PFIZER EX. 1088
`Page 1
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`

`

`MOLECULAR AND CELLULAR BIOLOGY
`
`VOLUME 11 • FEBRUARY 1991 • NUMBER 2
`
`Alan M. Weiner, Editor in Chief (1995)
`Yale University
`New Haven, Conn.
`
`Don W. Cleveland, Editor (1994)
`The Johns Hopkins University
`Baltimore, Md.
`
`James R. Broach, Editor (1995)
`Princeton University
`Princeton, N.J.
`
`Anita K. Hopper, Editor (1994)
`The Milton S. Hershey Medical Center
`Pennsylvania State University
`Hershey, Pa .
`
`Lawrence A. Chasin, Editor (1995)
`Columbia University
`New York, N.Y.
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`Tony Hunter, Editor (1993)
`The Salk Institute
`San Diego, Calif.
`
`David A. Clayton, ~ditor (1995)
`Stanford University School of Medicine
`Stanford, Calif.
`
`Steven L. McKnight, Editor (1992)
`Carnegie Institution of Washington
`Baltimore, Md.
`
`Charles J. Sherr, Editor (1995)
`Howard Hughes Medical Institute
`St. Jude Children's Research Hospital
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`The Johns Hopkins University
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`Shirley M. Tilghman, Editor (1993)
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`Owen N. Witte, Editor (1993)
`Molecular Biology Institute
`University of California
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`Frederick W. Alt (1993)
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`Arnold J. Berk (1991)
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`J. Michael Bishop (1993)
`Elizabeth H. Blackburn (1991)
`Jef D. Boeke (1991)
`Marjorie C. Brandriss (1991)
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`Milton J. Schlesinger (1992)
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`
`PFIZER EX. 1088
`Page 2
`
`

`

`MOLECULAR AND CELLULAR BIOLOGY , Feb . 1991, p. 979-986
`0270-7306/911020979-08$02.00/0
`Copyright © 1991, American Society for Microbiology
`
`Vol. 11 , No.2
`
`Regulation of Phosphorylation of the c-erbB-2/HER2 Gene Product
`by a Monoclonal Antibody and Serum Growth Factor(s)
`in Human Mammary Carcinoma Cells
`RAKESH KUMAR/* H. MICHAEL SHEPARD,2 AND JOHN MENDELSOHW·3
`Laboratory of Receptor Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York ,
`N ew York 10021\ Developmental Biology, Genentech Inc. , South San Francisco, California 940802
`;
`and Cornell University Medical College, New York, N ew York 10021 3
`
`Received 9 May 1990/Accepted 17 November 1990
`
`, the gene product of
`Monoclonal antibody (MAb) 4DS was used to analyze the phosphorylation of p185HER2
`c-erbB-2/HER2, in SK-BR-3 cells. Culture in the continuous presence of 4D5 reduced the in vivo steady-state
`levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4DS may have
`interfered with the activation of phosphorylation of p185HER2
`• The observed MAb-mediated reduction of
`p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were
`grown under serum-free conditions, the steady-state levels of p185HER2 phosphorylation were reduced by 56%,
`and addition of 4DS further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure
`to increasing concentrations of newborn calf serum in these cultures, there was a linear increase in
`tyrosine-specific phosphorylation of p185HERz, reaching a 5.4-fold increase with 10% newborn calf serum.
`Phosphorylation of pl85HER2 in the presence of newborn calf serum was not attributable to stimulation of the
`epidermal growth factor receptor by epidermal growth factor or by transforming growth factor-a. Extension
`of these observations to two other mammary carcinoma cell lines, MDA-MB-453 and BT -474, also demon(cid:173)
`strated a significant capacity of serum to induce p185HERZ phosphorylation. The demonstration of antibody(cid:173)
`mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma
`cells may also produce and secrete a factor or factors which may activate p185HERz. Our observation that
`growth-inhibitory MAb 4DS is able to reduce the phosphorylation of p185HERZ by newborn calf serum and by
`a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HERZ
`as a signal transduction pathway to regulate cell proliferation.
`
`Proto-oncogenes are a group of normal genes which play
`important roles in the regulation of cell proliferation and
`function (2 , 5) . Abnormalities in the expression, structure, or
`activity of proto-oncogene products contribute to the devel(cid:173)
`opment and maintenance of the malignant phenotype in
`complex but important ways (36, 37, 46) . Evidence that the
`gene products of several activated proto-oncogenes are
`either growth factors or growth factor receptors has sug(cid:173)
`gested a possible link between proto-oncogenes and growth
`factors (20). For example, the receptor for macrophage
`colony-stimulating factor is identical to the product of c-fms
`(35), and c-erbB-1 encodes the receptor for epidermal growth
`factor (13) and transforming growth factor-a (TGF-a) (43).
`Growth factor receptors encoded by proto-oncogenes are
`transmembrane glycoproteins with intrinsic tyrosine-kinase
`activity (22). Receptor tyrosine kinases are activated by
`binding of their respective ligands , the growth factors (48).
`This activity is thought to be an integral part of signal
`transduction processes involved in the regulation of cell
`proliferation (21). Overexpression of some growth factor
`receptors has been shown to induce transformed properties
`in recipient cells (11 , 32) , possibly because of excessive
`activation of signal transduction mechanisms. Furthermore ,
`a number of tumor cells with increased expression of growth
`factor receptors also produce ligands for these receptors
`(10).
`HER2 (also known as c-erbB-2 or c-neu) , the human
`
`* Corresponding author.
`
`979
`
`homolog of the rat proto-oncogene neu (9) , encodes a
`185-kDa transmembrane glycoprotein with intrinsic tyrosine
`kinase activity which is presumed to be the receptor for an
`as-yet-unidentified ligand (3, 39). p185HERz also has homol(cid:173)
`ogy to, but is distinct from , the epidermal growth factor
`receptor (EGF-R) , which is the product of c-erbB-1. Both
`proteins have a cysteine-rich extracellular domain , a trans(cid:173)
`membrane domain, and an intracellular tyrosine kinase (4,
`31 , 47). In spite of sequence homology between c-erbB-2 and
`c-erbB-1 , EGF does not bind to p185 17Enz (33) . p18Y-IERZ has
`been shown to be overexpressed or amplified or both in a
`number of human malignancies: breast (45), ovarian (38) ,
`thyroid (1), lung (7), salivary gland (34), and stomach (50). In
`addition , p185HERz is a potent oncogene capable of inducing
`transformation and tumorigenesis when overexpressed in
`NIH 3T3 cells (12, 19). Overexpression of p185HERz also
`induces tumor cell resistance to macrophage killing (15).
`Thus p185HERz may have an important role in the develop(cid:173)
`ment and maintenance of human tumors.
`These observations suggest that receptor-associated ty(cid:173)
`rosine kinase activity of overexpressed proto-oncogene pro(cid:173)
`tein products is important for the regulation of cell growth .
`We have developed a panel of monoclonal antibodies
`(MAbs) reactive with domains of the human EGF-R (23) and
`p185HERz (17 , 18) in intact cells and have demonstrated
`antiproliferative effects of these antibodies in vitro (16, 18,
`23) and in vivo (29). Antibody 4D5 , which is specifically
`directed against p185HERz, exhibits strong anti proliferative
`activity on cultured human breast tumor cell lines which
`overexpr.ess p185HERz (18). Since p185 17Enz is a receptor
`
`PFIZER EX. 1088
`Page 3
`
`

`

`980
`
`KUMAR ET AL.
`
`MoL . CELL. BioL.
`
`with intrinsic tyrosine kinase activity, we investigated the
`modula ion of p185HERz phosphorylation by MAb 4D5 . We
`report here that activation of phosphorylation of p185HERZ
`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-a or EGF.
`Furthermore, SK-BR-3 cell-conditioned medium contained a
`factor(s) that could activate p185HER2 phosphorylation and
`was partially inhibited by MAb 4D5 .
`
`MATERIALS AND METHODS
`
`Materials. MAbs 4D5 (18) and 906 (44) were raised against
`human p185H£Rz. 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. 32P; (carrier free ; 28.5 Ci/nmol)
`and 35S-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 (1 :1, vol/vol) (F-12/
`DMEM) supplemented with 10% fetal bovine serum.
`
`Labeling of p185HER2 with 32P; and e5S]cysteine. Cells (3 X
`105
`) 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 3 2P; per
`ml in the presence or absence of MAb and newborn calf
`serum . At desired times, cells were harvested in 400 J.Ll of
`lysis buffer (20 mM HEPES [N-2-hydroxyethylpiperazine(cid:173)
`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(cid:173)
`nyl fluoride , 10 J.Lg 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 J.Ll of Pansorbin was added as described elsewhere
`
`(42). For labeling with e5S]cysteine, the cells were washed
`F-12/DMEM containing 0.15 mCi of e5S]cysteine per ml
`
`with cysteine-free medium and refed with cysteine-free
`
`with or without 5% newborn calf serum.
`Immunoprecipitation and SDS-polyacrylamide gel electro(cid:173)
`phoresis. Aliquots (350 J.Ll) of the cell lysates (or equal
`amounts of trichloroacetic acid-precipitable counts per
`minute) containing 32P-labeled or [35S]cysteine-labeled
`p185HERz were subjected to immunoprecipitation with 10 J.Lg
`of MAb 906, 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 buffer (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 J.Ll of
`sample loading buffer (10 mM Tris HCI [pH 6.8], 1% sodium
`dodecyl sulfate [SDS], 0.2% 2-[3-mercaptoethanol , 10% glyc(cid:173)
`erol , 0.001% bromophenol blue) , heated at 95°C for 5 min ,
`and resolved on a 7% SDS-polyacrylamide slab gel (26). The
`efficiency of precipitating labeled receptor with MAb 906 is
`80 to 90% when this procedure is used . Low-molecular-mass
`colored markers (Amersham Corp.) were used as standards.
`Phosphoamino acid analysis. The band corresponding to
`
`2 3 4 5
`
`FIG . 1. Effect of MAb 405 on steady-state levels of p185HER2
`phosphorylation in SK-BR-3 cells. Subconftuent cultures were la(cid:173)
`beled with 32P; (400 f.LCi in 1 ml of phosphate free F-12/DMEM
`supplemented wi th 5% newborn calf serum) in the continuous
`presence of different amounts of antibod y for 15 h. Detergent
`extracts were made, and p185HERl was immunoprecipitated by using
`MAb 9G6 and then resolved by 7% SDS-polyacrylamide gel elec(cid:173)
`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 32P-labeled p185HER2
`. Lane 1, Control cells;
`lanes 2 to 4, cells treated with MAb 405 at 30, 150, and 300 nM,
`respectively ; lane 5, cells treated with 400 nM F(ab) frag ment of
`MAb 405 . The amounts (in counts per minute) of p185HERl 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(cid:173)
`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. 32P-labeled p185HERZ in a gel slice
`was partially hydrolyzed with 200 J.Ll of 6 N HCI at l10°C for
`1 h. Two portions (10 J.LI each) of the hydrolysate were taken
`for measurement of radioactivity in a liquid scintillation
`counter to determine the total incorporation of 32P into the
`p185HERl 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 32P-labeled materials were eluted
`with 0.5 N HCl and lyophilized. The recovery of radioactiv(cid:173)
`ity by this procedure was 78 to 85% . 32P-phosphoamino
`acids mixed with unlabeled carrier phosphoamino acids
`(phosphoserine, phosphothreonine , and phosphotyrosine
`[1:1:1]) were analyzed by thin-layer electrophoresis as de(cid:173)
`scribed elsewhere (8) .
`
`RESULTS
`MAb 405 reduces amount of 32P-Iabeled pl85HER2
`• MAb
`4D5 was used to investigate the regulation of phosphoryla(cid:173)
`tion . SK-BR-3 cells, which have an amplified c-erbB-2 gene
`(45), were cultured for 15 h in medium containing 32P; in the
`continuous presence of various concentrations of MAb 405.
`The p185HERz from these cells was immunoprecipitated wi.th
`another anti-p185H£Rl MAb, 906, which recognizes a diS(cid:173)
`tinct epitope of p185 17£RZ, and resolved by SDS-polyacryl(cid:173)
`amide gel electrophoresis. Results of such an experiment ~e
`shown in Fig. 1. Treatment of cells with 4D5 reduced in VIVO
`steady-state levels of 32P-labeled p185HERl up to 80% in a
`d, se-dependent manner (lanes 2 through 4) . There was 4~
`± 8% reduction in phosphorylation by 150 nM MAb 4D5 IR
`eight different experiments. When the F(ab) fragment of 4D5
`was used instead of intact antibody , comparable or greater
`reduction of 32P-labeled p185Ti£Rl 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 32P-labeled p185H£Rl (unf
`°5
`published data) . The reduction in steady-state levels
`0
`32P-Iabeled p185HERl was not due to interference by 4
`with MAb 906 during the immunoprecipitation reaction, as
`immunoprecipitation performed with another polyclonal an-
`
`PFIZER EX. 1088
`Page 4
`
`

`

`Abs
`
`Pl85
`
`EGF-R
`
`A.
`
`B.
`
`c.
`
`MODULATION OF PHOSPHORYLATION OF p185H£Rl
`
`981
`
`FIG. 2. Specificity of the reduction of 32P-labeled p185H£RZ by
`in SK-BR-3 cells in the presence or absence of MAb 405.
`bbc;ontltUent cells were labeled with 32P; for 15 h. The cells were
`600 JJ.l of extraction buffer and divided into two equal parts
`JJ.I each. Immunoprecipitation was performed with anti-p185
`1 to 3) or with anti-EGF-R MAb 528 (lanes 1' to 3'). An
`1Ulctra<iio1gram of a dried gel is shown here . Lane 1 and 1', Control;
`and 2', 30 nM MAb 4D5 ; Janes 3 and 3' , 150 nM MAb 405 .
`per minute: Jane 1, 5,985 ; lane 2, 3,798; lane 3, 3,120; Jane 1' ,
`Jane 2' , 779; Jane 3' , 932. Abs , Antibodies.
`
`recognizing the carboxy-termin(!l 17 amino acids
`n 1 ~, "~ .. -· · · gave similar results (unpublished data).
`we examined the possibility of general inhibitory
`of MAb 4D5 on the steady-state levels of other
`receptor proteins by analyzing the amount of
`p185HERz and 32P-Iabeled EGF-R in the same
`ment (Fig. 2) . These results indicated that there was
`reduction of 32P-Iabeled EGF-R during 15 h of treatment
`SK-BR-3 cells with 150 nM MAb 4D5, which had reduced
`amount of 32P-labeled p185H£R by 48% .
`of reduction of p185H£R2 phosphorylation. The
`reduction of steady-state levels of 32P-Iabeled p185H£nz by
`, shown in Fig. 1 and 2, could result from down(cid:173)
`regulaLtio'n of pl85HERZ and/or interference in the activation
`nll~"'"'"'" phosphorylation by a direct or indirect mecha(cid:173)
`In initial studies to explore these possibilities,
`cultures of cells were metabolically labeled with
`~J~iJc:yst.ei·, 1e or 32P; . During 11 h of concurrent incubation
`4D5 , there was a 45% reduction in 32P-Iabeled
`nl~:-.H.ERZ (Fig. 3A) and only a 14% reduction in 35S-Iabeled
`nlR:'\H.ERZ (Fig. 3B). This suggests that the reduced 32P label
`pl85HERz in the presence of MAb 4D5 can only partially
`attributed to reduced p185H£Rz content. Next , we per(cid:173)
`a similar experiment comparing the capacities of the
`alent F(ab) fragment of MAb 4D5 and an intact MAb
`to affect the reduction of 35S-Iabeled p185H£nz. There
`no change in 35S-labeled p185HERZ in the presence of
`, but there was a 26% reduction caused by MAb 4D5
`3C, lanes 3 and 2, respectively). The results obtained in
`immunoprecipitation experiments documented in Fig.
`3A through C were confirmed by immunoblotting (D). Immu(cid:173)
`Doblotting of the 32P-labeled SK-BR-3 cell extracts used in
`2 demonstrated only a marginal reduction in the content
`p185H£Rz protein when cells were cultured in the presence
`MAb 4D5 but a substantial reduction in the a mount of
`labeled p185H£Rz (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(cid:173)
`tent of pl85HERz by MAb 4D5 (Fig. 3D, experiment 2) .
`findings indicate that increased receptor catabolism
`· m'"'"·--' by a MAb cannot fully account for the observed
`reduction in 32P labeling and show [with F(ab)] that reduced
`labeling is dissociated from reduced content of p185H£nz.
`32 Next we addressed the possibility that the reduction in
`P-labeled p185H£R2 associated with exposure to MAb 4D5
`Could be related to a change in the level of expression of
`PlSSHERz on the plasma membrane or to the extent of
`
`405
`D.
`
`+
`
`EXP. l
`
`Abs Pl85
`
`EGF-R
`
`+
`
`2 3
`
`- +
`+
`+
`405
`FIG. 3. Analysis of the reduction ofp185H£RZ phosphorylation in
`SK-BR-3 cells treated with MAb 405 . Cell s were labeled with 32P;
`(A) or e5S]cysteine (B) in the presence or absence of MAb 4D5 (150
`nM) for 11 h. Samples were prepared and separated as described in
`Materials and Methods. The autoradiogram shown here was ob(cid:173)
`tained by 6 h of exposure. (C) Cells were labeled w'ith [35S]cysteine
`for 11 h in the presence of MAb 4D5 (150 nM , Jane 2) or F(ab) (400
`nM , Jane 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 p185H£Rz 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 p185HERZ bands in panels A and B gave results
`similar to those with densitometric scanning, and there was a 27% ±
`3% additional reductio
`·n 32P-labeled p185HERZ compared with
`35S-Jabeled pl85H£1<2 . (D) Immunoblotting of p185HERZ and EGF-R
`proteins. In experiment 1 (Exp.l) , 32P-labeled SK-BR-3 ceil extracts
`(50 J.l.g of protein) used in Fig. 2, Janes 1 and 2, were resolved on a
`7% SDS-polyacrylamide gel and then immunoblotted with anti-Pl85
`MAb 9G6 or anti-EGF-R polyclonal antibody RK-Il . Experiment 2
`shows the immunoblotting of unlabeled SK-BR-3 cell extracts
`prepared following culture for 15 h with or without 30 nM MAb 405.
`Since some of the extracts used here were radiolabeled , immuno(cid:173)
`blotted membranes were visualized by using a protein A-gold
`.:nhancernent kit (30). Abs, Antibodies.
`
`down-regulation of receptor protein . First, we determined
`what fraction of the 35S-labeled pl85fiERz is present on the
`cell surface at 37oC (Fig. 4A) . In these experiments,
`p185fiERz 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 35S-Iabeled pl85HERz is expressed on
`the cell surface under these experimental conditions ; thus
`p185fiERz is available for down-regulation by MAb 4D5.
`Down-regulation of EGF-R has been shown to be dependent
`on temperature (41). To confirm that down-regulation of
`surface pl 85H£Rz also is reduced at 4°C , experiments were
`performed to analyze the effect of temperature on the
`abundance of 35S-labeled p185HERZ on the cell surface.
`Results indicated that at 4°C the amount of total 35S-labeled
`p185HERz expressed on the surface increased to 35% ± 3%
`(data not shown) compared with 19% ± 4% of total 35S(cid:173)
`labeled p185H£Rz at 37°C.
`In orde r to define the contribution of down-regulation to
`MAb-induced reduction in p185HERZ phosphorylation, we
`
`PFIZER EX. 1088
`Page 5
`
`

`

`982
`
`KUMAR ET AL.
`
`A. EXP. 1
`EXP. 2
`~ I 3 4 1
`
`Antibody P185
`
`-
`
`MOL. CELL. BIOL,
`
`P-Tyr
`1' 2' 3' 1
`
`-
`
`FIG. 5. Partial agonist nature of F(ab). Subconfluent SK-BR-3
`cells were labeled with 32P; for 15 h. Some cultures were treated with
`400 nM F(ab) for the indicated times. The cells were lysed in 800 ~~ol
`of extraction buffer. The lysates were divided into two equal parts of
`350 fL( 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.
`
`405
`
`- +
`- +
`FIG. 4. (A) Quantitation of surface expression of 35S-Iabeled
`• Cells were labeled with [35S]cysteine for 11 h. At the end
`p185HER2
`of incubation , some cultures were lysed in 500 fLI of lysis buffer for
`the determination of total 35S-Iabeled p185H£Rl by immunoprecipi(cid:173)
`tation with 10 fLg of 405 (Materials and Methods). For measuring the
`surface expression of 35S-Iabeled p185HERl, cultures were was hed
`with phosphate-buffered saline and further incubated with F-12/
`DMEM-20 mM HEPES (pH 7.5) containing 20 J.Lg of high-affinity
`MAb 405 per ml for 1 hat 4°C. The cultures were washed , lysed in
`500 fLI of extraction buffer, and processed for immunoprecipitation
`by adding RAM-protein A-Sepharose beads but no more MAb 4D5
`during the immunoprecipitation procedure. The results of two
`representative · experiments are shown here. Lanes 1 and 3, Total
`35S-labeled p185H£R2 ; lanes 2 and 4, 35S-Iabeled p185HERl on the cell
`surface. (B) Effect of MAb 4D5 on surface expression of p185HERl in
`a temperature shift experiment. SK-BR-3 cells were first equili(cid:173)
`brated with 32P, for 4 h at 3rC (lane 1) and then further incubated
`with or without MAb 4D5 for an additional 11 h (in the continuous
`presence of 32P;) 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 32P associated with p185HERl bands was obtained by densitomet(cid:173)
`ric scanning of the autoradiogram and by determining the radioac(cid:173)
`tivity associated with p185HERl bands (A and B).
`
`analyzed the effect of incubation with MAb 4D5 at 4°C. In
`these studies, cells were first equilibrated with 32P; for 4 hat
`37oc (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 4D5 . A comparison of the labeled
`material in lanes 2 and 4 in Fig. 4B (ftuorographs exposed for
`24 and 96 h, respectively) showed a significant 85% reduc(cid:173)
`tion in 32P labeling of pl85H£Rl during 11 h at 4°C compared
`with labeling at 37°C. However, incubation of cells at 4°C did
`not prevent a further substantial MAb-mediated reduction in
`steady-state levels of pl85HER2 phosphorylation: there was a
`34% decrease at 4°C (compare lanes 4 and 5) and a 51%
`decrease at 37oC (compare lanes 2 and 3). Taken together,
`these observations indicate that MAb-induced reduction in
`pl85HER 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 slightly stimu(cid:173)
`lated in vivo tyrosine phosphorylation of p185HERl in cul(cid:173)
`tures labeled with 32P (Fig. 5, lane 2'). However, there was
`no activation in cultures exposed to F(ab) for a longer
`treatment of60 min (Fig. 5, lane 3'). The observation that the
`F(ab) fragment of 4D5 does not down-regulate the 35S-
`
`labeled p185HERl 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 p185HER2 in presence or
`absence of newborn calf serum. Next, we investigated the
`possible source of the factor(s)
`that might stimulate
`p185HERl phosphorylation. As shown in Fig. 6A, culturing
`the cells in serum-free medium resulted in a steady-state
`level of phosphorylation of pl85HER2 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 pl 85HERl
`
`A.
`
`2 3 4
`
`B.
`
`+ +
`+
`
`+
`
`+ +
`
`Serum
`405
`
`C.
`
`FIG. 6. (A) Detection of newborn calf serum-mediated phos(cid:173)
`phorylation of p185H£R2 • Subconfluent SK-BR-3 cells were labeled
`with 32P, 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 405.
`Samples were prepared and immunoprecipitated for assaying the
`amount of p185HERl as described in the Materials and Method~·
`Quantitation of the p185 bands was obtained by densitometnc
`scanning of the autoradiogram . (B) Control experiment showtn~
`effect of serum on the 32P, labeling of EGF-Rs for 15 h in SK-BR-.
`cell cultures. Cell extracts were immunoprecipitated with anti·
`EGF-~ MAb 528, which recognizes one distinct band wi~h an
`approximate molecular mass of 170 kDa (arrow) . (C) Two-dlm~n­
`sional thin-layer electrophoresis pattern of 32P-phosphoamino ac1~s
`in a hydrolysate of the p185H£Rl immunoprecipitated in panel A.
`'
`Phosphoserine; T , phosphothreonine; Y, phosphotyrosine. Number
`at lower left of each autoradiogram indicates the following cuttur~
`conditions: 1, with no serum ; 2, with serum; 3, with serum ~nd t.:fA d
`405. Tyrosine phosphorylation in control cells was v1suahze
`faintly on the autoradiogram but reproduces poorly.
`
`PFIZER EX. 1088
`Page 6
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`

`

`hm>ohonrlat.ion (Fig. 6A, lane 2) to 20% of the steady-state
`achieved in the absence of newborn calf serum (Fig.
`1). Experiments were done to examine the capacity
`fn4~WitJor·n calf serum to stimulate tyrosine phosphorylation
`~-111••~·--·· in short-term treatment. There was no increased
`of phosphorylation when serum-free cultures
`supplemented with newborn calf serum for 30 min at 37
`4°C (data riot shown).
`To determine the specificity of the capacity of newborn
`serum to stimulate activation of p185H£R2 phosphoryla-
`in SK-BR-3 cells, we investigated the potential for
`activation of another closely related molecule, the
`There was no potentiating effect of newborn calf
`phosphorylation of the EGF-R in SK-BR-3 cells
`
`shown an increase in the steady-state levels of
`phosphorylation induced by newborn calf serum
`its reduction by MAb 4D5, we determined the phos(cid:173)
`bOliml·tn·to acid content of p185HER2 under these conditions
`t\V():Cilrrtensional thin-layer e