~
Cancer Immunol Immunother(1991) 34:37 42 034070049100093D
ancer mmunology mmunothërapy
© Springer-Verlag 1991
Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site Rita Pellegrini, Filippo Centis, Stefania Martignone, Antonio Mastroianni, Elda Tagliabue, Emanuela Tosi, Sylvie Ménard, and Maria I. Colnaghi Division of Experimental Oncology E, Istimto Nazionale per lo Studio e la Cura dei Tumori, Via G. Venezian 1, 20133 Milan, Italy Received 10 December 1990/Accepted 29 May 1991
Summary, In this work a new monoclonal antibody (mAb), designated M G R 1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 x 104 receptors/cell of 10 g m diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.
Key words: Monoclonal antibody - Epidermal growth factor receptor
HER-2/Neu protein, expressed in many normal cells [4, 5, 12, 24, 25, 28], have been found to be overexpressed in some tumours [10, 12, 18, 25, 31, 38, 39]. These molecules cannot be considered tumour-specific from a qualitative point of view, but an abnormally high level of expression discriminates normal from tumour cells quite well. Attempts to exploit the EGF-R as a target for mAb therapy have been made [1, 13, 19, 27]. Different mAbs directed against the extracellular domain of EGF-R have been described in various studies [7, 15, 30, 32, 36]. Some of them are able to compete with the EGF binding and inhibit the tumour growth. Alternatively they have been used to construct immunoconjugates [2], especially immunotoxins [21, 26, 33, 35]. In order to allow exploitation of the quantitative differences in EGF-R expression between normal and tumour cells for a clinical application, an essential aspect is the availability of reagents capable of reacting functionally with cells with overexpression of the receptor only. In the present work we describe a new mAb directed against the EGF-R and explore its ability to discriminate cells expressing different amounts of EGF-R.
Materials and methods Introduction Monoclonal antibodies (mAbs) directed against tumourassociated antigens have been widely developed in order specifically to direct therapeutic agents against the tumour [8, 11, 29]. To date the tumour specificity of the reagent required for this approach has represented the main difficulty for achieving the goa! and in fact so far no tumourspecific markers have been identified by monoclonal antibodies. Some growth-factor receptors, such as the epidermal growth factor receptor (EGF-R) and probably the
Cell lines. The human cell lines used in this work include a vulvar epidermoid carcinoma (A431), a lymphoma (U937), a colon adenocarcinoma (HT-29), three breast carcinomas (MCF-7, DU 4475, MDAMB468), a lung adenocarcinoma (CaLu3) and a small-cell lung carcinoma (N592), all obtained from the American Type Culture Collection; two ovary carcinomas (OvCa 432 and IGROV1) and a melanoma (MeWo) kindly provided by Dr. Knapp (Dana Farber, Boston, USA), by Dr. Bénard (Inst. G. Roussy, Villejuif, France) and by the late Dr. Fogh (Sloan Kettering Cancer Center, N. Y., USA) respectively.The BALB/c myeloma NSO cell line was used in the somatic fusion. All cells were maintained in RPMI-1640 medium, supplemented with 10% fetal calf serum, penicillin (100 ~tg/ml) and streptomycin (100 [tg/ml). The A431 and the HT-29 cells were also grown for a particular experiment in a medium containing 0.5% fetal calf serum. lmmunization and somatic.fusion. A BALB/c mouse received three i.p.
Offprint requests to: M. I. Colnaghi
injections of 5x 100 A431 cells in complete Freund's adjuvant. The
38 a
b
MGR1 binding inhibition by EGF. The MGRI binding inhibition by EGF was evaluated on A431 cells seeded in 96-well culture plates and fixed with 0.1% glutaraldehyde. The incubation was performed at 37°C for 45 min in the presence of 100000 cpm 125I-MGR1 and different dilutions of murine purified EGF (Amersham International, Little Chalfont, UK).
c
Mr ×10 -3
200
--
W"'
-ù,-- 170 KD
92_5 --
Scatchard analysis. Live A431 cells (50 000/sample) were incubated for 3 h at 4°C with 50 gl containing the 125I-EGF or 125I-MGR1 at serial dilutions. After two washings the radioacUvity bound to the cells was counted directly in a ? counter. The data obtained were elaborated on a desk-top compact computer. The number of binding sites per cell was evaluated by calculating the molarity of the bound and the bound/free ratio of the ligand molecules, the significance of the curve and the values of the two intercepts.
69--
Fig. 1. Autoradiography of 125I-labelled antigens immunoprecipitated by MGR1 from A431 cell extract and analysed by 5 % - 10% sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The immunoprecipitation was performed with murine normal serum (a), the EGFRI mAb (b) and MGR1 (c) animal received a daily boost with the immunizing material three days before the fusion. The last two injections were performed both i.p. and i. v. The immune spleen cells were then fused with NSO myeloma cells as already described [22]. The supernatants of hybrid cell cultures were screened by indirect solid-phase radioimmunoassay on A431 and on MeWo cells as a positive and negative target respectively. The isotype of the selected mAb was determined by an indirect immunofluorescence test with fluoresceinated streptavidin and biotinylated anti-0nouse Ig heavy chain) against immunoglobulin of the classes IgM, IgG1, IgG2a and igG2b (Amersham).
MGRI purification and radioisotope labelling. The mAb was purified from ascitic fluid by affinity chromatography on a column of protein-A Sepharose CL4B (Pharmacia), as previously described [6]. The purification yield indicated that the MGR l concentration in the ascitic fluid was about 2.5 ing/ml. The labelling of the purified mAb was performed by lactoperoxidasecatalysed iodination [3]. Samples of 1 mCi NaU»I (Amersham International, Little Cbalfont, UK), 24 gg lactoperoxidase (Calbiochem Boehring, La Jolla, Calif.) and 8 gl 0.03% H202 were added to 50 gg purified MGRI. The mixture was finally desalted on an Econo-Pac TM 10DG (BioRad, Richmond, Calif.). The immunoreactivity of the labelled mAb was estimated as 68% by a direct radioimmunoassay performed at 37°C for 45 min, on serial dilutions of A431 cells in the presence of 10 000 cpm 125I-MGR 1. Solubilization of radiolabelled cells and immunoprecipitation. A431 cells at confluence were washed three times with phosphate-buffered saline (PBS) and used for membrane lactoperoxidase-catalysed iodination [23]. The cells were then solubilized at 0°C for 30 min by adding 1 ml PBS containing 1% NP40, 1% Antagosan (Behring, L'Aquila, Italy) and 0.001 M phenylmethylsulphonyl fluoride (Sigma, St. Louis, Mo.). The lysate was cleared by centrifugation (15 min, at 10000 g) and the soluble extract was immunoprecipitated [23] and then applied to a discontinuous ( 5 % - 10%) sodium dodecylsulpbate(SDS)/polyacrylamide slab gel [17], under reducing conditions. The mAh EGFRI (Amersham International, Little Chalfont, UK), directed against the EGF-R, was used as positive control. 1251-EGF binding. The EGF binding was evaluated on live cells of various cell lines. Samples containing 150 000 cells washed with Hank's solution, were ineubated for 45 min, at 37°C with 20000 cpm/100gl routine 125I-EGF (Amersham International, Little Chalfont, UK). The cells were then washed three times with Dulbecco's PBS and the bound radioactivity was counted on a 7 counter. To evaluate the MGR1 ability to inhibit EGF binding the incubation was performed in the presence of different dilutions of purified mAb.
lmmunocytochemical and immunohistochemical tests. Indirect immunofluorescence on live cells was performed using purified MGR1 (10 pg/ml) and isothiocyanate-conjugated goat anti-(mouse Ig) (Meloy Laboratories Inc. Springfield, Va.). Immunoperoxidase techniques were carried out on cryostatic sections, obtained from frozen surgical specimens and fixed for 10 min in cold acetone, using purified MGRI (10 gg/ml) and an avidin-biotin-peroxidase complex (ABC) kit (Vector, Burlingham, Calif.). These tests were performed as described in [22, 34]. Tumour cellproliferation assay in vitro. A431 or HT29 cells were seeded at a density of 5000 cells/well in 96-well culture plates in medium with 10% or 0.5% FCS alone or containing different amounts of purified MGR1. After 2 days the cells were stained with 3-(4,5-dimetbylthiazol2-yl)-2,5-diphenyltetrazolinm bromide 0.5 mg/tal and spectrophotometrically evaluated at 550 nm (reference at 620 nm). The growth inhibition was calculated as follows: 100 - (A treated sample/A control sample x 100) TumourcellprolifOration assay in vivo. Two types of in vivo assays were performed: a brief and a long-term experiment. In the first case athymic mice (Charles River Laboratories) were injected s. c. witb 5 × 106 tumour cells (A431, MeWo and HT-29) and injected i. p. daily for 10 days with 0.2 ml ascitic fluid containing MGR1, starting the treatment on the same day as tumour inoculation. As control groups, five mice injected with A431 cells were treated with the same dose of ascitic fluid containing the MOvl 6 mAb (2.5 mg mAb/ml ascitic fluid), reactive with A431 cells, or the MLuC9 mAb (2 mg mAb/ml ascitic fluid), unreactive with A431 cells. The effects of MGR1 on animals inoculated with A431 cells were also evaluated starting the mAb treatment 3 and 7 days after tumour injection. The long-term effects of MGR1 on A431 growth were observed by treating mice with the mAb 3 days after tumour injection for 10 days and controlling the growth for 3 months,
Results A somatic fusion was performed between mouse spleen cells, w h i c h w e r e i m m u n e a g a i n s t t h e e p i d e r m o i d c a r c i n o m a A 4 3 1 cell l i n e , a n d N S O m y e l o m a cells. A m A b d e s i g n a t e d M G R 1 , o f I g G 1 i s o t y p e , w a s s e l e c t e d f o r its s t r o n g reactivity on A431 cells and negativity on the MeWo cell line. T h e A 4 3 1 c e l l l i n e s o l u b l e e x t r a c t , l a b e l l e d b y lactoperoxidase-catalysed radioiodination, was immunoprecipitated by the purified antibody. The SDS electrophoresis of the radioimmunoprecipitate indicated that MGR1 recogn i z e d a m o l e c u l e o f 170 k D a (Fig. 1), as a c o r m n e r c i a l antibody directed against EGF-R. As regards the immunocytochemical characterization o n l i v e c e l l s f r o m t u m o u r c e l l lines, t h e m A b s t r o n g l y r e a c t e d w i t h M D A M B 4 6 8 c e l l s i n a d d i t i o n to t h e A 4 3 1 c e l l
39 Table 1. Correlation between immunocytochemicalreactivity of MGR1 and binding of 125I-labelledepidermal growth factor (EGF), in the presence or absence of MGR1, on different cell lines Cell line
A431 MDAMB468 SKBr3 HT-29 IGROV1 MCF-7 CaLu3 U937 MeWo DU4475 N592
MGR 1 reactivitya
+++ +++ + + -
(100%) (100%) (100%) (60%)
100
125I_EGFbinding (cpm bound) -MGR1
+MGR1
7100 7060 2170 990 1100 800 0 0 0 0 0
1100 530 300 300 200 280 N.D. N.D. N.D. N.D. N.D.
/ / /
80 c o
60
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20
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a The reactivity was determined by an indirect immunofluorescence test. The staining intensity was evaluated as: +++, strong; +, slight, -, negative. Parentheses indicate the percentage of labelled cells
0
+
'
'
0.026 0.130 0.6L0 3.2 16 ivlGR1 Concentrotion (t~g/ml)
80
Fig. 2. Competition test between purified MGR1 and 125I-labelled epidermal growth factor (EGF) on A431 (8) and HT-29 (+) live cells Table 2. Number of EGF receptors evaluated by Scatchard plot analysis on A431 and HT-29 cell lines Cell line
100
104 x Number ofEGF receptors/cell evaluated by binding of t25I_EGF
125I_MGR1
270 5
260 5
80
A
A431 HT-29
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60
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line, whereas it scored weakly positive on 100% of the breast carcinoma SKBr3 cells and on 60% of the colon carcinoma HT-29 cells and was found to be negative with all the other lines tested (Table 1). W h e n the same cell lines were analysed for the 125I-EGF binding, in the absence or presence of M G R 1 , similar but not completely superimposable results were obtained. In fact, on I G R O V 1 and M C F - 7 only the E G F b i n d i n g was detectable, though at a low level, whereas M G R 1 scored completely negative. In all cases, the growth factor b i n d i n g was always inhibited by a previous i n c u b a t i o n of the cells with M G R 1 . A Scatchard plot analysis of the b i n d i n g data of labelled M G R 1 and labelled E G F showed very similar results as regards the n u m b e r of b i n d i n g sites per cell, with approximately 50 times more E G F - R on A431 than on HT-29 cells (Table 2), and a relatively low Kaff of 6 x 107M -1. As shown in Fig. 2, the b i n d i n g of 125I-labelled E G F was completely inhibited by MGR1 on A431 and HT-29. The a m o u n t of antibody required for this inhibition was in the same range for both cell lines. Moreover, the 125I-MGR1 b i n d i n g on fixed A431 was inhibited by EGF, as shown in Fig. 3. I m m u n o p e r o x i d a s e tests performed on cryostatic sections revealed that M G R 1 was negative on all the n o r m a l tissues tested, with the exception of n o r m a l keratinocytes. A weak reaction was f o u n d on liver cells (Table 3). As regards the tumour tissues, the m A b scored strongly posi-
/
#
~5 4O c rn
ä: ~o
0 ~ 0.16
, , 0.8 z. 20 EGF Concentretion (l~g/ml)
, 100
Fig. 3. Competition test between purified EGF and 12SI-labelledMGRI on A431 fixed cells
Table 3. Binding reactivity of MGR1 on cryostatic sections of normal tissues by immunoperoxidase Tissue
No. cases evaluated
No. MGRl-reactive cases (intensity)
Skin (keratinocytes) Lung Breast Intestine Kidney Liver Spleen Ovary
3 7 3 3 4 6 3 2
3 (intermediate) _a 5 (weak) -
a
.
Negative
40 Table 4. Binding reacfivity of MGR1 on cryostatic sections of tumoral tissues by immunoperoxidase
Table 5. Effect of the mAbs MGR1, M O v l 6 and MLuC9 on the growth capacity of A431, MeWo and HT-29 cell lines in athymic mice
Tumours
No. cases evaluated
MGRl-positive cases (mean intensity) (%)
No. treated animals
Tumour cell lines inj ected
mAb inj ected
No. animals with tumour growth
Lung carcinomas Breast carcinomas Ovarian carcinomas
20 19 10
65 (strong) 37 (intermediate) 30 (intermediate)
10 10 10 10 5 5 5 5
A431 A431 A431 A431 MeWo MeWo HT-29 HT-29
MGR 1 MOvl6 MLuC9 MGR1 MGR1
10 0 l0 10 5 5 5 5
70
60
~1
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2.0
50 co
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30
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0.03
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I
I
0.5
0.3 3 30 MGR1 Concentrotion (tag/ml)
Fig. 4. Growth inhibition by MGR 1 on A431 ( I , []) and HT-29 ( 0 , C)) cells in vitro. The test was performed on cells maintained in RPMI medium + 10% fetal calf serum (FCS) ( [~, ©) or in RPMI + 0.5% FCS (mm, 0 ) . The cells were seeded in a 96-well culture plate (5000 cells/well) in the presence of MGR1 at different concentrations
tive on 30% of the ovarian carcinomas, 37% of the breast carcinomas and 65% of the lung carcinomas (Table 4). When MGR1 was added to the culture medium of A431 cells, an interference with the tumour growth was observed (Fig. 4). When the cells were maintained in RPMI medium supplemented with different amounts of growth factors (10% or 0.5% fetal calf serum), the growth inhibition varied from 37% to 60%. On the other hand, the EGFR1 m A b tested at 1.5 gg/ml did not mediate growth inhibition either in the presence or absence of fetal calf serum. The MGR1 molar concentration required to inhibit the cell growth was about six times more than the EGF molar concentration needed to obtain the same inhibition. On the other hand, MGR1 had no effect on HT-29 growth in vitro. The interference of MGR1 on the in vivo tumour growth was also investigated. In a short-term experiment, athymic mice were injected with A431 cells and treated, starting the same day, for 10 days with MGR1 or two control mAbs of the IgG1 isotype: M O v l 6 , reactive, or MLuC9, unreactive with these cells. As reported in Table 5, a complete inhibition of A431 tumour growth was observed in the MGRl-treated mice, whereas in the untreated control group and in the groups inoculated with the other two mAbs all the animals developed tumours. On the other hand, MGR1 showed no effect on the growth capaci-
0 A
B T=0
A B T=3days
A B T=7days
Fig. 5. Effect of MGR1 injected in athymic mice (five animals in every group), starting on different days after s. c. inoculation with 5 × 106 A431 cells. The mean tumour weights + SE are presented. A, MGRl-treated animals; B, untreated animals; T, day of begining of treatment after tumour inoculation
ty of colon carcinoma HT-29 and M e W o melanoma cell lines. Furthermore, the effect of MGR1 treatment on the growth of already implanted tumors was investigated in another short-term experiment. Athymic mice were inoculated with A431 cells and 0 or 3 or 7 days later three groups of five animals each were treated daily for 10 days with MGR1. These mice were sacrificed 30 days after tumour inoculation and the tumours were removed and weighed. As shown in Fig. 5, in the animals treated 7 days after tumour implantation the tumour growth was partially inhibited, whereas a complete growth inhibition was found when the mAb treatment was started on the same day or 3 days later. The tumour cells grown in the MGRl-treated animals were still able to bind 125I-EGF in a direct radioimmunoassay (data not shown), which indicated that no immunoselection of EGF-R-negative cells occurred. A further experiment was performed to investigate the long-term effects of the MGR1 treatment on already implanted tumours. Five athymic mice were treated for
41 100
8o
v
60
._(2 ~,0
2O
~,
[]
o 1:6/,oo
1:16oo I:Loo Serum dilution
1:1oo
1:25
Fig. 6. Competitiontest on A431 live cells between ]25I-labelledMGR 1 and serum of mice inoculated with A431 cells and treated with MGR 1 for 10 days. The serum was tested 7 (A), 14 (:~) and 45 (n) days after the mAb treatment was terminated
10 days with MGR1 starting 3 days after A431 tumour cell inoculation and controlled for tumour growth three times weekly for 3 months. In the control untreated group all five mice developed tumours 2 weeks after tumour injection. In the mAb-treated group only one mouse developed a tumour 2 months after tumour implantation. The sera of the treated mice were tested for the presence of MGR1 (Fig. 6). By using a competition test with 125I-MGR1 on A431 live cells, the antibody could be detected 7 and 14 days after the end of the treatment, whereas after 45 days these sera scored completely negative.
Discussion We have reported the production and characterization of a monoclonal antibody designated MGR1. The molecular mass of the recognized molecule, together with EGF crosscompetition for binding on reference cells, demonstrated that MGR1 recognizes the EGF receptor binding site. Many other antibodies produced against EGF-R have been shown to inhibit the growth factor binding to relevant target cells [7, 9, 30]. In keeping with the results obtained with these other mAbs [1, 16, 19], MGR1 inhibits tumour growth both in vitro and in vivo. The in vitro effect depends on the presence in the culture medium of growth factor supplementation. Increasing amounts of EGF required increasing doses of mAbs to obtain the same degree of inhibition. In addition, the amount of MGR1 required for growth inhibition was ten times more than that necessary to inhibit EGF binding, which might be due to the release of soluble EGF-R extracellular domain in the culture medium [37]. When injected in vivo in tumour-bearing nude mice, MGR1 was found to inhibit the growth of the cells that
overexpressed EGF-R, whereas it had no effect on cells with normal EGF-R levels, nor on cells that did not express this receptor at all. This inhibition is specific since other mAbs, either reactive or unreactive on A431 cells, were not able to mediate such an effect. Moreover, short and long-term experiments performed on already implanted tumours showed the strong efficacy of MGR1 to reduce the tumour growth partially or totally. The kind of mechanism involved in the anti-tumour activity induced by MGR1 has not been evaluated; however, blockage of the receptor function seems to be likely. An activation of the mouse effector cells was less probable since in the mice the IgG1 isotype is poorly efficient in mediating antibody-dependent cell-mediated cytotoxicity [20]. MGR1 seems to be quite tumour-restricted as far as its reactivity on normal tissues, detected by the immunoperoxidase technique, is concerned. In fact, it only recognizes the skin keratinocytes and shows a weak reaction on liver cells. This apparently tumour-restricted reactivity of MGR1 could have several explanations. First, MGR1 seems to recognize the ligand-free receptors better than the EGF-occupied ones, as demonstrated by the fact that its binding on fixed cells is inhibited by EGF, a finding not reported for other anti-EGF-R mAbs. Secondly, MGR1 is a relatively low-affinity m A b and may therefore require a high expression of the relevant antigen to obtain a detectable binding. The threshold detectability was established by immunocytochemistry on cell lines expressing a known amount of EGF-R. MGR1 showed a borderline reactivity on HT-29 cells, which expressed about 5 x 104 receptors/cell, as evaluated by Scatchard plot analysis. These cells were relatively small in diameter (10 gm). On the other hand, cells with the same amount of receptors per cell, such as IGROV1 and MCF7, but larger ( 1 2 - 1 4 g m diameter) appeared to be completely negative. The amount of EGF-R available for M G R l - b i n d i n g in most of the normal tissues is therefore below the detectability threshold of 5 x 104 EGF-R molecules/10 g m cell diameter, except for the skin keratinocytes, which have been reported to express 105 receptors/cell [14]. Considering that the growth of cells like HT-29, on which MGR1 is only weakly reactive, is not affected by the mAb, it can be expected that MGR1 would have no effect on normal cells. In conclusion, because of its ability only to recognize and inhibit the growth of cells with EGF-R overexpression, the MGR1 mAb can be considered a good candidate for immunotherapeutic approaches to tumours overexpressing EGF-R.
Acknowledgements. This work was partially supported by a grant from the Associazione Italiana per la Ricerca sul Cancro. We thank Mrs. P. Aiello, Mrs. A. Invernizzi and Mrs. M. Mazzi for their technical assistance, Ms. L. Mameli and Ms. M. Hatton for manuscript preparation and Mr. M. Azzini for the photographicreproduction.
References 1. Abound-PirakE, Hurwitz E, Pirak ME, Bellot F, Schlessinger J, Sela M (1988) Efficacy of antibodies to epidermal growth factor receptor against KB carcinoma in vitro and in nude mice. J Natl Cancer Inst 80:1605
42 2. Abound-Pirak E, Hurwitz E, Bellot F, Schlessinger J, Sela M (1989) Inhibition of human tumour growth in nude mice by a conjugate of doxorubicin with monoclonal antibodies to epidermal growth factor receptor. Proc Natl Acad Sci USA 86:3778 3. Canevari S, Orlandi R, Ripamonti M, Tagliabue E, Aguanno S, Miotti S, Ménard S, Colnaghi MI (1985) Ricin A chain conjugated with monoclonal antibodies selectively killing human carcinoma cells in vitro. J Natl Cancer Inst 75:831 4. Carpenter G (1987) Receptors for epidermal growth factor and other polypeptide mitogens. Annu Rev Biochem 56:881 5. DePotter CR, VanDaele S, VanDeVijver MJ, Pauwels C, Maertens G, DeBoever J, Vandekerckhove D, Roels H (1989) The expression of the neu oncogene product in breast lesions and in normal fetal and adult human tissues. Histopathology 15:362 6. Ey PL, Prowse SJ, Jenken CR (1978) Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 15:429 7. Fendly BM, Winget M, Hudziak RM, Lipari MT, Napier MA, Ullrich A (1990) Characterization of murine monoclonal antibodies reactive to either the human epidermal growth factor receptor or HER2/neu gene product. Cancer Res 50:1550 8. Foon KA (1989) Biological response modifiers: the new immunotherapy. Cancer Res 49:1621 9. Gill GN, Kawamoto T, Cochet C, Le A, Sato JD, Masui H, McLeod C, Mendelsohn J (1984) Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem 259:7755 10. Gullick W J, Marsden J J, Whittle N, Ward B, Bobrow L, Waterfield MD (1986) Expression of epidermal growth factor receptors on human cervical ovarian and vulvar carcinomas. Cancer Res 46:285 11. Harris AL (1990) The epidermal growth factor receptor as a target for therapy. Cancer Cells 2:321 12. Hendler FJ, Ozane BW (1984) Human squamous cell lung cancers express increased epidermal growth factor receptors. J Clin Invest 74:647 13. Kalofonos HP, Pawlikowska TR, Hemingway A, Courtenay-Luck N, Dhokia B, Snook D, Sivolapenko GB, Hooker GR, McKenzie CG, Lavender PJ, Thomas DGT, Epenetos AA (1989) Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase. J Nucl Med 30:1636 14. Kamata N, Chida K, Rikimaru K, Horikoshi M, Enomoto S, Kuroki T (1986) Growth-inhibitory effects of epidermal growth factor and overexpression of its receptors on human squamous cell carcinomas in culture. Cancer Res 46:1648 15. Kawamoto T, Sato JD, Le A, Polikoff J, Sato GH, Mendelsohn J (1983) Growth stimulation of A431 cells by epidermal growth factor: Identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc Natl Acad Sci USA 80:1337 16. Knowles AF (1988) Inhibition of growth and induction of enzyme activities in a clonal human hepatoma cell line (Li-7A): comparison of the effects of epidermal growth factor receptor antibody. J Cell Physiol 134:109 17. Laemmli VK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 18. Libermann TA, Nusbaum HR, Razon N, Kris R, Lax I, Soreq H, Whittle N, Waterfield MD, Ullrich A, Schlessinger J (1985) Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature 313:144 19. Masui H, Kawamoto T, Sato JD, Wolf B, Sato G, Mendelsohn J (1984) Growth inhibition of human tumour cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res 44:1002 20. Masui H, Moroyama T, Mendelsohn J (1986) Mechanism of antitumour activity in mice for anti-epidermal growth factor receptor monoclonal antibodies with different isotypes. Cancer Res 46:5592 21. Masui H, Kamrath H, Apell G, Houston LL, Mendelsohn J (1989) Cytotoxicity against human tumor cells mediated by the conjugate of
22.
23.
24.
25.
26. 27.
28.
29. 30.
31.
32.
33.
34.
35.
36.
37. 38.
39.
anti-epidermal growth factor receptor monoclonal antibody to recombinant ricin A chain. Cancer Res 49:3482 Ménard S, Tagliabue E, Canevari S, Fossati G, Colnaghi MI (1983) Generation of monoclonal antibodies reacting with normal and cancer cells of human breast. Cancer Res 43:1295 Miotti S, Canevari S, Ménard S, Mezzanzanica D, Porro G, Pupa SM, Regazzoni M, Tagliabue E, Colnaghi MI (1987) Characterization of human ovarian carcinoma-associated antigens defined by novel monoclonal antibodies with tumour-restricted specificity. Int J Cancer 39:297 Natali PG, Nicotra MR, Bigotti A, Venturo I, Slamon DJ, Fendly BM, Ullrich A (1990) Expression of the p185 encoded by HER2 oncogene in normal and transformed human tissues. Int J Cancer 45:457 Ozawa S, Ueda M, Ando N, Abe O, Shimizu N (1987) High incidence of EGF receptor hyperproduction in esophageal squamous-cell carcinomas. Int J Cancer 39:333 Ozawa S, Ueda M, Ando N, Abe O, Minoshima S, Shimizu N (1989) Selective killing of squamous carcinoma cells by an immunotoxin that recognizes the EGF receptor. Int J Cancer 43:152 Rodeck U, Herlyn M, Herlyn D, Molthoff C, Atkinson B, Varello M, Steplewski Z, Koprowski H (1987) Tumour growth modulation by a monoclonal antibody to the epidermal growth factor receptor: immunologically mediated and effector cell-independent effects. Cancer Res 47:3692 Sakai K, Mori S, Kawamoto T, Taniguchi S, Kobori O, Morioka Y, Kuroki T and Kano K (1986) Expression of epidermal growth factor receptors on normal human gastric epithelia and gastric carcinomas. J Natl Cancer Inst 77:1047 Sato GH, Sato JD (1989) Growth factor receptor monoclonal antibodies and cancer immunotherapy. J Natl Cancer Inst 81:1600 Schreiber AB, Lax I, Yarden Y, Eshhar Z, Schlessinger J (1981) Monoclonal antibodies against receptor for epidermal growth factor induce early and delayed effect of epidermal growth factor. Proc Natl Acad Sci USA 78:7535 Slamon D J, Press MF, Godolphin W, Ramos L, Haran P, Shek L, Stuart SG, Ullrich A (1989) Studies of the HER2/neu proto-oncogene in human breast cancer. Cancer Cells 7:371 Sobol RE, Astarita RW, Hofeditz C, Masui H, Fairshter R, Royston I and Mendelsohn J (1987) Epidermal growth factor receptor expression in human lung carcinomas defined by a monoclonal antibody. J Natl Cancer Inst 79:403 Taetle R, Honeysett JM, Houston LL (1988) Effects of anti-epidermal growth factor (EGF) receptor antibodies and an anti-EGF receptor recombinant-ricin A chain immunoconjugate on growth of human cells. J Natl Cancer Inst 80:1053 Tagliabue E, Ménard S, Della Torre G, Barbanti P, Mariani-Costantini R, Porro G, Colnaghi MI (1985) Generation of monoclonal antibodies reacting with human epithelial ovarian cancer. Cancer Res 45:379 Vollmar AM, Banker DE, Mendelsohn J, Herschman HR (1987) Toxicity of ligand and antibody-directed ricin A chain conjugates recognizing the epidermal growth factor receptor. J Cell Physiol 131:418 Waterfield MD, Mayes EL, Stroobant P, Bennet LPB, Young S, Goodfellow PN, Banting GS, Ozanne B (1982) A monoclonal antibody to the human epidermal growth factor receptor. J Cell Biochem 20:149 Weber W, Gill GN (1984) Production of an epidermal growth factor receptor related-protein. Science 224:294 Weiner DB, Nordberg J, Robinson R, Nowell PC, Gazdar A, Greene MI, Williams Wv, Cohen JA, Kern JA (1990) Expression of the n e u gene-encoded protein (p 185neu) in human non-small cell carcinomas of the lung. Cancer Res 50:421 Yamamoto T, Kamata N, Kawano H, Shimizu S, Kuroki T, Tayoshima K, Rikimaru K, Nomura N, Ishizaki R, Pastan I, Gamou S, Shimizu N (1986) High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines. Cancer Res 46:414