxicity.
`RESULTS: Scatohard analysis revealed that OVCA 420, OVCA 429 OVCA 432, and OVCA 433 cells had
`approximately 10" HER-2/neu receptors per cell, whereas the SKOvB and SKBrS cell lines expressed 10"
`and 10“ receptors per cell, respectively. Monoclonal antibody IDS caused significant inhibition of tritiated
`thymidine incorporation in SKBrS, SKOvs, and OVCA 420 cells (p < 0.002). The TA71~ricin immunotoxin
`significantly Inhibited the olonogenic growth of only SKBr3 and SKOV3 cellsi
`CONCLUSION: HER—2/neu may be a useful target for immunotherapy with unconjugated antibodies and
`immunotoxins in ovarian and breast cancers that overexpress this protooncogene.
`(AM J Ousrer GVNECOL
`1993;168:228-32.)
`
`Key words: Ovarian cancer, monoclonal antibody, IlER»2/11,(Iu oncogene, immunothcrapy
`
`The H ER-2/1Irm oncogene encodes a 185 kd ccll
`snrlitce protein (pl 85) that has I1 cysteine-rich extracel-
`lIIlzn‘ domain, a II1onIl)raIIo»spIuiIting region, and an
`intracellular domain that. has intrinsic tyrosine kinzisc
`activityil Because of its structural similarity to the epi-
`dermal growth factor receptor, l’lER-E/HL‘IL is thought to
`bc a peptide growth factor receptor. In this regard a
`ligand that binds to HER-When has been identified, but
`it has not yet been well characterized? Overcxprcssion
`of HERQ/mu, because of geneamplification, occurs in
`apponimately 2F1% Lo 30% of breast and ovarian cat—
`cinonias,‘ 'Ilnd overexprcssion of IlliR—2/neu has been
`
`From IIIe Divinan ofGynecoIogIc Oncology, Depttitiiumt ofOIIrietiIci
`mirl GyimmIngy," tIIr’ Dn/mihnent ofMerIIrIiir " lliilI [Ire DfljlfIiIIIII’III (If
`IIIIFIIJIIIIJIOg)‘ (In/I [’nllillltiolflt’fy {Hill IIH.’ DIIIM’ (tinI/IMIIIIIIrivt' CIIIIH’I
`Center, Du/Ie Uhiuerrz‘ty Medic/II Center,
`IIIl’
`Sup/Iowa] I'Ii [)mt by grams CA 39930 and CA 54116 fi'IIIII
`National Cancer Institute (Ind by AIIIeI'I'ImI C(ma'r Society CIIIIIHlI
`OIIcolagI Cmm IJKIIBIDINIII’HI Award 91-199 Io UI'. erIzucII
`Rtreitiedfm [IIIbIIrnIzon /lpIiI 14,1992jictiIxcrijIIIIe 24 1992;
`arcrplerljrmz 25,1992
`Reprint quumlx./III(II(!1U Ben/Inch, IUD, Dal/1071mm! (If Obxlehiu
`and Gynecology DIIIIL’ Untvmstt)‘
`IlIPlIlCIII Center,
`[501 3079,
`Durham, NC 27710
`6/1/4064]
`
`shown to correlate with poor survival in these cancers. "“
`It
`is
`thought
`that overexprcssion oi‘ l’ll‘lR-2/neu may
`confer :1 selective advantage to cells through augmen-
`tation oi pathways involved in stimulation of prolifcrm
`tion. In this regard transfcction olNational Institutcs of
`Health 3TB cells with the human HlfiR—Q/mm, gcnc
`results in induction and maintenance of It malignant
`phenotype.7 Conversely, antibodies that bind to and
`down-regulate cell
`surface levels of I-IER—Q/I'Ieu can
`inhibit
`the growth of cells that overexprcss this pro—
`tooncogcnc product.“ 1’
`We previously have studied a panel of antibodies
`reactive with the extracellular domain ol‘ the human
`
`HER-2mm oncogene product (13185).” In addition to
`mapping distinct antigenic epilopcs on the extracelltr
`lar domain, we demonstrated that several antibodics
`inhibited anchorage-indcpcndent growth of the SKBr?)
`breast cancer cell line, which overexpresscs p185. We
`also have shown that cell surface r1185—-antibody com~
`plexes are intetnalizcd by clathrinfgateidjits. " Ali
`though HER— 2/71611 is picscnton many n01mal coll—7T.2
`overcxprcsSion by some breast and ovarian cancers
`makes it an attractive target for iunnunothcrapy with
`monoclonal antibodies. The capacity of'13185 for inter-
`
`000279378/93 $1.00 + 0.20
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`IMMUNOGEN 2037, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
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`IIIMMIIi'de -
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`IMMUNOGEN 2037, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`‘r—-__—-———E ‘
`\'oltlllll‘ lti‘o
`Nulnbel
`1. Part
`
`l
`
`l-lEFl-Z/neu antibodies and ovarian cancer 229
`
`nalization makes it a particularly appealing target for
`immunotoxins, which must
`reach the ribosomes
`to
`exert
`their cytotoxic effect. In the culrent study, we
`have evaluated the effect ofsevcral anti-p185 monoclo—
`nal antibodies and an anti-i318?) ricin immunotoxin on
`the growth of breast and ovarian cancer cell lines that
`express different levels of p185.
`
`Material and methods
`
`Cell culture. Four epithelial ovarian cancer cell lines
`(OVCA 420, OVCA 429, OVCA 432, OVCA 1133) previ-
`ously established in our laboatory were maintained in
`monolayer culture it] tissue culture medium containing
`modified Eagle's medium (Gibco, Grand Island, N.Y.)
`supplemented with 10% h tat—inactivated fetal bovine
`serum (Gibco), sodium pyruvate, nonessential amino
`acids, glutamine, penicillin. and streptomycin. A human
`ovarian cancer cell line (SKOVB) that overcxpress HER-
`2/1Lr11t was obtained from the American Type Culture
`Collection (Rockville, Md.) and was maintained in
`monolayer culture in McCoy's medium supplemented
`with 10% fetal bovine serum. The human breast cancer
`cell line SKl’yr'd was maintained in RPM] [th0 medium
`supplemented with
`15% fetal bovine
`serum and
`2 mmol/L. L-glutamine. All cell lilies were maintained in
`a humidified incubator in an atn'losphel'e containing 5%
`carbon dioxide.
`
`Monoclonal antibodies and immunotoxins. Hybrid—
`omas known to produce murine monoclonal antibodies
`'l'A-l
`(immuuoglobulin Cl), PIS-3
`(immunoglobulin
`Gm), and ID—5 (immlluoglobulin GI), reactive Willi the
`external domain of the 185 kd human l’ll‘iR-2/‘ltr3u gene
`product were obtained from Applied Biotechnology
`(Cambridge, Mass.).':‘ Fab fragments of IDS were pre—
`pared by means of the Avidclu'om Fab kit (Bioprobe,
`Tustin, Calif). A murine monoclonal immunoglobnlin
`G. antibody not
`reactive with human cells (MOl’CZl)
`(Cetus, Iimolyville, Calif.) was used as a negative con-
`trol. M()l'(.121 does not bind to SKBrS cells, which are
`known to overexpress l’ll‘iR-Q/‘ttttll. Recolnbillantly de-
`rived ricin A chain was kindly provided by Cetus. 'l‘Ail
`was conjugated to ricin A chain, as previously described,
`by means of the imiuothiolane method (Cetus). An
`tlansl'errin
`against
`the
`antibody
`raised
`receptor
`
`
`
`(IlBLJ-Al2), obtained from Cetus, was similarly coniu~
`gated to ricin if.
`7
`7
`7,
`7
`.,
`Scatehard analysis. '1 he number of HERE/lieu mol
`ecules per cell
`ill each of
`the cancer cell
`lines was
`determined by means of Scatchard analysis. Cells
`(5 X 107‘) nerc seeded in Elli—well plates and grown to
`Confluence Cells then were incubated with 10% bovine
`
`serum albumin in Dulbecco’s modified Eagle's medium
`with 0.1% sodium azide for 1 hour at 37” (ll Iodine
`125—labeled 'l‘Ail antibody was then added to triplicate
`replicates at final concentrations of 000.3. 0.05, 0.25,
`
`0.5, and 2.5 rig/ml and incubated for 2 hours at 37° C.
`Cells were then washed four times with tissue culture
`medium, lysed with 2N sodium hydroxide, and counted
`in a Packard yeountcr. Nonspecific binding was deter
`mined at each concentration of radiolabeletl antibody
`by measuring residual radioactivity in washed wells that
`had contained media but not cells.
`lsotype—matched
`control antibody was used ill all experiments. The
`number of cells per well was counted in representative
`wells after trypsinization and staining with 0.1% tlypan
`blue.
`
`lines
`Thymidine incorporation assay. Cancer cell
`were seeded in 96—well plates (5 X 10' cells per well) in
`tissue culture meditlln containing 2% fetal bovine so
`ruin in different concentrations ol'anti~p [85 antibodies
`('l'A-I,
`ll)—5,
`ID—5 Fab, Pill-3). Alter .12 hours,
`i mCi of
`tritiated thylnidine (6.7 Ci/mmol) (New England Nu—
`clear, Wilmington, Del.) was added to Each well. Six
`hours later the culture medium was removed, and cells
`were washed with phosphate—billlbl'ed saline solution
`and solubilized with ?N sodium hydroxide. Cell-associ—
`ated radioactivity was determined by scintillation count
`ing. A nonspecific antibody (MOI’C‘Zl) was used ill each
`experiment as a negative control.
`Limiting dilution assay.
`/\
`limiting dilution clono-
`gellic assay was used to evaluate the ellcct of TA<l
`antibody and a TA-l~ricin A chain immunotoxin on
`growth of ovarian cancer cell lines." 5141518 cells were
`used as a positive control. Cell lilies were incubated at
`37° C at a comentration of l X 10” cells per milliliter
`with different concent'ations of'l"A~l antibody (0 to 10
`ug/ml) and TA—l ricin immunotoxin (0 to l() ug/inl) for
`3 hours. An equal mlinher ol‘cells
`'as incubated with a
`nonspecific antibody and with antitransl'erl'in immuno-
`toxin as controls. The cells then were washed with tissue
`
`culture lncdiilm, and serial fivefold dilutions were pre-
`pared. I\lltll|0l5 (l00 ul) of each dilution were then
`plated in each of six wells in a 96-well microtiter plate.
`An additional l00 pl of tissue culture medium was then
`added to each well, Cells were incubated for 14 days at
`37° C in 5% carbon dioxid ‘ and 95% humidified air.
`
`Clonogenic growth was then scored by visual inspection
`with an inverted phase microscope; the number ofwells
`with at
`least one surviving tunlor colony was noted.
`()nly colonies containing >30 cells were counted. Dil-
`uentilreated cultures were used as controls. Clonogcnic
`units were estimated according to the method ol'Speal -
`man and Karher.”s
`
`Results
`
`Scatchard analysis of ”Sli’l'Ail binding to the ovarian
`cancel cell lines and the SKP>r3 cell lint: revealed that
`HER-210m: was expressed by all of the cancer cell lines
`(fable 1).
`'[hc number of receptors per cell ranged
`from 1.3 X 10' to 3.2 X 10;,
`in the ovarian cancer cell
`
`
`
`IMMUNOGEN 2037, pg. 3
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2037, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

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`
`
`230 Rodriguez et al.
`
`jatiuan IEIEIII
`:\m [ Obslet Gvuecol
`
`
`
`Table I. Expression ofI-[ER-‘ZJWH in cancer
`cell lines
`*Z/JII’H
`H 'R
`Dinar iulitut rum/(nil
`
`
`
`
`
`
`
`[to [WI 11'”)
`(
`("ml/L)
`(Tr/I lim'
`r
`
`SKBrS
`4.5 X 10""
`1.2 X It)"
`SKvai
`3.2 X It)“
`2.} x 10 "
`OVCA 420
`1.3 X 10'
`0.8 X 10'”
`0ch 420
`2.3 x 10'
`0.8 x 10’”
`OVCA 432
`2.0 X It)1
`3.3 X 10‘“
`OVCA<133
`Mix 10'
`0.6 X 10'”
`
`lines, with the greatest number of receptors noted in
`the SKOVE cell line. The SKBrfi breast cancer cell line,
`which is known to tnarlLedly overexpress I'Il‘lR-Q/mu,
`had 1.2 X 10“ receptors per cell.
`In all cases linear
`Scatchard plots were obtained,
`indicative of a single
`class of high-affinity rcccplor (dissociation constant 0.6
`to 4.5 nmol/l.).
`The ”)5 antibody inhibited tritiated tliytnidine incor—
`poration oI'SKBrfi, OVCA 1120, and SKOV3 cells by 45%
`to 50% (j) < 0.002), whereas growth of OVCA 42E),
`OVCA 432, and OVCA 435 cells was not. afl'ectcd (Fig.
`l). Fab li‘agmenls ol‘ ll)—5 were similarly ell'ective in
`inhibiting cellulzu proliferation (data not shown). Anti-
`bodies "l‘A—l, I’B-E‘I, and MOPC2] did not all’ect thymi-
`dine incorporation in any ol‘ the. cell lines.
`An inunnnotoxin reactive with I—IER—‘Z/amt was pre—
`pared by conjugating TA-l antibody with ricin A chain.
`Inhibition of clonogenii growth by FI‘A-i—ricin immu-
`uotoxin at concentrations of] and It) ug/ml was tested
`on SKBrB cells and five ovarian cancer cell lines (Fable
`II). Unconjugated 'I‘A—l antibody (lid not significantly
`inhibit clonogenic tumor growth oI‘SKBr?) cells or any
`of the live ovarian cattcer cell lilies. 'I‘A—l—ricin immu-
`
`notoxin significantly inhibited the clonogenic growth of
`SKBrfi cells. Among the ovarian cancer cell
`lines the
`only cell
`line that was inhibited by the TAd—ricin
`immunotoxin was the SKOV3 cell line, which contained
`more receptors per cell (tenfold higher) than the other
`four ovarian cancer cell lines. The inhibition of SKBrS
`
`cells ivas approximately 1.5 logs greater than that seen
`in SKOV3 cells. Thus inhibition of clonogenic growth of
`the cancer cell
`lines by TA-l—ricin A chain immuno-
`toxin was lonnd to correlate with the number of p185
`receptors per cellI with the greatest degree ofinhibition
`noted 'in
`the SKBr3 breast cancer cell
`line. The
`
`4511A1271icin A chain imtnunotoxin significantly inhib-
`ited clonogenic growth OISKB1‘3, OVCA 420, OVCA
`429, OVCA 432, and OVCA 433 cells by.0.6 to 5.510gs.
`Interestingly, 454Al2—ricin A chain immunotoxin did
`not
`significantly inhibit
`the clonogenic growth of
`SKOVE cells (data not shown).
`'
`7
`
`Comment
`
`Imttiunotherapy directed against the HER—2/twu on—
`cogene product p185 represents a novel approach to
`
`setothcrapy in that antibodies. interact with a cell stir»
`lace receptor that probably contributes to maintenance
`of the malignant phenotype. Although I'll‘lR—Q/neu is
`expressed in normal tissues,” an acceptable therapeutic
`index may possibly be achieved in the traction of breast
`and ovarian cancers that ovetcxpress IIIiRQ/iwu.
`In
`this regard others have shown that antibodies reactive
`with rat p185 exhibit .potenl. antiluntor elTectsiinivivo
`against
`tumors that overcxpress this protooncogene
`while sparing normal tissues.“'
`""
`To explore the therapeutic use of H]1[l-HI‘:R<2/171?It.
`antibodies, we have obtaitted a panel ()I' ll antibodies
`reactive with the extracellular domain.” Cross»blocking
`experiments have been performed to enable us
`to
`construct an epilope map of antigenic determinants on
`this molecule 'I‘cn antibodies recognized cpitopes that
`are expressed in a linear array, whereas another anti-
`body recognizes a distinct epitope.
`In SKBI'S cells,
`which have strikingly elevated levels of PIER—When,
`seven of the antibodies inhibited anchorage-indcpcn-
`(lent. growth in soil agar, and two ol‘ the antibodies
`(RD—5
`and
`Il)»5)
`inhibited
`anchorage-(1ependent
`growth.” In studies perloi tiled in collaboration with Dr.
`Ruth l.upu these same two antibodies were [bond to
`block binding ol’ the putative $50 kd ligand to HER-
`2/turu,
`suggesting that
`these
`antibodies
`recognize
`epitopcs near the ligandbinding site.
`lu this study ID»?! was shown to inhibit anchorage—
`dependent growth in SKOvi’i and OVCA 420 cells. In
`contrast, 'l'A-l and PBS, which do not block binding of
`the 30 kd ligand, did not, inhibit growth of any 01‘ the
`ovarian cancer cell
`lines. 'l'hesc data suggest
`that the
`effect ol~ ID-5 on proliferation may be due to direct
`activation ofHER—2/ttett, similar to the natural ligand. If
`this is correct, HER-2mm may be a receptor for a
`growtlrinlfibitory.
`rather
`than
`growth—stimulatory,
`ligand. On the other hand, analogous to epidermal
`growth factor, which is a potent. mitogen for most cells
`but
`inhibits growth in cells that have markedly in-
`creased epidcrmal growth factor receptor levels,
`the
`effect of the HER—2km” ligand on proliferation may
`depend on the level ol‘rcceptor expression. To explore
`further the hypothesis that
`ID-Ei mimics the natural
`Hl‘:R»2/1LZU ligand, we are examining the effect of ID»5
`on molecular events, such as diacylglycerol production,
`that are known to occur after activation ofp185 and
`other receptor tyrosine kinases.
`Alternatively, inhibition ol‘prolifet’ation by ID—5 could
`result from interference with an intrinsic autocrine
`
`growth—stimulatmy loop because of competitive inhibi—
`tion of binding of secreted 3t) kd ligand to Iii‘:l{f2/jifilt.
`This hypothesis assumes that PIER—2mm is a growth-
`stimulatmy receptor If this was the case,
`Iii—5 sliiiiild
`inhibit proliferation in cells that both secrete the ligand
`into their culture medium and also express HERQ/nm.
`In this model overexpression of the receptor might not
`be requisite, however. In support of this model, SKBi-S
`
`
`
`IMMUNOGEN 2037, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2037, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`
`
`Volume 108
`Number 1. Putt 1
`
`l-lER-2/neu antibodies and ovarian cancer
`
`231
`
`
`
`OVCA433
`OVCAIIJZ
`
`OVCA4Z9
`
`
`
`[3H1ThymidineIncorporation,
`
`PercentofControl
`
`
`
`
`5K0v3*
`SKEr3*
`OVCA420*I
`1.5
`2.0 '
`2.5
`3.0
`6.5
`1.0
`ID5 Antibody tug/n11)
`
`0.0
`
`*p<.002
`
`,.
`
`Fig. 1.
`
`[illch ol ll)—5 anti—l ll‘lR—2/mttt antibody on growth 01‘ cancer cell lilies.
`
`Table II. Effect 01' uumnjugaled antibodies and imintinotoxins on log inhibition ol‘clonogenic growth 01'
`cancer cell lines
`
`7V1—Fririit A-c/trtin
`
`Haunting/11ml 'I'/l-l
`
`'-
`M()1’C2I
`Cell lint)
`I 0 (1'1;
`l () pig
`
`0.20 '1: 0.13
`0.507 + 0.443
`2.097 1': 0235*
`SKl’n'S
`0.077 3': 0.006
`0157 i 0.0021
`0.11 :t 0.12
`0.270 r 0.000 -
`0.5023 i 0.0181
`SKOvB
`0.077 + 0.000
`0,193 i 0.136
`0.01 t 0.06
`0.273 i 0.240
`0.103 t 0.178
`OVCA 420
`0.157 |_ 0.072
`0.153 1: 0.170
`0.16 :': 0.12
`0.1555 1' 0.000
`0.1021 i 0.178
`()V(IA<129
`0.0110 + 0.009
`0 i: 0
`0.04 :': 0.06
`0.103 i 0.001
`0.270 .I: 0.240
`OVCA 432
`0.34.11 1 0.202
`0.270 t 0.2/10
`0.08 + 0.138
`0 .l. 0
`0.0/10 i 0.060
`OVCA 433
`0.040 .t 0.009
`0.08 1— 0.1558
`
`
`
`llllCL‘ CXl)(jI1llllIlllS.
`til
`lTICilll
`VillthS ICIH'CSL‘nl
`”7! < 0.001. compared with I
`tip; 'l‘Arliticin [\Jchain, 10 up; unconjugated 'l'A»l, and l0 pg M()l‘(l2l.
`l]; < 0.05. compared with 1 pg 'l‘A-l-ricin I\-(ll:11lt, 10 at; uucouhtgaied 'l‘A-l, and 10 pg M()l'()2l.
`
`cells, which are inhibited by ID-S, have been shown to
`produce significant amounts of the 30 kd ligand.
`In
`addition, the finding that 1043 inhibited prulllbraliou ol'
`OVCA 1120 cells, which do not. overexpress Hl'llL‘ZIi'mu,
`could he explained by the autocriue hypothesis.
`The capacity [or
`inlcrualizatimi
`01' p185 through
`ClaLhriu—coaletl pits makes this receptor an ideal target
`for immunotoxins. Toxins such as ricin can be ellectivc
`
`only \fljtllLU'iln'iiClfl'CI.UQLllQL)1QPL<l5JUi Mttt'yeinllibi-
`tiort (it"protcin synthesis can take place’in the ribo«
`Sonics. Although in theory internalization of only one
`toxin molecule is stillicieul to kill a cell, eliectire inter?
`utilization of iuiutuuotoxins probably requires binding
`of a large ntnitber of molecules 01' the immuuocouju-
`gate to the cell surface.
`In one study evaluating =17
`different antibodyiricin A chain conjugates directed
`against breast cancer cell
`lilies. the most attire conju—
`gates used antibodies that had the highest alliuity for
`cell surface antigenic determinants and that exhibited
`the greatest density of binding to the cell surface.'7
`Similarly, we found that the efficacy oli'l‘A-l—ricin im-
`
`munotoxiu correlated with the number of p185 retep-
`tors per cell. 'l'A—l—ricin itumunotoxin was not cytotoxic:
`to ovarian cancer cells expressing normal
`levels of
`HER-2/m'u., whereas it was cytotoxic [or the SKOv?) and
`310513 cancer cell lines, which overexpress H ER-2/11m.i..
`The requirement of a threshold number 01‘ p185
`receptors per cell
`for effective t'ytOtUxicity with an
`iuunuuotoxiu has important therapeutic implications.
`l’rcvions clinical trials with ricin A chain and Pseudomo-
`uas exutoxiu iinmnnocouiugales in patients with ovar-
`ian cancer have resulted in unexpected severe central
`and periphefal uetuotuxicity.” Subsequently.
`the anti-
`bodies used in these trials have been found to bind
`weakly to nervous system tissues. HER-2/urtt
`is not
`expressed in peripheral nerves or in the central nenous
`system but
`is expressed by most epithelial cells.
`If
`overexpressiou is
`required for cytotoxicilr
`to occur.
`not mal cells may be spated, providing an acceptable
`therapeutic index for [he lrealiueut of ovarian cancers
`that merexpress this protooucogeue. The poor prog—
`nosis 01‘ this subgroup oftn’ariau cancer patients
`may
`
`
`
`IMMUNOGEN 2037, pg. 5
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2037, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`
`
`Ianurn) 1993
`
`.-\nr_] Obstcr (Lyneml
`.
`
`232 Rodriguez et at.
`
`justify the use ofirwestigational therapies. such as those
`involving anti—p185 immunotoxins, earlier in the course
`of treatment.
`
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`IMMUNOGEN 2037, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2037, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`