We have constructed genes expressing single-chain antigen binding proteins (scFv) which recognize the human erbB-2 receptor. These genes encode the heavy and light chain variable domains of an erbB-2 receptor specific monoclonal antibody, MAb FRP5, connected by a peptide linker. In order to express a bifunctional molecule, a bacterial alkaline phosphatase gene was fused 3' to the scFv gene. The scFv(FRP5) and scFv(FRP5)-alkaline phosphatase fusion protein (scFv(FRP5)-PhoA) expressed in E. coli specifically recognize the human erbB-2 protein and compete with MAb FRP5 for binding to the receptor. The bound scFv(FRP5)-PhoA protein can be detected directly on tumor cells using a substrate for alkaline phosphatase, showing that the chimeric protein retains both binding and enzymatic activity.

Summary Four monoclonal antibodies (MAbs) specific for the extracellular domain of the human erbB-2/ HER2 protein (FRP5, FSP16, FWP51 and FSP77) have been isolated (Harwerth et al., J. Biol. Chem., 267, 15160-15167, 1992). In this paper we describe the effects of erbB-2 specific MAb administration on the tumorigenic growth of human erbB-2 transformed NIH3T3 cells implanted into The erbB-2 protein is a member of the receptor tyrosine kinase family and is closely related to the epidermal growth factor (EGF) receptor (Schechter et al., 1984;Coussens et al., 1985;Yamamoto et al., 1986). The oncogenic potential of the erbB-2 receptor has been shown to be released through different mechanisms involving point mutation (Segatto et al., 1988;Suda et al., 1990) and overexpression. Amplification of the c-erbB-2 gene, leading to overexpression of the protein, has been observed in a high percentage of human breast and ovarian tumour cells (Slamon et al., 1987;Kraus et al., 1987;van de Vijver et al., 1987;Slamon et al., 1989). It is likely that the elevated levels of the erbB-2 protein contribute to the malignancy process. Overexpression of erbB-2 in cultured cells has been found to induce the malignant phenotype in fibroblasts (Di Fiore et al., 1987) as well as in mammary epithelial cells (Pierce et al., 1991). Overexpression of erbB-2 in breast and ovarian carcinomas has been correlated with an unfavourable patient prognosis (Slamon et al., 1987; Varley et al., 1987;Berger et al., 1988;Slamon et al., 1989;Wright et al., 1989).The use of monoclonal antibodies (MAbs) in diagnosis and treatment of cancer has many promising aspects. The tumour enriched expression and extracellular accessibility of the erbB-2 receptor make it a potential target for immunotherapy. We have recently described several MAbs which bind to the extracellular domain of the human erbB-2 protein (Harwerth et al., 1992). These MAbs are able to affect erbB-2 receptor phosphorylation and turnover, as well as the anchorage-dependent growth properties of erbB-2-expressing tumour cells. In this paper we describe the effect of these anti-erbB-2 monoclonal antibodies, alone and in combination, on the tumorigenic growth of erbB-2 expressing cells in athymic nude mice. Two of the MAbs, FWP51 and FSP77, inhibited the onset of tumour growth. In a parallel experiment the antitumour activity of a singleCorrespondence: N. Hynes.

The present study investigated the expression of c-erbB-2 in 59 meningiomas, including different histological subtypes and anaplastic variants, by immunocytochemistry and molecular biological techniques. Immunohistochemistry using the monoclonal antibody FWP-51 directed against c-erbB-2-encoded oncoprotein gp185 demonstrated variable degrees of immunoreactivity in all meningiomas. The intensity of immunostaining correlated with the degree of expression as assessed by Western analysis in 28 meningiomas using polyclonal antiserum 21N. There was no correlation between the degree of expression and histological variants. Immunoreactivity of all meningiomas was distinctly less intense, however, than that of the human breast cancer cell line SK-BR-3, and slightly lower than that of brain metastases of breast and ovarian carcinomas that served as positive controls for both methods. By Southern analysis all meningiomas showed a single copy of the c-erbB-2 gene. Non-neoplastic arachnoid cap cells also exhibited c-erbB-2 expression and the degree of immunoreactivity was comparable with the majority of meningiomas. These data argue against an overexpression of c-erbB-2 in meningiomas, but rather indicate a cell-type-specific constitutive expression of the c-erbB-2 gene product in meningiomas and their putative progenitor cells. Since a subgroup of meningiomas is known to express progesterone receptors (PR), gp185 immunoreactivity was compared to the hormone receptor status using monoclonal antibody KD68. Fifty-six percent meningiomas showed PR immunoreactivity, but there was no statistically significant correlation with the degree of gp185 expression.

The HC11 cell line was isolated from mammary gland cells of pregnant mice. The cells displayed a normal phenotype and retained some characteristics of mammary epithelial cell differentiation. After treatment with the lactogenic hormones prolactin and glucocorticoids, the HC11 cells expressed the milk protein beta-casein. Various oncogenes were transfected and expressed in HC11 cells. The oncogenes were tested for their transformation ability and for their effects upon the differentiation of the HC11 cells. All of the oncogenes tested, including activated human Ha-ras, human transforming growth factor-alpha, activated rat neuT, and human c-erbB-2 activated by a point mutation in the transmembrane domain, caused transformation of the HC11 cells, as shown by tumor formation in nude mice. HC11 cells expressing the neuT and activated c-erbB-2 genes synthesized beta-casein in response to lactogenic hormones, whereas those expressing the Ha-ras or transforming growth factor-alpha oncogenes were no longer able to respond to the lactogenic hormones. This inhibition of beta-casein production occurs at the transcriptional level and in the transforming growth factor-alpha-transformed cells is due to an autocrine mechanism involving the activation of the epidermal growth factor receptor. This suggests that, although the c-erbB-2 and epidermal growth factor receptors are structurally quite similar, their activation has different effects upon mammary epithelial cell differentiation.

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