Josef Brüggen

Use of an enzyme-linked immunosorbent assay (ELISA) for screening of hybridoma antibodies against cell surface antigens

A micro-ELISA for screening of antibodies from hybridoma cultures against surface antigens of human melanoma is described. The technique employs alkaline phosphatase-conjugated protein A and target cells attached to poly-L-lysine-coated microtiter plates. The micro-ELISA is equally sensitive as the radioimmunoassay. Mild glutaraldehyde treatment of cells did not lead to an appreciable loss of antigen activity. The fixed cells can be stored at 4 degrees C for at least 6 weeks. It is concluded that the ELISA is superior to the radioimmunoassay in the following aspects: (1) exclusion of radioactive hazards, (2) speed of performance, and (3) lower costs.

Heterogeneity of primary and metastatic human malignant melanoma as detected with monoclonal mntibodies in cryostat sections of biopsies

SummaryMonoclonal antibodies were generated against established melanoma cell lines and characterized by their reactivity with various sublines. The antibodies selected for their reaction with melanoma-associated antigens were tested on cryostat sections of melanoma tissue from various stages and on other tumors. The reactivity with normal tissues was also determined. Of 30 antibodies reacting with melanoma cell lines 11 did not react with melanoma biopsies. Of the remaining 19 antibodies nine displayed broad cross-reactivity with normal cells and structures and other benign or malignant tumor cells. Among the remaining antibodies five types were defined that detected antigens (nevocellular I, nevocellular II, neural, endothelial, basal cell) found on certain normal tissues and structures and on certain tumor phenotypes. Even though there seems to be a tendency for some antigens to be preferentially associated with certain stages of melanoma, it has not yet been possible to establish any clear-cut correlation between the expression of one of the differentiation antigens and a particular stage or malignancy potential of melanoma.

Oral Imatinib Mesylate (STI571/Gleevec) Improves the Efficacy of Local Intravascular Vascular Endothelial Growth Factor-C Gene Transfer in Reducing Neointimal Growth in Hypercholesterolemic Rabbits

Background—Platelet-derived growth factor (PDGF) antagonists have demonstrated beneficial effects on neointima formation, but in studies using PDGF inhibitors and extended follow-up, the lesions reoccur. These findings implicate a need to combine targeting of PDGF with other strategies. Stimulation of reendothelialization by treatment with endothelial cell mitogens of the vascular endothelial growth factor (VEGF) family counteracts restenosis, but there are also concerns regarding the durability of the effect with this approach. Methods and Results—To explore whether a combined use of PDGF antagonist and stimulation of reendothelialization confers better results than each therapy alone, we combined systemic administration of imatinib mesylate (STI571/Gleevec, 10 mg/kg−1 per d−1), a tyrosine kinase inhibitor with activity against PDGF receptors, with local intravascular adenovirus-mediated VEGF-C gene transfer (1.15×1010 pfu) in cholesterol-fed, balloon-injured rabbits. Throughout the course of the STI571 therapy, the circulating concentrations were able to suppress PDGF receptor phosphorylation. At 3 weeks, the treatment with STI571 led to a transient decrease in intralesion macrophages and to an increase in intimal smooth muscle cell apoptosis. VEGF-C application reduced neointima formation and accelerated reendothelialization. However, none of the therapies alone reduced intimal thickening at a 6-week time point, whereas the combined treatment led to a persistent reduction (55% versus control) in lesion size at this time point. Conclusions—Our study provides one of the first successful examples of gene therapy combined with a pharmacological treatment to modulate 2 distinct ligand-receptor signaling systems and suggests combination of local VEGF-C gene therapy with systemic inhibition of PDGF signaling as a novel principle to prevent intimal hyperplasia after vascular manipulations.

A Novel Potent Oral Series of VEGFR2 Inhibitors Abrogate Tumor Growth by Inhibiting Angiogenesis

This paper describes the identification of 6-(pyrimidin-4-yloxy)-naphthalene-1-carboxamides as a new class of potent and selective human vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitors. In biochemical and cellular assays, the compounds exhibit single-digit nanomolar potency toward VEGFR2. Compounds of this series show good exposure in rodents when dosed orally. They potently inhibit VEGF-driven angiogenesis in a chamber model and rodent tumor models at daily doses of less than 3 mg/kg by targeting the tumor vasculature as demonstrated by ELISA for TIE-2 in lysates or by immunohistochemical analysis. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Distribution of the Calcium-Binding Proteins MRP-8 and MRP-14 in Normal and Pathological Conditions: Relation to the Cystic Fibrosis Antigen

Eukaryotic cells require calcium ions for optimal growth and functioning. The intracellular actions of calcium as a biological second messenger appear to be the result of its interaction with a set of calcium-binding proteins referred to as calcium modulated proteins. (Persechini et al. 1989).

Cell-Surface Structure and State of Malignancy in Human Malignant Melanoma

Human malignant melanoma is a spontaneous tumor that progresses from the initial premalignant lesions to highly metastatic forms. Clinical and histological observations of this process and some recent knowledge gained on experimental tumor models suggest that tumor progression is a complex multistage process. In the course of this process, several distinct properties have to be aquired by the tumor cells either sequentially or in parallel (Poste and Fidler, 1980; Nicolson et al., 1977). Properties of prime importance are believed to be invasiveness, neovascularization, resistance to rejection mechanisms, and adaptation to and growth in a different organ or tissue. It has been shown in animal tumor models that the capacity to metastasize is a phenotypically expressed property of a tumor cell; moreover, even the target organ seems to be predetermined by the phenotype (Dexter et al., 1978; Hart and Fidler, 1980; Nicolson and Winkelhake, 1975; Nicolson et al.,1978; Nowell, 1976; Schirrmacher et al., 1979a; Susuki et al., 1978). The mechanisms by which primary tumors develop highly malignant variants have been circumscribed by the term retrodifferentiation and are unknown (Coggin and Anderson, 1974; Renselaer Potter, 1978; Uriel, 1979). That the process of retrodifferentiation might also be reversed is indicated in experiments wherein nonmetastasizing variants were isolated from metastases (Tao and Burger, 1977). It is logical to assume that phenotypic changes that are associated with the expression of different biochemical and biological properties are also associated with changes in the cell surface, which in fact has been demonstrated in several instances (Fogel et al., 1979; Killion and Kollmorgen, 1976; Nicolson and Winkelhake, 1975; Poste, 1977; Schirrmacher et al., 1979b; Sorg et al., 1978; Yogeeswaran et al., 1978, 1979).

Correlation between secreted and membrane-bound IgG in mouse myeloma cells transfected with chimeric immunoglobulin heavy and light chain genes.

Mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt) and the neomycin (neo) selection marker genes. A broad distribution in the level of mouse-human chimeric IgG expression was observed with series of independently isolated transfectoma clones. The relative amounts of secreted to membrane-bound antibodies correlated closely, which suggested, that fluorescence-activated cell sorting could be a valuable tool for the selection of high-yielding production cell lines. However, a single cycle of cell sorting did not steer the cloning process significantly toward cells that produce enhanced amounts of recombinant IgG. Only in cases in which the polyclonal transfectoma population contained a large percentage of nonproducing cells, these were successfully separated from the IgG-producing cell population. (c) 1996 John Wiley & Sons, Inc.