The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been shown to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extraceUlular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.HER21c-erbB-2, the human homolog of the rat protooncogene neu (4, 34), encodes a 1,255-amino-acid glycoprotein with extensive homology to the human epidermal growth factor (EGF) receptor (4,21,33,34,42). The HER21c-erbB-2 gene product, p185HER2, has all of the structural features and many of the functional properties of subclass I growth factor receptors (reviewed in references 43 and 44), including cell surface location and an intrinsic tyrosine kinase activity. However, the ligand for this putative growth factor receptor has not yet been identified.Amplification of the HER21c-erbB-2 gene has been found in human salivary gland and gastric tumor-derived cell lines (13, 34), as well as in mammary gland carcinomas (21,22,40,42). Slamon et al. (35) surveyed 189 primary breast adenocarcinomas and determined that the HER21c-erbB-2 gene was amplified in about 30% of the cases. Most importantly, HER21c-erbB-2 amplification was correlated with a negative prognosis and high probability of relapse. Similar although less frequent amplification of the HER21c-erbB-2 gene has been reported for gastric and colon adenocarcinomas (45,46). Experiments with NIH 3T3 cells also suggest a direct role for the overexpressed, structurally unaltered HER21 c-erbB-2 gene product p185HER2 in neoplastic transformation. High levels of HER21c-erbB-2 gene expression attained by coamplification of the introduced gene with dihydrofolate reductase by methotrexate selection (18) or by using a strong promoter (6) was shown to transform NIH 3T3 fibroblasts.Only cells with high levels of pl85HER2 are transformed, i.e., have an altered morphology, are anchorage independent, and will form tumors in athymic mice.

The HER2 protooncogene encodes a receptor tyrosine kinase, p185HER2. The overexpression of p185HER2 has been associated with a worsened prognosis in certain human cancers. In the present work we have screened a variety of different tumor cell lines for p185HER2 expression using both enzyme-linked immunosorbent and fluorescence-activated cell sorting assays employing murine monoclonal antibodies directed against the extracellular domain of the receptor. Increased levels of p185HER2 were found in breast (5/9), ovarian (1/6), stomach (2/3) and colorectal (5/16) carcinomas, whereas all kidney and submaxillary adenocarcinoma cell lines tested were negative. Some monoclonal antibodies directed against the extracellular domain of p185HER2 inhibited growth in monolayer culture of breast and ovarian tumor cell lines overexpressing p185HER2, but had no effect on the growth of colon or gastric adenocarcinomas expressing increased levels of this receptor. The most potent growth-inhibitory anti-p185HER2 monoclonal antibody in monolayer culture, designated mumAb 4D5 (a murine IgG1 kappa antibody), was also tested in soft-agar growth assays for activity against p185HER2-overexpressing tumor cell lines of each type, with similar results. In order to increase the spectrum of tumor types potentially susceptible to monoclonal antibody-mediated anti-p185HER2 therapies, to decrease potential immunogenicity issues with the use of murine monoclonal antibodies for human therapy, and to provide the potential for antibody-mediated cytotoxic activity, a mouse/human chimeric 4D5 (chmAb 4D5) and a "humanized" 4D5 (rhu)mAb 4D5 HER2 antibody were constructed. Both engineered antibodies, in combination with human peripheral blood mononuclear cells, elicited antibody-dependent cytotoxic responses in accordance with the level of p185HER2 expression. Since this cytotoxic activity is independent of sensitivity to mumAb 4D5, the engineered monoclonal antibodies expand the potential target population for antibody-mediated therapy of human cancers characterized by the overexpression of p185HER2.

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The HER2 protooncogene encodes a 185-kDa transmembrane protein (p185HER2) 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 p185HER2 present on the surface of overexpressing tumor cells may be a good target for receptor-targeted therapeutics. The anti-p185HER2 murine monoclonal antibody (muMAb) 4D5 is one of over 100 monoclonals that was derived following immunization of mice with cells overexpressing p185HER2. The monoclonal 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 protooncogene. MuMAb 4D5 has also been shown to enhance the TNF-alpha 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 the tumor site and can inhibit the growth of human tumor xenografts which overexpress p185HER2. Modulation of p185HER2 activity by muMAb 4D5 can therefore reverse many of the properties associated with tumor progression mediated by this putative growth factor receptor. Together 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 p185HER2.

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