Orith Leitner

Cytokeratin polypeptides expression in different epithelial elements of human salivary glands.

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.

Examination of HER3 targeting in cancer using monoclonal antibodies

Significance The human EGF receptor (EGFR/HER) family plays critical roles in tumor progression. Therefore, several therapies intercepting these receptors were developed and clinically approved. Importantly, patients treated with such therapeutics often develop resistance, and in some cases this resistance has been associated with activation of HER3. Potentially, HER3 blockade might overcome patient resistance. Hence, antibodies to HER3 have been developed by us and other researchers. However, it has remained unclear which antibody attributes are required for effective tumor inhibition. To address this issue, we generated several monoclonal antibodies, which were tested in vitro and in tumor-bearing animals. Our results suggest that anti-HER3 antibodies able to intercept stroma–tumor interactions, as well as accelerate HER3 degradation, might inhibit tumor growth better than other antibodies. 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.

Tailored cancer immunotherapy using combinations of chemotherapy and a mixture of antibodies against EGF-receptor ligands

Growth factors are implicated in several processes essential for cancer progression. Specifically, growth factors that bind to ErbB family receptors have been implicated in cell proliferation and in resistance of solid tumors to chemotherapy. We quantified ligand secretion by several human cancer cell lines, and generated mAbs against two ligands, namely TGF-α and heparin-binding EGF-like growth factor. These growth factors are frequently secreted by pancreatic tumor cell lines, including BxPC3 cells. The monoclonal antibodies were tested for their antigen specificity and ability to inhibit growth of BxPC3 cells in vitro. Combining the two antibodies resulted in enhanced inhibition of BxPC3 cell growth, both in vitro and in tumor-bearing animals. Hence, we combined the two antibodies with gemcitabine, an effective chemotherapeutic drug commonly used to treat pancreatic cancer patients. Because treatment with a combination of two monoclonal antibodies enhanced the ability of chemotherapy to inhibit BxPC3 tumors in mice, we propose a general cancer therapeutic strategy that entails profiling the repertoire of growth factors secreted by a tumor, and combining with chemotherapy several antibodies capable of blocking autocrine ligands.

A novel approach for evaluating tyrosine kinase activity based on the radioimmunological determination of phosphotyrosine

A novel technique was designed to conveniently determine substrate phosphorylation by tyrosine kinase. The technique is based on quantitation of phosphotyrosine content of the phosphoproteins, generated during the enzyme reaction, by radioimmunoassay. Here, we utilized high-titer monoclonal antibodies to phosphotyrosine, and radioiodinated bovine serum albumin-phosphotyrosine conjugate. The radiolabeled antigen was displaced from the complex formed in the assay by unlabeled phosphotyrosine, phosphotyrosine derivatives or phosphotyrosine-containing protein substrates. Half-maximal displacement was achieved at 0.4 +/- 0.05 microM by free phosphotyrosine, and at 40 +/- 3 and 45 +/- 4 nM by acetyl-phosphotyrosine and acetyl-phosphotyrosyl-glycine ethyl ester, respectively. Neither phosphoserine, phosphothreonine nor ATP cross-reacted with the phosphotyrosine antibodies. None of the components of the enzyme reaction interfered in the RIA. The method allows quantitation of the incorporated phosphate into tyrosyl residues without interference of serine/threonine phosphorylation. This technique avoids the use of short-lived [gamma-32P]ATP and omits the separation of the phosphorylated substrate from excess nucleotide.

Selective expression of cytokeratin polypeptides in various epithelia of human Brenner tumor

A human ovarian Brenner tumor presenting a wide spectrum of benign and malignant histologic features was studied for its patterns of intermediate filament expression. All epithelial elements of the tumor, regardless of their morphologic type, contained cytokeratins as their only intermediate filament component. Differences were detected, however, between tumor nests that displayed transitional epithelium and those with squamoid features. These differences were manifested by the presence of cytokeratin 18, in the former type only, and by the abundance of cytokeratins 10/11 in the latter. We also detected mixed epithelial nests in which both features were present, suggesting that the transitional epithelium transforms in polar fashion into squamous epithelium. Examination of cytokeratin patterns found in urothelium and in the surface epithelium of the ovary pointed to certain differences from the Brenner tumor epithelia. The significance of these latter findings with regard to cellular transformation and histogenesis of the Brenner tumor are discussed.

Immunocytochemical characterization of lung tumors in fine‐needle aspiration. The use of cytokeratin monoclonal antibodies for the differential diagnosis of squamous cell carcinoma and adenocarcinoma

In the current study, immunocytochemical typing of intermediate filaments was used for a differential diagnosis of human lung tumors from transthoracic fine‐needle aspiration biopsies (TFNAB). The authors have compared the cytologic diagnosis of 53 lung cancer cases with the immunofluorescence patterns obtained using a panel of monoclonal antibodies, five of which (KG 8.13, KM 4.62, Ks B.17, KS 8.12, KK 8.60) react with specific cytokeratin polypeptides and one with vimentin (VIM 13.2). Only in six of 23 samples cytologically diagnosed as squamous cell carcinoma did the immunocytochemical typing of cytokeratins (ICTC) confirm the cytologic diagnosis. In seven cases some of the tumor cells stained positively with antibody Ks B.17 specific for simple epithelial keratin (No: 18), suggesting the presence of some cells of glandular origin. In ten additional cases the ICTC was in conflict with the cytologic diagnosis of squamous cell carcinoma (i.e., antibodies Ks 8.12 and KK 8.60 were negative, and antibody Ks B.17, positive) supporting a diagnosis of adenocarcinoma. In 14 of 18 cases cytologically diagnosed as adenocarcinoma, the ICTC confirmed the diagnosis whereas in four cases additional presence of some squamous cells was noticed. The ICTC labeling of cases cytologically diagnosed as undifferentiated and large cell carcinomas was similar to that of the group of adenocarcinomas. Thus, the application of cytokeratin typing for TFNAB samples seems to provide a vital complementation to routine cytologic study, especially for cases cytologically diagnosed as squamous carcinoma.