`
`
`
`
`PHIGENIX
`PHIGENIX
`Exhibit 1011
`Exhibit 101 1
`
`

`

`.5
`
`[CANCER RESEARCH 51. 5361-5369. October 1. 1991]
`
`Requirements for the Internalization of a Murine Monoclonal Antibody Directed
`against the HER-Z/neu Gene Product c-erbB-Zl
`
`Lisa A. Maier, Feng J i Xu, Susan Hester, Cinda M. Boyer, Sara McKenzie, Arthur M. Bruskin, Yair Argon,
`and Robert C. Bast, J r}
`
`Departments ofMedicine IL. M., F. X., C. B., R. C. 8.] and Microbiology-Immunology /S. H., Y. A., R. C. 8.], Duke Comprehensive Cancer Center, Duke University
`Medical Center. Durham, North Carolina 27710, and Applied Biotechnology, Inc. [5. M., A. B.1, Cambridge, Massachusetts 02142
`
`ABSTRACT
`
`A murine monoclonal antibody, TA], is directed against an epitope on
`the extracellular domain of the HER-Zlneu (c—erbB-Z) gene product.
`Requirements for TAI-induced internalization of c—erbB-2 have been
`studied using the SKBr3 human breast cancer cell line and several rat
`fibroblast cell lines that express either wild-type or mutant human c-
`erbB-Z. Internalization of TA] was monitored by assaying protease—
`resistant uptake of ”SI-labeled TA], by electron microscopy of gold-
`labeled TA], and by inhibition of clonogenic growth of cells incubated
`with TA] that had been conjugated with blocked ricin. Similar rates of
`internalization of TA] were observed in SKBr3 and in rat fibroblasts that
`expressed human c-erbB—Z. The route of endocytosis was the same as
`that observed with antibodies against other membrane receptors. Anti-c-
`erbB-Z and anti-transferrin receptor cointernalized through clathrin-
`coated pits, coated vesicles, endosomes, and multivesicular bodies. Prod-
`ucts of mutant c-eth-Z that lacked a portion of the tyrosine kinase
`domain or that lacked most of the cytoplasmic domain were endocytosed
`in the presence of TA] as promptly as the wild-type c-erbB-Z product.
`Slightly more rapid internalization of TA] was observed in rat cells that
`expressed c—erbB—Z with a single point mutation in the transmembrane
`domain. Taken together, our data suggest that neither the intracyto-
`plasmic domain nor receptor phosphorylation is required for antibody-
`mediated endocytosis of c-erbB-2.
`
`INTRODUCTION
`
`The c—erbB-Z gene encodes a cell surface glycoprotein which
`ishomologous to the EGFR.3 This protein is composed of
`extracellular, transmembrane, and intracellular domains. The
`extracellular domain contains two cysteine-rich areas and is
`44% homologous to EGFR (1). The intracellular domain con-
`tains a tyrosine kinase which is 82% homologous to that of
`EGFR. Because of these similarities to the EGFR and to other
`tyrosine kinase receptors, investigators have suggested that the
`c—erbB-Z protein may function as a growth factor receptor. A
`putative ligand for this receptor-like protein has recently been
`reported (2).
`The oncogenic activity of the rat neu gene was initially
`associated with a point mutation from valine to glutamic acid
`in the transmembrane domain ofneuroblastomas (3). In human
`cancers this same mutation has not been found in the c-erbB—Z
`
`gene, although substitution of the glutamic acid for valine at
`the same position in the transmembrane domain of the human
`gene followed by transfer into murine cells results in their
`
`Received 2/27/91; accepted 7/16/91.
`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.
`' This work was supported in part by Grant 5-R01-CA 39930 from the National
`Cancer Institute, Department of Health and Human Services.
`"To whom requests for reprints should be addressed, at Box 3843. Duke
`University Medical Center. Durham, NC 27710.
`’ The abbreviations used are: EGFR. epidermal growth factor receptor; EGF,
`epidermal growth factor: FBS, fetal bovine serum: DMEM, Dulbecco’s minimal
`essential medium;
`lgG,
`immunoglobulin G; lgM,
`immunoglobulin M; FlTC.
`fluorescein isothiocyanate; PBS, phosphate-buffered saline; BSA, bovine serum
`albumin: DSP. dithiobis(succinimidylproprionate); EM, electron microscopy.
`
`transformation.4 In clinical specimens, the wild—type c-erbB—Z
`gene is amplified and overexpressed in adenocarcinomas from
`several different sites, including breast, pancreatic, and ovarian
`cancers (4—7). A number of studies have reported an association
`between poor prognosis and overexpression of the protein in
`breast cancer (8, 9) and ovarian cancer (10, 11). Because of its
`overexpression in these carcinomas, the c-erbB-Z protein is a
`potentially useful target for therapy with monoclonal antibody
`conjugates that react with its extracellular domain.
`Immunotoxins have been prepared that contain monoclonal
`antibodies conjugated to plant or bacterial toxins. Immunotox-
`ins that contain ricin A chain or blocked ricin appear to be
`internalized through clathrin-coated pits and vesicles. The toxin
`moieties must then be released from vesicles into the cytoplasm
`to inhibit protein synthesis by catalytic inactivation of the 605
`ribosomal subunit. The EGFR is an effective target for ricin A
`chain immunotoxins (12). During ligand-dependent and anti—
`body~dependent internalization, EGFR can be found in clath-
`rin-coated pits, vesicles, and endosomes (13, 14). Given the
`homology between EGFR and c-erbB-2, it seemed likely that
`the latter would be an effective target for an immunotoxinland
`that the conjugate would be internalized via the same pathway.
`To date, the internalization and subsequent fate of c—erbB-2
`have not been fully studied, largely due to the absence ofa well-
`defined ligand. In the case of EGF-mediated endocytosis of
`EGFR, the tyrosine kinase activity of the receptor may have
`some role, as receptors deficient in this activity may (14, 1,5) or
`may not (16. 17) be internalized efficiently. The intracellular
`domain of EGFR (18), as in the case of transferrin receptor
`(19—21).
`is important in facilitating the ligand-induced inter-
`nalization ofthese cell surface molecules.
`
`In this paper the antibody-dependent internalization of the
`c-erbB-2 protein, p185, was examined in a human breast cancer
`cell line that overexpressed c-erbB-2 as well as in different rat
`cell lines that expressed either wild—type or mutated human c-
`erbB-Z genes. These studies have helped to define the roles of
`the intracellular and transmembrane domains of c—erbB—2 in
`antibody-mediated endocytosis.
`
`MATERIALS AND METHODS
`
`Cell Lines. A human breast cancer cell line, SKBr3 (22), was main-
`tained in RPMI 1640 medium supplemented with 15% FBS and 2 mM
`L-glutamine. The rat 1A, 1174, and 711 cell lines were obtained from
`Applied Biotechnology (Cambridge, MA). Rat 1A is a rat fibroblast
`cell line. The cell line 1174 was derived from rat 1A cells that had been
`infected with a defective retrovirus that contained the pMX1112 plas—
`mid with the full-length wild-type human c-erbB-Z. lmmunoprecipita—
`tion (23) of 1174 cells with anti-c-erbB-Z antibodies yields the Mr
`185,000 wild-type protein. Rat 71 1 cells had been similarly derived by
`infection with a defective retrovirus that contained the pMX1112
`plasmid with the full-length human c-erbB-2 gene which had been
`mutated at codon 659 within the transmembrane domain substituting
`
`‘ A. Bruskin. unpublished data.
`
`5361
`
`“—4...—
`
`A—/~——..—,,_..-...._-_,,—_..._..
`
`
`
`
`..-_._—--.__....._..____...—..,—.._.
`
`IIIIS MATERIAL MAY BE PROTECTED
`B] COPYRIGHT LAW ( 17 11.8. CODE)
`
`PHIGENIX
`
`Exhibit 1011-01
`
`

`

`
`
`ANTIBODY-MEDIATED INTERNALIZATION OF HER-Z/neu
`
`glutamic acid for valine (see Fig. 1). lmmunoprecipitation of 711 cells
`with anti-c-erbB—Z antibodies yields a mutant M, 185,000 protein. Rat
`9-24c and 10-24k cells were derived from rat 1A cells by calcium-
`phosphate transfection with PMx1112 vector containing either a dele-
`tion of a portion of the tyrosine kinase domain (plasmid pABT9309)
`or deletion of all but 6 amino acids of the entire cytoplasmic domain
`(plasmid pABT9310), respectively. lmmunoprecipitation of cells con-
`taining the pABT9309 construct yields a Mr 150,000 mutant c-erbB-2
`protein.5 Cells containing the pABT9310 construct express a M,
`100,000 mutant c-erbB-Z protein. The rat cell lines were maintained in
`DMEM supplemented with 10% FBS and 2 mM L—glutamine. In addi-
`tion, medium for the 1 174, 71 1, 9-24c, and 10-24k cells contained 400
`jig/ml of G418 sulfate. All tissue culture reagents were obtained from
`GIBCO Laboratories, Grand Island, NY. The cells were cultured at
`37°C in 5% CO; and 95% humidified air. For experiments, cells were
`detached with 0.25% trypsin~0.02% EDTA.
`Antibodies and lmmunotoxins. Hybridomas that produced the murine
`monoclonal antibodies TA] (lgGl) and OD3 (lgM), known to react
`with the Mr 185,000 human c-erbB-Z product (23), but not with the rat
`neu, were obtained from Applied Biotechnology (Cambridge, MA).
`Three other murine monoclonal antibodies, MOPC21 (lgGl), 454A12
`(lgGl), and 9C6 (lgM), were obtained from Cetus Corporation (Emer-
`yville, CA). The 454A12 antibody reacts with the human transferrin
`receptor. MOPC21 and 9C6 antibodies failed to react with the rat cell
`lines or SKBr3 and were used as negative isotype-matched controls. An
`immunotoxin that contained the murine monoclonal antibody TA]
`conjugated to ricin with a chemically altered B-chain that blocked
`binding to galactose (TAl-Br) was prepared by lmmunogen (Cam-
`bridge, MA) and obtained through Applied Biotechnology. Unconju-
`gated ricin with a blocked B-chain (Br) was also used as a control.
`Plasmids. Plasmids pABT9309 and pABT9310 were obtained from
`Applied Biotechnology. pABT9309 was
`constructed from the
`pMX1112 plasmid and contained a human c—erbB—Z gene with a dele-
`tion of approximately 700 base pairs of the intracellular domain that
`included most of the coding sequence for the tyrosine kinase region
`(Fig. l). pABT9310 was also constructed from pMX] 112 and contained
`the coding sequence for all of the extracellular and transmembrane
`domain of the human c-erbB—Z gene, but‘lacked all but 6 amino acids
`of the intracellular domain. Plasmid DNA was purified by equilibrium
`centrifugation in CsCl-ethidium bromide gradients (24).
`Calcium Phosphate Transfection. Rat 1A cells were harvested and
`aliquots of 10" cells were plated in ]00-mm Petri dishes. Cell cultures
`were incubated at 37°C overnight. The purified pABT9309 or
`pABT9310 plasmid DNA was diluted in 0.25 M calcium chloride.
`Nitrogen was bubbled into 2 ml of a solution of 19.6 ml of 50 mM 4-
`(2-hydroxyethyl)—1-piperazineethanesulfonic acid with 280 mM sodium
`chloride and 0.425 ml of 70 mM dibasic sodium phosphate, pH 7.1.
`DNA was added dropwise to this mixture and incubated at room
`temperature for 40 min. A 2-ml portion of the DNA mixture was added
`to the rat 1A cultures (10 pg of DNA per 100-mm plate) and incubated
`at 37°C for 6 h. The medium was replaced, and the cultures were
`incubated for another 48 h. The cultures were split 1:4 in medium that
`contained 800 ug/ml of G418 sulfate. Individual colonies were selected
`in medium containing 800 ng/ml of G418 sulfate and subsequently
`maintained in medium containing 400 jig/ml of G418 sulfate.
`Selection of Transfected Cells with Indirect lmmunofluorescence and
`Flow Cytometry. Binding of murine monoclonal antibodies was meas-
`ured by flow cytometry using a FlTC-conjugated goat anti-mouse lgG
`(25). Transfected cells (5 x 105) were washed in PBS with 1% FBS and
`0.02% sodium azide, incubated with dilutions of different antibodies
`for 30 min on ice, and then washed 3 times with assay buffer. The
`fluorescein-conjugated goat anti-mouse immunoglobulin was added to
`the cells for 30 min on ice. The cells were washed 3 times in assay
`buffer, resuspended in PBS, and analyzed with an Epics 753 flow
`cytometer (Coulter Electronics, Hialeah, FL). Clones transfected with
`pABT9309 or pABT9310 were chosen that exhibited binding of TAl
`antibody comparable in fluorescence intensity to that of 1 174 and 71 1
`cells. The 9-24c clone contained pABT9309 that lacked a portion of
`
`5 S. McKenzie, unpublished data.
`
`the tyrosine kinase region, whereas the 10-24k clone contained
`pABT9310 that lacked all but 6 amino acids of the intracellular domain.
`Radiolabeling of Antibodies. Antibodies were conjugated with Na‘zsl
`obtained from Amersham (Arlington Heights, IL), using the lodogen
`method as previously described (26).
`Scatchard Analysis. The number of receptors per cell was estimated
`using different concentrations ofpeimmunoglobulins including those
`sufficient to saturate all binding sites (27). For the assay, 105 rat cells
`or 4 x 105 SKBr3 cells were seeded in 48-well plates and incubated
`overnight until cells were confluent. To block nonspecific binding of
`antibody, cells were incubated with 10% BSA in either DMEM or
`RPM] 1640 for 1.5 h at 37°C. ”51—Iabe1ed antibody was added to
`triplicate aliquots of cells at final concentrations of 2.5, 0.5, 0.25, 0.05,
`and 0.005 jig/ml. After 2-h incubation at 37°C, cells were washed 3
`times in DMEM or RPM] 1640, lysed with 2 N NaOH, and counted
`in a Packard gamma counter. Aliquots of ”51~labeled antibody were
`also counted. The number of cells in each microtiter well was estimated
`by detaching monolayers from 6 wells using trypsin and counting the
`cells in the presence of0.1% trypan blue using a hemocytometer.
`Internalization of ”SI-TA]. ”SI-labeled TA] (1 jig/ml) was added to
`aliquots of 5 X 105 rat cells. After incubation for l h at 4°C to allow
`binding, cells were washed 3 times with DMEM with 1% BSA and
`then either counted immediately to determine the total amount-of
`antibody bound or incubated at either 4°C or 37°C for l
`to 4 h to
`permit internalization of immunoglobulin. To remove antibody still
`bound to the cell surface, 2.5 mg/ml ofproteinase K (Sigma) was added
`to the cells for 1 h at 37°C. The cells were washed 3 times in DMEM
`with 1% BSA and 0.1% sodium azide. Radioactivity associated with
`cell pellets was counted in a Packard gamma counter. The amOunt
`(cpm) of antibody internalized was determined by subtracting the cpm
`obtained after incubation at 4°C followed by protease stripping from
`the cpm obtained after incubation at 37°C for the same time interval,
`followed by protease stripping. The percentage internalized was then
`calculated by dividing the cpm of the antibody internalized by the total
`cpm initially bound to the cell surface. Protease stripping immediately
`after binding removed an average of 94 to 98% of the surface-bound
`immunoglobulin from the various transfectant cell
`lines in three
`experiments.
`Assays of Clonogenic Growth. To study the effect of TAl-bR on
`growth of cells that expressed c-erbB-Z, a clonogenic assay was used.
`Rat fibroblasts (5 x 105) and SKBr3 cells (1 X 10") Were incubated at
`37'C on a rotator with different concentrations of the TAl-bR immu-
`notoxin conjugate or unconjugated bR as a control for 6, 12, 18, or 24
`h. The cells were washed twice with tissue culture medium and serially
`diluted 5-fold. A 100-111 portion of each dilution was plated in each of
`6 wells within a 96-well flat-bottomed microtiter plate. An additional
`100-111 tissue culture medium was added to each well. The cells were
`incubated for 7 days at 37°C. Clonogenic growth was determined using
`an inverted phase microscope, scoring the number of wells with at least
`one tumor colony that contained at least 10 cells. Estimates of the
`surviving clonogenic units were calculated according to a modification
`by Johnson and Brown (28) of the method of Spearman and Karber.
`Diluent-treated cultures were used as controls for the assay. Clonogenic
`elimination was calculated by subtracting the log clonogenic units of
`the diluent-treated cultures from the log clonogenic units ofthe treated
`cell culture for each individual cell line.
`Conjugation of Antibody with Gold Sols. Five- and 15-nm gold sols
`were obtained from Amersham (Arlington Heights, IL) for conjugation
`to monoclonal antibodies according to instructions of the manufacturer.
`Briefly, the antibodies were dialyzed against 2 mM sodium borate buffer,
`pH 9.0, and centrifuged at 100,000 X g for 1 h. The upper two-thirds
`of the supernatant was retained. The antibody concentration was esti-
`mated using Am. The pH ofthe gold sols was adjusted to approximately
`0.5 units above the p] of the antibody. To prepare the gold probe, 25
`ml of pH-adjusted beads were added to 17 to 40 ug/ml ofantibody and
`stirred for 2 min. The pH was adjusted to 9.0 with potassium carbonate.
`A final concentration of 1% BSA was added, using a solution of 10%
`BSA in distilled deionized water. To remove any unconjugated gold
`sol, the mixture was centrifuged at 4°C for 45 min at,45,000 X g for
`5362
`
`v
`
`~r
`
`PHIGENIX
`
`Exhibit 1011-02
`
`

`

`ANTIBODY-MEDIATED INTERNALIZATION OF HER~2/nru
`
`the 5-nm sol or at 12,000 x g for the 15-nm sol. The pellet was
`resuspended in the lower 10% of the supernatant and layered over a 10
`to 30% glycerol step gradient. The gradient was centrifuged for 45 min
`at 125,000 X g for the S-nm sol or at 15,000 X g for the 15—nm sol.
`The concentrated conjugated gold probes were then dialyzed against
`1% BSA—Tris buffer, pH 8.2, to remove the glycerol, and the absorbance
`was measured at 520 nm.
`lmmunoelectron Microscopy. Confluent SKBr3 cells in 60-mm Petri
`plates were incubated with 0.4 to 0.6 ml of gold-conjugated TA] and
`454A12 alone or in combination for 30 min at 4'C. Concentrations of
`conjugates were estimated by Am that ranged from 1.7 to 3.0 for 15-
`nm conjugates and from 0.8 to 1.4 for 5-nm conjugates. Confluent rat
`1A, 1174, 711, 9-24c, and 10-24k cells were incubated with 0.4 ml of
`TAl-gold conjugate for 30 min at 4'C. The cells were then washed
`with PBS to remove any nonadherent antibody and were warmed to
`37‘C for 0, 10, or 30 min to allow internalization. The cells were fixed
`for at least 1 h with 2% glutaraldehyde in 150 mM sodium cacodylate
`buffer at pH 7.4 with 2.5 mM calcium chloride. The fixed cells were
`scored with a blade and removed from the dishes with a rubber police-
`man. Monolayers were sedimented by centrifugation. The pellets were
`postfixed on ice with 2% osmium tetroxide and 0.5 to 1.0% potassium
`ferrocyanide in the same buffer. After washing in cacodylate buffer,
`cells were transferred to sodium acetate buffer, and the pellets were
`stained en bloc with 1% uranyl acetate in 0.2 M sodium acetate, pH
`5.2. Subsequently, cells were dehydrated with increasing concentrations
`of ethanol, incubated with mixtures of 100% ethanol and epoxy, and
`embedded in beem capsules with Embed 812 (EM Sciences).
`Pale gold to silver sections were cut on a Reichert-Jung Ultracut E
`microtome and stained with either saturated uranyl acetate and lead
`citrate or with lead citrate alone. The sections were examined on a
`Phillips 300 electron microscope at 80 kV.
`Cross-Linking and lmmunoprecipitation of Cell Surface Molecules.
`10-24k cells were radiolabeled overnight with [35$]cysteine (Amersham)
`and treated with either diluent or the reducible cross-linking agent DSP
`(Pierce). Cell
`lysates were immunoprecipitated (23) with either an
`irrelevant lgG (MOPC~21) or an anti-c-erbB-Z monoclonal antibody
`(TA]) and Protein A-Sepharose. lmmunoprecipitates were eluted with
`sodium citrate (pH 2.8), analyzed under reducing and nonreducing
`conditions on sodium dodecyl sulfate-polyacrylamide gel electropho-
`r'esis, and autoradiographed.
`
`RESULTS
`
`Expression of Wild-Type and Mutant c-erbB-Z in SKBr3 and
`in Rat 1A Fibroblasts. Requirements for antibody-mediated
`internalization of c—erbB-2 were studied with cell
`lines that
`eXpressed the wild-type (SKBr3, 1174) or modified gene prod-
`ucts (711, 9-24c, and 10-24k) (Fig. 1). Rat 1 fibroblasts lacked
`expression ofthe c-erbB-2 product and had served as the parent
`line for transfection of constructs. The 9-24c line expressed c-
`erbB-Z that lacked the tyrosine kinase coding region, whereas
`the 10-24k expressed c-erbB-Z from which all but 6 amino acids
`of the intracellular domain had been deleted. The 7|] line
`
`in which a point mutation had been
`contained a construct
`inserted at codon 659 within the transmembrane domain.
`Expression of c-erbB—2 was compared using indirect immu-
`nofluorescence after binding of the TA] murine monoclonal
`antibody to the extracellular domain of the gene product. As
`indicated in Fig. 2, the 10—24k cells displayed the highest level
`of fluorescence. The 711 cells, while displaying a significant
`level of fluorescence, were the least reactive with antibody. The
`1174 and 9—24c cells displayed levels of fluorescence interme-
`diate in intensity between the 10~24k and 7]] cells.
`The c-erbB-2 expression of each cell line was confirmed by
`immunoprecipitating the protein with TA] (data not shown).
`The SKBr3, l 174, and 71 ] cells expressed Mr 185,000 products
`when precipitated with TA]. The 9—24c line expressed a Mr
`150,000 product and 10-24k, a M, 100,000 product, consistent
`with predictions from the constructs.
`
`The number of c-erbB-2 molecules present per cell was esti-
`mated by Scatchard analysis for each of the rat fibroblast cell
`lines and the SKBr3 cell line. As indicated in Table 1, the K.
`for TA] was similar for the rat fibroblast cell lines. The SKBr3
`cells had the highest concentration of sites, with 1.3 X 10‘
`molecules per cell. Similar levels of c-erbB-2 have been reported
`for SKBr3 using different antibodies against c-erbB-Z to esti-
`mate antigen binding sites (29). The rat fibroblasts displayed a
`lower level of c-erbB-2 expression than did the SKBr3 cells,
`ranging from 2.3 X 10" sites/cell for the 1174 cells to 9.5 x 10‘
`sites/cell for the 10-24k cells. This analysis indicates that all 4
`transfected cell lines expressed similar concentrations of c-erbB-
`2 molecules per cell, with slightly higher expression by the 10—
`24k cells.
`
`Endocytosis of ”SI-labeled TA]. To determine if the 4 rat cell
`
`l2sl-labeled antibody
`lines internalized TA] at the same rate,
`was added to the cells for 1 h at 4°C to permit binding. The
`cells were subsequently warmed to 37°C for different intervals
`to allow internalization. Antibody that had not been internal—
`ized was removed with proteinase K. In preliminary experi-
`ments, minimal
`internalization of ”SI-labeled antibody was
`observed at 15- and 30-min intervals at 37°C. Therefore ]-h
`and 4-h intervals were tested. ”SI-TA] activity that resisted
`proteinase K digestion in 3 replicate experiments is plotted in
`Fig. 3. Rat [A cells do not express the human c-erbB-2 and did
`not internalize the TA] antibody. I25]-TA1 bound to each of
`the rat transfectants, but only 6 to 12% of the antibody that
`bound to cells was internalized after 4 h. There was no signifi-
`cant difference between the 4 rat cell lines in the percentage of
`the antibody internalized at 1 or 4 h (t test, P > 0.05). Among
`the 4 cell lines, uptake by 71 I was somewhat more prompt than
`uptake by the other lines. After 1 h at 37°C, 12% ofthe antibody
`that had bound to the 711 cells was internalized, whereas only
`
`Tranembrm dam-1n
`
`Fig. 1. The structures of the c-erbB»2 gene
`constructs in the pmx1112 expression vector
`from top to bottom include:
`the full-length
`wild-typec-erbBQ gene retrovirally transferred
`into 1174 cells: full-length c-erbB-2 with one
`amino acid mutation at codon 659 retrovirally
`transferred into the rat 1A cells to yield 711
`cells; kinase»negative c-erbB-Z with a deletion
`of amino acids 751 to 979 transfected into the
`rat [A cell to yield 9-24c cells; and a c-erbB-Z
`construct lacking most ofthe intracytoplasmic
`domain. leaving a cytoplasmic tail of 6 amino
`acids transfected into the rat 1A cell. to yield
`10-24k cells.
`
`E""“""" “mu“
`
`(Cylophlllk domain
`I
`I
`I
`I
`'
`_
`_
`
`185 Rd 1
`\
`1155 Norm“ 6 SEP- 3 2
`Tyrollnc kin-u
`don-In
`
`185 Kd I
`659
`11.5!
`
`Mutated 6'9!!! 3‘2
`.
`. _ ,
`(ammo “Id 659' val -. 8'“)
`.
`150 RdW Klnase-negative C'etb 3’2
`751-979
`(amino acids 751 -O 979 deleted)
`
`lOOKdml 693
`
`C9" Line
`1174
`
`711
`
`9‘24c
`
`10-24k
`
`5363
`
`Truncated 0ng 3-2 (amino acids
`as; -o 1255 deleted)
`
`
`
`
`
`—.——.._‘_—.w____....__,.___—/—.._.‘,.-..._._...__._..,
`
`
`
`PHIGENIX
`
`Exhibit 1011-03
`
`

`

`
`
`ANTIBODY-MEDIATED INTERNALIZATION OF HER-g/neu
`
`Table l
`
`c-erbB—Z sites per cell and association constants determined by Scale/lard
`analysis with '"l- TA1
`
`K. (M")
`Sites/cell
`Cell line
`ll.2X]0'
`2.3x IO‘
`H74
`5.1x10‘
`5.3 x l0‘
`7H
`8.2 x 10'
`4.2 X l0‘
`9-24c
`10-24k
`9.5 X IO‘
`3.8 X 10'
`
`SKBr3
`].3 X 10"
`8.7 X 107
`
`,
`
`‘
`
`_
`
`i
`
`
`
`Percentlnternalizad
`
`D
`
`0.0
`
`IO
`
`20
`Time (hrs)
`
`3.0
`
`4.0
`
`
`lA
`lntcrnalization of ”’l-Iahcled TA] by different rat cell lines: rat
`Fig. 3.
`)2 I l74( ----- ): 7| I
`(— ~ -): 9—24 C( ----- )2 and 10-24k (— — — —). Points. mean
`(
`percentage internalized of three experiments: bars. SE.
`'
`
`;_
`
`<71
`
`tl
`
`
`
`LogClonogenicElimination
`
`0.5
`0.05
`0.005
`TA1 lmmunotoxin Concentration (pg/ml)
`
`Fig. 4. Clonogenic elimination of rat transfectants with TAl-bR immunotoxin
`following a 24-h incubation. —. rat 1A; —————. H74: — - -. 7] l: ----- . 9-24c:
`- - — -. lO-24k. Points. mean log clonogenic elimination of three experiments:
`bars. SE.
`
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`TA1 lmmunotoxin Concentration (pg/ml)
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`Fig. 5. A. kinetics of elimination of clonogenic I I74 cells with TAI immuno-
`toxin. Points. mean oflog clonogenic elimination for three experiments: bars. SE.
`B. kinetics of elimination of clonogenic lO-24k cells with TAI immunotoxin.
`Points. mean of log clonogenic elimination for 3 experiments: bars. SE.
`5364
`
`PHIGENIX
`
`Exhibit 1011-04
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`TA1 lmmunotoxin Concentration (pg/ml)
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`
`Relative Fluorescence Intensity
`Fig. 2. Flow cytometric analysis ofTAl binding (5 ug/ml) to the rat cell lines.
`using a FITC labeled goal anti-mouse antibody and isotypc matched negative
`control. MOPCZ] (5 rig/ml). Rat IA cells with MOPCZ] (A; [.2972 cells positive)
`or TA] (F; 0.9% cells positive). | 174 cells with MOPCZ] (B; [.498 cells positive)
`or TA] (G; 81.5% cells positive). 7] l cells with MOPCZI (C: [7.2% cells positive)
`or TA] (H: 82.8% cells positive). 9-24c cells with MOPCZ] ((13.2% cells positive)-
`or TA] (1; 68.9% cells positive). and lO-Z4k cells with MOPCZI (E: 1.7% cells
`positive) or TA] (.1; 93.0% cells positive). MFI (Mean Fluorescence Intensity) is
`expressed as channel number on a linear scale.
`
`] to 2% of bound antibody was internalized by ] 174 cells. This
`difference at l h approached statistical significance (P = 0.065),
`but by 4 h there was no apparent difference in the amount of
`labeled TA] internalized by the two cell lines. The 9—24c and
`.10-24k cell lines showed a level of internalization intermediate
`between 1174 and 7]] cells at ] h, and uptake by each cell line
`was approximately the same by 4 h (Fig. 3).
`_
`-
`Endocytosis of TA1-blocked Ricin lmmunotoxin Conjugates.
`.To assess further the internalization of TA], each of the rat
`cell lines was treated with TA] immunotoxin. Only the antibody
`conjugate that was internalized would inhibit clonogenic growth
`in a limiting dilution. assay. Unconjugated blocked ricin was
`used on an equimolar basis as a control for the conjugate and
`did not affect grOwth of any of the cell lines at the concentra-
`tions of toxin used in this study (data not shown). As indicated
`in Fig. 4, a 24—h incubation with the TA] immunotoxin inhib-
`ited the growth of all 4 rat transfectant cell lines, but did not
`affect growth ofthe rat 1A cell line that lacked c-erbB-Z.
`Cells with more complete or more rapid internalization of
`the immunotoxin should display greater inhibition of clono-
`genic growth than cells with less complete or less rapid inter!
`nalization of immunotoxin. To study the kinetics of internali-
`zation and elimination of clonogenic cells by TA] immuno-
`toxin, the 1174 and 10-24k cell lines were treated with the
`TAl-bR conjugate fOr different intervals from 6 to 24 h. With
`each of the cell lines, the longer the incubation with immuno-
`toxin, the greater the fraction of cells that were killed (Fig. 5).
`Each ofthe cell lines including 9-24c and 7]] (data not shown)
`exhibited similar sensitivity to the immunotoxin, consistent
`with similar rates and levels of internalization of TAl-bR after
`
`binding to c-erbB-Z.
`
`

`

`ANTIBODY-MEDIATED INTERNALIZATION OF HER-Z/neu
`
`immunofiuorescence. After warming for 5 min, some TAl-gold
`was observed in clathrin-coated pits, vesicles, and superficial
`endosomes, although a significant portion of the tracer re-
`mained on the cell surface (Fig. 6, A and B). Anti—transferrin
`receptor antibody (454A12) conjugated to 5-nm gold particles
`was used as a marker for clathrin-coated pits and vesicles, as
`transferrin is known to be internalized through these structures.
`TAl-gold and 454A12-gold conjugates coexist in pits and ves-
`icles (Fig. 6A), further supporting the conclusion that the TA]
`and p185 were internalized through clathrin-coated structures.
`By 30 min at 37°C,
`the TAl-gold conjugates were seen in
`deeper cellular structures, including endosomes, lysosomes, and
`multivesicular bodies (Fig. 6C). Much TAl—gold was still evi-
`dent on the cell surface even after 30 min of endocytosis, a time
`when very little 454A12-gold remained at the cell surface (Fig.
`6C). Thus, c-erbB—Z was internalized at a slower rate and to a
`lesser extent than the transferrin receptor.
`The antibody-induced internalization of p185 in rat—1 fibro-
`blasts resembled that
`in SKBr3 breast carcinoma cells. As
`
`expected, 1174 cells that expressed the wild-type human c-
`erbB-Z bound much less TA1 gold than did SKBr3 cells, con-
`sistent with the Scatchard analysis, whereas no binding was
`observed to rat-1 cells that had not been transfected. After 5 to
`
`10 min at 37°C, TAl-gold was seen primarily on the surface of
`the 1174 fibroblasts. Occasionally, TAl-gold was present
`in
`clathrin-coated pits, vesicles, or superficial endosomes (Fig. 7).
`By contrast to the kinetics of 1174 endocytosis, after 5 min at
`37°C much of the TAl—gold conjugate was found within 711
`. cells, primarily in peripheral clathrin-coated pits, vesicles, and
`endosomes, but also in some deep endosomes (Fig. 8, A to C).
`By 30 min at 37°C, most of the TAl-gold was internalized by
`711 cells and was found in endosomes and in multivesicular
`
`bodies (Fig. 8C). After 30 min at 37°C, the TAl-gold in the
`other 3 cell lines was still primarily on the cell surface and in
`endosomal structures (Fig. 70). Although the tracer was present
`in some endosomes adjacent to the Golgi complex in all ofthe
`rat cell lines, no gold was evident in the Golgi cisternae of any
`of the cells.
`To determine whether the intracellular domain of the c-erbB—
`
`
`
`tor in SKBr3 breast carcinoma cells. SKBr3 cells were incubated with TAl-gold
`(15 nm) and 454A12~gold (5 nm), at 4°C, to label surface p185 and transferrin
`receptor. respectively. Endocytosis of the two receptors was followed at 37'C
`after 5 min (A and B) or 30 min (C). Clathrin-coated pits and endosomal vesicles
`contain both labels (curled arrows in A and B). Note the concentration of p185
`label around surface microvilli (B). C. after 30 min of endocytosis most surface
`label is due to pl85 (broad arrows). and multivesicular bodies are doubly labeled
`(‘). The c-erbB-Z product is also present in small. tubular endosomes (arrow-
`heads). PM, plasma membrane: M, mitochondria. Bars. 0.25 am.
`
`2 was necessary for internalization of the protein, we studied
`the 10—24k and 9-24c cell lines. As shown in Fig. 8, D and E,
`the truncated c-erbB-Z products were internalized in both cell
`lines, through the same route, and apparently with the same
`kinetics as the wild-type c-erbB-Z expressed in 1 174 cells.
`This observation was surprising, considering the role that the
`intracellular domain of EGFR may play in the endocytosis of
`that receptor (16, 17). Since p185 can, under some circum-
`stances, associate with EGFR, we investigated the possibility
`that the truncated c-erbB-Z gene product was internalized as a
`complex with the rat EGFR. The 10-24k cells were labeled
`uniformly with [35$]cysteine, and detergent lysates were immu-
`noprecipitated first with TAl and then with rabbit anti~rat
`EGFR (the generous gift of Dr. Sheldon Earp),
`to test for
`coprecipitation of proteins. We failed to detect M, 185,000 or
`Endocytosis of Gold-TA] Conjugates. To study the route by
`170,000 bands in the TAl—eluted material, even after gross
`which the c-erbB-Z gene product (p185) is internalized, TAl
`overexposure of the Mr 100,000 truncated c—erbB-Z band
`was conjugated to colloidal g