[CANCER RESEARCH 58. 2661-2666.
`
`June 15. 1998)
`
`erbB-2 Receptor-specific Tumor Toxin
`Systemic Treatment with a Recombinant
`Efficiently Reduces Pulmonary Métastasesin Mice Injected with Genetically
`Modified Carcinoma Cells
`
`Martina Maurer-Gebhard, Mathias Schmidt, Marc Azemar, Uwe Altenschmidt, Elisabeth Stöcklin,Winfried Wels,
`and Bernd Groner1
`
`Institute for Experimental Cancer Research. Tumor Biology Center, D-79106 Freiburg. Germany
`
`ABSTRACT
`
`Receptor-mediated targeted tumor therapy is an important applied con
`sequence of the studies on the genetic causes of cancer. These therapy
`concepts have to be evaluated in novel animal models that reflect the molec
`ular aberrations found in human tumors. Here we introduce an animal model
`that allows the evaluation of drugs directed against a surface receptor that is
`frequently altered in primary human adenocarcinomas. Tumor toxins are
`polypeptides
`in which a tumor cell-specific recognition domain and a toxic
`effector domain have been joined by DNA recombination in vitro. Tumor cell
`recognition is contributed by a single-chain antibody domain specific for the
`extracellular domain of the erbB-2 receptor [scFv(FRPS)] and cytotoxicity by
`the enzymatically active domain of a bacterial exotoxin (exotoxin A from
`Pseudomonas aeruginosa). The erbB-2 receptor
`is overexpressed
`in many
`primary human cancer cells and is a favorable target
`for directed tumor
`therapy. The fusion protein scFv(FRP5)-exotoxin A has previously been
`shown to be able to efficiently and specifically kill erbB-2 receptor-expressing
`tumor cells. We have investigated the potential of this tumor toxin to detect
`and eliminate metastasizing tumor cells upon systemic administration. Mu
`rine renal carcinoma cells genetically modified with human erbB-2 receptor
`and bacterial ß-galactosidasegenes form large pulmonary métastaseswhen
`injected into the tail vein of BALB/c mice. Administration of the tumor toxin
`over a 10-day time period starting 1 day after tumor cell transplantation
`totally suppressed the formation of métastases.The treatment of animals 11
`days after tumor cell transplantation,
`allowing the establishment of many
`pulmonary métastases,led to a drastic reduction in their number and size.
`
`INTRODUCTION
`
`cellular
`that underlie
`changes
`the genetic
`of
`The identification
`consequences
`that
`result
`from
`transformation
`and the biochemical
`structurally
`changed or
`inappropriately
`expressed
`genes are being
`used as a vantage point for the development of new anticancer agents
`(1). A promising strategy originates with the preferential
`recognition
`of tumor cells by the therapeutic agent when compared with normal
`cells. This can be achieved when the therapeutic agent
`is able to bind
`to a cell surface component
`that exhibits enhanced expression levels
`in tumor cells. This is frequently the case for the erbB-2 receptor
`(2).
`The erbB-2 receptor
`is a member of the type I/erbB receptor
`tyrosine
`kinase family (3) and is overexpressed in many primary human tumor
`cells of epithelial origin including those of the breast, ovaries, stom
`ach, and lung. erbB-2 receptor expression in normal adult cells is low;
`the molecule is extracellularly accessible and causally involved in the
`process of malignant
`transformation (2). These aspects have made the
`erbB-2 receptor a suitable target for directed tumor therapy (4-6).
`Tumor
`toxins are bifunctional polypeptides designed to recognize
`antigens overexpressed on the surface of tumor cells and to kill target
`cells via a cytotoxic effector domain (7-9). We previously described
`
`Received 12/16/97; accepted 4/21/98.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement
`in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`at Institute for Experimental
`1To whom requests
`for reprints
`should be addressed,
`Cancer Research, Tumor Biology Center, Breisacher Strasse 117. D-79106 Freiburg.
`Germany.
`Phone:
`49-761-206-1600;
`Fax: 49-761-206-1599;
`E-mail:
`groner@sunl.
`tumorbio.uni-freiburg.de.
`
`2661
`
`that
`[scFv(FRPS)]
`antibody fragment
`the fusion of a single-chain
`provides
`recognition specificity for the extracellular
`domain of the
`erbB-2 receptor
`(10, 11) to the enzymatically
`active portion of ETA2
`
`fusion protein,
`(9). This recombinant
`aeruginosa
`from Pseudomonas
`and efficiently
`kills human erbB-2-
`specifically
`scFv(FRP5)-ETA,
`expressing tumor cells in vitro and inhibits the growth of s.c. tumors
`in vivo (4, 9, 12).
`The treatment of disseminated métastasesby the systemic application
`of antitumor agents is still a most difficult task for the medical oncologist.
`In a novel mouse model, we investigated the systemic engagement of the
`erbB-2 receptor-specific tumor toxin in the prevention of formation and
`in the reduction of established pulmonary métastases.For this purpose,
`we genetically modified a murine renal carcinoma cell
`line. Renca,
`(13-16) with the human erbB-2 gene to confer sensitivity toward the
`recombinant
`toxin and with the bacterial ß-galactosidasegene to provide
`for a sensitive means to detect
`tumor cells and quantitate metastasis
`formation. These genetic alterations did not affect
`the potential of the
`Renca cells to efficiently cause pulmonary métastaseswhen injected i.v.
`into syngeneic BALB/c mice. The therapeutic efficacy of scFv(FRPS)-
`ETA on pulmonary métastaseswas investigated. When animals were
`systemically treated, starting 1 day after tumor cell inoculation, with 20
`fj.g of tumor toxin for 10 consecutive days, the establishment of pulmo
`nary métastaseswas completely suppressed. When treatment was started
`11 days after tumor cell inoculation, upon formation of many metastatic
`nodules in the lung, the tumor toxin drastically reduced the number and
`size of the pulmonary métastases.Our experiments show that the sys
`temic application of scFv(FRP5)-ETA has a very favorable therapeutic
`effect.
`
`MATERIALS AND METHODS
`
`Cell Culture. Mouse renal carcinoma cells (Renca cells) and transfected
`cell clones Renca-lacZ and Renca-lacZ/erbB-2
`were grown in RPMI 1640
`supplemented with 10% PCS, 2 mM L-glutamine, 100 units/ml penicillin,
`100
`/¿g/ml streptomycin,
`0.25 mg/ml Zeocin (for Renca-lacZ and Renca-lacZ/
`erbB-2), and 0.48 mg/ml G418 (for Renca-lacZ/erbB-2).
`Transfection
`of Renca Cells. Renca cells were transfected with vector
`pZeoSV2/lacZ
`encoding
`for
`/3-galactosidase
`(Renca-lacZ)
`and were subse
`quently cotransfected with the plasmids pSV2erbB-2N and pSV2neo (Renca-
`lacZ/erbB-2)
`using the calcium phosphate precipitation method. Stable trans-
`fectants were selected in medium containing Zeocin (Renca-lacZ)
`or Zeocin
`and G418 (Renca-lacZ/erbB-2).
`Staining of Stably Transfected Cells. The
`ß-Galactosidase-dependent
`medium was removed from the cell culture plates, and the cells were fixed with
`0.05% glutaraldehyde
`for 5-10 min in PBS. The fixative
`solution was re
`moved,
`and the cells were washed with PBS before
`staining with X-Gal
`solution containing
`5 mM K,Fe(CN)6,
`5 mM K4Fe(CN)6 X 3H2O, 1 mM
`MgCl2, and I mg/ml X-Gal. The cells were incubated for 6 h at 37°C.
`X-Gal Staining
`and Quantitation
`of Pulmonary Métastases. Excised
`lungs were fixed overnight
`at 4°Cin PBS containing
`2% formaldehyde
`and
`
`0.2% glutaraldehyde. The fixative solution was removed,
`
`and the lungs were
`
`2 The abbreviations
`fluorescence-activated
`
`used are: ETA, exotoxin A; mAb, monoclonal
`cell-sorting.
`
`antibody; FACS,
`
`
`
`Downloaded from on October 21, 2014. © 1998 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2065, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`SUPPRESSION OF METASTASES
`
`BY RI-.COMHINANT TOXIN
`
`Renca-lacZ Renca-lacZ/Ert>B2
`
`-208
`
`-107
`
`-45
`
`io2
`fluorescenceintensity
`
`in renal carcinoma (Renca) cells. A. immunoblot
`Fig. I. Expression of erbB-2 receptor
`analysis of cell extracts
`from Renca-lacZ (Lane I) and Renca-lacZ/erbB-2
`(Lane 2).
`SKBR3 human breast carcinoma cells served as a control
`(Lane 3). erbB-2 protein was
`detected with an anti-c-neu antibody (mAb 3; Calbiochcm). The position of the 185-kDa
`erbB-2 molecule is indicated by an arrow. B. FACS analysis of Renca-lacZ and Renca-
`lacZ/erbB-2 cells. erbB-2 surface expression was measured with erbB-2-specific mAb
`FRP5 and subsequent
`incubation with a FITC-labeled antimouse
`IgG (Serotec).
`
`washed with PBS. Staining with X-Gal solution was performed as described
`above at 37°Cin the dark for 10-12
`h. Metastatic
`surface nodules were
`
`counted under a dissecting microscope.
`Immunoblotting.
`Cell extracts were prepared by lysis of 5 X IO5cells in
`7(X) fil »Ia buffer containing
`\7c NP40. 150 rtiM NaCl, 50 mM Tris-HCl
`(pH
`7.5), and 5 mM EDTA. Proteins were separated by SDS-PAGE and electro-
`blotted onto polyvinylidene
`difluoride membranes
`(Millipore. Bedford, MA).
`The erbB-2 receptor was visualized
`by incubation
`of the membranes with
`c-neu antibody (mAb 3; reactive with the mouse and human receptors; Cal-
`hiochem).
`followed by incubation with a horseradish peroxidase-coupled
`an-
`timouse antibody (Sigma) and chemiluminescent
`detection with the enhanced
`chemiluminescence
`kit (Amersham, Aylesbury, United Kingdom).
`FACS Analysis. Cells
`(5 X !(/) were incubated for 45 min with the
`erbB-2-specific
`antibody FRP5 and subsequently
`incubated with a FITC-
`labcled antimouse
`IgG (Serotec) according to the manufacturer's
`recommen
`
`dations. Stained cells were washed twice in PBS supplemented with 5% PCS,
`and fluorescence was measured by FACScan (Becton Dickinson).
`Tumor
`Bacterial Expression
`and Purification
`of
`scFv(FRP5)-ETA
`Toxin. The erbB-2-specific
`antibody toxin scFv(FRP5)-ETA was expressed
`and purified under denaturing
`conditions
`as described
`previously
`(12. 17).
`Purified recombinant proteins were renatured by dialysis against PBS contain
`ing 4(X) mM i.-arginine and subsequently
`renatured by dialysis against PBS.
`Cell Viability Assay. Cells were seeded in 96-well plates at a density of
`IO4 cells/well
`in growth medium. Various concentrations
`of scFv(FRP5)-ETA
`
`were added to triplicate samples, and the cells were incubated for 40 h. Ten ¡JL\
`of 10 mg/ml
`3-(4.5-dimethyllhiazol-2-yl)-2,5-diphenyltetrazolium
`bromide
`(Sigma)
`in PBS were added to each well, and the cells were incubated for
`another 3 h. Cells were lysed by the addition of 90 /xl of 20% SDS in 50%
`dimethyl
`formamide
`(pH 4.7). The absorbance of each sample at 590 nm was
`determined in a microplate reader as a measure of the relative amount of viable
`cells
`in comparison
`to the number of cells grown without
`the addition of
`recomhinant
`protein.
`Metastatic Growth of Transplanted Renca-lacZ and Renca-lacZ/erbB-2
`Cells in BALB/c Mice. Renca-lacZ or Renca-lacZ/erbB-2 cells (IO5) in 100
`/il of PBS were injected into the lateral
`tail vein of female 4-6-week-old
`BALB/c mice. Five animals/group were sacrificed at weekly intervals for up to
`4 weeks after tumor cell
`injection, and the lungs were excised. The number of
`pulmonary métastaseswas determined
`as described above.
`Antitumor Activity
`of scFv(FRP5)-ETA.
`Female BALB/c mice (five
`mice/group)
`at 4-6 weeks of age were injected i.v. with IO5 Renca-lacZ or
`Renca-lacZ/erbB-2
`cells in I(X) fi\ of PBS at day 0. ScFv(FRP5)-ETA was
`administered
`systemically through the tail vein at a dose of 20 jig in 100 /il of
`PBS/day
`from either days 1-10 or days 11-20 after
`tumor cell
`injection.
`Control
`animals
`received PBS. Four weeks after
`tumor cell
`injection,
`all
`animals were sacrificed, and the lungs were excised and stained with X-Gal for
`visualization
`of the pulmonary métastases.
`in 4% buffered
`Histology. Tissues were fixed for histológica! analysis
`formalin,
`embedded
`in paraffin,
`and sectioned at 4 ¡an.The sections were
`stained with H&E and examined for pathological
`findings.
`
`Stable transfection of Renca cells with an expression vector encod
`ing the human erbB-2 receptor yielded genetically modified cell
`clones which persistently express the receptor of 185 kDa on their cell
`surface (Fig.
`\A, Lane 2 and Fig.
`Iß).These cells are very sensitive
`toward the tumor-toxin. An 1CMIof 6 ng/ml was found and the cells
`loose their viability entirely upon 40 h of exposure to 100 ng/ml (Fig.
`2). The cytotoxic activity of scFv(FRP5)-ETA was suppressed when
`a 100-fold molar excess of the parental mAb FRP5, which gave rise
`to the scFv domain, was included in the incubation (data not shown).
`The introduction of a second gene into the Renca cells, the lacZ gene
`encoding ß-galactosidase, enabled us to conveniently
`and quantita
`tively visualize the tumor cells in culture (Fig. 3) or in organs upon
`staining with an appropriate substrate.
`Pulmonary Metastasis Formation by Renca-lacZ and Renca-
`lacZ/erbB-2 Cells. The tumorigenic propertiesof Renca cells and their
`potential to induce metastatic growth after transplantation into mice have
`been well established (13, 16). To ascertain that the genetically modified
`derivatives Renca-lacZ and Renca-lacZ/erbB-2 maintain the properties of
`parental Renca cells, we investigated their ability to form métastasesin
`BALB/c mice. For this purpose, 10s Renca-lacZ or Renca-lacZ/erbB-2
`cells were injected into the lateral tail vein of mice at 4-6 weeks of age.
`In four weekly intervals after the injection of the tumor cells,
`five
`animals/group were sacrificed, and the lungs were excised,
`fixed, and
`stained with X-Gal solution to visualize the disseminated tumor foci.
`Tumors on the surface of the lungs can be easily recognized and counted
`under a dissecting microscope.
`The number of tumor nodules detected on the lung surface at 1,2,
`3, and 4 weeks after tumor cell
`injection is shown in Fig. 4. After 2
`weeks,
`-150
`(Renca-lacZ)
`and -200
`(Renca-lacZ/erbB-2) métasta
`ses were detected. The numbers increased to approximately
`280 and
`320 after 3 weeks and increased to approximately
`320 and 350 after
`4 weeks. Histopathological
`analysis did not reveal metastasis
`forma
`tion in any other major organ (data not shown). We also analyzed the
`sera of the mice for the presence of antibodies directed against
`the
`human erbB-2 receptor or the bacterial ß-galactosidase protein. No
`humoral
`immune responses against
`these proteins were found (data
`not shown). We conclude that the Renca-lacZ and Renca-lacZ/erbB-2
`cells retain their potential
`for organ-specific metastasis
`formation.
`Systemic Treatment
`of BALB/c Mice with scFv(FRP5)-ETA
`Prevents the Establishment of Métastases.Renca-lacZ/erbB-2 cells
`are able to metastasize in BALB/c mice, and they are sensitive to the
`action of scFv(FRP5)-ETA in vitro. These prerequisites enabled us to
`2662
`
`RESULTS
`
`Metastasis
`scFv(FRP5)-ETA-sensitive
`a
`of
`Establishment
`Model. Mouse renal carcinoma cells (Renca) were established from a
`spontaneously arising kidney tumor in BALB/c mice. These cells effi
`ciently form métastasesin the lung upon transplantation into BALB/c
`mice through the tail vein (13, 18). We have based our experimental
`approach on this extensively characterized and widely used metastasis
`model. No reactivity of a mAb specific for the mouse or human erbB-2
`receptor (mAb 3) was found when receptor expression was probed by
`Western blot or FACS analysis [Fig. \,A (Lane 1) and B\. In accordance
`with this result, we found that parental Renca cells are insensitive to the
`action of the recombinant
`tumor toxin scFv(FRP5)-ETA. The function of
`this toxin is dependent on the binding to the extracellular domain of the
`human erbB-2 receptor, receptor-mediated internalization, and the inhi
`bition of cellular protein synthesis through ADP ribosylation of elonga
`tion factor EF-2 (19, 20).
`
`
`
`Downloaded from on October 21, 2014. © 1998 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2065, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`SUPPRESSION OF METASTASES
`
`BY RECOMBINANT
`
`TOXIN
`
`treated animals died within 6 weeks, whereas more than 50% of the
`treated animals survived longer than 44 weeks.
`Regression of Established Métastasesby Treating BALB/c Mice
`with scFv(FRP5)-ETA. We also tested the efficacy of treatment with
`the erbB-2-specific
`tumor toxin upon establishment of métastases.For
`this purpose, animals were inoculated with IO5 Renca-lacZ or Renca-
`lacZ/erbB-2 cells. The animals were left alone for 10 days to provide
`time for the tumor cells to establish lung métastases.At
`this time,
`-150-200 métastaseshad formed (Fig. 4). Treatment with 20 jag/day
`scFv(FRP5)-ETA commenced
`on day 11 and continued to day 20
`after tumor cell
`injection. At day 28, surface lung métastaseswere
`quantitated. Treatment with the tumor toxin caused a strong reduction
`in the number of pulmonary métastasesin the mice inoculated with
`Renca-lacZ/erbB-2
`cells. Only 85 tumor nodules were counted on the
`lung surface as compared with 380 tumor nodules
`found in mice
`inoculated with Renca-lacZ cells and treated with scFv(FRP5)-ETA in
`an identical
`fashion (Fig. 5C). Lungs
`from each treatment
`group
`stained with X-Gal are shown in Fig. 5D.
`treatment groups were also
`The lungs of the animals in the different
`evaluated
`by histopathological
`examination
`(Fig. 6). A section
`through the lung of a noninoculated untreated control animal
`is shown
`in panel Fig. 6A. In the lungs of the Renca-lacZ/erbB-2-inoculated
`mouse treated with PBS from days 1-10, a large number of tumors
`can be seen (Fig. 65). No tumors can be detected in the section from
`the mouse inoculated with Renca-lacZ/erbB-2
`and treated with scFv-
`(FRPS)-ETA from days 1-10 after inoculation (Fig. 6C). The lungs of
`the mouse inoculated with Renca-lacZ cells not expressing the erbB-2
`receptor
`and treated with scFv(FRP5)-ETA from days 1-10 after
`inoculation (Fig. 6D) showed the same number and size of métastases
`as the PBS-treated
`controls
`(Fig. 6B). Fewer
`and smaller
`tumor
`nodules are present
`in the lungs of animals treated from days 11-20
`after inoculation of the tumor cells (Fig. 6E) when compared with
`those of the control animals treated with PBS during the same time
`period (Fig. 6F). We also compared the appearance
`of métastases
`before the onset of treatment with the situation found in animals
`treated from days 11-20. A larger number of métastaseswere found
`in the lungs of untreated animals 14 days after tumor cell inoculation
`(Fig. 6G) than in the animals
`treated from days 11-20 with scFv-
`
`Renca-lacZ
`Renca-lacZ/ErbB2
`
`400
`
`300-
`
`200-
`
`to
`CD
`
`3 1"
`
`55
`
`m
`_Q
`E
`
`100-
`
`Renca-lacZ/ErtB2
`Renca-lacZ
`
`120
`
`100-
`
`8s
`
`s
`
`20-
`
`10000
`
`100
`1000
`concentration(ng/ml)
`Fig. 2. In vitro cytotoxicity
`of scFv(FRP5)-ETA toward Renca-lacZ or Renca-lacZ/
`erbB-2 cells. The cells were incubated for 40 h with the indicated concentrations
`of
`erbB-2-specific
`toxin scFv(FRP5)-ETA.
`The relative number of viable cells was deter
`mined using an enzymatic
`assay as described previously.
`
`Renca-lacZ/ErbB2
`Renca-lacZ
`Renca
`of /3-galactosidase
`activity in transfected Renca cells in vitro.
`Fig. 3. Expression
`Untransfected Renca cells (A),
`lacZ-transfected Renca cells (ß),or lacZ/erbB-2-trans-
`fected Renca cells (O were fixed and incubated with X-Gal as a substrate (magnification,
`X50).
`
`in vivo.
`scFv(FRP5)-ETA
`of
`activity
`antitumor
`the
`investigate
`BALB/c mice (five animals/group) were injected i.v. with 1 X IO5
`Renca-lacZ or Renca-lacZ/erbB-2
`cells. Treatment with scFv(FRPS)-
`ETA was started 1 day after the tumor cell
`injection. A total of 20
`¿ng/daywere administered in 100 ^.1 of PBS by injection through the
`tail vein.
`Injections were repeated at daily intervals
`for 10 days.
`Control animals were treated for the same time period with PBS.
`Twenty-eight
`days after tumor cell
`inoculation and 18 days after
`termination of treatment,
`the animals were sacrificed,
`their lungs were
`excised and stained with X-Gal, and surface pulmonary métastases
`were counted. Animals
`inoculated with Renca-lacZ or Renca-lacZ/
`erbB-2 cells and treated with PBS exhibited ~360 pulmonary métas
`tases (Fig. 5A). The treatment of animals inoculated with Renca-lacZ
`cells served as a control
`for the toxin specificity.
`In this case,
`treat
`ment with scFv(FRP5)-ETA did not influence the number of detect
`able métastases.In sharp contrast, four of five animals inoculated with
`Renca-lacZ/erbB-2
`cells and treated with scFv(FRP5)-ETA were
`completely free of pulmonary métastases(Fig. 5A). Only two very
`small
`tumor foci were found in the lungs of the fifth animal. Lungs
`from each treatment group stained with X-Gal are shown in Fig. 5B.
`These results show that systemic administration
`of the tumor
`toxin
`efficiently prevents
`the establishment
`of métastasesby Renca-lacZ/
`erbB-2 cells in the lungs. When the follow-up period of the nontreated
`and treated mice was extended, we observed that 100% of the non-
`
`01
`
`2345
`weeksaftertumorcellinoculation
`Fig. 4. Kinetics of pulmonary metastasis
`formation by Renca cells in BALB/c mice.
`Renca-lacZ or Renca-lacZ/erbB-2
`cells (10s) were injected into the lateral tail vein of each
`animal. At weekly intervals, up to 4 weeks after tumor cell injection,
`five mice from each
`group were sacrificed, and the lungs were excised. After
`fixation and incubation of the
`organs with X-Gal solution,
`tumor
`foci on the organ surface were counted as described
`previously.
`2663
`
`
`
`Downloaded from on October 21, 2014. © 1998 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2065, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`SUPPRESSION OF METASTASES BY RECOMBINANT
`
`TOXIN
`
`PBS
`
`scFv(FRP5)-ETA
`
`B
`
`MOt
`
`a
`
`Cellline:
`B
`Renca-lacZ
`
`0
`
`Renca-lacZ/ErbB2
`
`Treatment:20ug/dayi.v.fromday1-10
`
`PBS
`
`scFv(FRP5)-ETA
`
`PBS
`
`scFv(FRP5)-ETA
`
`treatment
`
`Cellline:
`a
`Renca-lacZ
`Renca-lacZ/ErbB2 §
`0)
`CE
`
`NOt
`
`Treatment:20ug/dayi.v.fromday11-20
`
`450
`
`scFv(FRP5)-ETA
`treatment
`
`CMm
`
`0)oc
`
`toxin scFv(FRP5)-ETA. Mice of 4-6 weeks of age (five mice/group) were
`tumor
`Fig. 5. Systemic treatment of BALB/c mice injected with Renca cells with the erbB-2-specific
`injected with IO5 Renca-lacZ or Renca-IacZ/erbB-2
`cells in the lateral
`tail vein at day 0. Animals were treated i.v. with 20 ¿ig/doseof the erbB-2-specific
`antibody toxin
`scFv(FRP5)-ETA either
`from days 1-10 or from days 11-20. Control animals were treated with PBS. All mice were sacrificed at day 28, the lungs were excised, and pulmonary
`métastaseswere visualized and counted under a dissecting microscope. A, the number of pulmonary métastasesin mice treated with scFv(FRP5)-ETA or PBS from days 1-10 after
`tumor cell injection. B. lungs of animals treated with scFv(FRP5)-ETA or PBS from days 1-10 after tumor cell injection. C, the number of pulmonary métastasesin mice treated with
`scFv(FRP5)-ETA or PBS from days 11-20 after tumor cell
`injection. D, lungs of animals treated with scFv(FRP5)-ETA or PBS from days 11-20 after tumor cell
`injection.
`
`the majority of the métastasesthat
`(FRPS)-ETA. We conclude that
`have been established at the onset of treatment at day 11 are elimi
`nated by the tumor toxin. The residual
`tumor cells that remain after the
`termination of treatment at day 20 probably expand into the métasta
`ses seen in Fig. 6£.
`
`of death. For this reason it is mandatory to improve on antitumor agents
`that can be administered systemically and are able to detect and eliminate
`distant métastases.We have been developing targeted antitumor agents
`that are able to distinguish between normal cells and tumor cells and have
`previously shown that these agents are effective in model systems in vitro
`and in vivo (9, 22-26). We now extend the investigation of the usefulness
`of an erbB-2 receptor-specific tumor toxin to its effect on distant métas
`tases.
`Surgery and radiotherapy are effective means to treat primary tumor
`in the
`cells have been used extensively
`Mouse renal carcinoma
`lesions and result in the cure of more than 50% of cancer patients (21).
`influence metastatic growth and survival
`study of agents that might
`However, not all tumors can be efficiently operated upon. In particular,
`after
`transplantation
`into animals
`(16, 27, 28). We improved this
`metastatic disease is still difficult to treat and remains the primary cause
`2664
`
`DISCUSSION
`
`
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`Research.
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`IMMUNOGEN 2065, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`SUPPRESSION OF METASTASES BY RECOMBINANT
`
`TOXIN
`
`Fig. 6. Histopathological analysis of the lungs of mice injected with Renca cells and treated with scFv(FRP5)-ETA. Sections of lung tissue 28 days after tumor cell injection (A-F) from:
`A. an untreated control animal; B, a Renca-lacZ/erbB-2-injected mouse treated with PBS from days 1-10 after tumor cell injection (multiple, extensive métastasesof an undifferentiated
`carcinoma); C, a Renca-lacZferbB-2-injected mouse treated with scFv(FRP5)-ETA from days 1-10 after tumor cell injection (normal
`lung, no microscopically detectable tumor cells); D. a
`Renca-lacZ-injected mouse treated with scFv(FRP5)-ETA from days 1-10 after tumor cell injection (multiple, extensive métastases);E. a Renca-lacZ/erbB-2-injected mouse treated with
`scFv(FRP5)-ETA from days 11-20 after tumor cell injection (few métastases);F, a Renca-lacZ/erbB-2-injected mouse treated with PBS from days 11-20 after tumor cell injection (multiple,
`extensive métastases).G, an untreated Renca-lacZ/erbB-2-injected mouse. The lungs were excised 14 days after tumor cell injection (multiple, dispersed métastases).Scale bar. 200 firn.
`
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`Research.
`
`IMMUNOGEN 2065, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`SUPPRESSION OK MKT AST ASES BY RECOMBINANT
`
`TOXIN
`
`10.
`
`important model system by stable genetic modification. The introduc
`tion of the human crbB-2 receptor gene renders the cells sensitive to
`the human erbB-2-specific
`tumor toxin, and the bacterial ß-galacto-
`sidase gene allows a sensitive and quantitative detection of pulmonary
`métastases.The Renca cells transfected with the /3-galactosidase gene
`only, provided an appropriate control for tumor toxin specificity. The
`genetically modified cells retain their lung-specific metastasizing po
`tential, and no métastaseswere found in other organs. The cells are
`not rejected by a host
`immune response, and no erbB-2- or ß-galac-
`tosidase-specific
`antibodies were found in the sera after transplanta
`tion into BALB/c mice (data not shown).
`The erbB-2 receptor
`is a crucial
`target for directed tumor therapy in
`human patients. The genetically modified Renca cells were used to
`show that the erbB-2-specific
`tumor toxin scFv(FRP5)-ETA had very
`beneficial
`antitumor
`effects
`in two different
`treatment
`settings:
`(a)
`treatment was started 1 day after tumor cell
`inoculation through the
`tail vein. At this time, no macroscopic
`surface métastaseswere pres
`ent. In this setting,
`four of five animals did not develop pulmonary
`métastases18 days after the termination of treatment. The recombi
`nant
`toxin efficiently suppressed the establishment of metastatic foci
`by the tumor cells. Histopathological
`analysis did not reveal signs of
`micrometastases,
`indicating a quantitative
`elimination of the tumor
`cells; and (b) animals were treated 11 days after tumor cell
`inocula
`tion. At this time, approximately
`200 métastaseswere detectable on
`the surface of the lungs. The number of pulmonary métastasesob
`served 8 days after termination of treatment was reduced to 25% of
`the number found in the control animals, and they were much smaller
`in size.
`on
`efficacy was strictly dependent
`therapeutic
`In both settings
`erbB-2 receptor expression,
`indicating the tumor cell specificity of
`scFv(FRP5)-ETA. During the experiments,
`the body weights of the
`treated mice were monitored at 2-day intervals, and no abnormalities
`were observed. The therapy was well
`tolerated. An analysis of the
`spleen,
`liver, and kidney of mice treated with scFv(FRP5)-ETA did
`not reveal signs of systemic toxicity. This is expected, because scFv-
`(FRPS)-ETA specifically recognizes the human erbB-2 receptor. After
`tumor toxin treatment,
`the animals were sacrificed on day 28 of the
`experimental
`schedule and analyzed for the presence of antibodies
`against
`the recombinant
`protein in their
`sera. All animals
`tested
`positive for specific antibodies
`(data not shown). We suggest
`that the
`elimination of the tumor cells precedes the induction of the immune
`response.
`of tumor
`the therapeutic deployment
`show that
`Our experiments
`toxins is not limited to the treatment of localized disease but can be
`expanded to the targeted eradication of disseminated tumors infiltrat
`ing the lungs. They also show that transfected Renca cells serve as an
`efficient
`and affirmative model
`for
`the investigation
`of
`treatment
`strategies directed at the erbB-2 receptor. The effectiveness
`in immu-
`nocompetent mice opens new ways to study the benefits of other
`biological
`and chemical
`treatment
`strategies. T-cell modification or
`costimulation
`and gene transfer-mediated
`cytokine expression strate
`gies (29, 30) have been designed that can be decisively tested for their
`antimetastatic potentials
`in this animal model.
`
`ACKNOWLEDGMENTS
`
`We thank Prof. G. Mihatsch (Department of Pathology, University Hospital,
`Basel. Switzerland)
`for help with the histológica! analysis.
`
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