PETITIONER'S EXHIBITS
`
`Exhibit 1088 Page 1 of 8
`
`

`
`980
`
`KUMAR ET AL.
`
`MoL. CELL. BIoL.
`
`with intrinsic tyrosine kinase activity, we investigated the
`modulation of pl85”E”2 phosphorylation by MAb 4D5. We
`report here that activation of phosphorylation of p185”ER"
`by serum was reduced in the presence of an excess of MAb
`4D5 and that MAb-sensitive phosphorylation was mediated
`by a growth factor or factors other than TGF-at or EGF.
`Furthermore, SK-BR-3 cell-conditioned medium contained a
`faetor(s) that could activate p185”ER2 phosphorylation and
`was partially inhibited by MAb 4D5.
`
`MATERIALS AND METHODS
`
`Materials. MAbs 4D5 (18) and 9G6 (44) were raised against
`human p185"’E'". MAbs 528 and 225 bind to the human
`EGF-R (23). Antiphosphotyrosine MAb PY-69 was obtained
`from ICN Biochemicals, Inc. Rabbit
`immunoglobulin to
`mouse immunoglobulins G (RAM) was supplied by Accurate
`Chemicals, Westbury, N.Y. 32?, (canier free; 28.5 Ci/nmol)
`and “S-labeled L-cysteine (1,030 Ci/mmol) were purchased
`from New England Nuclear, Boston, Mass.
`Cell lines and cell culture. Human breast tumor cell lines
`SK-BR-3, BT-474, and MDA-MB-453 were obtained from
`the American Type Culture Collection. The A431 human
`epidermal carcinoma cell
`line was originally supplied by
`Gordon Sato. All cell lines except MDA-MB-453 (which was
`grown in L-15 medium) were maintained in Ham F-12-
`Dulbecco modified Eagle medium (lzl, vol/vol)
`(F-l2/
`DMEM) supplemented with 10% fetal bovine serum.
`Labeling of pl8S"£'" with ”P, and [”S]cysteine. Cells (3 x
`10’) were plated in F-12/DMEM in each well of a six-well
`dish. Twenty-four hours later, cultures were washed with
`phosphate-free medium and incubated for up to 15 h in
`phosphate-free F-12/DMEM containing 0.4 mCi of “Pi per
`ml
`in the presence or absence of MAb and newborn calf
`serum. At desired times, cells were harvested in 400 pl of
`lysis bufl'er (20 mM HEPES [N-2-hydroxyethylpiperazine-
`N’-2—ethanesulfonic acid; pH 7.5], 1% Triton X-100. 10%
`glycerol, 1.5 mM magnesium chloride, 1 mM ethyleneglycol
`bis-N,N,N’,N'-tetraacetic acid, 0.1 mM phenylmethylsulfo-
`nyl fluoride, 10 p.g of leupeptine per ml, 2 mM sodium
`orthovanadate) at 4°C for 20 min. The lysate was centrifuged
`at 10,000 rpm in an Eppendorf microfuge for 10 min, and
`then 60 p.l of Pansorbin was added as described elsewhere
`(42). For labeling with [3’S]cysteine, the cells were washed
`with cysteine-free medium and refed with cysteine-free
`F-12/DMEM containing 0.15 mCi of [”S]cysteine per ml
`with or without 5% newborn calf serum.
`Immunoprecipltation and SDS-polyacrylamide gel electro-
`phoresis. Aliquots (350 pl) of the cell
`lysates (or equal
`amounts of trichloroacetic acid-precipitable counts per
`minute) containing 32?-labeled or
`[ 5S]cysteine-labeled
`p185”ER’ were subjected to immunoprecipitation with 10 p.g
`of MAb 9G6, 528, or PY-69 at 4°C for 2 h.
`Immune
`complexes were collected by absorption to RAM-protein
`A—Sepharose beads at 4°C for 1 h. Beads were washed three
`times with 1 ml of bufier (20 mM HEPES [pH 7.5]. 150 mM
`NaCl, 0.1% Triton X-100, 10% glycerol, 2 mM sodium
`orthovanadate). Washed pellets were mixed with 40 pl of
`sample loading bufi'er (10 mM Tris HCl [pH 6.8], 1% sodium
`dodecyl sulfate [SDS]. 0.2% 2-B-mercaptoethanol, 10% glyc-
`erol, 0.001% bromophenol blue), heated at 95°C for 5 min,
`and resolved on a 7% SDS-polyacrylamide slab gel (26). The
`efliciency of precipitating labeled receptor with MAb 9G6 is
`80 to 90% when this procedure is used. Low—molecular-mass
`colored markers (Amersham Corp.) were used as standards.
`Pltosplioamino acid analysis. The band corresponding to
`
`12345
`
`I-
`
`~
`
`FIG. 1. Effect of MAb 4D5 on steady-state levels of pl85”E‘"
`phosphorylation in SK-BR-3 cells. Subeonfluent cultures were la-
`beled with ”Pi (400 p.Ci
`in 1 ml of phosphate free F-12/DMEM
`supplemented with 5% newborn calf serum) in the continuous
`presence of different amounts of antibody for 15 h. Detergent
`extracts were made, and p185”“’*’ was immunoprecipitated by using
`MAb 9G6 and then resolved by 7% SDS-polyacrylamide gel elec-
`trophoresis (Materials and Methods). An autoradiogram resulting
`from 16 h of exposure of the dried gel is shown here. The arrow
`indicates the position of ’2P-labeled pl85”5‘". Lane 1. Control cells;
`lanes 2 to 4. cells treated with MAb 4D5 at 30. 150, and 300 nM.
`respectively; lane 5. cells treated with 400 nM F(ab) fragment of
`MAb 4D5. The amounts (in counts per minute) of pl85””’" in each
`lane were 4,453 (lane 1), 1.967 (lane 2), 1,785 (lane 3). 1,040 (lane 4),
`and 335 (lane 5). Counts were corrected by subtracting the back-
`ground of 60 cpm. The results shown are representative of results in
`six different experiments.
`
`the 185-kDa HER2 protein. resolved as described above,
`was excised out of the gel. ”P-labeled p185”E’" in a gel slice
`was partially hydrolyzed with 200 pl of 6 N HCl at 110°C for
`1 h. Two portions (10 pl each) of the hydrolysatc were taken
`for measurement of radioactivity in a liquid scintillation
`counter to determine the total incorporation of “P into the
`p185”5”" receptor. The rest of the hydrolysate was dried.
`suspended in distilled water, and applied to a Dowex
`AG1-X8 column. The column was washed with distilled
`water. and the absorbed ”P-labeled materials were eluted
`with 0.5 N HCl and lyophilized. The recovery of radioactiv-
`ity by this procedure was 78 to 85%. ”P-phosphoamino
`acids mixed with unlabeled carrier phosphoamino acids
`(phosphoserine, phosphothreonine, and phosphotyrosine
`[1:l:1]) were analyzed by thin-layer electrophoresis as de-
`scribed elsewhere (8).
`
`RESULTS
`
`MAb 4D5 reduces amount of “P-labeled pl85”"’. MAb
`4DS was used to investigate the regulation of phosphoryla-
`tion. SK-BR-3 cells, which have an amplified c-erbB-2 gene
`(45), were cultured for 15 h in medium containing “P, in the
`continuous presence of various concentrations of MAb 4D5.
`The p185”E"’ from these cells was immunoprecipitated with
`another anti-p185”E'” MAb, 9G6, which recognizes a dis-
`tinct epitope of p185”"" . and resolved by SDS-polyacryl-
`amide gel electrophoresis. Results of such an experiment are
`shown in Fig. 1. Treatment of cells with 4D5 reduced in vivo
`steady-state levels of "P-labeled p185”E’” up to 80% in a
`dose-dependent manner (lanes 2 through 4). There was 49%
`1 8% reduction in phosphorylation by 150 nM MAb 4D5 in
`eight different experiments. When the F(ab) fragment of 4D5
`was used instead of intact antibody, comparable or greater
`reduction of ”P-labeled p185”E"’ was observed (lane 5). As
`a control, SK-BR-3 cells were incubated with another MAb,
`225 lgGl, specifically directed against the EGF-R, and there
`was no effect on the amount of “P-labeled p185”ER’ (un-
`published data). The reduction in steady-state levels of
`2P-labeled pl85"E"’ was not due to interference by 4D5
`with MAb 9G6 during the immunoprecipitation reaction, as
`immunoprecipitation performed with another polyclonal an-
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1088 Page 2 of 8
`
`

`
`VoL. 11.1991
`
`MODULATION OF PHOSPHORYLATION OF p185”"'"
`
`981
`
`Abs
`
`P185
`
`EGF-R
`
`1 2 3
`
`1' 2‘ 3'
`
`""43"
`
`4-
`
`A.
`
`B.
`
`C.
`
`FIG. 2. Specificity of the reduction of ”P-labeled pl85””" by
`MAb in SK-BR-3 cells in the presence or absence of MAb 4DS.
`Subconfiuent cells were labeled with “Pi for 15 h. The cells were
`lysed in 600 p.l of extraction buffer and divided into two equal parts
`of 250 p.| each. immunoprecipitation was performed with anti-p185
`MAb (lanes 1 to 3) or with anti-EGF-R MAb 528 (lanes 1' to 3'). An
`autoradiogram of a dried gel is shown here. Lane 1 and 1'. Control;
`lanes 2 and 2'. 30 nM MAb 4DS; lanes 3 and 3'. 150 nM MAb 4D5.
`Counts per minute: lane 1. 5,985; lane 2, 3.798; lane 3. 3.120; lane 1'.
`853; lane 2'. 779: lane 3’. 932. Abs. Antibodies.
`
`405
`
`—
`
`+
`
`—
`
`+
`
`l 2 3
`
`EXP.2
`EXP.)
`9-
`[CT Fiji
`P185
`EGF-R
`P185
`l—'-'_l
`l—"'T
`l——:jl
`
`Abs
`
`jit ‘
`
`""
`
`tibody (18) recognizing the carboxy-terminal 17 amino acids
`of pl85”E”’ gave similar results (unpublished data).
`Next. we examined the possibility of general inhibitory
`effects of MAb 4D5 on the steady-state levels of other
`’2P-labeled receptor proteins by analyzing the amount of
`’2P-labeled pl85”E’" and ”P-labeled EGF-R in the same
`experiment (Fig. 2). These results indicated that there was
`no reduction of ”P-labeled EGF-R during 15 h of treatment
`of SK-BR-3 cells with 150 nM MAb 4D5. which had reduced
`the amount of “P-labeled p185”ER by 48%.
`Analysis of reduction of pl85""’ phosphorylation. The
`reduction of steady-state levels of "P-labeled pl85”ER" by
`MAb 4DS. shown in Fig. 1 and 2. could result from down-
`regulation of p185”’“” and/or interference in the activation
`of p185”"‘’ phosphorylation by a direct or indirect mecha-
`nism(s).
`In initial studies to explore these possibilities.
`parallel cultures of cells were metabolically labeled with
`[”S]cysteine or 32?, During 11 h of concurrent incubation
`with MAb 4D5, there was a 45% reduction in ”P-labeled
`p185”’5R2 (Fig. 3A) and only a 14% reduction in 35S-labeled
`p185”ER’ (Fig. 3B). This suggests that the reduced "P label
`in p185”5R2 in the presence of MAb 4DS can only partially
`be attributed to reduced p185”ER’ content. Next. we per-
`formed a similar experiment comparing the capacities of the
`monovalent F(ab) fragment of MAb 4DS and an intact MAb
`4DS to affect the reduction of ”S—labeled p185”E"’. There
`was no change in ”S-labeled p185”E“" in the presence of
`F(ab). but there was a 26% reduction caused by MAb 4D5
`(Fig. 3C, lanes 3 and 2, respectively). The results obtained in
`the immunoprecipitation experiments documented in Fig.
`3A through C were confirmed by immunoblotting (D). Immu-
`noblotting of the ”P-labeled SK-BR-3 cell extracts used in
`Fig. 2 demonstrated only a marginal reduction in the content
`of p185”E"" protein when cells were cultured in the presence
`of MAb 4D5 but a substantial reduction in the amount of
`“P-labeled p185”£R’ (Fig. 2). The expression of EGF-R was
`not affected. immunoblotting of similar unlabeled SK-BR-3
`extracts also demonstrated very little reduction in the con-
`tent of p185”E’” by MAb 4DS (Fig. 3D. experiment 2).
`These findings indicate that increased receptor catabolism
`induced by a MAb cannot fully account for the observed
`reduction in “P labeling and show [with F(ab)] that reduced
`labeling is dissociated from reduced content of p185”ER".
`Next we addressed the possibility that the reduction in
`32P~labeled p185”ER’ associated with exposure to MAb 4D5
`could be related to a change in the level of expression of
`p185”E”" on the plasma membrane or to the extent of
`
`405
`
`— +
`
`— +
`
`—
`
`+
`
`FIG. 3. Analysis ofthe reduction of p185”E'” phosphorylation in
`SK-BR-3 cells treated with MAb 4DS. Cells were labeled with “P;
`(A) or [”S]cysteine (B) in the presence or absence of MAb 4DS (150
`nM) for 11 h. Samples were prepared and separated as described in
`Materials and Methods. The autoradiogram shown here was ob-
`tained by 6 h of exposure. (C) Cells were labeled with [”S]cvsteine
`for 11 h in the presence of MAb 4DS (150 nM. lane 2) or F(ab) (400
`nM. lane 3) or with culture medium (lane 1). Samples were prepared
`and immunoprecipitation was carried out as described in Materials
`and Methods. An autoradiogram of a dried gel
`is shown here.
`Quantitation of the pl85”E”" bands was obtained by densitometric
`scanning (A through C) or by determining radioactivity associated
`with bands (A and B). Quantitation by determining the radioactivity
`associated with p185"”" bands in panels A and 8 gave results
`similar to those with densitometric scanning, and there was a 27% :
`3% additional reduction in "P-labeled p185”“"" compared with
`"S-labeled pl85’”"”. (D) lmmunoblotting of pl8S”’”” and EGF-R
`proteins. In experiment 1 (Exp.l). ”P—labeled SK-BR-3 cell extracts
`(50 pg of protein) used in Fig. 2. lanes 1 and 2. were resolved on a
`7% SDS-polyacrylamide gel and then immunoblotted with anti-P185
`MAb 9G6 or anti~EGF-R polyclonal antibody RK-ll. Experiment 2
`shows the immunoblotting of unlabeled SK-BR-3 cell extracts
`prepared following culture for 15 h with or without 30 nM MAb 4DS.
`Since some of the extracts used here were radiolabeled. immuno-
`blotted membranes were visualized by using a protein A-gold
`.-nnancement kit (30). Abs, Antibodies.
`
`down-regulation of receptor protein. First. we determined
`what fraction of the “S-labeled p185”E“ is present on the
`cell surface at 37°C (Fig. 4A).
`In these experiments.
`p185’”""" expressed on the plasma membrane was identified
`by its capacity to bind MAb 4D5 prior to cell lysis. The
`results indicate that 19% : 4% (average from three different
`experiments) of total ”S—labeled pl85’"5R’ is expressed on
`the cell surface under these experimental conditions; thus
`p185”"""’ is available for down-regulation by MAb 4DS.
`Down-regulation of EGF-R has been shown to be dependent
`on temperature (41). To confirm that down-regulation of
`surface pi85”ER" also is reduced at 4°C. experiments were
`perfonned to analyze the effect of temperature on the
`abundance of “S-labeled p185”E"’ on the cell surface.
`Results indicated that at 4°C the amount of total “S-labeled
`p185”E"" expressed on the surface increased to 35% : 3%
`(data not shown) compared with 19% : 4% of total “S-
`labeled p185’"*’” at 37°C.
`ln order to define the contribution of down-regulation to
`MAb-induced reduction in p185”E"’ phosphorylation, we
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1088 Page 3 of 8
`
`

`
`982
`
`KUMAR ET AL.
`
`MOL. CELL. BioL.
`
`Antibody P185
`
`123
`
`P-Tyr
`r—.——%II
`123
`
`—~
`_
`_,
`FIG. 5. Partial agonist nature of F(ab). Subcontinent SK-BR-3
`cells were labeled with 32Pi for 15 h. Some cultures were treated with
`400 nM F(ab) for the indicated times. The cells were lysed in 800 u.l
`of extraction buffer. The lysates were divided into two equal parts of
`350 p.l each and then immunoprecipitated with MAb 9G6 (lanes 1 to
`3) or with antiphosphotyrosine MAb PY-69 (lanes 1' to 3'). An
`autoradiogram resulting from a 1-h exposure of dried gel is shown
`here. Lane 1. Control: lane 2. F(ab) incubation for 15 min; lane 3.
`F(ab) incubation for 60 min.
`
`labeled pl85"E"’ but can act for a short time as a partial
`agonist is interesting; however, we have not attempted to
`further characterize these properties in the present study.
`Activation of phosphorylation of pl85”E'” in presence or
`absence of newborn calf serum. Next, we investigated the
`possible source of
`the factor(s)
`that might
`stimulate
`p185”E‘" phosphorylation. As shown in Fig. 6A, culturing
`the cells in serum-free medium resulted in a steady-state
`level of phosphorylation of pl85”ER’ reduced 56% (lane 1)
`compared with that observed in the continuous presence of
`newborn calf serum (lane 3). The addition of MAb 4D5 in
`serum-free culture conditions further reduced p185”ER’
`
`A1234 3
`
`—-I-ofll»
`
`+ +
`Serum — —
`405
`— + — +
`
`—
`
`— ++
`
`C.
`
`A.
`
`EXP. 1
`
`EXP. 2
`
`l
`
`2
`
`3 4
`
`-> 1
`
`~ Q‘.
`
`3-
`
`4°C
`37°C
`fl Fl
`23 45
`
`-> -0'.
`
`4D5——+—+
`
`FIG. 4. (A) Quantitation of surface expression of ”S-labeled
`p185”“". Cells were labeled with [”S]cysteine for 11 h. At the end
`of incubation, some cultures were lysed in 500 p.l of lysis buffer for
`the detennination of total “S-labeled p185”E’" by immunoprecipi-
`tation with 10 pg of 4D5 (Materials and Methods). For measuring the
`surface expression of ”S-labeled pl85”5’”. cultures were washed
`with phosphate-buffered saline and further incubated with F-12!
`DMEM—20 mM HEPES (pH 7.5) containing 20 pg of high~aflinity
`MAb 4D5 per ml for 1 h at 4°C. The cultures were washed. lysed in
`500 pl of extraction buffer. and processed for immunoprecipitation
`by adding RAM-protein A-Sepharose beads but no more MAb 4DS
`during the immunoprecipitation procedure. The results of two
`representative experiments are shown here. Lanes 1 and 3. Total
`”S-labeled pl85”E”; lanes 2 and 4. ”S-labeled pl85”E’" on the cell
`surface. (B) Effect of MAb 4D5 on surface expression of p185”5’" in
`a temperature shift experiment. SK-BR-3 cells were first equili-
`brated with 321’. for 4 h at 37°C (lane 1) and then further incubated
`with or without MAb 4D5 for an additional 11 h (in the continuous
`presence of ”Pi) either at 37°C (lanes 2 and 3) or at 4°C (lanes 4 and
`5). The autoradiogram shown here was obtained by exposing lanes
`1 to 3 for 24 h and lanes 4 and 5 for 96 h. Quantitation of the amount
`of ”P associated with p185”5” bands was obtained by densitomet-
`ric scanning of the autoradiogram and by detennining the radioac-
`tivity associated with p185”E’" bands (A and B).
`
`analyzed the elfect of incubation with MAb 4D5 at 4°C. In
`these studies. cells were first equilibrated with “Pi for 4 h at
`37°C (Fig. 4B, lane 1) and then maintained at 37°C (lanes 2
`and 3) or shifted to 4°C (lanes 4 and 5) for an additional 11 h.
`with or without MAb 4DS. A comparison of the labeled
`material in lanes 2 and 4 in Fig. 4B (fluorographs exposed for
`24 and 96 h, respectively) showed a significant 85% reduc-
`tion in “P labeling of p185”ER’ during 11 h at 4°C compared
`with labeling at 37°C. However, incubation of cells at 4°C did
`not prevent a funher substantial MAb-mediated reduction in
`steady-state levels of pl85”E"2 phosphorylation: there was a
`34% decrease at 4°C (compare lanes 4 and 5) and a 51%
`decrease at 37°C (compare lanes 2 and 3). Taken together,
`these observations indicate that MAb-induced reduction in
`
`p185”E” phosphorylation cannot be completely accounted
`for by down-regulation.
`Experiments were performed to determine whether the
`F(ab) fragment might have the capacity to act as an agonist
`by activating tyrosine phosphorylation. The results in Fig. 5
`indicate that the addition of F(ab) for 15 min slight]
`stimu-
`lated in vivo tyrosine phosphorylation of p185 5" in cul-
`tures labeled with “P (Fig. 5, lane 2’). However, there was
`no activation in cultures exposed to F(ab) for a longer
`treatment of 60 min (Fig. 5, lane 3’). The observation that the
`F(ab) fragment of 4D5 does not down-regulate the “S-
`
`0
`
`0
`
`1
`
`,
`
`O
`_
`_Y
`-‘
`3
`2 .
`1
`FIG. 6. (A) Detection of newborn calf serum-mediated phos-
`phorylation of p185"E"". Subconfluent SK-BR—3 cells were labeled
`with "P. in the culture medium without (lanes 1 and 2) or with (lanes
`3 and 4) 5% newborn calf serum for 15 h. Cultures analyzed in lanes
`2 and 4 also were continuously exposed to 150 nM MAb 4DS.
`Samples were prepared and immunoprecipitated for assaying the
`amount of p185"E‘" as described in the Materials and Methods.
`Quantitation of the p185 bands was obtained by densitometric
`scanning of the autoradiogram. (B) Control experiment showing
`effect of serum on the “Pi labeling of EGF-Rs for 15 h in SK-BR-3
`cell cultures. Cell extracts were immunoprecipitated with anti-
`EGF-R MAb 528, which recognizes one distinct band with an
`approximate molecular mass of 170 kDa (arrow). (C) Two-dimen-
`sional thin-layer electrophoresis pattern of "P-phosphoamino acids
`in a hydrolysate of the p185”5”’ immunoprecipitated in panel A. S.
`Phosphoserine; T. phosphothreonine; Y, phosphotyrosine. Number
`at lower left of each autoradiogram indicates the following culture
`conditions: 1, with no serum; 2. with serum; 3. with serum and MAb
`4DS. Tyrosine phosphorylation in control cells was visualized
`faintly on the autoradiogram but reproduces poorly.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1088 Page 4 of 8
`
`

`
`VOL. 11, 1991
`
`MODULATION OF PHOSPHORYLATION OF p185”£”
`
`983
`
`phosphorylation (Fig. 6A, lane 2) to 20% of the steady-state
`levels achieved in the absence of newborn calf serum (Fig.
`6A, lane 1). Experiments were done to examine the capacity
`of newborn calf serum to stimulate tyrosine phosphorylation
`of p185”5"’ in short-tenn treatment. There was no increased
`activation of phosphorylation when serum-free cultures
`were supplemented with newborn calf serum for 30 min at 37
`or 4°C (data not shown).
`To determine the specificity of the capacity of newborn
`calf serum to stimulate activation of p185”"’ phosphoryla-
`tion in SK-BR-3 cells, we investigated the potential for
`serum activation of another closely related molecule, the
`EGF-R. There was no potentiating elfect of newborn calf
`serum on phosphorylation of the EGF-R in SK—BR-3 cells
`(Fig. 6B).
`Having shown an increase in the steady-state levels of
`p185’”
`phosphorylation induced by newborn calf serum
`and its reduction by MAb 4D5, we determined the phos-
`phoamino acid content of p185’’'“’ under these conditions
`by two-dimensional
`thin-layer electrophoresis (Fig. 6C).
`pl85”E‘” from cells cultured in the absence of newborn calf
`serum contained predominantly phosphoserine and phos-
`phothreonine with little phosphotyrosine (Fig. 6C, blot 1).
`The presence of some phosphorylation on tyrosine can be
`demonstrated by longer exposure of the autoradiogram but is
`not visualized well in the figure shown. Quantitation of the
`relative amount of label in each amino acid was obtained by
`scraping the ninhydrin-identified spots from the thin-layer
`plate for liquid scintillation counting. Activation by 5%
`newborn calf serum increased the total phosphoamino acid
`content 2.2-fold, while for phosphotyrosine,
`the increase
`was 3.9-fold (Fig. 6C, blot 2). Inhibition of newborn calf
`serum-mediated stimulation of p185”‘“'’ phosphorylation by
`MAb 4D5 resulted in a parallel reduction in the content of all
`three phosphoamino acids (Fig. 6C, blot 3).
`Tyrosine phosphorylation of p185"E"’. To further quanti-
`tate the relative increase in the steady-state phosphotyrosine
`content of p185"E"’ induced by newborn calf serum, cells
`were metabolically labeled with [”S]cysteine and assayed
`for the steady-state phosphotyrosine content of p185”E‘" by
`using antiphosphotyrosine MAb PY—69. MAb PY-69 was
`specific for phosphotyrosine in the immunoprecipitation
`reaction; i.e., we were able to show competion for binding
`with cold phosphotyrosine and not with phosphoserine in
`experiments with ”S-labeled EGF-R (data not shown). As
`illustrated in Fig. 7A, lanes 1’ to 4’, the amount of phosphor-
`ylation of pl85”E” on tyrosine increased with the concen-
`tration of newborn calf serum in the culture medium. The
`level of activation of tyrosine phosphorylation in serum-free
`medium was 18% of that observed in 10% newborn calf
`serum, and in medium containing 2.5% serum,
`tyrosine
`phosphorylation was 35% of that observed with a serum
`concentration of 10%. As a control, equal amounts of labeled
`cell extracts were immunoprecipitated with anti-pl85"'E‘”
`MAb 9G6 (Fig. 7A,
`lanes 1 through 4). There was no
`significant effect of serum on the levels of “S-labeled
`pl85”ER2 in the cells. To quantitate these results, the ratios
`of phosphotyrosine-associated counts to total counts asso-
`ciated with p185”E’” are presented in Fig. 7B, which shows
`a dose-dependent increase of up to 5.4-fold with 10% new-
`born calf serum in the culture medium.
`Partial depletion of activating factor from newborn calf
`serum. Since our results indicated the presence of some
`activating factor(s) for p185”5'” phosphorylation in new-
`born calf serum, we sought confinnation of this observation
`by determining whether newborn calf serum could be de-
`
`Antibody
`
`P185
`P-Tyr
`
`r& I
`I
`I
`I
`l
`2
`3
`4
`2
`l
`3
`4
`
`-*
`
`‘ft?’ H I! Q
`
`°/.Serum
`
`o
`
`2.5
`
`5
`
`10
`
`o
`
`2 5
`
`5
`
`10
`
`A.
`
`B.
`
`Lo_ 500
`2 3C
`‘as
`,‘._
`}— O
`C'U\" 100
`
`.
`
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`
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`l 2 3 4
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`~-Elli
`
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`
`5
`Percent Serum, V/V
`
`10
`
`FIG. 7. (A) Detection of tyrosine-specific phosphorylation of
`p185”“" by newborn calf serum. Subcortfluent SK-BR-3 cells were
`metabolically labeled with [”S]cysteine in the presence of diflerent
`concentrations of serum for 15 h. Cells were lysed in 650 pl of
`extraction buffer. The lysates were divided into two equal parts of
`300 pl each and then immunoprecipitated with anti-pl85’""‘” MAb
`9G6 (lanes 1 to 4) or antiphosphotyrosine MAb PY-69 (lanes 1' to
`4'). Other details of the assay were as described in the legend to Fig.
`5. To detect the phosphotyrosine (P-Tyr) signal in cells cultured in
`the absence of serum (lane 1'),
`it was necessary to expose the
`autoradiogram for 20 h, which resulted in overexposure of lanes 1 to
`4. (B) To qluantitate the data in panel A, the radioactivity associated
`with p185 9'’ was determined by counting the excised bands in a
`liquid scintillation counter. The ratios of counts in p185”5"" phos-
`photyrosine over total counts in pl85”‘“" were plotted as a percent-
`age of control with 0% newborn calf serum against the concentration
`of newborn calf serum used in the culture medium. (C) Depletion of
`the activator(s) of p185"5"’ phosphorylation in serum. SK-BR-3
`cells were labeled with “P, in the absence or presence of serum for
`15 h. Lane 1. Control without serum; lane 2, medium with 5%
`newborn calf serum; lane 3, medium with 5% newborn calf serum
`depleted of factor(s) by three repetitive adsorptions of 4 h each on
`SK-BR-3 cells at 4°C; lane 4, control medium adsorbed on A431
`cells. Cell extracts were prepared and p185”E"’ was assayed as
`described in Materials and Methods.
`
`pleted of such a factor(s). In these experiments, phosphate-
`free medium containing 5% newborn calf semm was treated
`by repetitive absorption with SK-BR-3 cells (three times, for
`4 h each time, at 4°C). Culture medium treated in an identical
`manner by adsorption with A431 cells, which do not express
`high levels of p185”E‘”, was used as control. Figure 7C
`shows that there was a 53% reduction in p185”E"’ phosphor-
`ylation in SK-BR-3 cells cultured in the presence of medium
`preadsorbed with SK—BR-3 cells (lane 3) compared with
`untreated medium (lane 2), and there was only a 16%
`reduction in p185”ER2 phosphorylation when SK-BR-3 cell
`cultures were supplemented with medium preadsorbed with
`A431 cells (Fig. 7C, lane 4).
`Activation factor(s) in newborn calf serum was not TGF-a
`or EGF. pl85”E"’ has been shown to be phosphorylated
`when EGF-Rs are activated by exggsure to EGF or TGF-a,
`which are not ligands for pl85”E (24, 40). To determine
`whether the newborn calf serum-mediated 2.2-fold enhance-
`ment of p185”"'“’ phosphorylation resulted from the activity
`of TGF-a or EGF. we used anti-EGF-R MAb 528, which has
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1088 Page 5 of 8
`
`

`
`984
`
`KUMAR ET AL.
`
`MoL. CELL. Bror..
`
`U‘
`
`J>
`
`units
`32F-IncorporatedintoP185.orbitory
`
`
`
`0|
`
`I\)
`
`Serum - - + + + + +
`4D5
`- + — + — - -
`528
`- - — — + — +
`TGF-a - — — — — + +
`
`FIG. 8. Activation of p185"“" by newborn calf serum was not
`due to TGF-a or EGF. Subconfluent SK-BR-3 cells were labeled
`with ”P, for 15 h in the presence or absence of 5% newborn calf
`serum, 150 nM MAb 4D5, and 300 nM MAb 528. As a positive
`control, some cultures received 40 nM TGF-cr either alone (column
`6) or with 300 nM MAb 528 (column 7) during the last 20 min of
`labeling. Cells were lysed and then immunoprecipitated with MAb
`9G6 to assay p185”'”". The insert displays the autoradiogram
`resulting from a 3-h exposure of the dried gel. To quantitate the
`amounts of pl85”"-"2. protein bands were excised for determining
`the radioactivity. which is represented here on an arbitrary scale.
`These experiments were performed three times.
`
`been shown to block the binding of EGF and TGF-0. to their
`receptors (23, 25). The experiment whose results are shown
`in Fig. 8 demonstrates that coincubation of SK-BR-3 with a
`saturating amount of anti-EGF-R MAb 528 did not signifi-
`cantly alter the cagacity of newborn calf serum to increase
`the levels of p185 5”’ phosphorylation. As a positive con-
`trol, some cultures received TGF-a with or without MAb 528
`during the last 20 rrrin of labeling before cells were harvested,
`and MAb 528 completely blocked the predicted and ob-
`served increases in phosphorylation of pl85””5’*’ induced by
`TGF-or (Fig. 8, column 6 and 7).
`In
`Observations with other mammary carcinoma cells.
`order to determine whether activation of pl85”5R’ phos-
`phorylation by newborn calf senrm and its inhibition by
`MAb 4D5 are phenomena restricted to SK-BR-3 cells or
`whether they can be demonstrated with other breast tumor
`cells overexpressing p18S”ER’, we extended our investiga-
`tion to two other lines: MDA-MB-453 and BT-474. The
`results in Fig. 9 indicate a significant capacity of newborn
`calf serum to activate p18S"E“2 phosphorylation and show
`that the addition of MAb 4D5 could reduce the steady-state
`levels of p185”“" phosphorylation both in the presence of
`newborn calf serum and in serum-free cultures.
`
`DISCUSSION
`
`Activation of kinase activity associated with receptor
`tyrosine kinase by ligand binding is thought
`to be the
`common mechanism by which transmembrane receptors for
`
`12 3 4.,
`
`"
`
`IN
`
`E fizowolll‘
`
`Serum — —- + +
`4D5
`- + - +
`
`FIG. 9. Etfects of MAb 4D5 and newborn calf serum on steady-
`state levels of p185”5’" phosphorylation in MDA-MB-453 mam-
`mary carcinoma cell line (A) and BT-474 mammary carcinoma cell
`line (B). Cells (4 X 10’) from each cell line were cultured with “P, for
`15 h in the presence or absence of 5% serum and/or MAb 4D5 (150
`nM). Cell lysates were prepared and processed for the detennination
`of radioactivity in p185”E” as described in Materials and Methods.
`The amounts of radioactivity in p185”E"’ (in counts per minute) in
`panel A were 420 (lane 1), 263 (lane 2), 1.237 (lane 3). and 804 (lane
`4). ln panel B, the counts per minute were 322 (lane 1), 268 (lane 2).
`508 (lane 3), and 225 (lane 4).
`
`growth factors transduce signals that activate cell prolifera-
`tion (6, 22, 27. 48). Support for this model is provided by our
`experiments demonstrating growth inhibition of cells over-
`expressing EGF-Rs by MAbs 225 and 528, which recognize
`these transmembrane receptors on intact cells, prevent
`binding of the growth factor-ligand, and block ligand-in-
`duced phosphorylation of EGF-Rs (16, 23, 29). A similar
`antiproliferative effect has been observed when SK-BR-3
`cells, which overexpress p185”E"’, are exposed to MAb 4D5
`(17, 18). The present work was undertaken to explore the
`modulation of phosphorylation of p185”’“" by an anti-
`p185”E"’ MAb, 4D5, and by newborn calf serum.
`The results presented here indicate that
`treatment of
`SK-BR-3 cells with MAb 4D5 significantly reduced the
`steady-state levels of phosphorylation of the c-erbB-2/HER2/
`c—neu gene product p185”E" on serine,
`threonine. and
`tyrosine residues. Anti-p185”E"2 MAb has been shown to
`cause enhanced down-regulation of p185”“-R2 from the sur-
`face of human and murine cells by reducing the half-life of
`pl85”5”’ (14, 18). Our conclusion that MAb 4D5-mediated
`inhibition of the steady-state levels of p185”E'" phosphory-
`lation was only in part attributable to the down-regulation of
`pl85”E”’ is supported by the following lines of evidence: (i)
`the reduction of phosphorylation was far in excess of the
`reduced content of 32S-labeled protein measured by immu-
`noprecipitation and also by immunoblotting; (ii) inhibition of
`phosphorylation was observed at 4°C under conditions in
`which MAb-induced down-regulation of p185”"5"’ would be
`limited; (iii) the inhibitory etfect of MAb 4D5 on p185”E"’
`phosphorylation could be mimicked by F(ab) fragment,
`which does not down-regulate p185"E‘”. The last observa-
`tion is similar to findings with the F(ab) fragment of another
`MAb-specific murine pl85’”‘”’ (14). Taken together, these
`data provide strong indirect evidence that MAb 4D5 may
`have interfered with the activation of phosphorylation of
`p185”E‘" by a growth factor(s).
`The observation that newborn calf serum contains an
`activator(s) of phosphorylation of p185”E”’ opens a new
`area of investigation for identifying an additional factor(s)
`
`PETITIONER'S EXHIBITS
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`Exhibit 1088 Page 6 of 8
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`PETITIONER'S EXHIBITS
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`Exhibit 1088 Page 7 of 8
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`PETITIONER'S EXHIBITS
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`Exhibit 1088 Page 8 of 8