Journal of Clinical Immunology, Vol. 11, No. 3, 1991 Special Article Monoclonal Antibody Therapy of Human Cancer: Taking the HER2 Protooncogene to the Clinic H, MICHAEL SHEPARD, ~'4 GAlL D. LEWIS, 1 JAY C. SARUP, ~ BRIAN M. FENDLY, l DANIEL MANEVAL,I JOYCE MORDENTI,1 IRENE FIGARI, 1 CLAIRE E. KOTTS, 1 MICHAEL A. PALLADINO, JR., 1 AXEL ULLRICH, 2 and DENNIS SLAMON 3 Accepted: January 22, t991 The HER2 protooncogene encodes a 185-kDa transmem- brane protein (p185 HER2) with extensive homology to the epidermal growth factor (EGF) receptor. Clinical and experimental evidence supports a role for overexpression of the HER2 protooncogene in the progression of human breast, ovarian, and non-small cell lung carcinoma. These data also support the hypothesis that p185 HER2 present on the surface of overexpressing tumor cells may be a good target for receptor-targeted therapeutics. The anti- p185 HER2 murine monoclonal antibody (muMAb) 4D5 is one of over 100 monoclonals that was derived following immunization of mice with cells overexpressing p185 hER2. The monoclonat antibody is directed at the extracellular (ligand binding) domain of this receptor tyrosine kinase and presumably has its effect as a result of modulating receptor function. In vitro assays have shown that muMAb 4D5 can specifically inhibit the growth of tumor cells only when they overexpress the HER2 pro- tooncogene. MuMAb 4D5 has also been shown to en- hance the TNF-a sensitivity of breast tumor cells that overexpress this protooncogene. Relevant to its clinical application, muMAb 4D5 may enhance the sensitivity of p185HER2-overexpressing tumor cells to cisplatin, a chemotherapeutic drug often used in the treatment of ovarian cancer. In vivo assays with a nude mouse model have shown that the monoclonal antibody can localize at ~Department of Developmental Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080. 2Max Planck Institute for Biochemistry, Martinsreid, Germany. 3Department of Hematology and Oncology, University of Cali- fornia, Los Angeles, California 90024. 4To whom correspondence should be addressed. 117 the tumor site and can inhibit the growth of human tumor xenografts which overexpress p185 rmR2. Modulation of p185 r~ER2 activity by muMAb 4D5 can therefore reverse many of the properties associated with tumor progression mediated by this putative growth factor receptor. To- gether with the demonstrated activity of muMAb 4D5 in nude mouse models, these results support the clinical application of muMAb 4D5 for therapy of human cancers characterized by the overexpression of p185 I-mR2. KEY WORDS: HER2; neu; TNF-et; monoclonal antibody ther- apy. BACKGROUND: THE HER2 PROTOONCOGENE AND HUMAN CANCER Cellular protooncogenes encode proteins that are thought to regulate normal cellular proliferation and differentiation. Alterations in their structure or am- plification of their expression lead to abnormal cellular growth and have been associated with car- cinogenesis (1-4). Protooncogenes were first iden- tified by either of two approaches. First, molecular characterization of the genomes of transforming retroviruses showed that the genes responsible for the transforming ability of the virus in many cases were altered versions of genes found in the genomes of normal cells. The normal version is the protoon- cogene, which is altered by mutation to give rise to the oncogene. An example of such a gene pair is represented by the EGF receptor and the v-erbB gene product. The virally encoded v-erbB gene product has suffered truncation and other alter- 0271-9142DU0501)-0117506.50/0 © 1991 Plenum Publishing Corporation
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`118 SHEPARD ET AL. ations that render it constitutively active and endow it with the ability to induce cellular transformation (5). The second method for detecting cellular trans- forming genes that behave in a dominant fashion involves transfection of cellular DNA from tumor cells of various species into nontransformed target cells of a heterologous species. Most often this was done by transfection of human, avian, or rat DNAs into the murine NIH 3T3 cell line (1-5). Following several cycles of genomic DNA isolation and re- transfection, the human or other species DNA was molecularly cloned from the murine background and subsequently characterized. In some cases, the same genes were isolated following transfection and cloning as those identified by the direct character- ization of transforming viruses. In other cases, novel oncogenes were identified. An example of a novel oncogene identified by this transfection assay is the neu oncogene. It was discovered by Weinberg and colleagues in a transfection experiment in which the initial DNA was derived from a carcino- gen-induced rat neuroblastoma (6,7). Characteriza- tion of the neu oncogene revealed that it had the structure of a growth factor receptor tyrosine ki- nase, had homology to the EGF receptor, and differed from its normal counterpart, the neu pro- tooncogene, by an activating mutation in its trans- membrane domain (8). The association of the HER2 protooncogene with cancer was established by yet a third approach, that is, its association with human breast cancer. The HER2 protooncogene was first discovered in cDNA libraries by virtue of its homology with the EGF receptor, with which it shares structural similarities throughout (5). When radioactive probes derived from the cDNA sequence encoding p185 hER2 were used to screen DNA samples derived from breast cancer patients, amplification of the HER2 protoon- cogene was observed in about 30% of patient sam- ples (9). Further studies have confirmed this origi- nal observation and extended it to suggest an important correlation between HER2 protoonco- gene amplification and/or overexpression and wors- ened prognosis in ovarian cancer and non-small cell lung cancer (I0-14). The association of HER2 amplification/overex- pression with aggressive malignancy, as described above, implies that it may have an important role in progression of human cancer; however, many tu- mor-related cell surface antigens have been de- scribed in the past, few of which appear to have a direct role in the genesis or progression of disease (15,16). The data which support a role of HER2 overexpression in the basic mechanisms of human cancer are summarized below. Amplified expression of p185 HERz can lead to cellular transformation as assessed by morphologi- cal alterations and growth of p185nERZ-overex - pressing cells in soft agar and in nude mice (17,18). In addition, NIH 3T3 fibroblasts overexpressing p185 ~IER2 have an increased resistance to cytotox- icity mediated by activated macrophages or recom- binant human TNF-et (19), the cytokine that ap- pears to be mainly responsible for macrophage- mediated tumor cell cytotoxicity (20). This observation extends also to breast tumor cells, which overexpress p185 nER2 (19), and suggests that high levels of p185 HER2 expression may be related to tumor cell resistance to at least one component of the host's antitumor surveillance armamentarium, the activated macrophage. This work has been reviewed previously (21), and similar data have recently been reported for ovarian tumor cell lines which overexpress p185 I-IER2 (22). Further support for a role of p185 HER2 or the related neu oncogene- encoded tyrosine kinase in tumorigenesis comes from work with transgenic mice that have been manipulated to overexpress one or the other of these two related genes. Transgenic mice express- ing the activated form of the rat neu protooncogene, under the control of a steroid inducible promoter, uniformly develop mammary carcinoma (23). In another transgenic mouse model the HER2 pro- tooncogene product, "activated" by point mutation analogous to the rat neu oncogene product, or an unaltered form of the HER2 protooncogene, has been expressed in mice (24). The main malignancies induced in this model were either lung adenocarci- noma or lymphoma but not mammary carcinoma. While it is not known why the different transgenic mouse models give such distinct results, the latter model may be of particular significance given the recent report of an association between p185 HER2 overexpression and poor prognosis in nonsmall cell lung cancer (14). These differing results suggest some difference in the activity of activated neu and HER2-encoded tyrosine kianses, although effects due to mouse strain differences cannot be excluded. The structural similarities between p185 HER2 and the EGF receptor suggest that function of p 185 HER2 may be regulated similarly to the EGF receptor. In particular, one expects that the tyrosine kinase activity associated with the cytoplasmic domain of Journal of Clinical Immunology, Vol. 11, No. 3, 1991
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`MONOCLONAL ANTIBODY THERAPY OF HUMAN CANCER 119 TNF R Ill[ - ,7 HER2 I11tt11 11111} HER2 I Ligandi ltll lltlllll I Iiib,._ ~ Tumor Cytotoxic Response Suppression of Cytotoxic Response Cellular "- "- "- Proliferation Fig. 1. Suppression of the TNF cytotoxic response by activation of p185 HER2. The schematic shows both the TNF cytotoxic pathway (top) and the p185HER2-stimulated cell proliferation/transformation pathway (bottom). Signaling from the TNF receptor following interaction with TNF has not been characterized. Binding of ligand to p185 r~ER2 is shown to activate the receptor-associated tyrosine kinase activity, resulting in stimulation of cellular proliferation and suppression of the tumor cell cytotoxic response to TNF. the receptor would be ligand activated. This pro- posal receives support from recent work describing a ligand for p185 hER2 (25). These data lead to a model (Fig. 1) wherein antagonists that downregu- late the function of p185 r~ER2 should have the effect of inhibiting growth of tumor cells dependent upon p185 rtER2 function and of increasing the sensitivity of such tumor cells to TNF-ct. By analogy with previous work done with two related tyrosine ki- nases, the EGF receptor (26) and the activated neu protooncogene product (27), we hypothesized that monoclonal antibodies targeted to the extraceUular domain of p185 HER2 may have the desired proper- ties. DERIVATION OF muMAb 4D5 A family of monoclonal antibodies focused against the extracellular domain of p185 HER2 were prepared (28). To do this, an NIH 3T3 fibroblast cell line that overexpresses p185 HER2 [NIH 3T3/HER2- 3-400 (18)] was used to immunize BALB/c mice. The mice were subsequently boosted with NIH 3T3/HER2-3-400 and, finally, with a preparation enriched for pi85 HER2 by wheat germ agglutinin chromatography of membrane extracts of this cell line. Following splenocyte fusion with a mouse myeloma partner, the hybridomas were cultured in 96-well microtiter plates. Hybridomas positive for anti-p185 HERa activity, but with little or no anti- EGFR activity, were detected by ELISA (Fig. 2). A critical property of an anti-p185 nERz monoclonal antibody with potential for therapy would be its lack of cross-reactivity with the closely related EGF receptor, which is expressed at elevated levels in multiple tissues (29). To select further monoclo- nal antibodies with this characteristic, a number of assays were performed, including immunoprecipita- tion assays utilizing in vivo labeled EGF receptor and p185 HER2 (Fig. 3A) and FACS analysis of antibody binding to tumor cells overexpressing ei- ther p185 ttER2 or the EGFR (Fig. 3B). The screen- ing results are summarized in Table I. Based upon these results, nine of the p185 HERE monoclonal antibodies were chosen for further characterization, including a cell growth inhibition assay utilizing the SK-BR3 human breast adenocarcinoma cell line, which greatly overexpress p185 HER2. The monoclo- Journal of Clinical Immunology, Vol. 11, No. 3, 1991
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`120 SHEPARD ET AL. t.8 t.4 1.2 E t- 1.0 03 0.8 d d 0.4 0.2 0.0 2C4 7C2 7F3 2Hll 3H4 3E8 4D5 588 6E9 703 5B6 40,1H1 MAb (1 ug/ml) Fig. 2. ELISA screening of anti-p185 HER2 monoclonal antibodies. Results shown measure the relative reactivities of the purified anti-p185 HER2 monoclonal antibodies (added to 1 I~g/ml) with membrane extracts enriched in EGF receptor (open bars; from A431 squamous carcinoma cells) or enriched in p185 HER2 (filled bars; from NIH 3T3/HER2-3-400). nal antibody, muMAb 4D5, was clearly the most effective of the group in this assay (Table II). The initial results characterizing the growth in- hibitory activity of these monoclonal antibodies were extended by comparing them for activity against a battery of human breast and ovarian tumor cell lines that expressed varying levels of p185 HER2. These results reveal that the monoclonal antibodies can be growth inhibitory, they may have no affect on cell proliferation, or they may stimulate the proliferation of breast tumor cells. Growth inhibi- tion appears to depend upon overexpression (Table III). This property, in particular, is shared by the monoclonal antibodies 4D5 and 3H4. These mono- clonal antibodies may exert their effects on cell growth by similar mechanisms since they compete for binding to the receptor (Tables I and III) (28) and, therefore, may recognize the same or overlap- ping epitopes. The other monoclonal antibodies vary in their ability to inhibit proliferation, but 7C2 and 6E9 are consistently less active in this respect, The potent growth inhibitory activity of 2C4 for MDA-MB-175 breast tumor cells is not understood at present but may represent cross-reactivity with another receptor expressed on these cells. Simi- larly, the properties that distinguish 7C2 from the other antibodies with regard to its ability to stimu- late the proliferation of several of the tumor cell lines shown in Table III has not been determined. The 6E9 monoclonal antibody has been shown to bind to the extracellular domain of p185 HER2, al- though only to a subset of receptors on the surface of SK-BR-3 tumor cells (30). The functional signif- icance of this subset of receptors is unclear. In addition to its activity on breast tumor cells, which overexpress p185 HERz, muMAb 4D5 is also clearly the most active of the monoclonal antibodies with respect to its ability to inhibit growth of SKOV-3, a human ovarian adenocarcinoma cell line that over- expresses p185 FIERz (Table III). Experiments are currently planned to try to understand in more detail how these monoclonal antibodies may exert Journal of Clinical Immunology, Vol. ll, No. 3, 1991
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`MONOCLONAL ANTIBODY THERAPY OF HUMAN CANCER 121 B. \ _--° ;j' 0 0 O~ E . £ 0.1 1 10 1'0 2 | ! 10 3 10 4 Relative Fluorescence Intensity Fig. 3. MuMAb 4D5 does not cross-react with the EGFR. (A) Immunoprecipitation of metabolically labelelled NIH 3T3 cells transfected by control plasmid (CVN), by a plasmid encoding the EGFR (HER1) or a plasmid encoding p185 HER2 (HER2). MuMAb 40.1.H1 is directed against hepatitis B surface antigen (lanes 1, 4, 7); muMAb 4D5 is directed against p185 HER2 (lanes 2, 5, 8); muMAb 108 is directed against the EGFR (lanes 3, 6, 9). (B) Fluorescence-activated cell sorter histograms of muMAb 40.1.HI (solid lines) or muMAb 4D5 (dotted lines) reacted with SK-BR-3 tumor cells (approx. 2 × 10 6 receptors per cell; upper panel) or the same antibodies reacted with the A431 squamous carcinoma cell line (approx. 2 × 106 EGFR per cell; lower panel). distinct effects on tumor cell proliferation. The in vitro results summarized in Table III clearly show that when the monoclonal antibodies are compared for efficacy, as measured by their abilities to inhibit growth of breast and ovarian tumor cells overex- pressing p185 HER2, muMAb 4D5 is usually the most potent and is therefore a good candidate for further characterization in other models that may be pre- dictive of its efficacy in human clinical trials. Inter- estingly, the most dramatic activity of the antibody is seen in cell lines that overexpress greater than fivefold the level observed in MCF-7 breast tumor cell lines [a low expressor control cell line; Table III (19)]. Patients who overexpress greater than five- fold the normal level of p185 HER2 have been shown to have a very poor prognosis (I0). These results will aid in choosing patients who are most likely to respond in clinical trials. The model depicted in Fig. 1 predicts that down- regulation ofp185 HER2 by a monoclonal antibody or other reagent should result in decreased cellular proliferation, as shown in Table III, but also in- creased sensitivity to TNF-o~. Results of experi- ments in which tumor cells overexpressing p185 HERz were treated with muMAb 4D5 or a control monoclonal antibody, alone and in combi- nation with TNF-ct, suggest the validity of this model (Fig. 4) (31). MuMAb 4D5 treatment of breast tumor cells overexpressing p185 HER2 re- sulted in enhanced sensitivity of these cells to TNF-m The growth and the TNF-tx sensitivity of normal cells or tumor cells that do not overexpress the receptor were unaltered. In addition to the relationship between TNF-a resistance and p185 HER2 overexpression, a possible relationship between protoncogene expression and resistance to the chemotherapeutic drug cisplatin has been investigated. A correlation between HER2 protooncogene overexpression and resistance to chemotherapeutic drugs rests on the grounds that Journal of Clinical Immunology, Vol. 11, No. 3, 1991
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`122 SHEPARD ET AL. Table I. Summary Table of Monoclonal Antibodies Described ELtSA a RIP b MAb Isotype EGFR p 185/-IER2 EGFR p185 HER2 Epitope c FACS a 4D5 IgG! ,k - + + - + + I(p/c) + + + 2C4 IgG1 ,k - + + + - + + F(p/c) + + + 2Hll IgG2a,k - + - + + H(p/c) + + 3E8 IgG2a,k - + - + + + H(p/c) + + + 3 H4 IgG 1,k - + - + I(p) + SB8 IgG1 ,k - + - + + nd(p) + 6E9 IgGl,k - + + - + nd(p) - 7C2 IgGl ,k - + + - + + G(p) + + + 7D3 IgGl,k - + + - + + F(p/c) + + + 7F3 lgGl,k - + + + - + + + G/F(p/c) + + + ~Summary of OD 492 rim: (-) <0.1; (+) 0.11-0.50; (++) 0.51-1.0; (+++) >1.0. bSummary of autoradiography from immunoprecipitations: (-) bands equal to negative control; (+) weak bands but darker than negative control; (++) moderately exposed bands; (+++) strongly exposed bands. CLetters were assigned to represent individual epitopes A through I (nd, not done). MAbs were considered to share an epitope if each blocked binding of the other by 50% or greater in comparison to an irrelevant MAb control. The epitope composition recognized by immunoprecipitations with each MAb from tunicamycin-treated cells is shown. The letters p, c, or p/c in parentheses indicate that the monoclonal antibody binds only to the polypeptide (p), the carbohydrate (c), or both (p/c) moieties in the extracellular domain of p185 HERz. dFluorescence staining of SK-BR-3 cells by the anti-pl85 HFR2 monoclonal antibodies: (-) MAbs equal to the negative control MAbs; (+) 1- to 9-fold higher than the negative controls; (+ +) 10- to 99-fold higher than the negative controls; (+ + +) > 100-fold higher than the negative controls. patients exhibiting overexpression appear to have a worsened prognosis, especially in ovarian cancer (10, 13). In addition, recent work with the EGF receptor (32) has indicated that when the anti- EGFR monoclonal antibody 108.4 was added to- gether with cisplatin, the antitumor effect of the antibody was greatly enhanced. Because the 108.4 monoclonal antibody and muMAb 4D5 appear to share the ability to inhibit soft agar growth of tumor Table lI. Inhibition of SK-BR-3 Proliferation by Anti-p185 HERz Monoclonal Antibodies ~ Monoclonal Relative cell antibody proliferation b 4D5 44.2 +_ 4.4 7C2 79.3 -- 2.2 2C4 79.5 +- 4.4 7D3 83.8 -+ 5.9 3E8 66.2 + 2.4 6E9 98.9 + 3.6 7F3 62.1 - 1.4 31-14 66.5 _+ 3,9 2Hll 92.9 + 4.8 40.1 H1C 105.8 ± 3.8 4F4 94.7 _+ 2.8 aSK-BR-3 breast tumor cells were plated at a density of 4 x 104 cells per well into 96-weU microdilution plates, allowed to adhere, and then treated with monoclonal antibody (10 ~g/ml). bRelative cell proliferation was determined by crystal violet staining of the monolayers after 72hr. Values are expressed as a percentage of results with untreated control cultures (100%). cControl monoclonal antibodies 40.1H1 and 4F4 are directed against hepatitis B surface antigen and human interferon-~, respectively (27). cells overexpressing their respective receptors, it seemed possible that such an interaction may also occur in the HER2 protooncogene system. The in vitro results (Fig. 5) show that treatment of SK- BR-3 breast tumor cells with muMAb 4D5 enhances their sensitivity to cisplatin. IN VIVO PRECLINICAL EFFICACY A critical part of the rationale supporting the application of muMAb 4D5 to human cancer ther- apy is its ability to inhibit the growth of tumor cells overexpressing p185 rIERz in vivo. A human tumor xenograft model was used to test this property of muMAb 4D5 and to compare its activities with those of the other monoclonal antibodies in a rele- vant model of human disease. In this model, a human breast tumor, characterized with respect to HER2 protooncogene amplification and expression, was grafted into the subrenal capsules of nude mice. Therapy was initiated 1 week postimplantation. In order to be active in this model, the monoclonal antibody must be able to localize to the overex- pressing tumor cells in the lesion and subsequently exert a growth regulatory effect mediated through p185 HER2. Growth inhibition occurs only with tu- mors that overexpress the receptor. Heterotrans- plants (approximately 1 nag) of Murray breast tumor [a high expresser of the HER2 gene product (I0)] were implanted into the subrenal capsule of 48 Journal of Clinical Immunology, Vol. 11, No. 3, 1991
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`MONOCLONAL ANTIBODY THERAPY OF HUMAN CANCER 123 Table IIL Inhibition of Human Breast and Ovarian Tumor Cell Growth by Monoctonal Antibodies Directed Against the Extracellular Domain of p185 HER2 Relative p185 HzRz Cell line expression ~ 4DU Cell proliferation (% control) b 3H4 c 2C4 't 7F3 a 7C2 e 6E9 r MCF7 1 94 101 101 97 I06 110 ZR-75-I 3 106 113 104 100 149 t 13 MDA-MB-175 4 61 84 24 48 87 103 MDA-MB-453 7 62 68 91 84 78 t01 MDA-MB-361 17 60 68 65 73 113 113 BT474 20 23 25 53 20 74 94 SK-BR-3 33 42 56 66 64 92 105 SK-OV-3 17 77 85 87 91 97 99 ~Based on FACS assay using muMAb 4D5 and fluorescence-labeled goat anti-murine IgG1 polyclonal antibody. °Five-day assay with 10 ~g/ml of indicated monoclonal antibody (SE, -10%). Other methods as described in the footnotes to Table II. C4D5 and 3H4 define epitope "I." d2C4 and 7F3 will partially block one another, 2C4 is assigned epitope "F," and 7F3 is assigned epitope "F/G." e7C2 defines epitope "G" and will partially block 7F3 binding. T6E9 epitope determination not done. athymic mice on day 0. Groups of eight animals were injected intravenously with tissue culture- derived muMAb 4D5 (36.4 mg/kg), PBS, or control monoclonal antibody, muMAb 5B6 (directed against gpl20; 36.4 mg/kg), as single agents in equally divided doses on days 7, 10, and 13. Four mice from each group were sacrificed on day 20, and the remainder of the animals were sacrificed on day 34. Tumor sizes were measured using ocular micrometer and gravimetric techniques. A sum- mary of the tumor weights (mean - SD) from animals sacrificed on days 20 and 34 is shown in Table IV. On day 20, average tumor weights of animals receiving muMAb 4D5 were significantly less than those receiving the same dose of the control antibody muMAb 5B6. Interactive effects between muMAb 4D5 and cisplatin have also been observed in this model (33). These studies in athy- mic mice bearing human breast tumor xenografts have demonstrated efficacy and suggested an en- hanced effect when muMAb 4D5 is given in combi- nation with cisplatin. MECHANISM OF ACTION The results described above are consistent with muMAb 4D5 having receptor antagonist activity. Surprisingly, however, muMAb 4D5 treatment of SK-BR3 tumor cells stimulates receptor tyrosine kinase activity (Table V) (30, 34). In addition, it can mediate the phosphorylation of intracellular sub- strates by p185 HER2 (34). Consistent with its ability to stimulate receptor activity, muMAb 4D5 treat- ment of SK-BR-3 or SK-OV-3 tumor cells results in a modulation of intracellular second messengers, including diacylglycerol. Diacylglycerol (DAG) is a product of phospholipase C breakdown of phos- phatidylinositol-4,5-bisphosphate. It is a cofactor Fig. 4. Monoclonal antibody 4D5 sensitizes breast tumor cells to the cytotoxic effects of TNF-a. Filled bars, cell number at initiation of the assay; dark cross-hatching, untreated control; dark stipples, TNF-<x alone; light cross-hatching~ MuMAb 4D5; open bars, MuMAb 4D5 combined with TNF-a. (B) Lack of growth inhibition of SK-BR3 tumor cells by muMAb 40.1 H1 (anti-hepatitis B antigen; light stipples) and failure of the 40.1 H 1 to enhance SK-BR-3 tumor cell sensitivity to TNF-ct (broken cross-hatching). SK-BR-3 and MDA-MB-175-VII overexpress p185HER2 (see Table III). MDA-MB-231 and HBL-100 are breast cell lines which do not overexpress p185 HEr~2, and T24 is a nonoverexpressing human bladder carcinoma cell line. The assay was performed as described in Ref. 31. Journal of Clinical Immunology, Vol. 11, No. 3, 1991
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`124 SHEPARD ET AL, t40- 12o- ~ 100 > ~ 80 o ~o 60 13. > 40 . ~ 20- rr A 0 .01 .1 1 10 100 Cisplatin (gg/ml) B .... I 11 I ........ ] ...... 1,] I fill i I ,r,,, I ........ ] 0 .01 .1 1 10 100 Cisplatin (gg/ml) Fig. 5. Treatment of SK-BR-3 breast tumor cells with muMAb 4D5 enhances sensitivity to cisplatin. MuMAb 4D5 (A) or muMAb 6E9 (control; B) and cisplatin were added at the indicated concentrations to SK-BR-3 breast tumor cells. The plate cultures were incubated for 3 days and relative cell proliferation was determined as described (31). No antibody ((3); 0.156 t~g/ml muMAb 4D5 or muMAb 6E9 (I); 0,625 ~g/ml muMAb 4D5 or muMAb 6E9 (~); 2.5 ~g/ml muMAb 4D5 or muMAb 6E9 (A). for activation of protein kinase C and has been closely associated with growth factor activity (35). As may be predicted from its effect on cell prolifer- ation, muMAb 4D5 treatment of SK-BR-3 tumor cells results in downregulation of intraceltular pools of DAG (Table VI) (30). This result is consistent with overall antagonist activity, as is inhibition of tumor cell proliferation. Other data suggest that muMAb 4D5 may inhibit association of ligand with the receptor (25). Similar monoclonal antibodies have been reported for the EGFR system (26, 28). Further work is under way to characterize the ligand(s) that binds p185 HER2 and the mechanism of action o f muMAb 4D5. While the ability of muMAb 4D5 to stimulate phosphorylation of p185 HER2 is consistent with an agonist of receptor function, it is important to note that our current data suggest that it does not behave as an agonist in our cell growth assays in vitro or in nude mice. The results of an experiment that com- pares the effects of muMAb 4D5 on the growth of MCF-7 and SK-BR-3 breast tumor cells are shown in Fig. 6. These data demonstrate that muMAb 4D5 has no effect on the growth of nonoverexpressing tumor cells (MCF-7; Tables III, VII) at any of the doses tested between 0.7 pM and 67 nM. Also, whatever allows the muMAb 4D5 to have differen- tial effects on overexpressing tumor cells, this dif- ference does not lie in different receptor affinities for the monoclonal antibody. Table VII clearly shows that SK-BR-3 and SK-OV-3, both pt85 HER2 overexpressors, which are growth inhibited by 4D5, and MCF-7, which is not, all have similar affinities for muMAb 4D5. The clearest difference between these cell lines is the number of binding sites per tumor cell. These data are consistent with other work that has been previously reported with tumor cells in monolayer culture or in soft agar (18, 19, 25). A possible mechanistic explanation, which takes many of our experimental observations into ac- count, is a model in which muMAb 4D5 binds Table IV. MuMAb 4D5 Inhibits the Growth of a Human Breast Tumor (Murray) in Athymic Mice" Tumor weight (mg) b Group (n = 4) Day 20 Day 34 PBS 6.79 +-- 9.79 36.0 ± 30.7 Control IgG (muMAb5B6) 7.11 + 5.48 88.1 ± 91.4 muMAb4D5 1.48 -+ 1.10 6.5 -+ 6.4 ~Administered as equally-divided intravenous doses on days 7, 10, and 13 post tumor implantation. bData are mean +- standard deviation (SD) (n = 4). Table V. Effect of muMAb 4D5 on Phosphoamino Acid HER2 Content of p185 in SK-BR-3 Cells Phospho- Phospho- Phospho- tyrosine serine threonine Treatment cpm" %b cpm % cpm % None 11 1.5 564 75 176 23.5 muMAb4D5 827 14.0 3,658 62.0 1,429 24.0 "Phosphoamino acids as cpm are expressed following back- ground subtraction (17 cpm for none, 21 cpm for muMAb 4D5). bPercentage of total phosphoamino acids. Journal of Clinical Immunology, Vol. II, No. 3, t991
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`MONOCLONAL ANTIBODY THERAPY OF HUMAN CANCER 125 Table VI. Effect of muMAb 4D5 or 6E9 Monoclonal Antibodies on sn-l,2-diacylglycerol levels in SK-BR-3 Cells pmol sn-l,2- Time Treatment DAG/106 cells % change 5 min Vehicle 111.0 + 10 0 5 min muMAb4D5 133.2 + 11.3 +20 5 rain muMAb6E9 133.3 -+ 12.9 +20 24 hr Vehicle 98.6 -+ 9.6 0 24 hr muMAb4D5 62.1 -+ 7.4 -37 24 hr muMAb6E9 92.0 +- 12.7 -7 aFollowing incubation with monoclonal antibody (33 nM) or vehicle (PBS) control, the reactions were terminated by aspi- rating the media and adding 1 ml of ice-cold 100% MeOH. Cells were scraped from the plates and transferred to 13 x 100-ram glass tubes containing 1 ml 100% chloroform. Plates were rinsed with an additional 1 ml of cold methanol, and the rinses combined and mixed thoroughly. Following phase separation at room temperature for 30 rain, 1 ml methanol and I ml NaCI were added, the samples were centrifuged at 3000 rpm for 5 rain, and the top aqueous layer was aspirated. The remaining organic phase was assayed for sn-l,2-diacylglycerol by standard proce- dures. tightly to p185 HEI~2, excludes ligand binding, stim- ulates receptor internalization, and downregulates receptor signaling pathways as a result of constitu- tive activation of tyrosine kinase activity that re- sults from nondissociation of the muMAB 4D5/ p185 HER2 complex during receptor cycling. This hypothesis has additional support from our obser- 11o! loo | c- O .e,.., I3. CD 0 ._> 132 90 4 80- 70- 60- 50- 40 30 o .ooo~ .oo~ .o~ .~ 4D5 (nM) 10 100 Fig. 6. MuMAb 4D5 does not stimulate proliferation of breast tumor cells. Cells were plated and the assay performed as described (31). MCF-7 breast tumor cells (squares) are compared with the SK-BR-3 tumor cell line (circles). MCF-7 expresses a low amount of p185 HErtz, while SK-BR-3 expresses about 33-fold more (Table llI). Coefficient of variation was less than 10%. Table VII. Monoclonal Antibody Binding to Cultured Human Adenocarcinoma Cells Receptor no. Growth Cell line Antibody Kd (nM) (sites/cell) inhibition a SK-BR-3 muMAb4D5 6.0 926,650 --* SK-OV-3 muMAb4D5 5.0 428,930 --* MCF7 muMAb4D5 1.2 5,525 - "Growth inhibition was measured as described in the footnotes to Table II. *Statistically significant inhibition of growth (P<0.05) as com- pared to an untreated control. vations that the monoclonal antibody is not de- graded following internalization (30) and, in the nude mouse experiments, localizes to and remains at the tumor site for more than 7 days following a single administration of antibody (D.M., personal communication). Such downregulation could result from activation of the serine-threonine protein ki- nase C, which is known to downregulate the func- tion of other receptor tyrosine kinases (5). The mechanism of action of muMAb 4D5 remains a subject for continuing work. SUMMARY AND CONCLUSIONS A convincing body of clinical and experimental evidence supports the role of p185 HER2 in the pro- gression of human cancers characterized by the overexpression of this protooncogene product. Im- portant aspects of this evidence include the wors- ened prognosis of breast, ovarian, and non-small cell lung carcinoma patients whose tumors overex- press p185 HER2, as well as observations that indi- cate that modulation of p185 HER2 activity by muMAb 4D5 can reverse many of the properties associated with tumor progression mediated by a growth factor receptor. The properties of muMAb 4D5 that indicate its potential usefulness for the therapy of human cancers characterized by the overexpression of p185 uE~2 are as follows: (i) downregulation of