No abstract

The ERBB2 (also called HER2, neu, and c-erbB-2) gene product, which encodes a growth factor receptor, was implicated in the malignancy of human adenocarcinomas. An antibody directed to the rat oncogenic receptor has been previously shown to have an antitumor effect in model systems. In an attempt to extend this observation to the protooncogenic human receptor and also to understand the underlying mechanism, we generated a panel of monoclonal antibodies specific to the extracellular portion of the ERBB2 protein. The effects of the antibodies on tumor growth were compared with their cellular and biochemical actions in vitro. Surprisingly, opposing in vivo effects were observed: although some antibodies almost completely inhibited the growth in athymic mice of transfected murine fibroblasts that overexpress Erbb-2, other antibodies either accelerated tumor growth or resulted in intermediate responses. When tested on cultured human breast carcinoma cells or ERBB2 transfectants, the tumor-stimulatory antibody was found to induce significant elevation of tyrosine phosphorylation of the ERBB2 protein. In contrast, only partial correlation was observed between the capacity to restrict tumor growth and the effects of the antibodies on receptor degradation and cellular proliferation in vitro. This suggests that the antitumor antibodies affect both receptor function and host-tumor interactions. Our results may help establish experimental criteria for the selection of specific antibodies for use either alone or in cogiunction with other molecules as pharmacological antitumor agents.Evidence has been accumulated in recent years for the involvement of growth factors and their receptors in the process of malignant transformation. The ERBB2 protein is a receptor tyrosine kinase (1), homologous to the epidermal growth factor (EGF) receptor (2, 3). The rat homologue ofthe gene undergoes oncogenic activation through a single point mutation (4). The ERBB2 protein was found to be overexpressed in several types of human adenocarcinomas, especially in tumors of the breast and the ovary (5-7), and the overexpression was correlated with short time to relapse and poor survival of breast cancer patients (5).The potential use of monoclonal antibodies (mAbs) in diagnosis and treatment of cancer has been studied extensively (8 (20), and the hybridomas were selected with hypoxanthine/aminopterin/thymidine medium. Supernatants of the growing cells were screened by using an indirect binding assay. CHO cells transfected with the ERBB2 gene (HCC cell line) were plated on a flexible 94-well plate, previously coated with polylysine (1 mg/ml). The cells were fixed with 3% (wt/vol) paraformaldehyde, and supernatants of hybridomas were incubated for 1 hr at 22°C with the fixed cells. The bound Abbreviation: mAb, monoclonal antibody. 8691The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The group of subtype I transmembrane tyrosine kinases includes the epidermal growth factor (EGF) receptor (ErbB-1), an orphan receptor (ErbB-2), and two receptors for the Neu differentiation factor (NDF/ heregulin), namely: ErbB-3 and ErbB-4. Here we addressed the distinct functions of the two NDF receptors by using an immunological approach. Two sets of monoclonal antibodies (mAbs) to ErbB-3 and ErbB-4 were generated through immunization with recombinant ectodomains of the corresponding receptors that were fused to immunoglobulin. We found that the shared ligand binds to highly immunogenic, but immunologically distinct sites of ErbB-3 and ErbB-4. NDF receptors differed also in their kinase activities; whereas the catalytic activity of ErbB-4 was activable by mAbs, ErbB-3 underwent no activation by mAbs in living cells. Likewise, down-regulation of ErbB-4, but not ErbB-3, was induced by certain mAbs. By using the generated mAbs, we found that the major NDF receptor on mammary epithelial cells is a heterodimer of ErbB-3 with ErbB-2, whereas an ErbB-1/ErbB-2 heterodimer, or an ErbB-1 homodimer, is the predominant species that binds EGF. Consistent with ErbB-2 being a shared receptor subunit, its tyrosine phosphorylation was increased by both heterologous ligands and it mediated a trans-inhibitory effect of NDF on EGF binding. Last, we show that the effect of NDF on differentiation of breast tumor cells can be mimicked by anti-ErbB-4 antibodies, but not by mAbs to ErbB-3. Nevertheless, an ErbB-3-specific mAb partially inhibited the effect of NDF on cellular differentiation. These results suggest that homodimers of ErbB-4 are biologically active, but heterodimerization of the kinase-defective ErbB-3, probably with ErbB-2, is essential for transmission of NDF signals through ErbB-3.Signals for growth and differentiation are mediated by binding of soluble growth factors to transmembrane receptors, that carry an intrinsic tyrosine kinase activity (1). The group of subtype I receptor tyrosine kinases includes four members that are characterized by ectodomains with two cysteine-rich sequences. Despite extensive structural homology, these receptors differ in their ligand specificities. Thus, ErbB-1 (also called HER-1) binds several distinct ligands whose prototype is the epidermal growth factor (EGF), 1 whereas ErbB-3 and ErbB-4 are the respective low and high affinity receptors for more than dozen isoforms of the Neu differentiation factor (NDF/heregulin) (2-4). The fourth member of the family, ErbB-2/Neu remains an orphan receptor because no fully characterized ligand of this receptor has been reported (5). Besides the interest in ErbB proteins as mediators of signal transduction, these receptors attracted attention due to their involvement in cancer development (6). Both ErbB-1 and ErbB-2 are oncogenic when overexpressed in murine fibroblasts (7,8), and their overexpression in human adenocarcinomas is associated with poor prognosis (9, 10). Likewise, ErbB-3 is overexpressed in some adenocarcinomas, but its prognostic s...

Immunofluorescent labeling of human salivary glands was carried out with a battery of monoclonal antibodies reactive with specific cytokeratin polypeptides. All the epithelial elements of the glands were positively labelled by a broad-spectrum cytokeratin antibody (KG 8.13) and by antibody Ks 18.18, which reacts with cytokeratin No. 18 exclusively. Labelling of frozen sections with antibody KM 4.62, which is reactive with the 40 Kd (No. 19) cytokeratin, was confined to the ductal system and apparently absent from the acini. Antibody KA-1, reactive with polypeptides 4, 5 and 6 stained both the myoepithelial cells and the basal cells of the large ducts. Antibody KS 8.58, however, reacted with the basal cells exclusively. It is thus proposed that the combined use of the various monoclonal antibodies may provide a most useful probe in studies on epithelial cell diversity in normal salivary glands as well as in pathological disorders of that gland.

The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells’ ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma–tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.