Cytotechnology 3: 279-293, 1990. 9 1990 KluwerAcademic Publishers. Printed in the Netherlands.

Monoclonal antibodies to epidermal growth factor receptors in studies of receptor structure and function

Tomoyuki Kawamoto, Gordon H. Sato 1, Kojiro Takahashi, Mieko Nishi, Shigehiko Taniguchi and J. Denry Sato 1 Department of Biochemistry, Okayama University Dental School, Okayama 700, Japan; a n d / W . Alton Jones Cell Science Center, 10 Old Barn Road, Lake Placid, N Y 12946, USA Accepted in revisedform 5 December 1989

Key words: EGF receptor, monoclonal antibodies, epidermal growth factor, receptor antibodies, growth factor receptors

Introduction The apparent similarities between oncogene products and growth factors [1,2] or growth factor receptors [3-6] underscore the importance of growth factor action in normal and pathological cell proliferation. Thus, knowledge of the mechanisms by which growth factors act on cells is crucial to understanding normal and abnormal physiological processes that involve cell growth. Epidermal growth factor (EGF) is a 53 amino acid polypeptide that is mitogenic for a variety of cells in vitro and in vivo [7,8]. Since its identification as a submaxillary gland factor that induced precocious eyelid opening and incisor eruption in neonatal mice [9], EGF has become one of the best characterized growth factors. Although the mechanism by which EGF acts on responsive cells is incompletely understood, interactions between EGF and EGF receptors and their attendant effects on cell physiology represent a useful model for growth factor action. One experimental approach that has been used to study the function of EGF is the production of EGF receptor monoclonal antibodies that stimulate or inhibit EGF-

induced cellular responses. In this article we will review the uses of EGF receptor monoclonal antibodies in studies of receptor structure and receptor function in EGF action. We will also review the application of these antibodies in modulating the growth of receptor-bearing malignant cells.

Production and binding specificities of EGF receptor monoclonal antibodies EGF receptors are M r 170,000 glycoprotein molecules found in the plasma membranes of EGFresponsive cells [ 10-12]. A number of monoclonal antibodies to EGF receptors have now been described in the literature [13-31]. These antibodies are classified in Table 1 [13-35] with respect to the source of the EGF receptors to which they were raised, their ability to inhibit EGF binding, and their receptor epitope. All of the hybridomas secreting EGF receptor monoclonal antibodies were produced by modifi-

280 cations of the cell fusion method devised by K o h l e r a n d M i l s t e i n [36]. T h e m o d i f i c a t i o n s

mainly involved the use of polyethylene glycol, as o p p o s e d to i n a c t i v a t e d S e n d a l v i r u s , as t h e

Table 1. EGF receptor monoclonal antibodies

Immunogen

Inhibits EGF binding

Receptor Epitope

IgM

A431

+ (13,15)a

N.D. b

IgG2b IgG2a IgG2a IgG2a IgG t IgGt IgG 3 IgG 3 IgG2a IgG 1 IgG3 IgM IgG 3 IgG3 IgM IgG3 IgG3 IgG3 IgG 3 IgG3 IgM IgG IgG IgG 1 IgG3 IgG2a IgG2a IgG2a IgG2a IgG t IgG2a IgGt IgG 1 IgG

A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 A431 PC12 A431 A431 A431 A431 A431 mouse liver A431 A431 A431 synthetic human peptide synthetic human peptide A431

- (14) - (15) + (16,17) + (17) + (17) - (16,17) - (18) - (19) + (20) - (21) N.D. (22) N.D. (22) N.D. (22) N.D. (22) N.D. (22) N.D. (22) N.D. (22) N.D. (22) N.D. (22) N.D. (22) - (23) + (24) + (25) + (26) - (26) - (26) + (27) - (28) + (29) + (29) + (29) - (30) - (30) - EGF/+TGFot (31)

peptide (32) A-related oligosaccharide (33) peptide (32) peptide (32) peptide (32) A-related oligosaccharide (32) Y antigen (34) ALeb/ALeY antigens (35)

Antibody

Ig Class

2G2 IgM EGF R1 TL5 IgG 528 IgG 579 IgG 225 IgG 455 IgG 101 EGR/G49 B 1D8 29.1 IgG IA 2A 3A 4A 8A 9A 10A 11A 13A 14A 2D1 IgM 151 IgG B4G7 2E9 2D 11 2G5 425 9D6 LA22 IgG LA58 IgG LA90 IgG F4 D10 13A9

anumbers in parentheses are references. bN.D., no data available.

peptide (20) A-related oligosaccharide (32) A antigen (22) A antigen (22) A antigen (22) A antigen (22) A antigen (22) A antigen (22) A antigen (22) A antigen (22) A antigen (22) A antigen (22) A-related oligosaccharide (23) N.D. N.D. peptide (26) A-related oligosaccharide (26) A-related oligosaccharide (26) peptide (27) N.D. Ala351-Asp364 (29) Ala351-Asp364 (29) Ala351-Asp364 (29) Asp985-Gln996 (30) Asp985-Gln996 (30) peptide (31)

281 fusogen [37] and the use of alternative MOPC 21-derived parent myeloma cells [38-41]. In each case the HAT (hypoxanthine/aminopterin/ thymidine) selection method of Littlefield [42] was used to eliminate unfused HPRT-negative parent myeloma cells. Recently, an alternative selection method exploiting the inability of NS1 cells to synthesize cholesterol was used to select hybridomas secreting EGF receptor monoclonals ([43]; Y. Myoken and T. Okamoto, pers. comm.). The usefulness of cholesterol-free medium as a selective agent for hybridomas follows from the observation that NS-1, X63, and X63-Ag8.653 myeloma cells cannot survive without an exogenous source of cholesterol owing to a deficiency in the activity of 3-ketosteroid reductase [44,45]. The advantage of this method of selection is that it increases hybridoma yields 3-fold or more over HAT selection [43]. Waterfield, et al. [14] have used a panel of human-mouse somatic cell hybrids to localize with antibody EGFR1 the human EGF receptor gene to chromosome 7; this finding led them to propose that monoclonal antibodies to human EGF receptors could be detected through binding to hybrid ceils containing human chromosome 7. This strategy, however, may not detect antibodies that recognize carbohydrate modifications of the receptor [22]. Another direct screening method that has been used successfully to detect EGF receptor monoclonal antibodies is that of antibody-mediated inhibition of EGF binding to target cells [13,16,17,20,24,29]. Although perhaps the simplest method of identifying receptor-specific antibodies, this approach selects by design only a subset of possible EGF receptor monoclonal antibodies. It is noteworthy that the antibodies obtained by this procedure whose epitopes have been characterized recognize peptide determinants (Table 1). In principle, once these antibodies are produced, antibodies specific for antigenic determinants remote from the EGFbinding region of the receptor could be identified by the ability to inhibit binding of EGF-competitive monoclonals to target cells. Finally, the molecular cloning of human EGF receptor cDNA [46] has made possible the production of peptidespecific EGF receptor antibodies. Rabbit antisera

have been raised against cytoplasmic [47,48] and extracellular [49] receptor peptides, and two monoclonal antibodies have been made to a synthetic peptide corresponding to amino acid residues 985 to 996 located C-terminal to the putative receptor kinase domain [30]. The advantage of using synthetic immunogens is that antibodies can be raised against highly defined pre-selected determinants. The great majority of the EGF receptor antibodies were raised against intact or fractionated A431 human vulval epidermoid carcinoma cells [50,51]. This cell line is a particularly useful immunogen because it expresses an overabundance of EGF receptors (1-3 • 106/cells) [52,53]. However, the choice of A431 cells as a source of EGF receptors has placed several functional restrictions on the receptor antibodies that were produced. With the exceptions of 2G2 IgM [13] and 29.1 IgG [21], which bound to A431 and Swiss 3T3 receptors, and TL5 IgG [15], which was reported to react with human, murine, rat, bovine and canine receptors [21], the monoclonal antibodies generated against A431 EGF receptors do not cross-react with receptors from other species. While eleven antibodies recognize peptide epitopes [20,26,27,29-31], the reactivity of the majority of antibodies to A431 EGF receptors is further limited by their specificities for blood group antigen-related oligosaccharides [22,2326,32-35]. The extracellular domain of A431 EGF receptors is highly glycosylated with 11 of 12 potential N-linked glycosylation sites (AsnXSer/Thr) being occupied by high mannoseor complex-type oligosaccharides [54-56]. The carbohydrate structures identified thus far on A431 EGF receptors are type 1-associated blood group antigens A, ALe b, Le a, sialated Le a type 2-associated ALeY and Y antigens, and unsubstituted and monofucosylated type 2 structures [34, 35,56,57]. Since the glycosylation of cellular components reflects the complement of glycosyltransferase and glycosidase activities present in the cell, the monoclonal antibodies generated against carbohydrate determinants on A431 EGF receptors cannot be expected to react with all human EGF receptors. Thirteen of these antibodies

282 that were tested for reactivity on non-A431 human cells were reported not to bind [18,19,22,23], and none of the oligosaccharide-specific antibodies inhibited EGF binding to A431 cells (Table 1). In addition, antibodies with carbohydrate specificities are likely to react with other cellular glycoproteins, proteoglycans, and glycolipids [58,59]. Two of the antibodies in Table I were raised against EGF receptors of non-human cells. The immunogen for 151 IgG [24] was membrane protein from the PC12 rat pheochromocytoma cell line, and the 9D6 antibody [28] was generated to affinity-purified mouse liver receptors. 9D6 reacted very poorly with human EGF receptor [28], while 151 IgG cross-reacted with bovine, rabbit, human, rat and murine EGF receptors [24]. The reactivities of the antibodies in Table 1 indicate that EGF receptors possess both speciesspecific and species-nonspecific epitopes with human, bovine, murine, rat, rabbit and possibly canine receptors sharing at least one epitope. All of the antibodies characterized thus far as recognizing peptide epitopes are apparently speciesspecific [14,16,17,20,26,27,29], and all but EGFR1 and 13A9 [31] have the additional property of inhibiting EGF binding. 13A9 [31] has the intriguing property of inhibiting the binding of TGFo~ but not EGF to A431 EGF receptors. As the EGF-competitive antibodies presumably inhibit EGF binding by steric hinderance, these results suggest that the EGF-binding region of the human receptor includes at least one unique antigenic determinant. In addition, it is apparent that EGF receptors may have highly immunogenic cell type-specific epitopes that are carbohydrate in nature [18,19,22,23] However, the two species-nonspecific antibodies whose epitopes have been characterized [ 15,21] recognize different human blood group A-related oligosaccharides [32,33,60]. From this discussion it is evident that the hybridoma screening method chosen in addition to the source of the EGF receptor immunogen strongly influences the binding specificities of the resulting receptor monoclonal antibodies. Antibodies selected for the ability to inhibit EGF-

binding tend to recognize species-spec'fic peptide determinants [ 16,17,20,27,29]; 2G2 IgM [ 13 ] and 151 IgG [24] are the only EGF-competitive antibodies reported to react with receptors from more than one species. Those antibodies selected on the basis of high levels of binding to target cells tend to recognize carbohydrate determinants which are often cell type-specific [17,22,23] EGF receptor monoclonal antibodies with greater cross-reactivity between species could be made to synthetic peptides once the primary structures of EGF receptors from additional species are determined. At present only the amino acid sequences of human [46] chicken [61] and Drosophila [62,63] EGF receptors are known.

E G F receptor structure The EGF receptor was first identified as a M r 170,000 plasma membrane protein that could be covalently cross-linked to [125I]EGF [64,65], and it was purified to near homogeneity from A431 cells [10,11]. Subsequently, sequenced peptides from antibody-purified A431 EGF receptors were used to clone A431 and human placental EGF receptor cDNAs [46] such that the complete primary structure of the human EGF receptor was deduced from a series of overlapping cDNA clones [46]. The receptor is synthesized in the form of a 1,210 amino acid precursor, which contains a 24 amino acid N-terminal signal peptide. The mature receptor polypeptide (Fig. 1) is divided into a 621 amino acid extracellular domain and a 542 amino acid cytoplasmic domain by a single 23 residue hydrophobic transmembrane region. The extracellular domain of the receptor, which binds a single molecule of EGF [66], contains 51 cysteine residues arranged in two cysteine-rich regions and 12 potential Nlinked glycosylation sites of which 11 are modified [54,55]. In addition to binding EGF the EGF receptor binds transforming growth factor c~ (TGFo0 [67,68] and an EGF-related protein from vaccinia virus [69,70]. The cytoplasmic domain contains a tyrosine-specific protein kinase [12], which is a member of the src family of kinases

283

1

134

313

446

612

622644

690

940

1186

Fig. 1. Schematic representationof the mature human EGF receptor. Amino acid residues are numbered at the top. In the extracellular

portion of the receptor the cysteine-richregions are hatched, and potential N-linked glycosylationsites are denoted by closed circles. The 23 residue transmembrane region is shaded. The region of the cytoplasmicdomain homologousto the tyrosine kinase of pp60src is stippled. The following amino acid residues are marked: T, protein kinase C substrate Thr654; K, ATP-binding site Lys721; Y, autophosphorylated Tyr residues 1068, 1148 and 1173. The data are from references 12, 46 and 71-73.

[71,72], and three C-terminal autophosphorylation sites [73]. Detailed descriptions o f the structure o f the E G F receptor are provided in references 12 and 72. Monoclonal antibodies are potentially powerful reagents in their ability to yield information about the conformation of molecules in a nondestructive manner. As most o f the E G F receptor monoclonal antibodies that have been made bind to determinants in the extracellular portion of the molecule, the antibody-derived information on receptor structure relates mainly to the ligandbinding domain. This region of the receptor is relatively resistant to protease activity when in intact cells [10,71], and its ability to bind E G F is sensitive to extremes in p H [10]. These characteristics suggested that the native conformation of the extracellular domain o f the receptor included a high degree o f tertiary structure that was important for ligand binding. Further evidence for this idea was provided by cloned receptor c D N A [46], which revealed the two large clusters of extracellular cysteine residues at positions 134-313 and 446-612. Consistent with these results are the observations that a n u m b e r o f the monoclonal antibodies to extracellular peptide determinants do not recognize denatured receptors [14,16,17,27] whereas carbohydrate-specific antibodies react with denatured receptors in immunoblots [ 18,22,23,29,56]. This trend would suggest that 9D6, a rat anti-mouse E G F receptor antibody with an undefined epitope [28], reacts with a carbohydrate determinant. As the antigens for the E G F receptor monoclonal antibodies were characterized (Table 1), it

became apparent that antibodies that inhibited E G F binding recognized peptide determinants while those antibodies with carbohydrate specificities did not inhibit E G F binding [32-35]. Several o f the peptide-specific antibodies, 151 [24], 528 [16,17,75], L A 2 2 [29] and LA58 [29], have been shown to inhibit E G F binding in a competitive manner. EGR/G49, which recognized A L e b and ALeY blood group antigens [35], reduced the affinity for E G F of the predominant population o f receptors but did not block binding. These results indicated that carbohydrate moieties were not directly involved in mediating the binding of E G F to its receptor. The same conclusion was reached by Slieker e t al. [76] who examined the effects o f glycosylation inhibitors and glycosidases on the ligand binding activity of E G F receptors in A431 cells. W h e n N-linked glycosylation was completely inhibited by tunicamycin, the receptors did not acquire the ability to binding EGF. However, the EGF-binding function was acquired prior to the receptors becoming resistant to endoglycosidase H, which occurred as high mannose oligosaccharides were processed to complex structures [77]. In addition, core glycosylated high mannose forms of the receptor retained the ability to bind E G F after treatment with endoglycosidase H. Thus, core glycosylation, but not processing to complex oligosaccharides [76,78], is essential for the acquisition o f ligand binding activity and receptor translocation to the plasma m e m b r a n e [76,78], yet oligosaccharides do not play an intrinsic role in the binding o f EGF. The E G F receptor monoclonal antibodies char-

284 acterized as inhibiting EGF binding (Table 1) were presumed to recognize determinants within or in close proximity to the ligand binding domain and thus to act as steric inhibitors of EGF binding. In support of this supposition, several of the antibodies were inhibited by EGF in binding to cells [17,26,29], and four were demonstrated to be competitive inhibitors of EGF binding [24,29,75]. Most of these inhibitors of binding, however, could not be used to localize the EGF binding domain in the extracellular region of the receptor because their epitopes were conformation-dependent. Recently Wu e t al. [29] made three peptide-specific monoclonal antibodies to A431 EGF receptors that were competitive inhibitors of EGF binding, were inhibited in binding to A431 cells by EGF, and bound to both native and denatured receptors. The epitopes for these antibodies were localized to a sequence of fourteen amino acids (Ala351-Asp364) between the cysteine-rich areas of the extracellular domain of the receptor. The mutually-inhibitory binding properties of the antibodies and EGF suggested that the fourteen amino acid epitope was involved in the formation of the EGF-binding site of the human receptor. Further evidence for this conclusion was provided by the finding that [125I]EGF could be covalently cross-linked to residues within twenty amino acids of the antibody epitope (D. Wu e t al., in press). These results confirmed and extended the previous findings of Lax et al. [79] who used synthetic peptide antibodies to isolate a 250 amino acid CNBr-generated receptor l~agment covalently cross-linked to [125I]EGF. This fragment corresponded to receptor residues 294 to 543, which spanned the entire region between the two cysteine-rich domains. Subsequently, these investigators demonstrated that the EGFbinding properties of the human receptor segregated with the region between the cysteine domains in chimeric chicken/human receptors [80]. This approach was based on the observation that the chicken receptor bound murine EGF with 100-fold lower affinity than did the human receptor [81]. Thus, by several different experimental approaches the region of the receptor between the clusters of cysteine residues, and in particular the

N-terminal portion of this region, has been found to play an important role in binding EGF. In addition to the EGF, the EGF receptor also binds TGFct with high-affinity [67,68]. Although it was found that TGFo~ could be covalently cross-linked to a M r 60,000 molecule as well as the M r 170,000 EGF receptor in A431 cells [82], Carpenter et al. [83] found that TGF~x induced anchorage-independent growth of NRK fibroblasts could be blocked by polyclonal EGF receptor antibodies. These results suggested that the growth effects of TGFct were mediated solely by EGF receptors. However, two recent reports indicate that TGFtx and EGF, which are structurally similar [84] but are only 35% identical in sequence [85,86], may not bind to the EGF receptor in precisely the same manner. Lax e t al. [81] found that although murine EGF bound to the human EGF receptor with 100-fold greater affinity than it did to the chicken EGF receptor, human TGFtx bound equally well to both receptor species. The differences in affinities of EGF and TGFo~ for the chicken receptor were reflected in their abilities to stimulate DNA synthesis in transfectants expressing chicken EGF receptors. Winkler et al. [31 ] raised a monoclonal antibody to A431 EGF receptors that slightly inhibited the binding of EGF to A431 cells but largely inhibited the binding of TGFct by decreasing the number of high-affinity receptors to 70% and reducing receptor affinity for TGFo~ 5 to 10-fold. These authors proposed that the EGF receptor has either spatially distinct binding sites for EGF and TGFct or a single ligand binding site that assumes different conformations. Antibody 13A9 could sterically inhibit TGFct binding under both models, but EGF could act as a steric inhibitor of TGFct binding only under the latter model. The two-site model is less plausible as EGF and several EGF-competitive monoclonal antibodies completely inhibit the binding of [125I]TGFtx to paraformaldehyde-fixed A431 cells, and both [125I]TGFct and [125I]EGF could be cross-linked to monoclonal antibody-reactive EGF receptor fragments of similar sizes (D. Wu and J.D. Sato, unpublished results). Nonetheless, the interesting possibility that receptor binding of EGF and

285 T G F ~ may be mutually exclusive but not equivalent, could explain differences in the biological activities of these growth factors [87,88].

EGF receptor function The binding of EGF to its receptor initiates a variety of immediate and delayed cellular responses which culminate in cell proliferation [7,12]. The delayed responses of DNA synthesis and cytokinesis require cells to be exposed to EGF for at least 8 hours [89-91], and are only detected after 15-24 hours. By contrast, the immediate responses to EGF occur within seconds or minutes of exposure to the growth factor. These responses include amino acid [92] and glucose [93] uptake, increased glycolytic activity [94], ouabain-sensitive N + influx [95], amiloridesensitive Na+/H+ exchange leading to cytoplasmic alkalinization [96-99], Ca 2+ influx [100] and transient increases in intracellular Ca 2+ levels [101-103], phosphatidylinositol synthesis and turnover [ 100,104,105], tyrosine-specific protein phosphorylation [106-110] including receptor autophosphorylation [10,11], and the transient expression of c-fos and c-myc proto-oncogenes [111-114]. The causal relationships, if any, between these events are unclear, however several of these rapid responses such as increases in cellular Ca 2+ levels followed by cytoplasmic alkalinization, phosphatidylinositol breakdown, tyrosine kinase activation and proto-oncogene expression are potential mechanisms by which the mitogenic signal of EGF might be transduced. Upon binding EGF, the distribution of EGF receptors on the cell surface becomes nonhomogeneous within minutes as EGF-receptor complexes diffuse in the plane of the membrane aggregating to form microclusters or patches [53,115-117]. A large proportion but apparently not all of the receptor clusters form over coated pits in the membrane [116,118]. The EGF-receptot complexes are then internalized into endocytotic vesicles [53,115-120] and are delivered to lysosomes where they are degraded [121]. As a consequence of receptor intemalization, the num-

ber of receptors available to EGF on the cell surface is down-regulated by as much as 80--90% [121]. Concomitant with receptor aggregation and internalization cells undergo morphological changes [122,123] in association with changes in the distributions of cytoskeletal proteins [124,125]. To induce a mitogenic response EGF must bind to high-affinity EGF receptors, which represent a minority of the total receptor population. Shechter, et al. [126] observed DNA synthesis in human fibroblasts that were exposed briefly to low concentrations of EGF, washed and incubated in EGF-free medium under conditions in which more than 90% of the cell-bound EGF dissociated; the mitogenic effect of the residual bound EGF was inhibited by EGF antibodies added within an 8 hour period. These results suggested that a very small proportion of EGF receptors were relevant to the biological effects of EGF. King and Cuatrecasas [127] described the existence of high- and low-affinity EGF receptors on KB epidermoid carcinoma cells, and they found that the appearance of high-affinity receptors was temperature-dependent and inhibited by cycloheximide, which suggested that lowaffinity sites were converted into high-affinity sites. The EGF-competitive receptor monoclonal antibody 528 IgG was used to identify a small pre-existing population of high-affinity EGF receptors on fixed A431 cells [16]. The two affinity classes of receptors were found to be functionally distinct in these cells as EGF binding to the high-affinity receptors correlated with increased A431 growth [16] while occupation of the lowaffinity sites, at higher EGF concentrations, correlated with growth inhibition [ 16,128,129]. These two classes of A431 EGF receptors could also be distinguished on the basis of mobility in the plasma membrane: low-affinity receptors labelled with rhodamine-EGF conjugates exhibited rapid lateral diffusion in photo-bleached membranes, whereas the diffusion of high-affinity receptors was undetectable [130]. These differences in mobility may be accounted for by the finding that high-affinity receptors detected with monoclonal antibody 2E9 [26] were preferential-

286 ly associated with Triton X-100-insoluble cytoskeletal elements [131]. A report by Landreth, et al. [132] suggested that more than 50% of the A431 EGF receptors covalently labelled with [125I] EGF were constitutively associated with the cytoskeleton both before and after internalization. One method by which low-affinity EGF receptors could be converted into biologically important high-affinity receptors is receptor oligomerization [133,134]. This hypothesis follows from the observation that CNBr-treated EGF retained receptor-binding activity but did not induce receptor clustering or DNA synthesis in 3T3 cells [ 135]; however, cross-linking EGF-receptor complexes with EGF antibodies restored both cellular responses. The importance of receptor crosslinking in EGF action was further supported by the finding that some EGF receptor monoclonal antibodies induced DNA synthesis in target cells under conditions in which receptor clustering occurred [13,15,23]. Thus, 2G2 IgM [13,15] or 2D1 IgM alone and 2G2 Fab fragments or TL5 IgG cross-linked with anti-mouse Ig antibodies [15] elicited receptor clustering and stimulated D N A synthesis. Neither 2G2 Fab fragments nor TL5 IgG alone were biologically active. These observations raised the possibility that the receptor itself contained the biochemical information necessary to induce the cellular responses triggered by EGF [15,136]. 151 IgG [24] alone stimulated DNA synthesis in quiescent human fibroblasts but had the paradoxical effects of enhancing EGF-induced DNA synthesis while inhibiting EGF binding. This antibody, however, blocked EGF binding to bovine endothelial cells and inhibited EGF-stimulated D N A synthesis in these cells. It should be noted that other receptor antibodies that were tested, including those with carbohydrate [ 17,19,26] or peptide [ 14,16,17,20,26,27,29] determinants, were without intrinsic mitogenic activity. The results obtained with receptor monoclonal antibodies that pointed to a significant role for receptor aggregation in EGF action were complemented by biochemical studies indicating that EGF-induced receptor oligomers had increased affinities and increased kinase ac-

tivity [137-139]. Since its discovery [140,141], the EGF receptor tyrosine kinase has been thought to play a central role in EGF signal transduction [12,142]. Once an article of faith, the requirement of the receptor kinase in EGF action has been demonstrated in functional assays of transfectants bearing EGF receptors made kinase-defective with site-specific mutations (reviewed in [134]). Although receptor kinase activity was indispensable for transducing a mitogenic signal, the three major autophosphorylation sites were found to be unnecessary [143,144] (Fig. 1). These residues, however, may affect the magnitude of the response to EGF (145,146). Receptor monoclonal antibodies with intrinsic mitogenic activity that were examined for their effects on the receptor kinase were found to stimulate kinase activity [13,15,23], while antibodies without mitogenic activity did not activate the receptor kinase [17,19,20,26,27,29]. These results are consistent with the receptor kinase being necessary for mitogenicity. Antibody 13A9 [31] which inhibited the binding of TGF~ but not EGF, stimulated the receptor kinase but was not tested for mitogenic activity. The mitogenic monoclonal antibody 2D1 IgM [23] had the interesting property of enhancing EGF-stimulated receptor kinase activity in A431 cells; this antibody recognized a carbohydrate determinant and did not inhibit EGF binding. Thus, the properties of 2D1 IgM and EGF/G49 [19], which did not inhibit EGF binding but reduced the affinity of low affinity EGF receptors, indicate that receptor activity can be modulated by ligands that do not interact with the EGF-binding site. Although the EGF receptor tyrosine kinase is manifestly essential for the induction of a mitogenic response, receptor kinase activity in itself is not a sufficient signal. Monovalent 2G2-Fab fragments [15], and antibodies 2E9 and 2 D l l [26] activated the receptor kinase but were not mitogenic. In contrast to 2G2-Fab fragments, 2E9 and 2D11 remained non-mitogenic for human fibroblasts even after being cross-linked with antimouse Ig antibodies [26]. These results were in accord with the earlier findings that CNBr-

287 cleaved EGF stimulated receptor kinase activity but was not mitogenic [135,147,148]. Three receptor monoclonal antibodies 2E9, 2 D l l and 2G5 [26], have been tested for the ability to induce in A431 cells the immediate EGF effects of raising intracellular Ca 2+ concentrations, increasing cytoplasmic pH and stimulating phosphatidylinositol turnover. All three antibodies were negative, but 2E9, which inhibited EGF binding, was able to block the induction of these responses by EGF. As each of the antibodies stimulated receptor kinase activity, albeit with differential effects on several known substrates, and were not mitogenic for human fibroblasts, these results suggested that these responses were also required for the transduction of a mitogenic signal. They further suggested that these cellular responses were not direct consequences of receptor kinase activation. The role of the EGF receptor kinase in receptor intemalization is the subject of conflicting experimental results. Two kinase-defective EGF receptor mutants produced by site-directed mutagenesis, one bearing a four amino acid insertion in the kinase domain [149] and the other with an alanine residue substituting for Lys 721, in the ATP binding site [150] (Fig. 1), were reported to be internalized normally by EGF in transfected NIH 3T3 cells. By contrast, a kinase-defective mutant containing a methionine residue in place of Lys 721 and an alanine residue in place of Thr 654, a substrate for protein kinase C (Fig. 1), formed receptor dimers but was not internalized in response to EGF in CHO and mouse L cell transfectants [151]. In addition, anti-phosphotyrosine antibodies injected into individual cells prevented EGF-induced internalization of wild type receptors [151]. The latter results suggested that the phosphorylation of non-receptor substrates was required for EGF receptor internalization. Four EGF receptor monoclonal antibodies have been reported to induce receptor internalization in the absence of a crosslinking antibody without activating the receptor kinase [19,27,152,153]. These results would appear to support the idea that EGF receptor kinase activity is not necessary for receptor internalization. However,

as antibody-induced receptor internalization usually requires saturating concentrations of antibodies [154], this process cannot be considered equivalent to EGF-induced internalization and may occur by a different mechanism.

Effects o f E G F receptor m o n o c l o n a l antibodies o n c a n c e r cell g r o w t h

The idea of using monoclonal antibodies to cell surface receptor molecules, including the EGF receptor, to inhibit or prevent the growth of malignant cells has received support from two lines of research. Studies on the growth requirements of cells in defined serum-free media have shown that individual cell types proliferate in response to different but overlapping sets of growth factors and hormones, in supplemented nutrient medium [155,156]. This knowledge suggested that it was feasible to select receptor monoclonal antibodies that would inhibit the function of required growth factors in a relatively cell type-specific manner. Studies on viral oncogenes have demonstrated homologies between some oncogene products and growth factor receptors [3-6]. The human EGF receptor is homologous to the v-erbB gene product of avian erythroblastosis virus [3,46] and, more distantly, to the neu oncogene product [5,6]. In addition, EGF receptors were found to be overexpressed in squamous cell carcinomas [157-159] and nonneuronal brain tumors [160,161], and the expression of EGF receptors was correlated with malignancy in melanomas [162], breast cancers [163], bladder cancers [164], and gastric carcinomas [165]. Thus, EGF receptors may represent useful target antigens in some types of malignancies. Seven unmodified EGF receptor monoclonal antibodies have been examined for effects on human tumor cell growth in vitro. Four peptidespecific antibodies that inhibited EGF binding to human EGF receptors (528,225,425, and 579) inhibited the growth of A431 cells in culture [16,17,166]. 528 IgG [16,17] also inhibited the growth of Li-7A human hepatoma cells [167] in vitro but had no obvious growth inhibitory effect

288 on four slowly growing glioma cell lines [168]. Antibody 425 [166] had no effect on the growth of colorectal carcinoma cell lines in vitro. Three receptor antibodies to A antigen-related oligosaccharides were also tested on A431 cells with diverse results: 455 IgG had no effect on A431 growth [17]; EGR/G49 inhibited growth [19]; and 2D1 IgM stimulated DNA synthesis by A431 cells [23]. The mechanism by which EGF receptor monoclonal antibodies inhibit tumor cell growth in vitro is unknown. It is possible that the inhibition of A431 growth by receptor antibodies and by EGF [169,170] is related to the overexpression of EGF receptors by these cells [166,171]. However, this possibility would not account for the lack of inhibition by 528 IgG of the growth of glioma cells expressing in excess of 106 EGF receptors per cell [168]. Another possibility is that the EGF receptor antibodies interfere with an autocrine mechanism of tumor cell growth stimulation. The expression of TGFct mRNA has been noted in A431 cells [172], but although low levels of EGF stimulate A431 growth [ 16], TGFtx has not yet been demonstrated to be an autocrine factor for these ceils. However, the TGFtx gene acted as a potent oncogene in NIH 3T3 transfectants expressing high levels of EGF receptors [173], and a correlation between the ectopic expression of EGF and the expression of EGF receptors was found in advanced gastric carcinomas [165]. Five unmodified EGF receptor monoclonal antibodies have been tested for tumoricidal activity in vivo. Antibodies 528, 225 and 455 [16,17] administered from the time of tumor initiation prevented the formation of subcutaneous A431 tumors in Balb/c nude mice [174]. When 528 antibodies were administered from the seventh day following inoculation with A431 cells, tumor growth was inhibited but not abolished. 528 antibodies additionally prevented the formation of tumors by T222 lung epidermoid carcinoma ceils but had no effect on the growth in vivo of Li-7 hepatoma cells or HeLa adenocarcinoma cells [174]. Antibody 425 [27] suppressed the formation of tumors in nude mice by A431 epidermoid

carcinoma cells and SW948 colorectal carcinoma cells but not by SW707 colorectal carcinoma cells [166]. Antibody 108.4 [175] inhibited the growth of KB epidermoid carcinoma tumors in nude mice and prolonged the lifespan of treated animals; this anti tumor activity was enhanced by cisplatin. These results indicate that carbohydrate-specific receptor antibodies such as 455 that do not interfere with EGF binding in addition to those antibodies that block EGF binding can mediate tumoricidal activity in vivo. They further indicate that not all EGF receptor-bearing tumor cells are subject to antibody-mediated cytotoxicity. The mechanisms by which EGF receptor antibodies exert their tumoricidal effects in vivo have been investigated. 528, but not 225 [176] or 425 [166], directed complement-dependent lysis of A431 cells in vitro. Thus, this mechanism of cytotoxicity cannot be solely responsible for the tumoricidal effects of these antibodies. 528 [ 176] and 425 [166] but not 225 [176], effectively mediated the lysis of tumor cells by activated mouse peritoneal macrophages. Antibody 425 also directed the killing of A431 cells and, to a lesser extent, SW948 cells by human monocytes and lymphocytes [166] while 528 did not mediate the lysis of A431 cells by natural killer cells from mouse spleen [176]. These results suggest that host cellular immune responses are involved in the tumoricidal effects of the EGF receptor antibodies, and they are consistent with the finding that antibodies of the IgG2a isotype are most effective in antibody-directed macrophage-dependent cytotoxicity [177]. As 225 IgG 1 did not mediate the lysis of A431 cells by activated macrophages [176], it is possible that effector cells other than macrophages, natural killer cells, or cytotoxic T cells were responsible for the cytotoxic effects of 225 in athymic mice [174]. Recent evidence suggests that EGF receptor monoclonal antibodies of the IgG 1 isotype direct the lysis of A431 cells by polymorphonuclear cells (K. Kishimoto, et al., in preparation). IgG l antibodies 225 [17] and LA1 (L. Wang and J.D. Sato, unpublished results) at 50 nM directed the lysis of A431 cells by polymorphonuclear cells

289 five-fold better than did the IgG2a antibodies 528 and 579 [16,17] at the same concentration; converse results were obtained with macrophages as the effector cells. The significance in preventing t u m o r growth o f the capacity o f some E G F receptor antibodies to block ligand binding and to induce receptor internalization is unclear. E G F receptor m o n o c l o n a l antibodies have also been used to deliver covalently linked toxins to receptor-bearing cancer cells. The toxins that have been conjugated to receptor antibodies were the A subunit o f ricin [25,178,179], doxorubicin [180], and gelonin [181]. All of the receptor antibodies used either partially (B4G7,108) or fully (528,225) inhibited the binding of E G F to h u m a n receptors. The antibody-toxin conjugates were shown to kill a n u m b e r o f h u m a n cancer cell lines in vitro including A431 [179,181] and KB [179,180] epidermoid carcinomas, H e L a adenocarcinoma [178], 547 ovarian carcinoma [179], M D A - 4 6 8 breast carcinoma [179] and NA, Ca922 and TE5 squamous cell carcinomas [181]. In each case but one toxin-induced cell death was specifically mediated by antibody-receptor binding. Doxorubicin conjugated to antibody 108 [180] was less effective than doxorubicin alone in killing KB cells in vitro. However, a highly substituted conjugate was more effective in inhibiting KB tumor growth in vivo than either 108 alone, doxorubicin alone or a mixture o f antibody and toxin 180. In two studies [179,181] the toxicity of the conjugates was correlated with the n u m b e r o f E G F receptors on the target cells, and cells expressing low numbers of receptors or no receptors were relatively unaffected by the immunotoxins. The results described here suggest that E G F receptor m o n o c l o n a l antibodies, either in an unmodified form or covalently linked to toxins, can be used to inhibit or kill some types of malignant cells in vivo. The efficiencies o f these reagents m a y vary according to the antigenic specificities, the binding affinities, and the isotypes of the antibodies and the degree to which the antibodies are internalized by target cells. The cells most susceptible to these agents m a y be those expressing very high levels of E G F receptors or those in

which E G F receptors are involved in autocrine growth stimulation. Although E G F receptor monoclonal antibodies are unlikely to be effective against all malignant cells bearing E G F receptors, the potential of these antibodies as therapeutic agents is b e c o m i n g more firmly established.

Acknowledgements W e thank Julie Lamb, Valerie Oliver and Marilyn Hauer for secretarial assistance and Mari~,a LaDuke for graphic art work. The authors are supported by grants C A 4 0 2 9 4 and C A 3 7 5 8 9 from the National Cancer Institute, and by a Grant-inAid for Scientific Research (61480388) and a special project research grant to O k a y a m a University from the Japanese Ministry of Education, Science and Culture.

References 1. Doolittle RF, Hunkapiller HW, Hood LE, Devare SG, Robbins KC, Aaronson SA and Antoniades HN (1983) Science 221: 275-277. 2. Waterfield MD, Scrace GT, Whittle N, Stroobant P, Johnsson A, Wasteson A, Westermark B, Heldin CH, Huang JS and Deuel TF (1983) Nature 304: 35-59. 3. Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ullrich A, Schlessinger J and Waterfield MD (1984) Nature 370: 521-527. 4. Sherr CJ, Rettenmier CW, Sacca R, Roussel MF, Look AT and Stanley ER (1985) Cell 41: 665-676. 5. Bargmann CI, Hung MC and Weinberg RA (1986) Nature 319: 226-230. 6. YamamotoT, Ikawa S, Akiyama T, Semba K, Nomura N, Miyajima N, Saito T and ToyoshimaK (1986) Nature 319: 230-234. 7. Carpenter G and Cohen S (1979) Ann. Rev. Biochem. 48: 193-216. 8. Cohen S (1987) In vitro Cell. Dev. Biol. 23: 239-246. 9. Cohen S (1962) J. Biol. Chem. 237: 1555-1562. 10. Cohen S, Carpenter G and King L Jr (1980) J. Biol. Chem. 255: 4834-4842. 11. Cohen S, Ushiro H, Stoscheck C and Chinkers M (1982) J. Biol. Chem. 257: 1523-1531. 12. Carpenter G (1987) Ann. Rev. Biochem. 56: 881-914. 13. SchreiberAB, Lax I, Yarden Y, Eshhar Z and Schlessinger J (1981) Proc. Natl. Acad. Sci. USA 78: 7535-7539.

290 14. Waterfield MD, Mayes ELV, Stroobant P, Bennet PLP, Young S, Goodfellow PN, Banting GS and Ozanne B (1982) J. Cell. Biochem. 20: 149-161. 15. Schreiber AB, Libermann TA, Lax I, Yarden Y and Schlessinger J (1982) J. Biol. Chem. 258: 846-853. 16. Kawamoto T, Sato JD, Le A, Polikoff J, Sato GH and Mendelsohn J (1983) Proc. Natl. Acad. Sci. USA 80: 1337-1341. 17. Sato JD, Kawamoto T, Le A, Mendelsohn J, Polikoff J and Sato GH (1983) Mol. Biol. Med. 1: 511-529. 18. Richert ND, Willingham MC and Pastan I (1983) J. Biol. Chem. 258: 8902-8907. 19. Gregoriou M and Rees AR (1984) EMBO J. 3: 929-937. 20. Kudlow JE, Khosravi MJ, Kobrin MS and Mak WW (1984) J. Biol. Chem. 259:11895-11900. 21. Schlessinger J, Lax I, Yarden Y, Kanety H and Libermann TA (1984) In: MF Greaves (ed) Receptors and Recognition (pp. 279-303, vol. 17). Chapman and Hall, London. 22. Parker PJ, Young S, Gullick WJ, Mayes ELV, Bennett P and Waterfield MD (1984) J. Biol. Chem. 259: 99069912. 23. Femandez-Pol JA (1985) J. Biol. Chem. 260: 5003-5011. 24. Chandler LP, Chandler CE, Hosang M and Shooter EM (1985) J. Biol. Chem. 260: 3360-3367. 25. Behzadian MA and Shimizu N (1985) Somatic Cell. Mol. Genet. I 1: 579-591. 26. Defize LHK, Moolenaar WH, van der Saag PT and de Laat SW (1986) EMBO J. 5: 1187-1192. 27. Murthy U, Basu A, Rodeck U, Herlyn M, Kose AH and Das M (1987) Arch. Biochem. Biophys. 252: 549-560. 28. Weller A, Meek J and Adamson ED (1987) Development 100: 351-363. 29. Wu D, Wang L, Sato GH, West KA, Harris WR, Crabb JW and Sato JD (1989) J. Biol. Chem. 264: 17469-17475. 30. Gullick, WJ, Marsden JJ, Whittle N, Ward B, Bobrow L and Waterfield MD (1986) Cancer Res. 46: 285-292. 31. Winkler ME, O'Connor L, Winget M and Fendly B (1989) Biochemistry 28: 6373-6378. 32. Gooi HC, Hounsell EF, Lax I, Kris RM, Libermann TA, Schlessinger J, Sato JD, Kawamoto T, Mendelsohn J and Feizi T (1985) Biosci. Rep. 5: 83-94. 33. Gooi HC, Schlessinger J, Lax I, Yarden Y, Libermann TA and Feizi T (1983) Biosci. Rep. 3: 1045-1052. 34. LePendu J, Fredman P, Richert ND, Magnani JL, Willingham MC, Pastan I, Oriol R and Ginsburg V (1985) Carbohydrate Res. 141: 347-349. 35. Gooi HC, Picard JK, Hounsell EF, Gregoriou M, Rees AR and Feizi T (1985) Mol. Immunol. 22: 689-693. 36. Kohler G and Milstein C (1975) Nature 256: 495-497. 37. Galfre G, Howe SC, Milstein C, Butcher GW and Howard JC (1977) Nature 266: 550-552. 38. Kohler G, Howe SC and Milstein C (1976) Eur. J. Immunol. 6: 292-295. 39. Kearney JF, Radbruch A, Liesgang B and Rajewsky K (1979) J. Immunol. 123: 1548-1550. 40. Schulman M, Wilde CD and Kohler G (1978) Nature 276: 269-270.

41. Kawamoto T, Sato JD, Le A, McClure DB and Sato GH (1983) Anal. Biochem. 130: 445-453. 42. Littlefield JW (1964) Science 145: 709-710. 43. Myoken Y, Okamoto T, Osaki T, Yabumoto M, Sato GH, Takada K and Sato JD (1989) In vitro Cell. Devel. Biol. 25: 477-480. 44. Sato JD, Welsh CJ, Cao HT, Okamoto T and Cabot MC (1988) FASEB J. 2: A579. 45. Sato JD, Cao HT, Kayada Y, Cabot MC, Sato GH, Okamoto T and Welsh CJ (1988) In vitro Cell. Devel. Biol. 24: 1223-1228. 46. Ullrich A, Coussens L, Hayflick JS, Dull TJ, Gray A, Tam AW, Lee J, Yarden Y, Libermann TA, Schlessinger J, Downward J, Mayes ELV, Whittle N, Waterfield MD and Seeburg PH (1986) Nature 309: 418-425. 47. Kris RM, Lax I, Gullick W, Waterfield MD, Ullrich A, Fridkin M and Schlesslnger J (1985) Cell 40: 619-625. 48. Gullick WJ, Downward J and Waterfield MD (1985) EMBO J. 4: 2869-2877. 49. Lax I, Burgess WH, Bellot F, Ullrich A, Schlessinger J and Givol D (1988) Mol. Cell. Biol. 8: 1831-1834. 50. Girard DJ, Aaronson SA, Todaro G J, Amstein P, Kersey JH, Dosik H and Parks WP (1973) J. NCI 51: 1417-1423. 51. Stoscheck CM and Carpenter G (1983) J. Cell. Biochem. 23: 191-202. 52. Fabricant RN, DeLarco JE and Todaro G (1977) Proc. Natl. Acad. Sci. USA 74: 565-569. 53. Haigler HT, Ash JF, Singer SJ and Cohen S (1978) Proc. Natl. Acad. Sci. USA 75: 3317-3321. 54. Mayes ELV and Waterfield MD (1984) EMBO J. 3: 531537. 55. Cummings RD, Soderquist AM and Carpenter G (1985) J. Biol. Chem. 260:11944-11952. 56. Childs RA, Gregoriou M, Scudder P, Thorpe S J, Rees AR and Feizi T (1984) EMBO J. 3: 2227-2233. 57. Basu A, Murthy M, Rodeck U, Heryln M, Mattes L and Das M (1987) Cancer Res. 47: 2531-2536. 58. Feizi T (1984) Biochem. Soc. Trans. 12: 545-549. 59. Feizi T (1985) Nature 314: 53-57. 60. Yarden Y, Harari I and Schlessinger J (1985) J. Biol. Chem. 260: 315-319. 61. Lax I, Johnson A, Howk R, Sap J, Bellot F, Winkler M, Ullrich A, Vennstrom B, Schlessinger J and Givol D (1988) Mol. Cell. Biol. 8: 1970-1978. 62. Livneh E, Glazer L, Segal D, Schlessinger J and Shilo BZ (1985) Cell 40: 599-607. 63. Wadsworth SC, Vincent WS III and Bilodeau-Wentworth D (1985) Nature 314: 178-180. 64. Das M, Miyake T, Fox CF, Pruss RM, Aharonov A and Herschmann HR (1977) Proc. Natl. Acad. Sci. USA 74: 2790-2794. 65. Wrann M and Fox CF (1979) J. Biol. Chem. 254: 80838086. 66. Weber W, Bertics PJ and Gill GN (1984) J. Biol. Chem. 259: 14631-14636. 67. Todaro GJ, Fryling C and DeLarco JE (1980) Proc. Natl. Acad. Sci. USA 77: 5258-5262.

291 68. Massague J (1983) J. Biol. Chem. 258: 13614-13620. 69. Twardzik DR, Brown JP, Ranchalis NE, Todaro GJ and Moss B (1985) Proc. Natl. Acad. Sci. USA 82: 5300--5304. 70. Stroobant P, Rice AP, Gulllck WJ, Cheng DJ, Kerr JM and Waterfield MD (1985) Cell 42: 383-393. 71. Hunter T and Cooper JA (1985) Ann. Rev. Biochem. 54: 897-930. 72. Yarden Y and Ullrich A (1988) Biochemistry 27: 31133119. 73. Downward J, Parker P and Waterfield MD (1984) Nature 311: 483--485. 74. O'Keefe El, Battin TK and Bennett V (1981) J. supramol. Struct. Cell. Biochem. 15: 15-27. 75. Gill GN, Kawamoto T, Cochet C, Le A, Sato JD, Masui M, McLeod C and Mendelsohn J (1984) J. Biol. Chem. 259: 7755-7760. 76. Slieker LJ, Martensen TM and Lane MD (1986) J. Biol. Chem. 261: 15233-15241. 77. Komfeld R and Kornfeld S (1985) Ann. Rev. Biochem. 54: 631-664. 78. Soderquist AM and Carpenter G (1984) J. Biol. Chem. 259: 12586-12594. 79. Lax I, Burgess WH, Bellot F, Ullrich A, Schlessinger J and Givol D (1988) Mol. Cell. Biol. 8: 1831-1834. 80. Lax I, Bellot F, Howk R, Ullrich A, Givol D and Schlessinger J (1989) EMBO J. 8: 421--427. 81. Lax I, Johnson A, Howk R, Sap J, Bellot F, Winkler M, Ullrich A, Vennstrom B, Schlessinger J and Givol D (1988) Mol. Cell. Biol. 8: 1970-1978. 82. Massague J, Czech MP, Iwata K, DeLarco JE and Todaro GH (1982) Proc. Natl. Acad. Sci. USA 79: 6822-6826. 83. Carpenter G, Stoscheck CM, Preston YA and DeLarco JE (1983) Proc. Natl. Acad. Sci. USA 80: 5627-5630. 84. Montelione GT, Winkler ME, Burton LE, Rindernecht E, Sporn MB and Wagner G (1989) Proc. Natl. Acad. Sci. USA 86: 1519-1523. 85. Marquardt H, Hunkapiller MW, Hood LE and Todaro GJ (1984) Science 223: 1079-1082. 86. Derynck R, Roberts AB, Winkler ME, Chen EY and Goeddel DV (1984) Cell 38: 287-297. 87. Schreiber AB, Winkler ME and Derynck R (1986) Science 232: 1250-1253. 88. Ibbotson KJ, Harrod J, Gowen M, D'Souza S, Winkler ME, Derynck R and Mundy GR (1986) Proc. Natl. Acad. Sci. USA 83: 2228-2232. 89. Carpenter G and Cohen S (1976) J. Cell. Physiol. 88: 227-238. 90. Aharonov A, Pruss RM and Herschman HR (1978) J. Biol. Chem. 253: 3970-3977. 91. Shechter Y, Hernaez L and Cuatrecasas P (1978) Proc. Natl. Acad. Sci. USA 75: 5788-5791. 92. Hollenberg MD and Cuatrecasas P (1985) J. Biol. Chem. 250: 3845-3853. 93. Bames D and Colowick SP (1976) J. Cell. Physiol. 89: 633-640. 94. Diamond I, Legg A, Schneider JA and Rozengurt E (1978) J. Biol. Chem. 253: 866--871.

95. Rozengurt E and Heppel LA (1975) Proc. Natl. Acad. Sci. USA 72: 4492-4495. 96. Rothenberg P, Reuss L and Glaser L (1982) Proc. Natl. Aead. Sci. USA 79: 7783-7787. 97. Moolenaar WH, Yarden Y, de Laat SW and Schlessinger J (1982) J. Biol. Chem. 257: 8502-8506. 98. Rothenberg P, Glaser L, Schlessinger P and Cassel D (1983) J. Biol. Chem. 258: 4883-4889. 99. Moolenaar WH, Tsien RY, van der Saag PT and de Laat SW (1983) Nature 304: 645-648. I00. Sawyer ST and Cohen S (1981) Biochemistry 20: 62806286. 101. Moolenaar WH, Tertoolen LGJ and de Laat SW (1984) J. Biol. Chem. 259: 8066-8069. 102. Hesketh TR, Moore JP, Morris JDH, Taylor MV, Rogers J, Smith GA and Metcalfe JC (1985) Nature 313: 481484. 103. Moolenaar WH, Aerts RJ, Tertoolen LGJ and de Laat SW (~c~86) J. Biol. Chem. 261: 279-284. 104. Smith KB, Losonczy I, Sahai A, Pannerselvam M, Fehnel P and Solomon DS (1983) J. Cell. Physiol. 117: 91-100. 105. Wahl M and Carpenter G (1988) J. Biol. Chem. 263: 7581-7590. 106. Hunter TA and Cooper JA (1981) Cell 24: 741-752. 107. Pepinsky RB and Sinclair LK (1986) Nature 321: 81-84. 108. De BK, Misono KS, Lukas TJ, Mroczkowski B and Cohen S (1986) J. Biol. Chem. 261: 13784-13792. 109. Gould KL, Cooper JA, Bretscher A and Hunter T (1986) J. Cell Biol. 102: 660-669. 110. Wahl MI, Nishibe S, Suh PG, Rhee SG and Carpenter G (1989) Proc. Natl. Acad. Sci. USA 86: 1568-1572. 111. Greenberg ME and Ziff EB (1984) Nature 311: 433-438. 112. Kruijer W, Cooper JA, Hunter T and Verma IM (1984) Nature 312: 711-716. 113. Muller R, Bravo R, Burckhardt J and Curran T (1984) Nature 312: 716-720. 114. Bravo R. Burckhardt J, Curran T and Muller R (1985) EMBO J. 4: 1193-1197. 115. Schlessinger J, Shechter Y, Willingham MC and Pastan I (1978) Proc. Natl. Acad. Sci. USA 75: 2659-2663. 116. Gordon P, Carpentier JL, Cohen S and Orci L (1978) Proc. Natl. Acad. Sci. USA 75: 5025-5029. 117. Haigler HT, McKanna JA and Cohen S (1979) J. Cell Biol. 81: 382-395. 118. Carpentier JL, Gordon F, Anderson RG, Goldstein JL, Brown MS, Cohen S and Orci L (1982) J. Cell Biol. 95: 73-77. 119. Hanover JA, Willingham MC and Pastan I (1984) Cell 39: 283-293. 120. Carpentier JL, White MF, Orci L and Kahn RC (1987) J. Cell Biol. 105: 2751-2762. 121. Carpenter G and Cohen S (1976) J. Cell Biol. 71: 159171. 122. Chinkers M, McKanna JA, Cohen S (1979) J. Cell Biol. 83: 260-265. 123. Chinkers M, McKanna JA and Cohen S (1981) J. Cell Biol. 88: 422-429.

292 124. Schlessinger J and Geiger B (1981) Exp. Cell Res. 134: 273-279. 125. Bretscher A (1989) J. Cell Biol. 108: 921-930. 126. Shechter Y, Hemaez L and Cuatrecasas P (1978) Proc. Natl. Acad. Sci. USA 75: 5788-5791. 127. King AC and Cuatrecasas P (1982) J. Biol. Chem. 257: 3053-3060. 128. Gill GN and Lazar CS (1981) Nature 293: 305-309. 129. Barnes DW (1982) J. Cell. Biol. 93: 1-4. 130. Rees AR, Gregoriou M, Johnson P and Garland PB (1984) EMBO J. 3: 1843-1847. 131. Wiegant FAC, Blok FJ, Defize LHK, Linnemans WAM, Verkley AJ and Boonstra J (1986) J. Cell. Biol. 103: 87-94. 132. Landreth GE, Williams LK and Rieser GD (1985) J. Cell. Biol. 101: 1341-1350. 133. Schlessinger J (1986) J. Cell. Biol. 103: 2067-2072. 134. Schlessinger J (1988) Biochemistry 27: 3119-3123. 135. Schechter Y, Hernaez L, Schlessinger J and Cuatrecasas P (1979) Nature 278: 835-838. 136. Hapgood J, Libermann TA, Lax I, Yarden Y, Schreiber AB, Naor Z and Schlessinger J (1983) Proc. Natl. Acad. Sci. USA 80:6451-6455. 137. Yarden Y and Schlessinger J (1984) Biochemistry 26: 1434-1442. 138. Yarden Y and Schlessinger J (1987) Biochemistry 26: 1443-1451. 139. Boni-Schnetzler M and Piltch PF (1987) Proc. Natl. Acad. Sci. USA 87: 7832-7836. 140. Carpenter G, King L Jr and Cohen S (1978) Nature 276: 409-410. 141. Carpenter G, King L Jr and Cohen S (1979) J. Biol. Chem. 254: 4884-4891. 142. Gill GN, Bertics PJ and Stanton JB (1987) Mol. Cell. Endocrinol. 51: 169-186. 143. Livneh E, Prywes R, Kashles O, Reiss N, Sasson I, Mory Y, Ullrich A and Schlessinger J (1986) J. Biol. Chem. 261: 12490-12497. 144. Clark S, Cheng DJ, Hsuan JJ, Haley JD and Waterfield MD (1988) J. Cell. Physiol. 134: 421-428. 145. Bertics PJ, Chen WS, Hubler L, Lazar CS, Rosenfeld MG and Gill GN (1988) J. Biol. Chem. 263: 3610-3617. 146. Velu TJ, Vass WC, Lowy DR and Beguinot L (1989) Mol. Cell. Biol. 9: 1772-1778. 147. Schreiber AB, Yarden Y and Schlessinger J (1981) Biochem. Biophys. Res. Comm. 101: 517-523. 148. Yarden Y, Schreiber AB and Schlessinger J (1982) J. Cell. Biol. 92: 687-693. 149. Livneh E, Reiss N, Berent E, Ullrich A and Schlessinger J (1987) EMBO J. 6: 2669-2676. 150. Honegger AM, Dull TJ, Felder S, Van Obberghen E, Bellot F, Szapary D, Schmidt A, Ullrich A and Schlessinger J (1987) Cell 51: 199-209. 151. Glenney JR Jr, Chen WS, Lazar CS, Walton GM, Zokas LM, Rosenfeld MG and Gill GN (1988) Cell 52: 675684.

152. Le A, Mendelsohn J, Sato GH, Sunada H and MacLeod C (1985) In: Murakami H, Yamane I, Barnes DW, Mather JP, Hayashi I and Sato GH (eds) Growth and Differentiation of Cells in Defined Environment (pp. 303-306), Springer-Verlag, New York. 153. Sunada H, Magun BE, Mendelsohn J and MacLeod CM (1986) Proc. Natl. Acad. Sci. USA 83: 3825-3829. 154. Sato JD, Le A and Kawamoto T (1987) Meth. Enzymol. 146: 63-81. 155. Barnes DW and Sato GH (1980) Cell 22: 649-655. 156. Barnes DW (1987) Biotechniques 5: 534-542. 157. Hendler FJ and Ozanne BW (1984) J. Clin. Invest. 74: 647-651. 158. Cowley G, Smith JA, Gusterson B, Hendler F and Ozanne B (1984) In: Levine A, Vande Woude G, Topp W and Watson JD (eds) Cancer Cells (pp. 5-10), Cold Spring Harbor Laboratory, New York. 159. Hunts J, Ueda M, Ozawa S, Abe O, Pastan I and Shimizu N (1985) Jpn. J. Cancer Res. 76: 663-666. 160. Libermann TA, Razon N, Bartal AD, Yarden Y, Schlessinger J and Soreq H (1984) Cancer Res. 44: 753-760. 161. Libermann TA, Nusbaum HR, Razon N, Kris R, Lax I, Soreq H, Whittle N, Waterfield MD, Ullrich A and Schlessinger J (1985) Nature 313: 144-147. 162. Koprowski H, Herlyn M, Balaban G, Parrniter A, Ross A and Nowell P (1985) Somat. Cell. Mol. Genet. 11: 297302. 163. Sainsbury JRC, Famdon JR, Sherbet GV and Harris AL (1985) Lancet 1: 364-366. 164. Neal DE, Marsh D, Bennett MK, Abel PD, Hall RR, Sainsbury JRC and Harris AL (1985) Lancet 1: 366-368. 165. Yasui W, Hata J, Yokozaki H, Nakatani H, Ochiai A, Ito H and Tahara E (1988) Int. J. Cancer 41:211-217. 166. Rodeck U, Herlyn M, Herlyn D, Molthoff C, Atkinson B, Varello M, Steplewski Z and Koprowski H (1987) Cancer Res. 47: 3692-3696. 167. Knowles AF (1988) J. Cell. Physiol. 134: 109-116. 168. Wemer MH, Humphrey PA, Bigner DD and Bigner SH (1988) Acta Neuropathol. 77: 196-201. 169. Gill GN and Lazar CS (1981) Nature 293: 305-307. 170. Barnes DW (1982) J. Cell. Biol. 93: 1--4. 171. Kawamoto T, Mendelsohn J, Le A, Sato GH, Lazar CS and Gill GN (1984) J. Biol. Chem. 259: 7761-7766. 172. Derynck R, Goeddel DV, Ullrich A, Gutterman JU, Williams RD, Bringman TS and Berger WH (1987) Cancer Res. 47: 707-712. 173. DiMarco E, Pierce JH, Fleming TP, Kraus MH, Molloy CJ, Aaronson SA and DiFiore PP (1989) Oncogene 4: 831-838. 174. Masui H, Kawamoto T, Sato JD, Wolf B, Sato G and Mendelsohn J (1984) Cancer Res. 44: 1002-1007. 175. Aboud-Pirak E, Hurwitz E, Pirak ME, Bellot F, Schlessinger J and Sela M (1988) J. Natl. Cancer Inst. 80: 16051611. 176. Masui H, Moroyama T and Mendelsohn J (1986) Cancer Res. 46: 5592-5598.

293 177. Steplewski Z, Herlyn D, Maul G and Koprowski H (1983) Hybridoma 2: 1-5. 178. Vollmar AM, Banker DE, Mendelsohn J and Herschman HR (1987) J. Cell. Physiol. 131: 418-425. 179. Taetle R, Honeysett JM and Houston LL (1988) J. Natl. Cancer Inst. 80: 1053-1059. 180. Aboud-Pirak E, Hurwitz E, Bellot F, Schlessinger J and Sela M (1989) Proc. Natl. Acad. Sci. USA 86: 3778-3781. 181. Ozawa S, Ueda M, Ando N, Abe O, Minoshima S and Shimizu N (1989) Int. J. Cancer 43: 152-157.

Address f o r offprints: J.D. Sato, W. Alton Jones Cell Science Center, 10 Old Barn Road, Lake Placid, NY 12946, USA Note added in proof: Defize et al. (J. Cell Biol. 109: 2495-2507, 1989) and Bellot et al. (J. Cell Biol. 110: 491-501, 1990) have used monoclonal EGF receptor antibodies to confirm the functional significance of high affinity EGF receptors.