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`PHIGENIX
`PHIGENIX
`Exhibit 1006
`Exhibit 1006
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`© The Macmillan Press Ltd 1989
`Oncogene (1989) 4 543-548
`
`
`Generation and characterization of monocllonall antibodies specific for the
`human new oncogene product, [[1185
`
`Sara J. McKenzie, Paula J. Marks, Terence Lam‘, Jon Morganl, Dennis L. Panicali
`Trimpe2 & Walter P. Carney2
`
`‘, Kevin L.
`
`lApplied bioTechnology, Cambridge, Massachusetts 02142. 2E]. DuPont de Nemours & Co., No Billerica, Massachusetts 01862,
`USA
`
`A series of monoclonal antibodies specific for the extra-
`cellular domain of the human neu gene product (p185)
`have been produced. The generation of these monoclonal
`antibodies, and their biochemical and immunological
`characterization is described. The immunization protocol
`utilized a series of injections of Nllll-ll3'll‘3 celis, cyclophos-
`phamide, and a neu transfected NlIlHl3T3 cell
`line
`(designated 18-3-7) which expressed the full
`length
`human neu-encoded protein. This immunization regimen
`induced an immune response to the extracellular portion
`of p185 on the 18-3-7 cells. A panel of ten hybridomas
`were identified which secreted monoclonal antibodies with
`a variety of epitope specificities, and reacted with p185'In
`a number of different experimental formats. As the neu
`gene product has been associated with human breast
`cancers, a series of monoclonal antibodies such as these
`could prove useful
`in the diagnosis, prognosis and/or
`treatment of these human malignancies.
`
`
`Introduction
`
`The neu oncogene (HER--2 or c--erbB-2), one ofthe erbB-
`like oncogenes, was first describedIn association with a
`neuroblastoma after treatment of rats with the chemical
`carcinogen ethylnitrosourea (Schechter et al., 1984). The
`human homolog of the rat neu oncogene has been iso-
`lated (Schechter et al., 1985; Yamamoto et al., 1986;
`Mazzara & Morgan, unpublished), and evidence has
`been presented which suggests that amplification of‘the
`neu oncogene may be associated with a poor short-term
`prognosis for women diagnosed with breast cancer
`(Cline et al., 1987; Slamon et al., 1987; van de Vivjer et
`al., 1987; Varley et al., 1987; Venter et al., 1987; Zhou et
`al., 1987; Berger et al., 1988).
`The human neu oncogene encodes a glycoprotein of
`185000kd (p185) which is closely related to, but distinct
`from, human epidermal growth factor receptor (EGFR).
`Both proteins have a cysteine-rich extracellular domain,
`a transmembrane domain, and an intracellular tyrosine
`kinase (TK) domain. Although the two genes are also
`closely related in sequence, EGFR is only 170000 Rd in
`size; furthermore, EGF does not bind to the neu gene
`product. A ligand for neu has not yet been described.
`Polyclonal and monoclonal antibodies which recog-
`nize the protein product of the rat and human neu
`oncogenes have been used to determine the presence or
`absence‘of the oncogene product in different cell types
`(Drebin et al., 1984; Gullick et al., 1987; Kraus et al.,
`
`
`Correspondence: Sara J. McKenzie
`Received IS November 1988; accepted 2 February I989
`
`1987; Venter et al., 1987; Berger et al., 1988) and to
`study the potential anti-tumor effects of antibodies both
`in vitro and in vivo (Drebin et al., 1985,1986, 1986a and
`1988b). These antibodies have been generated by immu-
`nization with peptides representing small portions of
`the intact protein, or with tumor cells which express the
`rat neu oncogene.
`We have generated a series of monoclonal antibodies
`(mAbs) which are specific for the extracellular domain
`of the human neu oncogene product. Mice were immu-
`nized using a protocol which consisted of adminis-
`tration of an NlH3T3 cell line, cyclophosphamide, and
`a transfected NIH3T3 cell line which expressed the full
`length human neu gene product (differential hybridoma
`technique, William Matthew, Harvard University, per-
`sonal communication). Using this protocol we were suc-
`cessful in producing monoclonal antibodies of different
`isotypes with a variety of epitope specifieites, all of
`which recognize the extracellular domain of p185. The
`generation of these monoclonal antibodies, their bio-
`chemical characterization and their
`immunological
`reactivities are described.
`
`Results
`
`A number of cell lines were produced by the successful
`transfection of NIH3T3 cells with a retroviral vector
`
`containing a cDNA copy of the human neu gene
`(Mazzara & Morgan, unpublished results). A cell line,
`designated l8-3-7, that was determined to express the
`highest relative level of neu mRNA was used as the
`immunogen for the production of monoclonal anti-
`bodies.
`
`Ten hybridomas were isolated following two PEG
`fusions of spleen cells obtained from two experiments
`using 18-3-7 cells as immunogen,
`in concert with the
`administration of NIH3T3 cells and cyclophosphamide.
`It was hoped that
`this immunization regimen would
`preferentially elicit antibodies specific for the extracellu-
`lar portion of pl85. Hybridomas secreting; anti-p185
`antibodies were initially identified by ELISA on lysates
`of 18-3-7 cells, and were then cloned twice by limiting
`dilution. The isotype and subclass of the resulting
`monoclonal antibodies are shown in Table i. As can be
`seen, the majority of the mAbs are of the IgGl isotype,
`although two are IgM, and one is an IgGZ, antibody.
`Table 1 also summarizes the reactivities of the ten
`mAbs with the neu protein product, p185, in a variety of
`assay formats. These assays include Western blot,
`immunoprecipitation,
`immunofiuorescence, and flow
`cytometry. All mAbs
`react with native antigen, as
`demonstrated in the immunoprecipitation (IP) experi-
`
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`544 SJ. MCKENZIE et al.
`
`Table 1 Characteristics of anti-p185 mAbs
`__________________———————-
`Reactivity‘
`Isotype ———-————-————’—_
`and
`Western
`Flow
`Fluorescencef
`IP"
`mAl:
`subclass
`blot
`cytometry
`_________.______—-————-——
`
`+
`+
`+
`—
`lgG l/tc
`BDS
`+
`+
`+
`—
`lgG ,/x
`RC1
`+
`+
`+
`—
`lgG l/i<
`TAl
`ND
`ND:
`+
`——
`IgM/x
`NA3
`+
`ND
`+
`+
`lgM/K
`OD3
`+
`ND
`+
`+
`lgGin).
`PB?
`ND
`ND
`+
`—-
`lgG l/tc
`RC6
`+
`ND
`+
`—
`lgG , /x
`N83
`+
`ND
`+
`—
`lgG l/tc
`IDS
`133
`lgG l/x
`—
`+
`ND
`ND
`___________________________________________._______________
`
`‘ All assays were performed as described in the text
`” 1P — immunoprecipitation
`1' Fluorescence — immunofluorescence
`1 ND - not done
`
`ments. However, only two of the mAbs, OD3 and PB3,
`are capable of reacting with the denatured form of the
`p185 molecule as it
`is presented in Western blot
`analysis. Three mAbs tested, TA1, RC1, and EDS,
`yielded positive immunofluorescence on fixed, non-
`permeabilized cells which are known to express neu.
`Furthermore, seven mAbs (BDS, RC1, TAI, OD3, PB3,
`NB3 and IDS) have been examined by flow cytometry,
`and all stain viable cells which are known to produce
`p185. Finally, competitive binding assays done by
`ELISA suggest that all of the mAbs recognize difierent
`epitopcs of the p185 molecule, as there is no com-
`petition for binding to p185 between any of the anti-
`bodies (data not: shown). Monoclonals which exhibited
`the highest relative binding affinity for p185, namely
`TAl, BD5, PB3, NB3, OD3 and NA3, were examined
`further to verify that the binding was specific for the neu
`protein product.
`
`
`
`lmmunoprecipitation of radioactively labeled cells with
`Figure 1
`anti—p185 monoclonals. All immunoprecipitations were performed
`using 3 pCi of [”Skysteine labeled lysates of various cell lines. In
`each case, 5 pg of purified antibody was used. along with 20 pg of
`either rabbit anti-mouse lgG or rabbit anti—mouse lgM. Panel (a)
`immunoprecipitation of 18-3-7 and NIH3T3 cell lysates with the
`lgG monoclonal antibodies. Lane 1 contains molecular weight
`standards. Lanes 2, 4. 6. 8 and 10 contain 18-3-7 lysates and lanes
`3, 5. 7, 9 and ll contain NIH3T3 lysates. Lanes 2 and 3: precipi-
`tation with TAI. Lanes 4 and 5: precipitation with BDS. Lanes 6
`and 7: precipitation with NB3. Lanes 8 and 9: precipitation with
`P133. Lanes 10 and 11: precipitation with MOPC-21. Panel (b)
`immunoprecipitation of 18-3-7 and NIH3T3 cell lysates with the
`lgM monoclonals. Lane 1 contains molecular weight standards.
`Lanes 2, 4 and 6 contain 18-3-7 lysates; lanes 3, 5 and 7 contain
`NIH3T3 lysates. Lanes 2 and 3: precipitation with OD3. Lanes 4
`and 5: precipitation with NA3. Lanes 6 and 7: precipitation with
`TEPC 183. Panel (c) immunoprecipitation of SKBR-3 and A-43l
`cell
`lysates with lgG monoclonals. Lane 1 contains molecular
`weight standards. Lanes 2, 4, 6, 8, 10 and 12 contain SKBR-3
`lysates. Lanes 3, 5. 7. 9. 11 and 13 contain A-431 lysates. Lanes 2
`and 3: precipitation with TAl. Lanes 4 and 5: precipitation with
`BDS. Lanes 6 and 7: precipitation with P83. Lanes 8 and 9: pre-
`cipitation with N83. Lanes 10 and 11: precipitation with MOPC
`21. Lanes 12 and 13: precipitation with rabbit anti-EGFR. Panel
`((1) immunoprecipitation ol' SKBRJ and A-431 cell lysates with
`IgM monoclonals. Lane 1 contains molecular weight standards.
`Lanes 2, 4, 6 and 8 contain SKBR-3 lysates. Lanes 3. 5, 7 and 9
`contain A431 lysates. Lanes 2 and 3: precipitation with OD3.
`Lanes 4 and 5: precipitation with NA3. Lanes 6 and 7: precipi-
`tation with TEPC l83. Lanes 8 and 9: precipitation with rabbit
`anti-EGFR
`
`[35$]cysteine
`immunoprecipitation of
`Results of
`labeled 18-3-7 cells and NIH3T3 cells are shown in
`Figures la and 1b. All mAbs precipitate a major band
`of 18S kd from the 18-3-7 lysates (sec lanes 2, 4, 6 and 8
`in Figure 1a and lanes 2 and 4 in Figure 1b). There is
`no reaction with a protein of that size from NIH3T3
`cells (lanes 3, 5, 7 and 9 in Figure 1a and lanes 3 and 5
`in Figure 1b). Furthermore, precipitation with isotype-
`matched control mouse myeloma proteins MOPC 21
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`MONOCLONAL ANTIBODIES SPECIFIC FOR HUMAN NEU 565
`
`matched mouse myeloma proteins MOPC 21 (Figure
`1c, lanes 10 and 11) and TEPC 183 (Figure 1d, lanes 6
`and 7) do not react with proteins in the 170 to 185 kd
`region.
`also demonstrated in
`for p185 was
`Specificity
`Western blot analysis by testing the reactivities of OD3
`and PB3 on lysates of SKBR-3 and A431 cells (Figure
`2). Proteins in lysates of both cell types were separated
`by SDS-PAGE, transferred to nitrocellulose, and then
`incubated with OD3 (Figure 2a), PB3 (Figure 2b), or
`with an anti-EGFR monoclonal antibody 291~3A
`(Figure 2c; a gift from Randall Schatzmann, Syntex
`Research, Palo Alto, CA). As can be seen in panels a
`and b, the mAbs OD3 and PB3 react with the p185
`molecule (lane 1) but do not cross-react with the human
`EGFR expressed by the A-431 cells (lane 2). Conversely,
`as shown in panel c, the anti-EGFR antibody recog-
`nizes the 170 kd protein expressed in the A-43l cells, as
`well as the p185 expressed in the SKBR-3 cells. This can
`be attributed to thedifference in the epitope specificities
`of the anti-p185 mAbs and the anti-EGFR mAb: the
`anti-p185 monoclonals were elicited with an antigen in
`which only the external domain of p185 was exposed,
`whereas 291—3A was developed using a peptide derived
`from the TK domain of EGFR.
`
`immunofluorescence was performed using
`Indirect
`TAl ascites fluid on 18-3-7 and NIH3T3 cells, and on
`SKBR-3 and A-43l cells. All cells were fixed with for-
`malin, but were notpermeabilized, before reaction with
`the antibody. Results are shown in Figure 3. Positive
`fluorescent staining is observed on 18-3-7 and SKBR-3
`cells (Figures 3a and 3c), and is absent
`from the
`NIH3T3 and A-43l cells (Figures 3b and 3d). These
`results indicate that TAI recognizes; the extracellular
`domain of p185 on the neu—expressing cell' lines, and
`that it does not bind to EGFR on the A431 cell line.
`Although the A-431 cells do express a 1x level of the
`human neu gene, this asay is apparently not sensitive
`enough to detect this level of protein expression. Other
`unrelated ascites fluid gave no staining of any of the
`cells (data not shown).
`Various transformed murine cell lines were examined
`by flow cytometry, also using the mAb TAl. The cell
`lines used included l8'~3-7, and two cell
`lines co-
`transfected with the neu and ras oncogenes, designated
`17-7-8 and X-3-5 (Morgan, unpublished results). An
`NIH3T3 cell
`line transfected with the ras oncogene
`alone (3T3/ras) was used as a control. Results are
`shown in Figure 4. These results indicate that greater
`than 95% of the viable ll8-3-7 and X-3-5 cells and 75%
`of the viable 17-7-8 cells were positively stained with
`TAl. Fewer than 5% of the 3T3/ras control cells were
`positive. The p185-positive cell
`lines demonstrated a
`mean-fluorescence intensity which was approximately
`10-fold greater than background. These observations
`support the other studies which suggest that TAl recog-
`nizes an epitope on p185 which resides on the extra-
`cellular domain of the molecule.
`
`As a final proof of antibody specificity for the extra-
`cellular portion of p185, all monoclonals were tested in
`a number of assay formats on vaccinia recombinants
`which expressed the human neu gene. These recombin-
`ants were made using a neu gene in which the entire
`intracytoplasmic domain of p185 was deleted, leaving
`only the transmembrane domain and the extracellular
`portion of the molecule. All ten antibodies which were
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`lines using
`Figure2 Western blot of SKBR-3 and A-43l cell
`OD3, PB3 and 29l-3A. In all cases,
`lane 1 contains 80ug of
`SKBR-3 cell
`lysate, and lane 2 contains Song of A—43l
`lysate,
`separated by SDS—PAGE on a 7% SDS-polyacrylamide gel, and
`electrophoretically transferred to nitrocellulose. Panel (a) detection
`with 0.5 ug ml'l purified OD3; panel (b) detection with Zug ml"l
`purified PB3; panel (c) detection with ZOugml'l 29l-3A
`
`(Figure la, lanes 10 and 11) and TEPC 183 (Figure lb,
`lanes 6 and 7) showed no reactivity with a 185 kd mol-
`ecule in either l8-3~7 or NIH3T3 lysates. These results
`indicate that the binding observed when the anti-p185
`mAbs were used for
`the immunoprecipitation was
`indeed specific for the p185 antigen.
`on
`done
`also
`Immunoprecipitations
`were
`[358]cysteine labeled SKBR-3 and A-431 cells (Figures
`lo and 1d). SKBR-3, a human cell
`line derived from
`breast adenocarcinoma, SKBR-3, was used as a source
`of human neu, as it was previously shown to express
`high levels of p185 (Kraus er al., 1987). The cell
`line
`A-431, which is derived from a human epidermoid
`carcinoma, and has been demonstrated to overproduce
`EGFR (Gullick et al., 1987), was used as a source of this
`molecule. Precipitation with the anti-p185 monoclonals
`results in a major band of 185 kd in the SKBR-3 immu-
`noprecipitations (Figure 1c,
`lanes 2, 4, 6 and 8 and
`Figure 1d, lanes 2 and 4). There is a second minor band
`present at approximately 150 kd in the same immuno-
`precipitations. This reactivity is attributable to either a
`processed product of the p185 molecule, or possibly to
`non-specific binding of a cellular protein to the Sep-
`harose matrix. It is not likely that it is the EGFR mol—
`ecule, as there is a notable absence of reactivity at
`170 kd in the A-431 cells (Figure 1c, lanes 3, 5, 7 and 9
`and Figure 1d, lanes 3 and 5). A polyclonal rabbit anti-
`serum which reacts with the tyrosine kinase domain of
`human EGFR was used in these experiments to indicate
`the migration of both p185 and EGFR (a gift of Stuart
`Decker, Rockefeller University, New York). This anti-
`serum precipitates a band at 185 kd from SKBR-3 cells
`(lanes 12 and 8, Figures 1c and 1d respectively) and a
`major band of 170 kd from A-431 cells (lanes 13 and 9,
`Figures 1c and 1d respectively). The anti-EGFR anti-
`serum also appears to bring down a band of I85 kd in
`the A-43l cells (the extended diffuse band present in
`lanes 13 and 9 of Figures 1c and 1d, respectively). This
`is consistent with the observation that the A-431 cells
`
`do express a [X level of neu mRNA (Gullick et 01.,
`I987). It should also be noted that the control isotype-
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`Indirect immunofluorescence of human neu-producing
`Figure 3
`cell lines using TAl. Cells were grown on 8-chambered microscope
`slides, and were fixed briefly with 3% formalin. All were incubated
`with a l
`: 50 dilution of TALascites fluid, washed, and then incu-
`bated with FlTC-label'éd'gda'i anti-mouse ‘lgG. Panel (a) 18-3-7
`cells. Panel (b) N1H3T3 cells. Panel (e) SKBR-3 cells. Panel (d)
`A-431 cells
`
`generated were capable of binding to this form of the
`neu gene product when tested by immunoprecipitation,
`or by an in situ ELISA performed on viral plaques. The
`mAbs OD3 and_PB3 also recognized this truncated
`form of the protein by Western blot analysis. (Data not
`shown.)
`
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`Discussi n
`
`Monoclonal antibodies specific for the protein product
`of the human neu oncogene have been generated using
`viable, non-tumor forming cells which express the full
`length neu-encoded protein as the immunogen. The
`hybridomas were produced following an immunization
`regimen which combined injection of NIH3T3 cells,
`treatments with cyclophosphamide, and injection of an
`NIH3T3 cell
`line transfected with the neu oncogene.
`The majority of the monoclonals were IgG,, one was
`IgGZa, and two were lgM antibodies. A variety of
`binding affinities and epitope specificities was generated,
`and all of the monoclonals bind epitopes on the extra-
`cellular domain of the neu protein.
`Data generated indicates that the mAbs are specific
`for the neu encoded protein, and do not cross-react with
`human EGFR. The monoclonals work in a variety of
`formats,
`including Western blot, ELISA,
`flow cyto-
`metry, immunoprecipitation, and immunofluorescence.
`Thus-these monoclonals will. be useful as reagents to
`study the expression of the neu oncogene product using
`a number of experimental procedures. Studies are
`underway to determine the ability of the mAbs to detect
`expression of
`the oncogene product by immuno-
`peroxidase staining of human tumors, and to correlate
`this expression with tumor status.
`The possibility of using flow cytometry as a gener-
`alized method for the study of tumor status is also being
`examined. This approach may afford a level of sensi-
`tivity which would be capable of detecting low numbers
`of metastatic cells present in lymph nodes.
`Another possible application of these monoclonal
`antibodies is as immunotherapeutic reagents. This is
`based on the observations that antibodies directed
`against the rat neu gene product have been used to kill
`tumor cells in vitro (Drebin et al., 1985) and in vivo in a
`nude mouse model (Drebin et al., 1986,
`198821 and
`l988b). Since the neu oncogene has been found in
`association with some human breast cancers (Cline et
`al., 1987; Slamon et al., 1987; van de Vivjer et al., 1987;
`Varley et al., 1987; Venter et al., 1987; Zhou et al., 1987;
`Berger et al., 1988), use of these monoclonal antibodies
`in a variety of formats may prove important in the diag-
`nosis, prognosis, or potential therapy of these malig-
`nancies.
`
`Materials and methods
`
`Transfection of N l H3T3 cells with human neu
`The pLJ
`retroviral vector was modified to remove the
`polyoma early region, eliminating the endogenous
`trans-
`forming activity of the pLJ vector. A cDNA copy of the full
`length coding region of the human neu oncogene was inserted
`(Mazzara & Morgan, unpublished results), and the resulting
`plasmid was used to transfect NIH3T3 cells by a standard
`calcium phosphate precipitation procedure (Graham & van
`der Eb, 1973; Stowe & Wilkie, 1976). Transfected cells were
`selected by their ability to grow in the presence of G418 and
`were screened for the expression of neu mRNA using RNA
`dot blots.
`
`Immunization ufmice
`Female Balb/c mice were immunized intrapcritoneally (l.P.)
`with 1 x 10" viable N1H3T3 cells per' animal on Day 0. This
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`MONOCLONAL ANTIBODIES SPECIFIC FOR HUMAN NEU 547
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`
`Figure 4 Flow cytometry on human neu producing cell lines using TAl. Cells were harvested from culture, washed and resuspended
`
`to 2 x 10‘S viable cells per sample. They were incubated at 4°C for 1h with TAl, washed and then incubated at 4°C for 1h with
`FITC-labeled goat anti-mouse IgG. Cells were analyzed using an EPICS V flow cytometer. In all panels: ‘
`-
`-
`~ MOPC 21.
`TAl. Upper left: NIH3T3/ras, an NIH3T3 cell line transfected with the ras oncogene. Upper right: 17-7-8, an NIH3T3 cell line
`co-transfected with the ms and neu oncogenes. Lower left: X—3-5, an NIH3T3 cell line co—transfected with the ms and neu oncogenes.
`Lower right: 18-3-7, an NIH3T3 cell line transfected with the neu oncogene alone
`
`was followed approximately 2h afterwards, by an LP. injec-
`tion of cyclophosphamide, 100mg kg“, in H20. The cyclo-
`phosphamide treatment was repeated on Days 1 and 2. On
`day 14 following immunization, the mice were injected l.P.
`with l x 105 viable 18—3-7 cells. The animals were allowed to
`rest for 14 days, at which time the entire sequence, NIH3T3
`cells, 'cyclophosphamide, and 18-3—7 cells, was repeated. Four
`days following the second injection of 18-3-7 cells, the animals
`were sacrificed and their spleens removed for fusion.
`
`Hybridoma methodology
`Hybridomas were produced by fusing spleen cells from immu-
`nized mice with SP2/0 myeloma cells by a polyethylene glycol
`(PEG) method. Spleen cells and SP2/0 cells were fused at a
`ratio of 5:1 (spleenzmyeloma). Following fusion,
`the cells
`were plated at a concentration of 5 x 105 cells ml" on perito-
`neal macrophages in the presence of HAT media (0.2mm
`hypoxanthine, 0.4 pM aminopterin, and 0.032 mM thymidine,
`in DME plus 20% fetal bovine serum). All hybridomas which
`yielded positive results in the initial ELISA screen for anti-
`plSS'antibody (see below) were subcloned twice by limiting
`dilution.
`
`NIH3T3 cells was prepared similarly for use as a negative
`control. Microtiter plates (Nunc, Immunoplate II) were coated
`overnight at room temperature with lysate, at a total protein
`concentration of 20 ugml",
`in 0.05M carbonate bulTer,
`pH9.6. The ELISA was performed by incubating culture
`supernatant obtained from hybridoma colonies for 2h at
`37°C. This was followed by a 1h incubation at 37°C with
`horseradish
`peroxidase
`labeled
`goat
`anti-mouse
`IgG + IgA + IgM (Kirkegaard & Perry Labs). The assay was
`developed for 5min using tetramethylbenzidine (TMB), and
`the enzymatic reaction was stopped by the addition of 2N
`stO‘. The optical density (OD) of the resulting yellow color
`was read at 450nm on a microtiter plate reader (Biotek
`EL309). Supernatants were considered positive for the pre-
`sence of anti—p185 antibody if the reactivity on 18-3-7 cells
`was at least twice the reactivity on NIH3T3 cells.
`
`Antibody isotype and subclass determination
`ELISA assays to determine the isotype and light chain class of
`the antibody produced were done using a ScreenType kit
`(Boehringer Mannheim), as per manufacturer‘s suggested pro-
`cedure.
`
`ELISA procedure
`Lysates of 18-3-7 cells were prepared for use as antigen by
`incubating freshly harvested 18-3-7 cells in the presence of a
`hypotonic lysis buffer (lOmM Tris, 10 mM KCI, SmM EDTA,
`pH 8.0), followed by Dounce homogenization, sonication, and
`centrifugation to remove
`insoluble matter. A lysate of
`
`Purification of mAbs
`
`All lgG monoclonals were purified from ascites fluid by affin-
`ity chromatography on Protein A-Sepharose (Pharmacia) (Ey
`et al., 1978; Seppala et al., 1981). The IgM monoclonals were
`purified by ammonium sulfate precipitation followed by gel
`sieve chromatography.
`
`
`
`
`
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`PHIGENIX
`
`Exhibit 1006-05
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`
`S48 SJ. MCKENZIE et al.
`
`\
`Western blot
`
`Lysates of SKBR—3 cells (ATCC HTB 30) and A—431 cells
`(ATCC CRL 1555) were electrophoresed on 1.5mm thick 7%
`SDS-polyacrylamide gels, using a 4.5% stacking gel. The
`separated proteins were
`transferred onto nitrocellulose
`(Schleicher & Schuell) using the BioRad Transblot apparatus.
`The nitrocellulose filter was then blocked for 1 h in Blotto (3%
`dry milk, 2% normal goat serum, 0.1% Tween-20 in PBS) and
`incubated for 311 at room temperature with either 0.5 pg ml"
`0D3, or 2 ugmli“ PBS (both diluted in Blotto), or with
`20 pg ml“ 291-3A (in culture supernatant). Filters were rinsed
`3 times in a High Salt Wash buffer (20min Tris-HCI, 1M
`NaCl, 0.05% Tween-20, pH 7.6) and were then incubated
`with
`alkaline
`phosphatase
`labeled
`goat
`anti—mouse
`IgG + IgA + lgM (Kirkegaard & Perry Labs) for l h at room
`temperature. They were washed again three times with the
`High Salt Wash buffer, and the bands were visualized using a
`BCIP/NBT substrate kit (Kirkegaard & Perry Labs).
`
`Immunoprecipitation
`
`A sub-confluent monolayer of cells in a 10cm petri dish was
`incubated overnight in cysteine-free media containing 500 aCi
`of [3SS]-labeled cysteine. The cells were harvested the follow-
`ing morning, and were lysed in a detergent bufier (IP buffer:
`1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10mM
`Tris, 0.65M NaCl, pH 7.2) containing the protease inhibitors
`PMSF and soybean trypsin inhibitor. Approximately 3 pCi of
`this labeled cell preparation was then used for each sample.
`The lysates were incubated overnight at 4°C with either 5 pg
`of purified antibody, with 511g of isotype-matched mouse
`myeloma protein, or with 10):] of rabbit anti—EGFR anti-
`serum. For each sample, 20 pg of purified rabbit anti-mouse
`IgGl or rabbit anti-mouse IgM (Organon Tekmika) was mixed
`with 50m of a 1
`2 1 slurry of Protein A-Sepharose (Pharmacia)
`in IP buffer am! was incubated overnight at 4°C. The excess
`rabbit antibody was removed by washing the Protein A-
`Sepharose once with IP buffer, and the slurry was then added
`to the mixture containing labeled cell lysate and monoclonal
`antibody. This mixture was allowed to react for 5h at 4°C.
`The Protein A-Sepharose was pelleted by centrifugation and
`was washed four times with [P buffer, and once with TBS
`(10 mM Tris, 150mm NaCl, pH 8.2). The pellet was allowed to
`dry at 37°C. Each pellet was resuspended in 50 ll] of sample
`
`buffer for SDS gels and heated for 5min at 100°C. A volume
`of each sample containing 10000 cpm of material was loaded
`onto an SDS-polyacrylamide gel, using a 4.5% acrylamide
`stacking gel, and a 7% separating gel. The gels were fixed,
`treated with EN’HANCE (New England Nuclear), dried and
`then autoradiographed.
`
`lmmunofluorescence
`
`Cell lines were grown to confluence on 8-chambered LabTek
`tissue culture slides (Miles Scientific) overnight. They Were
`briefly washed in Dulbecco’s PBS (containing Ca“ and
`Mg+ +) and were fixed with 3% formalin for 30min at room
`temperature. A l :50 dilution of TA] ascites fluid (diluted in
`50% normal goat serum) was incubated with the cells for 1h
`at room temperature. The slides were washed again with PBS,
`and were then incubated for 1h at room temperature with
`fluorescein labeled goat anti-mouse IgG (Cappel).
`
`Flow cytometry
`
`resuspendedtoaconcentration'of2x10°viablecellsper
`
`Cells were harvested from culture, washed once,
`
`and
`
`sample in Leibovitz L-15. They were then incubated with 1 pg
`of purified TAl
`or with
`the
`isotype-matched control
`MOPC-Zl for 1h at 4°C. The cells were washed three times
`with PBS and incubated with 1 ug of goat anti-mouse
`Ig-FITC for 1 h at 4°C. This incubation was followed by three
`additional washes in PBS. The cells were analyzed using an
`EPICS V flow cytometer with an argon laser tuned to 488 nm.
`Discriminators were set such that <5% of the cells were posi—
`live with the isotype-matched control antibody. The percent-
`age of cells positive and the mean fluorescence intensity for
`each histogram was determined using the Easy 88 software
`(Coulter).
`
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`
`Acknowledgements
`The authors would like to acknowledge Dr Gail Mazzara and
`Ms Sandra Katz for their contribution to the production of
`the cDNA clone of the human neu oncogene, Ms ”Ethel
`Gordon for her assistance in the transfection and isolation of
`the human neu-producing NIH3T3 cell
`lines, and Drs Sam
`Yin and Arthur Bruskin for their review of the manuscript.
`
`.~
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`
`References
`
`i
`I
`
`(l lt
`
`l
`
`(
`I"
`
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`Drebin, J.A., Link, V.C., Stern, D.F., Weinberg, R.A. &
`Greene, M.l. (1985). Cell, 41, 695-706.
`Drebin,
`.l_.A., Link, V.C., Weinberg, R.A. & Greene, M.l.
`(1986). Proc. Natl. Acad. Sci., 83, 9129—9133.
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`
`:2;..:.‘...:,:'‘.
`
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`
`PHIGENIX
`
`Exhibit 1006-06
`
`