19 August 1993
`
`Vol. 55 No.
`
`l
`
`
`INTERNATIONAL
`
`Journa/
`0/ Cancer
`
`K/J ‘r1
`
`-‘7
`
`EC
`
`{.3 .J
`
`
`
`JOURNAL INTERNATIONAL
`
`Editor-in-Clricf:
`Associate Editor:
`Assistant Editor:
`
`Editorial Oflice:
`
`DU CANCER
`
`'
`
`N. Odartchenko
`P. Cerutti
`S. Carrel
`
`International Journal of Cancer
`Swiss Institute for Experimental Cancer Research
`1066 Epalinges/Lausanne (Switzerland)
`Fax: 41—21-652 7705
`
`PUBLICATION STAFF
`Equipe de publication
`
`Technical Editors:
`Coordinating Manager:
`Editorial Secretary:
`
`P. Oberson, S. FitzGerald
`E. Lornan
`M, Cisshinsky-Inhm
`
`EDITORIAL BOARD
`Camité zle rédacliml
`
`S.A. AARONSON, Bethesda, MD (USA)
`G.I. ABLLEV, Moscow (Russia)
`RW. BALDWIN, Nottingham (UK)
`M. BARBACID, Princeton, NJ (USA)
`W.F. BENEDICF, The Woodlands, TX (USA)
`R.L.H. BOLHUJS, Rotterdam (The Netherlands)
`RR. BRENTANI, sao Paulo (Brazil)
`M.M. BURGER, Basel (Switzerland)
`W.K. CAVENEE, La Jolla, CA (USA)
`P. CHAMBON, Strasbourg (France)
`NH. COLBURN, Frederick, MD (USA)
`NE. DAY, Cambridge (UK)
`L. DENIS, Antwerp (Belgium)
`P.B. DESAI, Bombay (India)
`Sir Richard DOLL, Oxford (UK)
`M. FELDMAN, Rehovot (Israel)
`T. GRAF, Heidelberg (Germany)
`C.C. HARRIS, Bethesda, MD (USA)
`OH. HELDJN, Uppsala (Sweden)
`P. IIERRLlCH, Karlsruhc (Germany)
`
`ISSN 0020-7136
`
`'
`
`D. ME'ICALF, Melbourne (Australia)
`M. MIWA, Ibaraki (Japan)
`R. MONTESANO, IARC, Lyons (France)
`NP. NAPALKOV, St. Petersburg (Russia)
`J, PUN'r'EN, Uppsala (Sweden)
`M.F. RAJEWSKY, Essen (Germany)
`A.B. RICKINSON, Birmingham (UK)
`JD. ROWLEY, Chicago, IL (USA)
`E. RUOSLAHTI, La Jolla, CA (USA)
`K. SHANMUGARATNAM, Singapore (Singapore)
`K. SHIMOTOHNO, Tokyo (Japan)
`EJ. STANBRIDGE, Irvine, CA (USA)
`J. SVUBODA, Prague (Czechoslovakia)
`A. TAVITlAN, Paris (France)
`D.
`'I‘RICHOPOULOS, Boston, MA (USA)
`A. VAHERI. Helsinki (Finland)
`J.M. VASILIEV, Moscow (Russia)
`U. VERONESI, Milan (Italy)
`A.O. WILLIAMS, lbadan (Nigeria)
`H. ZUR HAUSEN, Heidelberg (Germany)
`
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`
`IMMUNOGEN 2063, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2063, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

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`Int. J. Cancer: 55, 122- 127 (1993)
`© 1993 Wiley—Liss, Inc.
`
`Publication of the International Union Against Cancer
`Publication de I'Union Internationale Centre Ie Cancer
`
`CHARACTERIZATION OF CYTOTOXIC ACTIVITY OF SAPORIN
`
`ANTI-GP185 /HER—2 IMMUNOTOXINS
`
`R. TECCEl, G. DIGIE311,A. SAVARESEQ, D. TRIZio3 and P.G. NATALIM
`
`Laboratories of lIrnrrzanology, and 2Pie-clinical Experimental Chemotherapy, Regina Elena Cancer Institute, Rome;
`and 3Department of Immunology, Farmitalia-Carlo Erba, Milan, Italy.
`
`The oncogene HER-2/neu encodes a trans-membrane recep-
`tor of 185 kDa with tyrosine-kinase activity. Over-expression of
`this molecule has been reported in a significant proportion of
`human breast and ovarian carcinomas, characterized by a poor
`clinical prognosis. Two monoclonal antibodies (MAbs), recogniz-
`ing distinct epitopes of the gp l85 extracellular domain, have
`been utilized in the present study for the production ofimmuno-
`toxins (lTs) by conjugation to the type-l RIP (ribosome-
`inactivating protein) plant toxin saporin 6 (SAP). These lTs have
`been shown to retain tumor-specificity and specifically to inhibit
`protein synthesis in the gp IBSHER‘1(+) SK-BR-3 breast-carcinoma
`cell
`line with leo values lower then I nM. Kinetics of the
`cytotoxic activity of the ITS are characterized by a slow rate.
`since incubation times ranging from 24 to 60 hr. depending on
`the different degree of expression of the receptor, are required
`to determine >90% inhibition in the incorporation of radiola-
`beled Ieucine. However. the cytotoxic activity of these lTs, as
`evaluated by a more sensitive clonogenic assay, appears highly
`potent, since we have observed that 3 to 4 logs of cells are killed
`upon exposure to the ITs for short times at concentrations
`ranging from I toS x IO’8 M.
`@1993 Wiley-Liss, Inc.
`
`HER-Z/neu is a proto-oncogcnc of the epidermal—growth-
`factor (EGF) family of tyrosine—kinase receptors. Similarly to
`the EGF receptor, the HER-Z/neu gene product gp185“ER'2
`consists 0f3 major domains characteristic of a trans—membrane
`receptor (Slcrn e: mi, 1985).
`indeed. a candidate ligand for
`gplBSHER'Z has been rcpoi'lcd (l’clcs et al., 1992). Over-
`expression ol' the l-llER-P. oncogene with or without gene
`amplification has been shown lo be associated with poor
`prognosis in patients bearing brcast (Dc Potter et a/., 1990;
`Slamon et al., 1989) and ovarian (Slamon et al., 1989) carcino-
`mas. Tissue-distribution studies performed with polyclonal 0r
`monoclonal antibodies to gplSSHER‘Z, have shown restricted
`expression of gplSSHER‘2 in normal human adult tissues (Press
`et al., 1990; Natali et (11., 1990). Of major interest, these studies
`have demonstrated that expression of gp185“‘3R‘2 in primary
`tumors is almost invariably ucunupnnicd by the expression of
`the receptor in concomiiuni or subsequent inclnstnsos (iglo—
`hart et al., 1991)).
`‘i‘hcsc findings clearly ilnlicalc Ihul
`lhc
`gpISSHF-R'2 may be u lurgcl ol inunnnothci'zlpculic strailcgics.
`To vcril'y the feasibility of such an approach, wt:
`luwc
`conjuguicrl 2 MAbs recognizing 2 dislinct cpitopcs ol
`the
`gpih'fi'u'“ -‘ cxll'nccllnlm' don-ruin (Digicsi u'r ril._. 1992). will: the
`plum toxin si-Iporin b from .S'riponrrr'irr oflir-inriliir, which requires
`less cumbersomc puriliculion procedures.
`is suinr to humllc,
`and is loss cylninxlclmll]in1-‘l1-‘(JJIUtllJIVil/‘0 (Stirpe et al., 1983).
`This type-l Rll' is endowed with advantageous properties such
`us ubscncc ol‘ czuhohydru to residues. extremely high pl (> 9.5),
`which is likely Io he. cli'ccliv: in prcwnting degradation of the
`toxin in the cndolysosomal compartment, absence of specific.
`receptor structures on animal cells, and optimal stability and
`I‘Ihurrnucokim-lic propcrlics in [lic conjugated l'orm (Slirpc ('i
`at, 1953: Luppi er at. 1983]. Furthermore, snporin huh been
`succussl'nily utilized int the production or l'l's to Iicmutulogic
`and solid tumors ("I'Iioi'pceiul.. J‘JSS'Sicnn will.
`IUHH: lircgni
`r: at.
`l‘ib'b'; liurbicri (It at. 1989: chcc ct at. 1991rr.h], which
`hovc shown potunl cytotoxic properlics ussocnucd with ulc-
`vatcd target specificity. The results of a Phase—I clinical trial by
`Falini er al. (1992) which cvaluatcd the effect of the systemic
`
`administration of an irnmunoconjugate with Saporin in pa—
`tients with Hodgkin’s disease have demonstrated a rapid
`anti—tumor effect and no serious toxicity, suggesting a potential
`therapeutic role for immunoconjugatcs using saporin 6 as toxin
`for clinical purposes.
`We report the development of 2 ITS to gpltSSHER'2 and the
`analysis of their in vitro cytotoxic activity.
`
`MATERIAL AND METHODS
`
`Cell lines
`
`lines SK—BR-3,
`The human brcast-adcnocarcinoma cell
`MDA»MB-365 and MCF7 were obtained from the ATCC
`(Rockvillc, MD). The T94 cell
`line derived from NIH 3T3
`mousc lihrohlnsl
`lrunsicclcd with a plasmid containing :I
`full-length i-lER-2/ncu human cDNA [Di Fiorc e! at, 198?)
`was kindly supplied by Dr. SA. Ani‘onson (NCl, Bethesda,
`MD). All cell
`lines were grown in medium D-MEM (Flow,
`lrvinc, UK) supplcmcntcd with 10% hcat-inuclivntud FCS
`containing 2 mM L—glutaminc.
`
`Construction of immunotoxins
`isolypc, Wfilh‘litlfln and
`The 2 muriuc MAbs ol’ lhc lgfl.
`Wfil‘JllUIlI, recognizing 2 distinct cpitopcs of the 3;]1185'"5“'3
`extracellular domain, won: obtained by immunization of
`BALBlc mice will] the T9-4 cell
`line us ticscrihcd (Digicsi er
`oi, 19192). The 2 reagents, displaying an nlliuity {11"}.1’18 x 10”
`and 3.26 x 10" rcspcclivcly, were purilicd from ascilic fluids by
`DEAE Alli—gel bluc chrom:dogrnphy as described by 'l'cccc. nr
`til. (1991(1). Fraclions containing purified unlibody, nsjudgcd
`by SDS-PAUE analysis, were (liulyscd against PBS. pli 7.2.
`um] stored at ~21]“C_ All protein conccnlrations were delet-
`mined by BCA protein assay (Pierce, Rockford, IL).
`SAP, puril‘lccl I'rom seeds of S. oflicinuiis [sorlpworU by the
`method of Slitpc or of. [1983) wilh modifications (Lappi at at.
`[985], was kindly provided by Dr
`I).
`'l‘rizio (Fm‘milalia,
`Nurviuno, Holy). Chomicui conjugution ol' WMSUUIEB and
`Wol‘Jllillll MAbs to SAP was carried-out according to mth-
`ods described by Thorpe er al. (1985) using the lictcrobil‘unc-
`tiooul cross'linltcr Nusuccinimitlyl 3-(2-pyridy|dilhio)propi:r
`nutc (SPO?) (l’inu'mucin. lippsulu. Swadcn). according to tho
`munulhctnrcfis instructions. The (.lcgrcc ol‘dcrivulion was 2.114
`(W’ti/Hlllllfib) and 1472 (Wbl‘illiilrlll moles Hl’Dl’lmol MAIL
`while the degree of derivation of SAP was 1.16. Conjugation
`was performed following reduction of derived SAP with 20
`mM DTT and recovely of thiol derivative by gel filtration on
`Scphadcx 625. The conjugation reaction was monitored by
`measurement of 0D. absorbancc at 343 nm. Immunotoxin
`moi-:culcs wcrc puriiicd by gel chroimnography on a Scphacryl
`SAfllli} column and [heir purity usscsscd by SDS—PAGE;
`ITS
`wcrc visuulizcd :n. 2 pi'utcin bunds of apparent molecular
`wciglu of about 185 K132: untl 2l5 K05: respectively, and of
`ziluiosl cquul inicnsily [darn not shown).
`
`
`4To whom correspondence and reprint requests should be ad
`dressed at CRSAIRE, Laboratory of Immunology, Via dolls MCSSl
`d’Oro 156, 00158—R0me, Italy. Fax: 3964180473.
`
`
`Received: February 9, 1993 and in levised form May 5,1993.
`
`
`
`‘—_—._._._—_
`
`IMMUNOGEN 2063, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2063, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`BREAST-CARCINOMA IMMUNOTOXINS
`
`123
`
`Ribosome inactivation in presence of ITs was evaluated by
`measuring incorporation of radiolabeled methionine in a
`nuclease—treated rabbit reticttlocyte lysate (N150, Atnersham,
`Aylcsbury, UK) using tobaccoemosaicevirus RNA as standard
`message. Serial dilutions of unreduced IT were added to
`incubation mixtures containing 2.5 uCi 3‘S-methionine (Amer—
`sham). Samples were incubated for 45 min at 30,°C and then
`processed according to the manufacturer’s instructions.
`Two different irrelevant ITs containing the saporin emie
`toxin, the first one, Ep2/SAP, directed to a melanoma associ—
`ated antigen and the second one, LAM3/SAP, directed to a
`monocyte—specific determinant, have been utilized as toxin—
`matched controls of cytotoxic specificity and have been de-
`scribcd in detail elsewhere (Tecce et (11., 199]o,b).
`Binding assay
`To compare the degree of expression of gp185““” in the
`different cell lines, the MAb W6/800E6 was radiolabeled with
`”I using the cltinraminc~T method at a specific activity of
`1.6 X 106 cpm/cg MAI). [IEILE gleS ("'l orl 'J cells (2 X 105)
`were plated in U-bollont 96-well rnicrotitcr plates and incu-
`bated for 45 min at 4°C with tt, [5 pg radiolabclctl MAb. At the
`end of the incubation, cells were washed 7 times in PBS and
`counted in a gamma counter. Results were expressed as mean
`cpm from triplicate samples.
`Serological studies
`Reactivity of purified MAbs and ITs with transfected and
`human tumor cell lines expressing various levels ot'gp185“'-"-""2
`was analyzed by indirect
`imnnmoltttnrcsccncc (IIF) on cell
`suspensions using as second antibody an atiinity—ptn'ii‘icd
`FITC-labclcd Half): rabbit antiserum to mouse lgCi (Cuppcl,
`'l‘cknika. Tttanhottt Itclgium]. Fltiorcsccncc intensity and dis-
`tribution were evaluated by How cylonlctry using FACStat'
`equipment (licctttn Dickinson, Mountain View, (IA). Statisti»
`cat! analysis ul‘quantitativc data obtained by [low cytontctry was
`performed utilizing PI-lARMA/PCS software (Springer. New
`York).
`
`Cytotoxicity assay
`Cells were seeded (2 X104/well) in flat-bottom 96—well
`microtiter plates (Costar, Cambridge, MA) and 24 hr later
`serial 10—fold dilutions in complete medium of MAbs,
`the
`corresponding immunotoxins and free saporin were added in
`triplicate wells and incubated 4 hr at 37°C. Plates were then
`washed twice with PBS containing 5% “(135, and tltc cells were
`incubated itt con'tplctc medium for an additional 2i] hr, after
`which I uCi -‘I-l-|cttcinc (Antcrsltutn) was then added to each
`\Vcll. Attcr
`IS-Itt‘
`incubation and extensive washing with
`Culture. medium. cells were harvested following ED'I‘A treat—
`ment, spotted onto 3 MM (Whatman, Maidstone, UK) chroma—
`tography paper dishes processed as described (Tecce et al.,
`1991a) and counted in a liquid scintillation counter. Results
`were expressed as mean cpm from triplicate wells. Analysis of
`the kinetics of the protein synthesis inhibition by th was
`evaluated following the same protocol and incubating cells
`\vtth IT for dichrcnt time periods. At tltc end of the incubation
`““10. cells were pulsed for .1 hr with Icucinc‘l'rcc medium
`Fontttining 5 uCifwcl] V‘I'l-lcttcinc, then chilled in'tntcdialcly on
`Ice and processed as indicated above.
`
`Clonogenic assay
`. SK-BR—3 cells grown at confluence were cultured for 1.5 hr
`in presence of dilicrcnl concentrations of Wbi’t‘iltllEb, W6!
`Until”. the cot't'cspmiding innnut'totoxins. tltc irrclcvant itnmtt-
`Halos-ins EpZ/SAP and LAMSKSAP. and free saporin. At The
`cud of the incubation. cells were washed with medium,
`'lL'talchcd from flasks with EDTA “.01 M. rte-suspended in
`melctc medium and countcd in a cell counter (Coulter ZM.
`L310", UK). Control and treated cells were then plated in T25
`llttsits (4 x It]1 cclls/l‘lnsk) and grown in complete medium,
`
`After 15 days the medium was aspirated and colonies were
`stained with 1% methylene blue [or 3 min and counted.
`"l‘rcnlcd cells tvcrc plated in triplicate, while (i cultures of
`untreated control cells were plated as well. To obtain a
`quantitative EISSCSSIIIL‘M ut‘ tltc cytotoxic activity otthc l'l' at tltc
`higher doses tested,
`treated cells were piztlcd. always
`in
`triplicate. at higher densities ranging from :t X lit" to | x
`|ll"/ll:tslt.
`
`Serological reactivity of [Ts
`
`RESULTS
`
`A preliminary radio—binding assay on live cells using 125I»
`labelled MAb W6/800E6 t0 the extracellular domain of the
`oncogene product was performed to select cull targets cxprctis-
`iltg dill'ct'cnl
`levels of gpliiS”':”'3. High binding values were
`observed on the lIER-Z-transl‘cctcd nnu'inc cells 'i'lP-t-t. Among
`the breastcarcinoma cell
`lines analyzed, SK-l3R-3 cells dis-
`played tltc Itigltcst binding: ML)A—Ml$~3tifi cclls gave low
`binding values, while the MCI-‘7 Wcrc consistently nnrcactive
`{'I'ablc l). On tltc basis of this information, puriticri prepara-
`tions 01‘ th/Rtltttitt MAb and tltc corresponding W6!8tttlEti~
`SAI'
`[T at tlii‘tcrcnl cquimular concentrations were assaycd
`comparatively by FACS analysis on SK-BR—J cells (Fig. 1). By
`linear
`regression analysis ol'
`the binding curves, a slope
`cnctlicicnt value of 0.386 [or Wh/fllitltio MAb and ”.955 for
`WMROUEGSAP IT was calculated. A parallelism test between
`the binding curves indicated a non-significant t value of [1.832
`(:95?!) = 4.3), demonstrating that tltc chemical conjugation of
`tltc MAI) to tltc tnsin does not alter its binding properties.
`Analogous results were obtaincd with the olltcr MAb—1T pair
`(data not shown).
`
`Inhibition ofprotein synthesis in breast—carcinoma cell lines
`by anti-HER-Z gp185 ITS
`Figure 2 summarizes tltc dose-response curves of the cytn~
`toxic activity at th as measured by il‘l-Icucinc incorporation in
`the cell
`targets dil‘l’cring for
`the degree ol‘ expression of
`gp'lSS”“'-“'3. While tltc ccll lilies '1‘9-4 and SK—BR—J (Fig. 2rt,c}
`with high density ot‘ the receptor were sensitive to the cytotoxic
`activity of thc I'I's. no significant inhibition of protein synthesis
`was observed with cell lines MDA-MI3-3b5 and MCF-7 (Fig.
`EM!) expressing low or Ito level of gplfifi”'"“'3. Furthermore.
`no inhibition ol‘ Jl-l-Icucinc incorporation was detected in any
`of
`tltc cell
`lines
`incubated with unconjugated MAlis, at
`concentrations as high as l X it] '7 M (not shown]. The
`cytotoxic activity of both 11‘s was partially blocked by co—
`incubation with a “Hold molar excess of ttncottjugztlcd anti-
`body; as an example,
`in the T944 cell
`line Mr-‘tb Wo/HtlUEb
`reduced protein synthesis inhibition from 94% to 5 l % at an IT
`concentration corresponding to 7 X it] 'i‘ M (Fig. 2a). Cont-
`plele inhibition occurred at Mitt—fold molar excess of antibody,
`as exemplified in tltc SK~BR-3 cell line. when: protein synthc~
`sis inhibition was reversed from ?2% to 9% at a WtiffitlflEo-
`SAP concentration ofl x 10’8 M (Fig. 20). Moreover. slightly
`higher cytotoxic activity (a 3-fold difference itt ICSU value) was
`observed in cell line T9—4 with IT W6/800t36-SAI’ endowed
`with higher affinity (ICSO = 4.3 X 10‘10 M), as compared with
`IT W6/900HLSAP (ICSU = 1.1 X 10‘9 M). Although cell lines
`
`TABLE] — BINDING OF 12*‘l-LABI—ZLLFJ') ANTI-gplSSHER'Z MAb W6/800E6 TO
`DIFFERENT CELL LINhS
`-
`
`
`Cell lines
`(cpm X 10—5/105 cells)I
`Tg-4
`102.2
`SKvBR-3
`58.7
`MDA—MB-365
`5.5
`MCF-7
`1.4
`
`NIH 3T3 1.2
`
`_
`
`lValues represent the mean of triplicate samples.
`
`
`
`
`
`IMMUNOGEN 2063, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2063, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`
`
`124
`
`TECCIZE7"AL.
`
`_..- .g-Is. -.:-p. um. wan.“ aux-c-n-‘vn...
`
`U3:RIDGEBBSEFL1\FITD
`U3:RIDGEBBé\FL1\FITC
`
` 1‘1
`
`1 U
`
`3:RIDGEBB4\FL1\FITC
`
`
`
`A
`
`FIGURE 1 — Serological reactivity of Wo/SUUEfi/SAP and of parental antibody W6/800E6 with human breast-carcinoma SK—BR—B cell
`line at different concentrations: (a) 0.50 ug/rnl; (b) 0.10 ug/ml; (c) 0.02 ug/m], evaluated by FACS analysis. Fluorescence profiles of
`MAb and corresponding IT coincide in (a) and in (b), while in (c) MAb shows slightly greater reactivity than the l']‘. Fluorescence profile
`of SK—BR-3 cells not incubated with MAb or IT is represented in (d).
`
`T9—4 and SK-BR-3 displayed comparable sensitivity to free
`saporin and were both intoxicated by MAb—saporin conjugates,
`a complete block of protein synthesis was observed in the T9-4
`cell
`line transfected with HER—2 gene (Fig. 2a) at an IT
`concentration of 7 X 10‘8 M, while at the same dose a residual
`26 to 28% 3Hrlcucine incorporation was observed in the
`breast-carcinoma cell line SK-BR-3 (Fig. 2c).
`
`Inhibition of anchorage—dependent growth by anti-HER-Z gpl85
`ITs assay
`Because in the conventional 24—ln' cyloloxicity assay the
`residual 3H—leucine incorporation ohsctvcd in SKeBR—3 cells
`could be indicativc oi
`incotnplélc killing of a cell
`sub—
`population, we investigated the long—term effects of ITs by a
`different and more sensitive assay. Since anchorage-dependent
`in vitro growth of SK—BR—3 colonies was not influenced by the
`presence of unconjugated antibodies at doses ranging front 0.1
`to 5.0 ug/ml (data not shown), we performed a clonogenic
`assay to measure the cytotoxic effects of the conjugate by
`evaluating colony growth following plating of a variable num—
`ber of cells, ranging from 4 X 103 to l X 10". Results of this
`analysis are reported in Figure 3; it should be noted that the
`log of colony—number reduction has been determined by a
`conservative estimate, assuming that absence or reduction Ofin
`vitro growth (as observed and quantified experimentally) had
`to be corrected taking in account a plating ‘cfliciency value
`corresponding to 1 colony/400 cells seeded, even if in un-
`treated control cells this value was >1/10 when plating >100
`cells/flask. By these criteria we calculated that 1.5—ht' treat-
`ment of SKeBR73 cells with W6/800E6—SAP at a concentration
`of S X 10‘8 M is capable of killing >3.65 log cells, while a
`5»fold lower dose of W6/900H1—SAP kills 2.39 log of cells. This
`highly efficient toxicity is specific since (i) exposure to the
`unrelevant ITS EpZ/SAP and LAM7/SAP (l, X 10—8 M) or to
`free saporin 6 (1 X 10c7 M) had no significant effect on colony
`
`growth in SK-BR-3 cells and (ii) pie-incubation of the cells
`with a 100—fold molar excess of the unconjugated antibody
`resulted in almost complete suppression of cytotoxic activity of
`ITs.
`
`Kinetics of immunotoxin activity
`Because of the somewhat discordant results obtained on
`SK-BR-fi cell
`lines
`in the short‘term protein-synthesis-
`inhibition assay and in the long—term clonogcnic assa , we have
`tntatlyxcd the cytotoxic activity ol. the anti-gplr‘s‘im*'2 ITs by
`evaluating the kinetics of prolcitnsynthcsis inhihilion in thc
`gpISSI'E-‘li'l "1 cell lines over £1 wide: rangc of exposure times
`(Fig. 4). This analysis indicutctl
`that both cell
`targets were
`intoxicated at a relatively slow rate by Ihc 2 Hit. While an n lit'
`in thc T9-4 cell line a residual 30% incorporation of 3H-leucine
`was observed at 1 X 10‘8 M IT concentration, 24—hr incubation
`indicated complete inhibition of protein synthesis at this same
`concentration (not shown). By contrast, in the SK-BR-3 cell
`line, longer incubation times (an to (:0 hr) in the presence of l'l"
`at
`the same molar concentration were required In attain
`complete inhibition ol‘ radiomtclitlc incorporation (Fig. 5). In
`this cell line, protein synthesis inhibition proceeded very slowly
`during the that
`r: In ol [T exposure, and ali‘if: and 32% or
`reduction was attained in the presence of W6/800E643AP and
`Whi'lOtllthAl’ IT respectively (Fig. dl). These slow kinetics,
`however. resulted in complete inhibition of protein synthesis,
`with no evidence of a minor fraction of cells cscaping: ccll
`death. as
`likewisc indicated by the clunogcnic assay,
`It
`is
`noteworthy that til. the samc molar concentration employed
`with ITS, 3.12.
`l X lll' “ M.
`l'I‘cc snporin was able to retlttcc
`protein synthcsis by only 5055?. and this titaxitnal cytotoxic
`activity was obscn-‘cd after 12 hr from exposure, while at longer
`incubation times no further increase in cytotoxicity was Inca-
`sul‘Ctl (Fig. 5).
`
`IMMUNOGEN 2063, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2063, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`BREAST-CA RCINOMA [MMUNO’I'OXINS
`
`
`125
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`3
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`E
`tn
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`
`125 p
`100 -
`
`75 -
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`50
`
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`
`0
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`
`.‘
`
`3
`
`,,
`
`-10
`
`__I_
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`-3
`-8
`Concentration ( Iogto M)
`
`. _
`-6
`
`.
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`50
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`41,
`I
`_
`v11
`-10
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`-8
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`-5
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`___
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`.5
`
`Concentration ( lento M)
`
`
`-5
`
`'.
`
`A
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`t
`
`‘-
`
`'.
`
`.
`
`‘I
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`'-
`
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`7
`
`I
`
`‘A
`
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`>6
`
`-5
`
`Concentratlon (legit) M)
`
`
`
`‘
`
`._
`
`‘A
`
`
`I.—
`6
`
`Concentration ( logtD M)
`
`in
`'6
`m
`E
`‘E
`E
`a
`23
`D.
`s“
`
`>m
`
`m
`"-7;
`>n
`
`2E
`
`z E
`
`§
`I1
`u\"'
`
`125
`
`100 '
`
`75 -
`
`_
`50 -
`
`25 '
`
`D
`-11
`
`
`
`FIGURE 2 — Inhibition of protein synthesis by immunotoxins in (a) T9-4-transfected, (c) SK—BR—S, (b) MDA—MB—365 and (d) MCF—7
`
` or presence O of (a)
`breast-cancer cell lines. Cells were incubated with different concentrations of W6/800E6/SAP in the absence
`10-fold or (c) IOU—fold excess of unconjugated antibody, W6/900H1/SAP, I, and SAP, A. Mean values of triplicate samples are
`expressed as percentage of controls.
`
`DISCUSSION
`
`The multidisciplinary approach to cancer treatment clearly
`justifies the current effort in developing therapeutic agents
`such as immunotoxins, which act through mechanisms differ
`cut from conventional chemotherapeutic and radiotherapeutic
`regimens, may be highly tumor specific, and do not require
`host accessory factors.
`This field has received renewed interest with progress in (i)
`the area of tumor radiolocalization demonstrating the ability
`0f MAbs to act as specific carriers, (ii) improved technology in
`antibody production, and (iii)
`the emergence of valuable
`alternatives to the native A chain of ricin toxin, such as
`Slngle-chain type—I RIP toxins (2.3., saporin 6, gelonin and
`momordin).
`A number of immunotoxins to breast and ovarian carcinoma
`Utilizing different toxic moieties and different MAbs have been
`generated (Pirkcrcr at, JUSS; Bjorn et al., 1985, 1988; Yu et at,
`lfl9lt). The major constraints ol‘these immunotoxins have been
`{1) limitations in the potential in viva application due to their
`hI'Oiuil reactivity with normal
`tissues, as in the ease of the
`i{Ini-lt'attst‘crrin receptor inttnunotoxins (Pirker at 111., 1985),
`r“) their reactivity with a low percentage of tumors (Bjorn et
`at, I985) and (iii) low levels of cytotoxicity despite prolonged
`Incubation times (Bjorn curl, 1988; Yu et (11., 1990).
`The choice of saporin-é-Iikc toxins as suitable for clinical
`“505 has been demonstrated by l‘alini et a1. (1992), who have
`been able to achieve a clinically ohjct‘l ive response in a phase—I
`
`trial conducted in Hodgkin patients using CD30-
`clinical
`saporin IT.
`The 2 antibodies to gplttfimiu'2 [Digicsict all. 1992). rccngniw
`ing 2 distinct cpilopcs of the extracellular domain and lacking
`coll-growlh-inhibiting activity, were selected in the present
`study for conjugation with stlporin l3
`taking into account a
`Ill-fold dilicrcncc in their ailinily constants (in, 7.63 x Il)‘I
`ntoI/L for MAI) WMSOUE'EF: and 3.2(1 ><
`It]S tool/L for MAb
`W6/9OUH1).
`The conjugation procedure employed the heterobifunc—
`tional reagent SPDI’ at a low degree of substitution. This has
`allowed the generation of ITs that retain unaltered the binding
`properties ol‘ their parental MAbs.
`Both ITs possess highly specific and potent cytotoxic activity
`by determining a 400 to 1000 fold increase in toxicity of
`uneonjugated saporin 6. While in HER72etransfected T9—4
`fibroblasts, protein synthesis could be inhibited > 97%, in the
`breast carcinoma SEC-BIL"! a residual 25% incorporation of
`radiolabclled Icucinc was observed at comparable concentra
`tions of ITs, despite the lac! that the 2 cell targets were shown
`by FACS analysis to express hon‘logcnnusly the ngSSm-‘R': in
`about 08% ol' the cell population. This has raised the question
`as to whether the incomplete block in protein synthesis could
`rcltccl a heterogeneous response of the tumor cells to IT
`exposure and whether this would result in major limitation of
`the potential therapeutic applications of the 2 ITs. In order to
`explore this possibility, 2 experimental approaches were uti—
`
`
`
`qr.
`
`.5 .._... .- ..mwmw_m 4-4" "maa- ’
`
`. -‘1v—.'_"_MM-f “w '
`
`IMMUNOGEN 2063, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2063, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`
`
`mum-- —Ti-,n.may-_nm. 1-.m hfi.¥”i"fl":-—-
`
`._
`
`126
`
`TECCE ETAL.
`
`
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`
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`
`125
`
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`
`U
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`
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`
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`
`\\
`
`x
`
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`
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`'—
`8‘
`7
`:
`:i
`c
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`
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`
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`
`
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`.
`I
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`._._
`_
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`-a
`
`J
`
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`
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`3
`.c
`i
`.2
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`0
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`3
`
`100 -
`
`75
`so -
`
`25
`
`0
`
`
`-
`—
`—
`
`3‘
`
`..
`
`A
`
`A
`--------.-------.
`
`
`
`N:-
`
`A___-_l
`o
`12
`
`a4
`
`\\
`
`I
`I
`Jib—‘25: .
`24
`36
`48
`so
`72
`
`Concentration (loglo M)
`
`TIME (hours)
`
`FIGURE 3 — (filmingcnic assays. SK-iiR-fi breast-carcinoma cells
`were treated for 3!] min at 37°C will] Wt'iflilllllllfSAP (1 X 10’11
`M to | K 1“"“Ml. I. orWMSUUl'ifi/SAPfi x 10 ‘OMtOS X 10’7
`M). Ll. counter! and plated in T25 llnsks: al'lcr 15 days, culture-cell
`colonies were counted after slaining wilh 1% methylene blue.
`
`FIGURE 5 — Long-term kinetics of IT cytotoxic activity in SK-
`BR-3 cells. The cel s were exposed for 12 to 72 hr to 1 X 10‘8 M
`W6/800E6/SAP, E, W6/900H1/SAP, I, 1 X 10’7 M SAP, A, and
`then pulsed for 1 1r with 3H-leucine. Results are expressed as
`mean % inhibition of 3Heleucine incorporation in triplicate sam-
`ples.
`
`
`
`levels of gp] SSHER'Z, and the ditference in kinetics of cell killing
`between T9-4 and SK—BR—3 suggest that the degree of antigen
`density may also play a role in determining the pharmacologic
`activity of the IT.
`The finding that anti—gtflSSHER'2 ITs are powerful cytotoxic
`agents characterized by delayed kinetic activity is reminiscent
`of the results reported by Maier er a1. (1991), who investigated
`antibody-mediated internalization of gplSSHER'Z and done-
`genie growth inhibition by anti-gp185”m’2 TAl —riein IT. While,
`in fact, only 12% of the radiolabellcd antibody was internal-
`ized following 4—hr incubation, the immunotoxin was neverthe—
`less capable of eliminating from 1 to 4 logs of gplSSHER'Z-
`positive cells, cytotoxicity increasing with longer incubation
`times, ranging from 6 to 24 hr. This behavior of the anti—
`gplSSHER'Z ITs could be explained by observations reported by
`Kyriakos et
`a1.
`(1992), who investigated the intracellular
`routing of different MAbs slowly internalized following bind-
`ing to the tumor-cell surface. High antigen density (>3 X 105
`sites/cell), as is observed where 1.3 X 106 gp1851'ER'2sites/cells
`are present
`in SK—BR—3 cells (Maier er al., 1991), favors
`divalent and irreversible binding (Kyriakos et al, 1992), result-
`ing in a slow access of the antibody to the cytoplasmic
`compartment. With ITs that are resistant to degradation, as
`reported for saporin-6 ITs (Bregni at (11., 1988), even a short
`binding time should be sufficient
`to produce the cytotoxic
`activity. On the contrary,
`low antigen density (<1 X 105
`sites/cell), as likely to occur in MDA—MB-365 breast-tumor
`cells, favors monovalent and reversible binding of the anti»
`body, which is released intact within 3 or 4 hr from the cell
`surface, Under these conditions, length of incubation in the
`presence of the ITs could become critical, with a low rate of
`internalization. Toxicity could be significantly reduced or even
`absent following a relatively short
`incubation time, while
`becoming progressively higher with prolonged incubation.
`Our study has demonstrated that highly specific and potent
`l’l's can be generated utilizing saporin—(j toxin and MAbs to
`different epitopes of gp185l"ER'3, thus widening our potential
`immunotherapeutic control of malignancies expressing the
`HER-Zoncogene product.
`ACKNOWLEDGEMENTS
`
`RT. is at present attached to the Department of Molecular
`Pathology, Regina Elena Cancer Institute, Rome. We grate—
`
`IMMUNOGEN 2063, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`- -— -— — —-—— —
`
`ahhit‘” ‘
`'hr
`.
`“'h—n.‘"""""‘------A
`. I'M—Ki
`In“.
`J“--L
`“‘1...
`.
`
`_______________ A
`""~-I-_..___
`*““-v-l
`.
`L1
`
`H
`
`m
`
`I
`
`a
`
`I
`
`I
`
`I
`
`‘ _
`
`l
`
`1"
`a
`2
`E
`3‘
`C
`.5
`8
`a
`“\u
`
`125
`
`100
`7
`
`5
`
`50
`
`25
`
`o
`
`TlME (hours)
`
`FIGURE 42 Short-term kinetics of IT cytotoxic activity in SK-
`
`
`BR—3 cells, SK—BR—3 cells were exposed for indicated times 0 to 6
`
`hr) to 1 X 10‘8 M W6/800E6/SAP,
`,W6/900H1/SAP,
`,1 X
`
`10 7 M SAP, A, and then pulsed for 1 hr with 3H-leucine. Results
`are expressed as mean % inhibition 0‘. 3H-leucine incorporation in
`triplicate samples.
`
`the protein—
`time-course evaluation of
`a
`lized, namely,
`inhibitory activity of the ITs, and the analysis of their capacity
`to interfere with clonogenic growth of SK—BRS cells.
`The results of these studies indicate that incomplete cytotoxe
`icity of ITs at 24 hr in SK-BR—3 can be related to a slow rate of
`internalization. Complete block of protein synthesis is in fact
`attained in SK—BR—3 cells after 48 to 60 hr of exposure to IT,
`suggesting that, in T9—4 and SK-BR-3 cells, no sub—population
`is constitutively resistant
`to anti-gplSSHER’Z/SAP ITS, but
`rather that the kinetics of intoxication are likely to be influ—
`enced by a ratealimiting stcp(s) that affects entry and/0r
`internalization of the [TS in reaching the ribosomal compart—
`merit.
`
`This is also supported by the potent and specific cytotoxic
`activity observed in the clonogenic assay, which does not
`require continuous exposure to the ITS. Furthermore,
`the
`obseivation that no cytotoxic activity was discernable in the
`MDA—MB-365 cell
`line, which expresses barely detectable
`
`IMMUNOGEN 2063, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`BREAST—CA RCINOMA IMMIJNOTOXtNS
`fullyacknowledge the technical assistance of Mrs. C. Full and
`Mr.’ T. Candiloro,
`the secretarial assistance of Miss M.V.
`Sarcone, and the graphic and photographic expertise of Mr. L.
`
`I. Zardin. This work was supported by
`Dall’Oco and Mrs.
`grants from PF CNR ACRO, AIRC and the Italian Ministry of
`Public Heal th.
`
`127
`
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`
`
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`'I-AZZNRI.
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`r\..
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`ttqutn-tn.
`
`I5t)Nl.-\IJI:\I. r\.. I.I.I.\lUl.l. R.M..
`l'tttt.-\
`.nnd S'l'llll’lt, l".. Imttutnotoxins
`containing saporin (I and monoclonal antibodies recognizing plasma
`cell“ . socitttctl antigens: ull'rcts on target cells and on normal myt‘loid
`prcutrsut'tt ((_.'I"‘U-(iM}. Entry). .I. Hrtr-itmml, 42, 238—245 [ “239].
`BJORN, M.J., RING, D. and FRANKEL, A., Evaluation of monoclonal
`antibodies for the development of breast—cancer iInm unotoxins. Ctll‘l-
`cerRes.. 45,1214(1985).
`
`BJORN. M.J., SMITH, HS, and DAIRKEE, S.I-I.. Response of primary
`human mammary—tumor cell cultures to a monoclonalantihody—
`recombinant ric1n—A»chatn untnunotoxin. Cancer Immunol. Immuno-
`then, 26, 121—124(1988).
`Btu ma. M., IAI'I'I, D.A., 31mm. 8., FORMOSA, A., VILLA, S_, SORIA,
`M., Bonaprmna. (i. and ('EIANNI. A.M., Activity of a monoclonal
`:mtilmtly-sztpnrinao conjugate against B—lymphoma cells. J. not. Cancer
`My!" HI], SI
`-SI 7" ( I933).
`
`I)»: I'tl'l'l'I-ZR. (LIL. BEGHIN, ‘C., MAKAR, A.P., VAN