BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 226, 59—69 (1996)
`ARTICLE N0. 1311
`
`ErbB Receptor Activation, Cell Morphology Changes, and Apoptosis
`
`Induced by Anti-Her2 Monoclonal Antibodies
`
`Yoshiko Kita,""1 Julia Tseng,* Thomas Horan,'l' Jie Wen,'l' John Philo,'l' David Chang,*
`Barry Ratzkin,:l: Robert Pacifici,:l: David Brankow,§ Sylvia Hu,§ Yi Luo,§ Duanzhi Wen,§
`Tsutomu Arakawa,'l' and Margery Nicolson*
`
`*Deparrment of Immunology, TDepartment of Protein Chemistry, iDepartment of Molecular Biology, and
`§Department of Mammalian Cell Molecular Biology, Amgen Inc., Amgen Center, Thousand Oaks, California 91320
`
`Received July 12, 1996
`
`A panel of mAbs were generated against the purified soluble form of erbB2/Her2 receptor, corresponding
`to the extracellular region of the receptor, and examined for their ability to mimic the receptor ligand.
`Some of the mAbs strongly induced tyrosine phosphorylation of 180-185 kDa proteins, including not only
`Her2 but also Her3 and Her4 receptors, when they were expressed on the surface of breast cancer cells.
`These mAbs do not cross-react with Her3 or Her4 as demonstrated by competition study. Receptor phosphor-
`ylation was also observed with the cell lines transfected with Her2 or a chimeric receptor consisting of the
`extracellular domain of Her2 and the transmembrane and cytoplasmic domains of epidermal growth factor
`receptor. Selected mAbs were tested for their ability to change cell morphology, and one specific mAb,
`mAb74, induced cell morphology changes and apoptosis.
`© 1996 Academic Press, Inc.
`
`There have been numerous studies showing that high expression of erbB2/I-Ier2 tyrosine
`kinase receptor correlates with poor prognosis in patients with breast cancer (1,2,3,4). Her2
`is a member of the epidermal growth factor (EGF) receptor subfamily, which includes EGF
`receptor, and Her3 and Her4 receptors (5,6). EGF, transforming growth factor-a, amphiregulin,
`heparin binding EGF and betacellulin are known as ligands of the EGF receptor. Neu differenti—
`ation factor (NDF) or heregulin and other structurally related ligands including p25 from MDP-
`activated macrophage conditioned media, NAF, p75 from SKBR3 conditioned media, NEL—
`GF, ARIA and GGF all have been shown to increase tyrosine phosphorylation of the Her2
`receptor and,
`therefore, were initially assumed to be ligands
`for
`the Her2 receptor
`(7,8,9,10,11,12,13,14). There is now convincing evidence that NDF neither binds directly to
`Her2 nor stimulates its kinase activity (15) but rather binds to Her3 or Her4 and stimulates
`tyrosine phosphorylation of these receptors (6,16,17).
`The conventional approach to circumvent the absence of ligand is to generate a ligand—like
`monoclonal antibody (mAb). In fact, several groups have generated anti-Her2 mAbs using
`cells expressing high levels of p185Her2 for immunizations (18,19,20,21). The p185Herz overex-
`pressing cells possibly coexpress p180Her3 and /or pISOHH“, and therefore the mAbs using those
`cells as an antigen may cross-react to p180Har3 and/or p180He'4. In addition, Her2 expressed on
`
`1 To whom correspondence should be addressed.
`Abbreviations used: EGF, epidermal growth factor; NDF, neu differentiation factor; MDP, muramyl dipeptide;
`NAF, neu protein-specific activating factor; NEL-GF, neu erbBZ ligand-growth factor; ARIA, acetylcholine receptor
`inducing activity; GGF, Glial growth factor; mAb, monoclonal antibody; CHO, Chinese hamster ovary; sHer2, soluble
`Her2 receptor; HEG, a chimeric receptor consisting of the extracellular domain of Her2 and the transmembrane and
`intracellular domains of EGF; EGFR, EGF receptor; PBS, Dulbecco’s phosphate-buffered saline; HRP, horseradish
`peroxidase; SDS, sodium dodecyl sulfate; SDS—PAGE, SDS-polyacrylamide gel electrophoresis.
`
`59
`
`0006-291X/96 $18.00
`Copyright © 1996 by Academic Press, Inc.
`All rights of reproduction in any form reserved.
`
`IMMUNOGEN 2034, pg. 1
`Phigenix v. Immunogen
`|PR2014-OO676
`
`IMMUNOGEN 2034, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Vol. 226, No. 1, 1996
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`cell surface may have limited accessibility of epitopes compared to its free molecule in solution.
`Although these mAbs induced increased tyrosine phosphorylation in Her2 overexpressing cells,
`they were not fully characterized in terms of the binding to each of Her2, Her3 or Her4 or in
`terms of the kinase activation in Her2 transfected cells in which no other members of EGFR
`
`family exist. Using Her2 specific mAbs, which were extensively characterized and will be
`described elsewhere, we show here their ability to induce kinase activation using various cell
`lines either overexpressing Her2, or cell lines transfected with Her2 or chimeric receptor
`(HEG or Her2-EGFR). We also show here one specific mAb that induces cell apoptosis and
`morphologic change.
`
`.
`
`-
`
`MATERIALS AND METHODS
`
`Cells. SKBR3 and MDAMB453 cells were grown in DMEM and RPM11640, respectively, supplemented with 10%
`heat-inactivated fetal bovine serum (FBS) and 2mM glutamine. Her2/CHO cells were prepared by co-transfection of
`dihydrofolate reductase-deficient CHO cells with two vectors: pJT2 carrying the genes coding for Her2 and dihydrofo-
`late reductase (dhfr) in pDRa2. Her2-transfected CHO cells were grown in selective medium without nucleosides
`(DMEM containing 5% dialyzed FBS, 2mM glutamine and 0.1mM non-essential amino acids). A hematopoietic cell
`line, 32D, was transfected with a chimeric receptor consisting of the Her2 extracellular ligand binding region and
`EGFR intracellular and transmembrane regions (designated HEG), or with a full length Her2. Construction of a
`chimeric receptor cDNA and gene transfection were carried out as described (22). HEG/32D, Her2/32D and 32D
`cells were grown in RPMI, supplemented with 10% heat inactivated FBS and 1 ng/ml lL-3. Her2/MCF7 were grown
`in MEMa containing 10% inactivated FBS, 0.1mM non-essential amino acids and lmM sodium pmvate.
`Assay of receptor tyrosine phosphorylation. Adherent cells (SKBR3 or MDAMB453) were grown in 48 well plates
`and washed with DMEM 2-3 times. Suspension cells (32D, Her2/32D or HEG/32D) were pelleted by centrifugation
`and washed with PBS. mAb solution or ligand solution was added to the well or to the pelleted tube and incubated
`for 5 min at 37°C. The solution was removed and the cells were solubilized with SDS sample buffer. The samples,
`with or without immunoprecipitation, were subjected to SDS-PAGE followed by Western blotting and probing with
`anti-phosphotyrosine antibody.
`Cell morphologic change. Cells were seeded in 5 cm dishes to about 20% confluency and mAbs added after 18 h.
`After 5 days, cells were observed with light microscopy, photographed, and counted.
`Cell apoptosis assay. Cells were seeded in 8-well Chamber Slides (Nunc) at 60-70% confluency and after 18 h,
`culture media was changed to 1% FBS-containing media with or without mAb. On day one, cells were fixed with
`4% neutral-buffered formalin (NBF) followed by 3 washes with PBS. After cells were dried, apoptosis assay was
`done using a modified TUNEL (terminal deoxynucleotidyl transferase, TdT, mediated dUTP-biotin nick end-labeling)
`method (23). TUNEL detects 3’-OH DNA ends generated by DNA fragmentation, after labeling digoxigenin-conj ugated
`dUTP with TdT followed by incubating with HRP-conjugated anti-digoxigenin. Bound HRP was detected with the
`substrate, 3—amino-9-ethylcarbazole (Sigma). Most of the reagents used were from Apop Tag in situ apoptosis detection
`kit (Oncor). HRP-conjugated antibodies were from Boehringer Mannheim.
`
`RESULTS AND DISCUSSION
`
`Twelve clones of anti—sHer2 mAbs were tested for stimulation of receptor tyrosine phosphor-
`ylation in SKBR3 cells. As shown in Figure la, mAb74, 52, 58 and 83 at 250 nM strongly
`stimulated the tyrosine phosphorylation of 180-185 kDa proteins in SKBR3 cells in which
`both Her2 and Her3 were identified. Stimulation was much weaker for mAb42b, 86, 80 and
`73, and not much different from the basal level.
`
`Dose dependence of stimulation was examined for mAb74 and 83, as shown in Figure lb.
`As the concentration of mAb74 was increased from 10 nM to 250 nM, the phosphorylation
`increased dose—dependently, approaching a level observed with 2 nM NDFa2. mAb83 also
`exhibited dose—dependent increase in tyrosine phosphorylation, but to a much smaller extent
`than the level with mAb74 when compared at the same concentration.
`Next, specificity of Her2 stimulation by the mAbs was tested by competition experiments.
`SKBR3 cells were incubated with 250 nM mAb83, 74 and 50 in the presence of increasing
`concentration of sHer2. As shown in Fig. 2-a it is evident that tyrosine phosphorylation was
`dose-dependently inhibited by sHer2. As expected, 10 nM sHer2 exhibited little inhibition
`
`60
`
`IMMUNOGEN 2034, pg. 2
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2034, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Vol. 226, No. 1, 1996
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`A
`
`SKBR3
`
`
`
`Western
`Blot
`
`B
`
`PTY
`
`SKBR3
`
`mAb 74
`
`mAb 83
`
`l l
`
`NDFa2,2nM
`
`10nM
`
`
`
`
`
`
`
`
`
`
`
`DMEMcontrol
`
`DMEMcontrol
`
`25nM
`
`50nM
`
`100nM
`
`250nM
`
`25nM
`
`50nM
`
`100nM
`
`250nM
`
`
`
`
`
`p185»
`
`Western
`Blot
`
`FIG. 1. Her2 and Her3 tyrosine phosphorylation induced by mAb stimulation in SKBR3. SKBR3 cells were seeded
`in a 48-well plate for 5 min at 37°C for 18 h before mAb stimulation. Cells were solubilized with SDS sample buffer.
`Solubilized samples were electrophoresed on 6% polyacrylamide gels, followed by Western blotting and probing with
`anti-phosphotyrosine. (a) All mAb concentrations were 250 nM in DMEM and 2 nM NDFa were used as a positive
`control. (b) mAb dose dependence of tyrosine phosphorylation.
`
`while 1.3 or 2.5 pM sHerZ showed a nearly complete suppression of stimulation by these
`mAbs. Competition with sHer3 was tested with SKBR3 stimulated by mAb52. sHer3 concentra-
`tion was varied from 1.5 nM to 1.6 pM in the presence of 250 nM mAb52. As shown in
`Figure 2-b, the phosphorylation was not inhibited with sHer3 even at the highest concentration.
`Although these mAbs are highly specific for Her2, both Her2 and Her3 in SKBR3 cells and
`
`61
`
`IMMUNOGEN 2034, P9. 3
`Phigenix v. Immunogen
`|PR2014-00676
`
`
`
`IMMUNOGEN 2034, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Vol. 226, No. 1, 1996
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`A
`
`SKBFI3
`
`(D
`L
`‘—
`"
`(D
`L
`L
`‘—
`L
`E‘.‘ N
`<_\_I
`N N 9‘. N
`N N (E N
`I. %
`£7
`if
`i‘.’
`3:
`a?
`£3
`if f e
`Competedby wwwd,_>_wwgu',_>wgaa_>
`E E 5 2 c
`9 E 2 E E
`2 E 2
`SolubIeHerz
`c
`c
`:x.
`:L
`E
`0
`~
`:1.
`:1. o C
`o
`:L o
`c
`o
`l". m 8 5 D
`C
`‘0
`(q
`to o
`.D
`m. m o
`.o
`N s- N s- <
`8
`(\i
`v— N v— <
`v— N v— <
`+
`+
`+
`+
`E
`E
`+
`+
`+
`+
`E
`+
`+
`+
`E
`mAb as
`E
`mAb 74
`mAb 52
`250nM
`D
`250nM
`250nM
`l—_—I
`r———l r—l
`
`'.5-.'II
`
`|____—____—__,
`PTY
`
`Western
`Blot
`
`B
`
`Competed
`bySolubleHera
`
`SKBRS
`
`E Q 9 Q Q Q Q Q
`f if
`j? in f f f g
`u', w w u';
`0')
`ch
`d,
`(b
`21 E E g g g E E E
`‘9. 3 B an
`an
`no
`'1
`‘0.
`.o
`‘-
`(‘3
`1- " (O
`1-
`00
`1— <
`+
`+
`+
`+
`+
`+
`+
`+
`E §
`mAb52
`B
`250nM
`Z
`
`:3
`8
`O
`2
`§
`D
`
`
`
`l_____—__|
`PTY
`
`Western
`Blot
`
`FIG. 2. Inhibition by soluble receptor of receptor tyrosine phosphorylation induced by mAb. Phosphorylation assay
`is similar to that described in Figure 1. Cells were incubated with 250 nM mAb with different concentrations of sHer2
`(a) or sHer3 (b).
`
`62
`
`'
`
`l
`
`
`
`IMMUNOGEN 2034, P9. 4
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2034, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Vol. 226, No. 1, 1996
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`A
`
`Her2/32D
`HEG/32D
`l l
`
`HE
`gamem
`5
`“EWH”
`53222 n.
`EEEEE u:
`[w
`
`E
`ii
`2
`3
`2
`
`9?
`BE
`mvNB
`35
`“2532225
`5311<<<3
`JEEmEEEE
`[—1
`
`B
`
`SKOV3
`
`123
`
`h-I.‘
`
`«p132
`
`I
`
`i
`
`I
`
`i
`
`‘3. a:m
`
`”H..
`
`
`PTY
`
`Western
`Blot
`
`.—
`
`FIG. 3. Receptor tyrosine phosphorylation of cell lines. (a) Transfected cell lines Her2/32D and HEG/32D, induced
`by mAb stimulation. For phosphorylation assay, cells were pelleted by centrifugation, washed with PBS, and then
`incubated with 100 pl of 250 nM mAbs in RPMI for 5 min at 37°C, followed by quenching with the addition of 1
`ml ice cold PBS and centrifugation at 4°C. Supernatant was removed and SDS sample buffer added to the centrifuged
`pellet. The sample was subjected to 6% SDS-PAGE followed by Western blotting and probing with anti—phosphotyro—
`sine antibody. A431 basal phosphorylated sample was used as a positive control. (b) Wild type cells, SKOV3. Phosphory-
`lation assay is similar to that described in Figure 1. Higher MW bands are p180. Lane 1, 2nM NDFa; Lane 2, DMEM
`control; Lane 3, 250nM mAb74.
`
`all Her2, Her3 and Her4 in MDAMB453 cells were found to be phosphorylated when the cells
`were stimulated with mAb52 and irnmunoprecipitated with specific antibodies (data not shown).
`A similar assay was done with transfected cell lines, Her2/CHO and Her2/32D to study direct
`interaction of the mAbs and cell-surface Her2. All the mAbs tested, mAb83, 74 and 52 failed at
`
`250 nM to phosphorylate Her2 in the transfected cells, Her’Z/CHO (data not shown) and Her2/
`32D (Figure 3-a). As the transfection may have caused receptor inactivation and changed the
`interaction between mAb and Her2, we prepared a cell line, HEG/32D, transfected with a chimeric
`receptor (HEG), whose extracellular domain comes from Her2 and intracellular and transmembrane
`domains come from EGF receptor. As shown in Figure 3-a, when HEG/32D cells were stimulated
`with the same mAbs, HEG is phosphorylated over basal level. mAb74 showed the strongest
`activity among the three tested. These results show that Her2 kinase does not phosphorylate itself
`whereas EGFR kinase could autophosphorylate HEG upon mAb incubation under the experimental
`conditions used. However, when the expression of H612 was increased in 32D, it was phosphory-
`lated in the presence of mAb (data not shown). We also examined tyrosine phosphorylation in
`SKOV3 cells. SKOV3 cells that naturally overexpress Her2 were not phosphorylated by NDF/
`hereguljn (24) but were phosphorylated by mAb74 as shown in Fig. 3-b. These results strongly
`suggest that the Her2 kinase was activated by homodirnerization, and Her3/or Her4 kinase were
`not required for Her2 kinase activation.
`Cell morphology change by mAb. Her2/ MCF7 cells were incubated with 250 nM mAb42b,
`mAb83 and mAb74. After 5 days incubation, mAb74 caused extensive cell death and a
`dramatic cell morphology change as shown in Figure 4-a,b,c,d. mAb83 caused a moderate
`cell morphology change and 42b resulted in little change. The viable cell number 5 days after
`mAb74 incubation was only 36% of the control grown without mAb incubation. mAb74 also
`induced cell morphology change in MDAMB453 (Fig. 4-e,f).
`
`63
`
`IMMUNOGEN 2034, pg. 5
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2034, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Vol. 226, N0. 1, 1996
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`
`
`IMMUNOGEN 2034, P9. 6
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2034, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Vol. 226, No. 1, 1996
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`
`
`FIG. 4. Cell morphologic change induced by mAbs. Cells (a—d, Her2/MCF7; e,f, MDAMB453) were grown in 1%
`FBS in culture media with or without mAb. After 5 days, cells were observed and photographed. (a,e) control (without
`mAb). (b) 250 nM mAb74. 65. 250 nM mAb83. (d) 250 nM mAb42b. (I) 100 nM mAb74.
`
`65
`
`IMMUNOGEN 2034, P9. 7
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2034, pg. 7
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`- .
`
`i
`
`J.
`
`-.
`
`.“j'.
`1.
`.
`
`a}
`
`L?
`
`66
`
`IMMUNOGEN 2034, pg. 8
`Phigenix v. Immunogen
`|PR2014-OO676
`
`IMMUNOGEN 2034, pg. 8
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Vol. 226, No. 1, 1996
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`-1,- wwrw '
`-i'
`I
`1‘ ._.-ml"
`
`I
`
`.
`
`I"
`
`~
`
`-
`
`-
`
`1.-
`
`
`
`
`
`-
`
`
`
`FIG. 5. Detection of apoptotic cells with a modified TUNEL method. MDAMB453 (a-d) cells or Her2/MCF7 (6,1)
`cells were incubated with or without mAbs in 1% FBS culture media for one day followed by an apoptosis assay.
`(a,e) control (without mAb). (b) 50 nM mAb74. (c,t) 500 nM mAb74. (d) 500 nM mAb42b.
`67
`
`IMMUNOGEN 2034, P9. 9
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2034, pg. 9
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Vol. 226, No. 1, 1996
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`Apoptosis with mAb74. We have found that mAb74 has the strongest effect on receptor
`tyrosine phosphorylation (Fig. 1a), cell morphology change (Fig. 4) and cell death. To clarify
`the mechanism of the cell death caused by mAb74, we examined apoptosis by a modified
`TUNEL method (terminal deoxynucleotidyl transferase mediated dUTP—biotin nick end-label-
`ing). Apoptosis is characterized by a nuclear collapse accompanied by a fragmentation of the
`cellular chromatin to single or multiple mononucleosomal units which is mediated by an
`endogenous endonuclease. TUNEL detects DNA strand breaks in cells undergoing apoptosis
`since the nuclei of apoptotic cells incorporate exogenous nucleotides (dUTP) in the presence
`of TdT. The UTP—containing fragmented DNAs are detected by a staining procedure. As shown
`in Figure 5, cells incubated with mAb74 for 1 day showed apoptosis as detected by red color,
`while incubation with mAb52 and mAb42b were barely apoptotic in MDAMB453 (Fig. 5a-
`d) and Her2/MCF7(Fig. 5e,f). The number of apoptotic cells induced by 50 nM mAb74 was
`significantly less than that induced by 500 nM mAb74 (~10% that at 500 nM), indicating
`that the apoptosis is mAb74 dose dependent. On day 5, we could not detect staining (data not
`shown), suggesting that all apoptotic dead cells were detached and live cells were not undergo-
`ing an apoptotic process.
`
`CONCLUSION
`
`We have explicitly shown that the mAbs, generated here against the purified soluble Her2
`corresponding to the extracellular region of the receptor, are highly specific for Her2, do not
`cross-react with the purified soluble Her3 or Her4. We observed phosphorylation of 180-185
`kDa proteins corresponding to Her2, Her3 and Her4 receptors by these mAbs, as has been
`observed by other investigators (25). While Tagliabue et al., 1991 and others (26,27,28,19,29)
`showed growth inhibitory effects on breast cancer cells, one of the mAbs in our study exhibited
`a novel apoptotic activity against breast cancer cells and induced cell-morphology changes.
`An anti-EGF receptor mAb also exhibited an apoptotic activity on the human colorectal
`carcinoma cell line, DiFi, which overexpresses EGF receptor, and induced morphological
`changes at concentrations of 5 to 20 nM (30). These effects were interpreted in terms of both
`blockage of EGF binding to the cognate receptor by the competing mAb and lack of the mAb
`mitogenic activity. Whether the observed apoptosis by the anti-Her2 mAbs is due to competition
`with the unknown Her2 ligand or direct effects on the cells is not clear. However, difficulty
`in identifying the source of Her2 ligand suggests the latter possibility as being more likely.
`Apoptosis, or programmed cell death, is a form of cell death and characterized by DNA
`fragmentation and cell shrinkage rather than the swelling seen in necrotic cell death, which
`causes release of intracellular components (31,32,33). Apoptotic cells, without releasing such
`components, are phagocytosed and hence degraded (34). Therefore, development of agents with
`the ability to induce apoptosis in tumor cells is promising and under extensive investigation.
`
`REFERENCES
`
`l. Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. B., Levin, W. J., Stuart, S. 0.,
`Udove, J., Ullrich, A., and Press, M. F. (1989) Science 244, 707—712.
`2. Wright, G, Angus, B., Nicholson, 5., Sainsbury, J. R., Cairns, J., Gullick, W. J., Kelly, R, Harris, A. L., and
`Home, C. H. (1989) Cancer Res. 49, 2087—2090.
`3. Gullick, W. J., Love, S. B., Wright, G, Barnes, D., Gusterson, B., Harris, A. L., and Altman, D. G. (1991) Br.
`J. Cancer 63, 434—438.
`4. Hartmann, L. C., Ingle, J. N., Wold, L. B., Farr, G. H. In, Grill, J. P., Su, J. Q., Maihle, N. J., Krook, J. EW.,
`Witzig, T. E., and Roche, P. C. (1994) Cancer 74, 2956—2963.
`5. Kraus, M. H., Issing, M., Popescu, N. C., and Aaronson, S. A. (1989) Proc. Natl. Acad. Sci. USA 86, 9193—
`9197.
`6. Plowman, G. D., Green, J. M., Culouscou, J. M., Carlton, G. W., Rothwell, V. M., and Buckley, S. (1993) Nature
`366, 473—475.
`
`a
`
`68
`
`IMMUNOGEN 2034. pg. 10
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2034, pg. 10
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Vol. 226, No. 1, 1996
`
`BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
`
`7. Peles, E., Bacus, S. S., Koski, R. A., Lu, H. S., Wen, D., Ogden, S. G., Ben-Levy, R., and Yarden, Y. (1992)
`Cell 69, 205—216.
`8. Wen, D., Peles, E., Cupples, R., Suggs, S. V., Bacus, S. 5., Lou, Y., Trail, G., Hu, S., Silbiger, S. M., Ben-Levy,
`R., Luo, Y., and Yarden, Y. (1992) Cell 69, 559—572.
`9. Holmes, W. E., Sliwkowski, M. X., Akita, R. W., Henzel, W. J., Lee, J., Park, J. W., Yansura, D., Ababi, N.,
`Raab, H., Lewis, G. D., Shepard, M., Wood, W. I., Goeddel, D. V., and Vandlen, R. L. (1992) Science 256,
`1205—1210.
`10. Tarakhovski, A., Zaichuk, T., Prassolov, V., and Butenko, Z. A. (1991) Oncogene 6, 2187—2196.
`11. Dobashi, K., Davis, J. G., Mikami, Y., Freeman, J. K., Hamuro, J., and Green, M. I. (1991) Proc. Natl. Acad.
`Sci. USA 88, 8582—8586.
`12. Huang, S. S., and Huang, J. S. (1992) J. Biol. Chem. 267, 11508—11512.
`13. Falls, D. L., Rosen, K. M., Corfas, G., Lane, W. S., and Fishbach, G. D. (1993) Cell 72, 801—815.
`14. Marchionni, M. A., Goodearl, A. D. J., Chen, M. S., Bermingham-McDonough, 0., Kirk, C., Hendricks, M.,
`Denehy, F., Misumi, D., Sudhaiter, J., Kobayashi, K., Wroblewski, D., Lynch, C., Baidassare, M., Hiles, 1., Davis,
`J. B., Hsuan, J. J., Totty, W. F., Otsa, M., McBury, R. N., Waterfield, M. D., Stroobant, P., and Gwynne, D.
`(1993) Nature 362, 312—318.
`15. Tzahar, E., Leukowitz, G., Karunagaran, D., Yi, L., Peles, E., Lavi, S., Chang, D., Liu, N., Yayon, A., Wen, P.,
`and Yarden, Y. (1994) J. Biol. Chem. 269, 25226—25233.
`16. Kita, Y., Barff, J., Luo, Y., Wen, D., Brankow, D., Hu, 5., Liu, N., Prigent, S. A., Gullick, W. J., and Nicolson,
`M. (1994) FEBS Lett. 349, 139—143.
`17. Carraway 111, K. L., Sliwkowski, M. K., Akita, R., Platko, J. V., Guy, P. M., Nuijins, A., Diamonti, A. J., Vandlen,
`R. L., Canfley, L. C., and Cerione, R. A. (1994) J. Biol. Chem. 269, 14303—14306.
`18. Yarden, Y. (1990) Proc. Natl. Acad. Sci. USA 87, 2569—2573.
`19. Harwerth, I.-M., Wek, W., Schlegel, J., Muller, M., and Hynes, N. E. (1993) Br. J. Cancer 68, 1140—1145.
`20. Srinivas, U., Tagliabue, E., Campiglio, M., Menard, S., and Colnaghi, M. I. (1993) Cancer Immunol. Immunother.
`36, 397-402.
`21. Stancovaski, J., Hurwitz, E., Leitner, 0., Ullrich, A., Yarden, Y., and Sela, M. (1991) Proc. Natl. Acad. Sci. USA
`88, 8691—8695.
`22. Pacifici, R. E., and Thomason, A. R. (1994) J. Biol. Chem. 269, 1571—1574. 12, 961—971.
`23. Surh, C. D., and Sprent J. (1994) Nature 372, 100—103.
`24. Peles, E., Ben-Levy R., Tzahar D., Liu N., Wen D., and Yarden, Y. (1993) The EMBO J.
`25. Tagliabue, E., Gentis, F., Campiglio, M., Mastroianni, A., Martignone, 5., Pellegrini, R., Casalini, P., Lanzi, C.,
`Ménard, S., and Colnagli, M. I. (1991) Int. J. Cancer 47, 933—937.
`26. Hudziak, R. M., Lewis, G. D., Winget, M., Fendly, B. M., Shepard, H. M., and Ullrich, A. (1989) Mol. Cell
`Biol. 9, 1165—1172.
`27. Drebin, J. A., Link, V. C., and Green, M. I. (1988) Oncogene 2, 387—394.
`28. Fendly, B. M., Winget, M., Hudziak, R. M., Lapari, M. T., Napier, M. A., and Hudziak, R. M., Lewis, G. D.,
`Winget, M., Fndly, B. M., Shepard, H. M., and U11ich, A. (1989) Mol. Cell Biol. 9, 1165—1172.
`29. Vitetta, E. S., and Uhr, J. W. (1994) Cancer Res. 54, 5301—5309.
`30. Wu, X., Fan, Z., Masui, H., Rosen, N., and Mendelsohn, J. (1995) J. Clin. Invest. 95, 1897—1905.
`31. Kerr, J. F. R., Wyllie, A. H., and Currie, A. R. (1972) Br. J. Cancer 26, 239—257.
`32. Wyllie, A. H. (1980) Nature 284, 555—556.
`33. Wyllie, A. H., Kerr, J. F. R., and Currie, A. R. (1980) Int. Rev. Cytol. 68, 251—306.
`34. Savill, J. S., Drawfield, I., Hogg, N., and Haslett, C. (1990) Nature 343, 170—173.
`
`69
`
`IMMUNOGEN 2034, pg. 11
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2034, pg. 11
`Phigenix v. Immunogen
`IPR2014-00676
`
`