THE JOURNAL OF BIOLOGICAL
`CHEMISTRY
`0 1994 hy The American Society for Biochemistry and Molecular Biology, Inc.
`Intracellular Expression of Single Chain Antibodies Reverts
`ErbB-2 Transformation*
`
`Vol. 269, No. 39, Issue of September 30, pp. 23931-23936, 1994
`Printed in U.S.A.
`
`(Received for publication, June 6, 1994, and in revised form, July 8, 1994)
`
`Roger R. Beerli, Winfried WelsS, and Nancy E. HynesP
`From the Friedrich Miescher-Znstitut, I? 0. Box 2543, CH-4002 Basel, Switzerland
`
`bodies (23) and Fab fragments (24) was possible. Recent tech-
`We report a novel approach for specific in vivo inacti-
`
`vation of the ErbB-2 receptor tyrosine kinase and sup-
`the cloning and
`nological advances have made possible
`expression of Fv molecules, the smallest high affinity binding
`pression of ErbB-2-induced transformation. Genes en-
`coding single chain antibodies that specifically bind to
`domain of an antibody (25-27). cDNAs specific for the variable
`the extracellular domain of human ErbB-2 were con-
`domains of Immunoglobulin heavy and light chains are cloned
`structed and expressed intracellularly in NIW3T3 fibro-
`by polymerase chain reaction and joined via a n oligonucleotide
`blasts transformed by activated ErbB-2. The single
`linker to yield a gene encoding a single chain antibody. Stable
`chain antibodies are derived from monoclonal antibod-
`alteration of a cellular phenotype by intracellular scFv expres-
`ies FRPS and FWP51 (Harwerth, I. M., Wels, W., Marte, B.
`sion in a mammalian system has been described recently. A
`M., and Hynes, N. E. (1992) J. Biol. Chern. 267, 15160-
`single chain antibody directed against the human immunodefi-
`15167) and are composed of heavy and light chain vari-
`ciency virus-1 envelope protein was expressed in the endoplas-
`able domains connected by a flexible peptide linker. The
`
`mic reticulum and shown to inhibit the processing of the enve-
`antibodies were provided with: 1) an N-terminal hydro-
`lope protein precursor (28).
`phobic leader sequence to target their synthesis to the
`We report here on
`the expression in mammalian cells
`
`lumen of the endoplasmic reticulum, and 2) a C-terminal
`scFvs directed against human ErbB-2. ScFvs derived from
`retention signal to prevent secretion. When expressed in
`mAbs FRP5 and FWP51, which bind to the extracellular do-
`ErbB-2-transformed cells, the single chain antibodies
`main of the receptor (20, 21) were used for this purpose. The
`
`bound to the receptor and prevented its transit through
`scFvs were targeted to the lumen of the ER’ of NIW3T3 fibro-
`the endoplasmic reticulum. This resulted in the func-
`blasts transformed by oncogenically activated ErbB-2. We show
`tional inactivation of the receptor and reversion of the
`that both scFvs were stably expressed at high levels and func-
`transformed phenotype. This is the first demonstration
`tional in binding and inhibiting the ER transit of ErbB-2. Re-
`of a targeted and stable inactivation of a cellular onco-
`tention of the receptor in the ER caused its functional inacti-
`protein via intracellular antibody expression. The use of
`such a strategy represents a simple and powerful ap-
`vation and reversion of the transformed phenotype.
`in vivo function of receptors and
`proach to study the
`other cellular proteins.
`
`of
`
`The abbreviations used are: ER, endoplasmic reticulum; FACS, flu-
`orescence-activated cell sorting; mAb, monoclonal
`antibody; PAGE,
`polyacrylamide gel electrophoresis; PDGF, platelet-derived growth fac-
`tor; scFv, single chain antibody; VH, heavy chain variable domain; VL,
`light chain variable domain.
`23931
`
`EXPERIMENTAL PROCEDURES
`Cloning and Construction ofthe Single Chain Antibodies-RNAfrom
`hybridoma cells producing mAbs FRP5 and FWP51(20,21) was reverse
`transcribed and the heavy chain (VH) and the light chain (VL) variable
`The erbB-2 gene encodes a 185-kDa transmembrane glyco-
`domain cDNAs were isolated by polymerase chain reaction, sequenced,
`protein that is a member of the subclass I, epidermal growth
`and used to construct genes encoding the single chain antibodies scFv
`(14). Amplification
`factor receptor-related tyrosine kinases
`FRP5 and scFv FWP51, as described (22, 29, 30). In order to obtain
`a n d o r overexpression oferbB-2 is observed in tumors arising at
`genes for intracellular expression in mammalian cells, cDNAs encoding
`many sites including breast and ovary where it correlates with
`scFvs-5S, 51S, 5R, and 51R were constructed. Two pairs of complemen-
`tary oligonucleotides encoding sequences specific for a human Immu-
`an unfavorable patient prognosis (5-10). The oncogenic poten-
`noglobulin heavy chain signal peptide (VH 71-5‘CL) (31) were de-
`tial of the ErbB-2 protein can be activated by different mecha-
`signed. The oligonucleotides were assembled, ligated, and cloned as a
`nisms including point mutation
`(11-14) and overexpression
`HindIIIIPstI fragment into pWW15 (291, upstream of the scFv FRP5
`(15, 16). ErbB-2 as a target for cancer therapy is an area of
`(22, 29, 30) and scFv FWP51 (30) cDNAs. To produce
`scFv-5S and
`intense research. ErbB-2-specific monoclonal antibodies
`(17-
`scFv-51S, the proteins were tagged with a C-terminal FLAG epitope
`21) and recombinant immunotoxins (22) both of which inhibit
`(DYKD). To produce scFv-5R and scFv-51R an additional EL was added
`to the C terminus. The DYKD and DYKDEL peptides were encoded by
`in vitro or in vivo growth of ErbB-2 overexpressing tumor cells
`synthetic oligonucleotides that were assembled into the cDNAs as
`have been described. Here we present a novel approach using
`BglIIIXbaI fragments. The resulting open reading frames were flanked
`intracellular expression of single chain antibodies (scFv) to
`by upstream Hind111 and EcoRI sites and downstream Sal1 and XbaI
`suppress ErbB-2 transformation.
`sites. The cDNAs were cloned into the retroviral vector pBabePuro (32)
`The concept of using antibodies to alter cellular physiology
`as EcoRIISalI fragments.
`was initially tested in yeast where it was shown that expres-
`Cell Culture-NIWErbB-2 cells are a clone of NIW3T3 cells (clone
`sion and assembly in the cytoplasm of functional intact anti-
`no. 3.7) expressing an oncogenically activated human erbB-2 under the
`control of the SV40 early promoter (21). NIW3T3 and NIWErbB-2*
`cells were maintained in Dulbecco’s modified Eagle’s medium contain-
`ing 8% fetal calf serum. For the latter, the medium was supplemented
`with 0.5 mg/ml G-418.
`
`~
`
`* The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`“aduertisement” in accordance with 18 U.S.C. Section 1734 solely to
`indicate this fact.
`$ Present address: Tumor Biology Center, Breisacher Strasse 117,
`D-79106 Freiburg, Germany.
`0 To whom correspondence and reprint requests should be addressed:
`Friedrich Miescher-Institut, P. 0. Box 2543, CH-4002 Basel, Switzer-
`land. Tel.: 41-61-696-6869; Fax: 41-61-696-3835.
`
`IMMUNOGEN 2066, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`/'\T\
`
`I
`
`/" MDWIWRILFLVGAATGAHS
`
`/ \
`
`R DYKDEL
`
`(GGGGS),
`
`scFu Mediated Inactivation of ErbB-2
`~ e ~ r o u i r a l Gene ~ansfeF-Ecotropic virus was prepared basically as
`described (33). The amphotropic packaging cell line PA317 (34) was
`transfected with 10 pg each of pBabePurolscFv-BS, 51S, 5R, 51R, and
`empty vector using the calcium phosphate precipitation method. M e r
`24 h, conditioned medium containing transiently produced virus was
`harvested and used for infection of the ecotropic packaging cell line CtE
`(32) in the presence of 8 pdml polybrene. Two days after infection, the
`cells were placed in 2 pg/m1 puromycin (Fluka). Virus-containing me-
`dium collected from pools of puromycin-resistant RE was used to infect
`NIW3T3 and NIH/ErbB-2* cells, and after 3 days the cells were placed
`in 2 pg/ml puromycin. Pools of puromycin-resistant cells were analyzed
`in all the experiments.
`lysates were prepared by adding 500 p1 of
`Western Blotting-Cell
`lysis buffer 150 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 m~ NaCl, 5
`m~ EGTA) supplemented with protease inhibitors ( 10 pgiml aprotinin,
`10 pglml Ieupeptin, 1 mM phenylme~ylsulfonyl fluoride) and phospha-
`tase inhibitors (2 mix sodium o~hovanadate, 50 m~ sodium fluoride, 10
`mM sodium molybdate, 20 p~ phenyl arsine oxide) to cells in a 10-cm
`dish and incubating on ice for 10 min. The lysates were clarified by
`centrifugation at 10,000 x g for 10 min. 50 pg of protein were separated
`by SDS-PAGE, transferred t o polyvinylidenedifluoride membranes, and
`assayed by Western blot analysis with either an anti-scFv antiserum
`(29), 21N antiserum against ErbB-2 (35), or an anti-phosphotyrosine
`monoclonal antibody (36). Bound antibody was detected with horserad-
`ish peroxidase-coupled anti-rabbit or anti-mouse polyclonal antibodies
`using enhanced chemiluminescence (Amersham).
`Immunoprecipitations-For the detection of scFv proteins in culture
`supernatants, conditioned medium was harvested, a polyclonal rabbit
`anti-mouse I&
`(ICN Immunobiologicalsf was added and allowed to
`bind for 1 h on ice after which the immune complexes were collected by
`the addition of protein A-Sepharose. Bound proteins were released by
`boiling in sample buffer and analyzed by Western blotting with an
`anti-scFv antiserum (29). For coimmunoprecipitation of SCFVS with
`ErbB-2*, the receptor from 250 pg of protein was immunoprecipitated
`with the 21N antiserum (35) and analyzed by Western blotting with an
`anti-scFv antiserum (29).
`monitor growth, 3 x lo3 cells in culture
`Cell Growth Assays-To
`medium were plated/well of a 96-well dish and growth was monitored
`aRer 24,48, and 72 h. To examine growth induction by PDGF and basic
`fibroblast growth factor, the cells were placed 24 h in serum-free me-
`dium. Then, they were either induced with 20 ng/ml PDGF or 50 ng/ml
`basic fibroblast growth factor, or grown in the absence of factors.
`Growth induction as compared with the untreated cells was measured
`after 2 days. The experiments were performed using the Cell Titer 96TM
`kit (Promega), and all points were prepared in triplicate.
`
`Z ~ m ~ n o c y t ~ ~ m i s t ~ - F o r indirect immunofluorescence, cells were
`grown 2 days on chamber slides (Nunc), fixed 30 min with 3.7% form-
`aldehyde, and permeabilized with 0.5% TritonX-100 for 3 min. Staining
`for SCFVS was done using a scFv-specific antiserum (29) in combination
`with a rhodamine-linked anti-rabbit polyclonal antibody (Sigma).
`Staining for ErbB-2 was done using mAb FSP77 (201 in combination
`with a fluorescein-linked anti-mouse polyclonal antibody (Amersham).
`The cells were mounted and observed by fluorescence microscopy.
`Flow Cytometrie Analysis-Cells were trypsinized and counted prior
`to staining. 5 x 10" cells were washed with 2 ml of FACS buffer (phos-
`phate-buffered saline containing 0.1% sodium azide and 1% bovine
`serum albumin) and resuspended in 50 pl of FACS buffer containing 20
`pg/ml fluorescein isothiocyanate-coupled mAb FSP77 (20). Following
`incubation on ice for 1 h, the cells were washed twice with 2 ml of FACS
`buffer, resuspended in 300 pi of FACS buffer, and analyzed for their
`fluorescence in a Becton-Dickinson FACScan.
`Soft Agar Growth-To examine anchorage-independent growth, 5 X
`lo* cells were plated in duplicate in 6-cm dishes in 6 ml of culture
`medium supplemented with 0.35% noble agar overlying a 0.7% agar
`layer. The plates were incubated 14 days at 37 "C after which colonies
`were stained by adding 2 ml of phosphate-buffered saline containing 0.5
`mgiml nitro blue tetrazolium for 24 h. Colonies were counted using an
`Artek 880 colony counter (Dynatech Laboratories, Inc.).
`
`23932
`
`RESULTS
`Construction and Expression of scFvs FRPS and FWP51-
`Hybridoma cells producing mAbs FRP5 and FWP51 (20, 21)
`which bind to the extracellular domain of human ErbB-2 were
`used to construct genes encoding SCFVS, as described previously
`(22, 29, 30). For expression in eukaryotic cells, two versions of
`each cDNA were created. The scFv-5s and SCFV-51s cDNAs are
`
`S: DYKD
`FIG. 1. Diagram of the secreted ( S ) and ER-retained (R) forms
`of scFvs FRPS and m 6 1 . The N-terminal signal peptide (Sf'), the
`heavy chain variable domain (VH), the linker peptide (L), the light
`chain variable domain (VL), and the C-terminal tags (2') are indicated.
`DYKD: FLAG epitope; DYKDEL: FLAG epitopeiER retention signal.
`
`predicted to encode secreted proteins, whereas the scFv-5R and
`scFb-SlR cDNAs encode variants which are expected to be lo-
`calized to the lumen of the ER (Fig. 1). The scFv proteins have
`an N-terminal Ig heavy chain-derived signal peptide which
`
`directs them to the secretory ~ m p a ~ m e n t of the cell, the same
`
`compartment through which ErbB-2 passes on its way to the
`plasma membrane. The FLAG epitope tag DYKD is present at
`the C terminus of all scFv proteins. The scFv-R proteins have
`an additional EL at their C terminus, thereby providing these
`versions with the ER retention signal, KDEL. This sequence is
`predicted to cause the retention of soluble proteins in the lumen
`of the ER (37).
`We investigated effects of intracellular scFv expression in
`ErbB-2-transformed fibroblasts. NIW3T3 clone 3.7 expresses
`an oncogenically activated form of the receptor, designated
`ErbB-2*, that carries a single amino acid substitution (valine to
`glutamic acid) in the transmembrane domain (21). The kinase
`activity of ErbB-2* is constitutive and ligand-independent (14).
`This cell line, referred to as NIH/ErbB-2*, shows the typical
`phenotype of transformed fibroblasts, as judged by morphology,
`focus formation, ability
`to undergo anchorage-independent
`growth, and tumor formation in nude mice (21).
`We have used retroviral gene transfer to express the scFvs
`FRPB and FWP51 in control NIW3T3 and in NIWErbB-2"
`cells. First, we examined expression and localization of the
`scFvs. In both cell lines the proteins were produced at high
`levels as shown by Western blotting with a scFv-specific anti-
`serum. The SCFVS targeted for secretion were found in the con-
`ditioned medium of the cells (Fig. %), whereas the SCFVS tar-
`geted to the lumen of the ER were found intracellul~ly (Fig.
`223). Both in control NIW3T3 and in N I ~ r b B - 2 * , t h e
`scFv-5R
`was detected as a double band (Fig. 2B, lanes 3 and 8). The
`upper band corresponds to a N-glycosylated form and was not
`detected after treatment of extracts with peptidyl-N-glycosi-
`dase F2. In extracts of NIWErbB-2* cells, more of the secreted
`SCFVS was detected than in extracts of control NIW3T3. This
`difference is probably due to scFvs bound to the ErbB-2* on the
`cell surface.
`Intracellular expression or secretion of scFv proteins did not
`affect the general metabolism of the cells. Morphology and
`growth rate of scFv expressing and vector control NIW3T3 cells
`
`was indistinguishable, indicating that the antibodies were non-
`toxic to the cells (Fig. 31.
`of scFus and ErbB-
`
`~ ~ b c e Z l ~ ~ a r Localization and Ass5c~at~5n
`next examined the subcellular distribution of the
`2*-We
`scFv-R proteins and ErbB-2* in NIH/ErbB-2* cells expressing
`scFvdR and scFv-5lR. In both cell lines, immunofluorescence
`using a scFv-specific antiserum revealed the staining of a tu-
`bular network throughout the cytoplasm, typical of ER resident
`proteins. Staining of control NIH/ErbB-2* cells with a mAb
`specific for human ErbB-2 revealed that the major part of the
`receptor was located intracellularly (Fig. 4A). Since it has been
`shown that most of the constitutively activated ErbB-2 is de-
`graded before reaching the plasma membrane (381, we assume
`that this is the reason for the weak plasma membrane staining.
`Signi~cantly, compared with control NIH/ErbB-2* cells, there
`
`IMMUNOGEN 2066, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`A
`
`B
`
`SCFV Mediated Inactivation
`
`
`
`culture supernatant
`
`NIH/3T3 NIH/erb&P'
`
`
`
`of ErbB-2
`
`23933
`
`A
`
`NIH/erbB-2' C
`
`x F v 5 R
`
`xFv5 1 -R
`
`38.0 >
`33.5 >
`
`proteln extracts
`
`NIH/3T3 NIH/erb&2'
`
`
`
`MW(kDa)
`38.0 >
`33.5 >
`
`B
`
`NIH/3T3 NIH/&B-2'
`
`s
`
`0
`
`33.5 >
`
`log fluorescence
`
`FIG. 2. Expression of scFvs FRP5 and FWP51 in control MW
`3T3 and in NIH/ErbB-2* cells. A, secretion of the scFvs. Conditioned
`medium of cells infected with vector control, scFv-5S, scFv-5R, scFv-
`51S, and scFv-51R virus was collected, immunoprecipitated with rabbit
`anti-mouse IgG (ICN Immunobiologicals), subjected to 15% SDS-PAGE,
`and analyzed by Western blotting with a scFv-specific antiserum (29).
`B, intracellular expression of scFvs. Virus-infected cells were lysed in
`Triton X-I00 lysis buffer, and 50 pg of protein were subjected to 15%
`SDS-PAGE and analyzed by Western blotting as above.
`,
`
`1.0
`
`I
`
`*Control
`
`0.8 - +scFv-SR
`--scFv-SlS
`*scFv-SlR
`
`-
`5
`In 0.6 -
`2
`0
`0
`0.4 -
`
`0.2 -i
`12
`
`24
`
`60
`
`72
`
`48
`36
`time (hours)
`FIG. 3. Growth of NW3T3 cells infected with vector control,
`SCFV-~S, scFv-5% scFv-SlS, and scFv-51R virus. Cell growth was
`monitored at the indicated times using the Cell Titer 96TM kit.
`
`was an increased ER staining of the NIH/ErbB-2* cells express-
`ing scFv-5R and scFv-51R with the ErbB-2 specific mAb (Fig.
`4A 1. This suggests that there was colocalization of ErbB-2* and
`antibodies in the lumen of the ER. As an additional indication
`for a physical interaction in vivo, the scFvs could be coimmu-
`noprecipitated with
`the receptor from total extracts of NIW
`ErbB-2* cells using an ErbB-2 specific antiserum (Fig. 4B).
`However, it is noteworthy that in the case of the scFv-5R ap-
`parently only the unglycosylated protein bound to the receptor.
`A flow cytometric analysis revealed that the amount of ErbB-2*
`on the cell surface was dramatically decreased in the cells
`expressing scFv-5R and scFv-51R (Fig. 4C). This demonstrates
`that there was a stable association of the scFvs with ErbB-2*
`within the lumen of the ER which inhibited transit of the re-
`ceptor to the cell surface.
`Antibody expression did not affect the ER transit of other
`growth factor receptors. Both PDGF, as well as basic fibroblast
`
`log fluorescence
`FIG. 4. Subcellular localization and association of scFvs and
`ErbB-2* in NIH/ErbB-2* cells. A, immunofluorescence of scFv ex-
`pressing NIH/ErbB-2'? cells. Cells infected with scFvdR, scFv-51R. and
`vector control virus were stained for expression of scFvs using a scFv-
`specific antiserum (29) and for human ErbB-2:* using mAb FSP77 (20).
`B, coimmunoprecipitation of scFvs and ErbB-2". Control NIH/3T3 and
`NIWErbB-2I' cells infected with scFv-BR, scFv-51R. and vector control
`virus were lysed in Triton X-I00 lysis buffer. ErbB-2" was immunopre-
`cipitated from 250 pg of lysate the with 21N antiserum (351, subjected
`to 15% SDS-PAGE, and analyzed by Western blotting with a scFv-
`specific antiserum (29). C, cell surface staining
`for ErbB-2*. NIW
`ErbB-2" cells infected with scFv-5R (solid line, uppergraph), scFv-51R
`(solid line, lower graph), and vector control virus (narrow dotted lines),
`as well as control NIW3T3 cells (wide dotted lines) were stained using
`mAb FSP77 (20) and subjected to flow cytometric analysis.
`
`growth factor, stimulated growth of the scFv-5R and scFv-51R
`expressing NIW3T3 cells to an extent similar to that seen for
`the vector control cells (Table I), showing that the effect of the
`scFv-R proteins is specific for ErbB-2.
`ErbB-2* Function in scFv Expressing Cells-To
`assess the
`effects of the scFv-mediated ER retention on ErbB-2*, we ana-
`lyzed whole cell lysates by Western blotting (Fig. 5). An anal-
`ysis using an ErbB-2-specific antiserum revealed that the ER-
`retained ErbB-2* showed an increased mobility on SDS-PAGE
`(Fig. 5A), suggesting that there are changes in its post-trans-
`lational modifications. ErbB-2 is a glycoprotein (4, 38) which
`undergoes N-linked glycosylation in the ER followed by modi-
`fication and extension of the carbohydrate side chains in the
`Golgi apparatus. Thus it is likely that glycosylation of the ER-
`retained ErbB-2* is impaired which may account for the differ-
`ent apparent molecular weight. The level of ErbB-2:% was
`strongly elevated in the scFv-5R and scFv-51R expressing cells
`the ER might be due to an
`(Fig. 5A). This accumulation in
`increased rate of synthesis or a decreased rate of degradation of
`the protein. A Western analysis using a phosphotyrosine-spe-
`cific mAb showed that the total phosphotyrosine content of
`ErbB-2* did not change dramatically in any of the cell lines
`
`IMMUNOGEN 2066, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`23934
`
`
`
`
`
`SCFV Mediated Inactivation
`
`TARLE I
`Growth stimulation by PDGF and basic fibroblast growth factor in
`scFv expressing NIHl3T3 cells
`NIW3T3 cells infected with vector control virus, scFv-5R virus, and
`scFv-51R virus were serum-starved for 24 h prior to induction by PDGF
`(20 ng/ml) or basic fibroblast growth factor (50 ng/ml) for 2 days. The
`percentage of growth stimulation (2 S.D.) in comparison to untreated
`cells was calculated and is ~ v e n below.
`
`NIIU3T3
`
`Control
`SCFV-~R
`S C F V - ~ ~ R
`
`A
`
`10.8 (e0.6)
`13.8 (k1.5)
`10.3 (e1.0)
`
`NIH/erbE2'
`
`Basic fibroblast
`growth factor
`12.6 (k1.9)
`15.5 (k1.6)
`12.4 (k0.4)
`.&
`
`C
`
`1'
`
`1
`
`58 >
`48.5 >
`
`<€
`
`. c
`
`c
`
`E
`
`of ErbB-2
`
`NIH/3T3
`
`NIH/erbB-2'
`
`control
`
`NIH/erbB-2' scFv51-R
`NIH/erbB-2' scFv5-R
`FIG. 6. Morphological reversion of cell transformation. The
`morphology of NIWErbB-2* cells infected with SCFV-~R, scFv-51R, and
`vector control virus, as well as control NIW3T3 cells is shown.
`
`2*-transformed cells reverted to the more flattened appearance
`of normal fibroblasts (Fig. 6). In addition, while the control-
`infected cells and those expressing the secreted scFvs formed
`foci a t a high efficiency, the cells expressing the ER-retained
`scFvs failed to do SO.^ To further investigate the changes in the
`transformed phenotype of NIWErbB-2'% cells, we analyzed
`their anchorage-independent growth in soft agar. ER expres-
`sion of both scFvs dramatically reduced colony formation as
`compared to the control-infected cells and to cells expressing
`the scFv-51s. Expression of the scFv-5S caused up to 44%
`inhibition on soft agar growth (Table 11). Anchorage-independ-
`ent growth of cells expressing the scFv-5R was similar to nor-
`indicating complete reversion of the trans-
`mal NIW3T3;
`formed phenotype.
`
`I
`I
`FIG. 5. Phosphotyrosine analysis and level of ErbB-2* in NIW
`ErbB-2* cells. A , Western blot analysis of ErbB-2*. NIWErbB-2* cells
`DISCUSSION
`infected with vector control, scFv-5S, scFv-BR, scFv-51S, and scFv-51R
`Our results show the stable inactivation of an oncoprotein,
`virus, as well as control NIW3T3 cells were lysed in Triton X-100 lysis
`accompanied by reversion of a transformed phenotype, via in-
`buffer, 50 pg of total protein were separated on 7.5% SDS-PAGE, and
`tracellular single chain antibody expression. As a model system
`the ErbB-2" content was analyzed by Western blotting with the 21N
`antiserum (35). B , phosphotyrosine content of ErbB-2". 50 pg of total
`we have chosen the mAbs FRP5 and FWP51 which bind to the
`protein were subjected to
`9% SDS-PAGE and analyzed by Western
`extracellular domain of human ErbB-2. We have studied effects
`blotting with a phosphotyrosine specific mAb (36). C, pattern of phos-
`of ER lumenal expression of scFvs FRP5 and FWP51 in NIW
`photyrosine containing proteins. 50 pg of total protein were subjected to
`ErbB-2* fibroblasts transformed by point-mutated, activated
`5-12% gradient SDS-PAGE and analyzed by Western blotting with a
`phosphotyrosine specific mAb (36). Arrows indicate bands that show
`human ErbB-2. We have shown that both scFvs, which recog-
`different intensity in the individual cell lines.
`nize different epitopes on the ErbB-2 extracellular domain,
`were very potent in affecting the ER transit and transforming
`
`ability of ErbB-2*. Our results demonstrate (i) the feasibility of
`expressing functional antibodies intracellularly, (ii) the possi-
`bility of inhibiting the transit of an integral membrane protein
`through the ER by means of scFv expression, and (iii) that
`retention of a constitutively active receptor tyrosine kinase in
`the ER interferes with its transforming ability.
`We have observed that single chain antibodies directed to the
`secretory pathway are
`found in the
`conditioned medium,
`whereas scFvs containing a C-terminal ER retention signal are
`stably retained in the ER, irrespective of coexpression of ErbB-
`2*. This is in contrast to a recent publication showing that a
`single chain antibody against gp160, the human immunodefi-
`ciency virus-1 envelope protein precursor, which was designed
`for secretion, was stably retained in the ER, whereas a variant
`carrying an additional C-terminal ER retention signal, yielded
`an unstable protein that was rapidly degraded if not coex-
`
`(Fig. 5B). Due to the strongly increased level of ErbB-2*, the
`receptor appears to be phosphorylated at a much lower stoichi-
`ometry in the cells expressing the retained versions of the
`scFvs, indicating a decrease in its kinase activity. It should be
`noted that the phosphotyrosine content of ErbB-2" was lower in
`cells expressing scFv-5R than in cells expressing scFv-SlR,
`while the ErbB-2* level was slightly higher. Most importantly,
`compared to the control-infected NIWErbB-2* cells and to cells
`expressing the secreted scFvs, the intensity of bands with ap-
`parent molecular masses of 145, 84, 56, 52, and 48 kDa was
`markedly reduced in cells expressing scFv-5R and scFv-51R
`(Fig. 5C). The pattern of phosphotyrosine-containing proteins
`in these cells was very similar to the one in control NIW3T3
`cells. These observations suggest that the retention of ErbB-2"
`in the ER led to its functional inactivation.
`of
`Reversion of
`the Dansformed Phenotype-Retention
`ErbB-2* in the ER resulted in a drastic change in the morphol-
`ogy of the NIWErbB-2* cells. The rounded shape of the ErbB-
`
`Roger R. Beerli, unpublished results.
`
`IMMUNOGEN 2066, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`
`
`
`
`
`
`SCFV Mediated Inactivation
`TABLE I1
`Soft agar growth of scFv expressing NIHIErbB-2” cells
`NIWErbB-2* cells infected with vector control, scFv-5S, scFv-5R,
`scFv-51S, and scFv-51R virus were plated in culture medium supple-
`mented with 0.35% agar. After 14 days viable cells were stained and
`colonies >50 and >200 pm were counted using an Artek colony counter.
`The experiment was repeated three times in duplicate, and the colony
`numbers of one typical experiment (k S.D.) are shown below.
`>200 pm
`NIH/ErbB-2*
`inhibition
`% inhibition
`>50 pm
`Control
`239 (k7.1)
`71 (20.0)
`40 (24.2)
`SCFV-~S
`163 (k7.8)
`4 (k0.7)
`SCFV-5R
`11 (k0.7)
`S C F V - ~ ~ S 231 (k9.9)
`68 (22.8)
`14 (k2.8)
`S C F V - ~ ~ R
`59 (k0.7)
`
`23935
`of ErbB-2
`ER-located ErbB-2* displayed a lower phosphotyrosine content
`in the scFv-5R, than in the scFv-51R expressing cells. Reten-
`tion of proteins in the lumen of the ER is mediated by a recep-
`tor-like protein recognizing the KDEL peptide present on the C
`terminus of ER resident proteins and is achieved by their con-
`tinual retrieval from the cis-Golgi (or a pre-Golgi) compartment
`(37, 39, 40). In the case of scFv-5R expressing cells, the final
`configuration of the transiently formed, trimeric complex con-
`sisting of the KDEL receptor, scFv, and ErbB-2* might interfere
`with dimerization and kinase activity. Second, binding of the
`the scFv FRP5, but not the scFv Fwp51, per se seems to affect
`the activity of ErbB-2*. The secreted form of the scFv FRP5
`also repressed the soft agar growth of NIH/ErbB-2* cells, with-
`out affecting ErbB-2* receptor trafficking2 or phosphotyrosine
`content, possibly by altering the receptor conformation. It was
`pressed with its binding partner (28). Single chain antibodies
`noted in a recent publication (41) that an ErbB-2-specific mAb
`consist of Immunoglobulin heavy and light chain variable do-
`was growth inhibitory, without affecting ErbB-2 tyrosine phos-
`sequence. This is likely to
`mains and are, therefore, variable in
`phorylation. This suggests that for the inhibition of growth,
`cause biochemical differences between individual scFv proteins
`which might explain their different in vivo behavior.
`antibody induced effects such as alteration of receptor confor-
`mation could be as important as effects on kinase activity.
`ER expression of both scFv-5R and -51R led to a strongly
`It has been suggested that constitutively active receptor ty-
`elevated level of ErbB-2*. This could either be due to an in-
`rosine kinases, such as ErbB-2* or the PDGF receptor in sis-
`creased synthesis rate or a decreased turnover rate of ErbB-2*.
`transformed cells, might at least partially exert their trans-
`We favor the latter explanation
`since in the transfectants,
`forming activity intracellularly, in the ER and/or the Golgi
`ErbB-2* expression is under the control of the SV40 early pro-
`compartment (38,42). The experiments presented in this paper
`moter making transcriptional or translational control unlikely.
`show that the intracellular retention of an activated ErbB-2*
`
`Due to its constitutive kinase activity, the half-life of ErbB-2* is
`short, approximately 1.5 h, while the normal ErbB-2 has a
`causes reversion of transformation. Cells expressing the se-
`half-life of more than 7 h (14). Compared to the transformed
`creted version of scFv FWP51 are not affected in their growth,
`indicating that the binding of scFv F W 5 1 does not affect re-
`control cells, the ER-retained ErbB-2* has, on a molar basis,
`lower amounts of phosphotyrosine, suggesting that its kinase
`ceptor activityper se. In contrast, cells expressing the scFv-51R
`are inhibited by 80% in their soft-agar growth. Thus, we con-
`activity is decreased which may in turn alter the kinetics of
`turnover. Indeed, the level of ErbB-2* found in scFv-5R and
`clude that the activated ErbB-2* must be on the cell surface, or
`at least in a cellular compartment distinct from ER and cis-
`scFv-51R-expressing cells suggests that there is a close link
`Golgi, to cause transformation.
`between half-live and kinase activity. In the scFv-5R express-
`ing cells where ErbB-2* kinase activity was inhibited to
`a
`of a
`The data presented here are the first demonstration
`greater extent than in the scFv-51R expressing cells, higher
`targeted and stable inactivation of a cellular oncoprotein via
`levels of ErbB-2* were found. In this context it is noteworthy
`intracellular antibody expression. The potential for inactivat-
`that in cell lines expressing normal ErbB-2, scFv-mediated ER
`therapy approach has
`ing malignant cell growth by a gene
`retention of the receptor did not affect its phosphorylation or
`implications for the treatment of cancer. In a more general
`result in an increased level of ErbB-2.2
`sense, our results and the recent work of other groups (28,43)
`Why are the NIH/ErbB-2* cells expressing scFv-5R and
`illustrate how potent engineered antibodies are in altering the
`in vivo function of selected cellular or viral target proteins. This
`scFv-5lR reverted? There are differences in the post-transla-
`tional modifications, activity, and subcellular
`location of
`approach should also be useful in studying complex receptor-
`ErbB-2* in the reverted cells. As discussed above, the ER-re-
`
`ligand systems such as the one that has become appare