Epidermal growth factor receptor, but not c-erbB-2, activation prevents lactogenic hormone induction of the beta-casein gene in mouse mammary epithelial cells.

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.

Selective inhibition of tumor cell growth by a recombinant single-chain antibody-toxin specific for the erbB-2 receptor.

A high percentage of human breast and ovarian tumors display amplified c-erbB-2 gene copies, leading to overexpression of the growth factor receptor. Its membrane location and elevated expression make the erbB-2 protein an appropriate target for a directed tumor therapy. We have used recombinant DNA technology to produce a single-chain antibody-exotoxin A (scFv-ETA) fusion protein which specifically binds the human erbB-2 receptor. The scFv portion is composed of the heavy- and light-chain variable domains of a monoclonal antibody which recognizes the extracellular domain of the human erbB-2 receptor. The bacterially produced scFv-ETA protein was shown to bind specifically to cells expressing the human erbB-2 protein. The scFv-ETA inhibits protein synthesis in erbB-2-expressing tumor cells at doses ranging from 2 to 200 ng/ml and is cytotoxic for these cells at equivalent doses. In athymic nude mice, administration of the scFv-ETA inhibited the growth of erbB-2-overexpressing human ovarian carcinoma cells.

Diminution of antibodies directed against tumor cell surface epitopes: a single chain Fv fusion molecule specifically recognizes the extracellular domain of the c-erbB-2 receptor.

We are evaluating strategies for the inhibition of growth or the selective killing of tumor cells. Cell surface antigens which are exclusively expressed or which are enhanced in their expression in tumor cells might provide the means to target cytotoxic or cytostatic agents to these cells. Few tumor specific cell surface antigens have been found, but the enhanced expression of growth factor receptors has been described for several types of tumors. A prominent example is the overexpression of the c-erbB-2 receptor in a high percentage of primary breast and ovarian carcinomas. We have derived monoclonal antibodies against the extracellular domain of the c-erbB-2 receptor. The antibody molecules were genetically engineered to minimize their size and to allow for their functional modification. For this purpose the cDNA sequences corresponding to the variable domains of one monoclonal antibody (FRP5) were molecularly cloned and joined by a short linker. The resulting single chain antibody molecule (scFv) was expressed in bacteria and purified. We show in an immunoprecipitation experiment that this molecule retains its ability to recognize the c-erbB-2 extracellular domain. This molecule could become a valuable vehicle to specifically transport anti-tumor agents to breast cancer cells.

Construction, bacterial expression and characterization of a bifunctional single-chain antibody-phosphatase fusion protein targeted to the human erbB-2 receptor.

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.

Monoclonal antibodies directed to the erbB-2 receptor inhibit in vivo tumour cell growth.

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 athymic nude mice. Two antibodies, FWP51 and FSP77, inhibited the onset of tumour growth, while the administration of FRP5 and FSP16 did not affect tumour growth. In addition, administration of MAbs FWP51 and FSP77 led to a retardation in the growth of established tumours. Treatment was not curative in that tumours regrew within two weeks of the final treatment. The administration of a combination of MAbs FWP51 and FSP77 which react with two distinct regions on the erbB-2 molecule was more effective than treatment with either MAb alone. The two growth-inhibitory antibodies were also effective in the treatment of tumours established from SKOV3 cells, a human ovarian tumour cell line with high levels of the erbB-2 protein. The effect of the MAbs on the anchorage-independent growth of erbB-2 transformed cells and on erbB-2 receptor turnover was also measured.

Monoclonal antibodies against the extracellular domain of the erbB-2 receptor function as partial ligand agonists.

In this paper we describe the isolation and characterization of four monoclonal antibodies (FRP5, FSP16, FWP51, and FSP77) which specifically recognize the human erbB-2 protein. All of the antibodies recognize epitopes on the extracellular domain of the receptor protein. FRP5 and FSP16 compete with one another for binding while FWP51 and FSP77 each recognize a different epitope. The effects of the antibodies on the erbB-2 receptor protein have been analyzed. Two different erbB-2-expressing cell lines, SKBR3 breast tumor cells and HC11 R111 cells, were examined. The SKBR3 cells express approximately 1 x 10(6) molecules of the erbB-2 protein/cell; HC11 R111 cells, a clone of mouse mammary epithelial cells derived by transfection of a human erbB-2 expression plasmid, contain 10-fold less erbB-2 protein than the SKBR3 cells. Treatment of the two cell lines with FRP5, FSP16, and FWP51 led to a rapid increase in the phosphotyrosine content of the erbB-2 protein. Three of the antibodies, FRP5, FSP16, and FSP77, stimulated the turnover of the erbB-2 protein. Binding of the antibodies did not stimulate DNA synthesis in HC11 R111 cells. Thus, the erbB-2-specific monoclonal antibodies behave as partial ligand agonists. The antibodies were examined for their effects upon the growth of SKBR3 and HC11 R111 cells. The growth of SKBR3 cells was inhibited by 90% following long term treatment of the cells with FSP77.

Expression of the c-erbB-2-encoded oncoprotein and progesterone receptor in human meningiomas.

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.