Sakuji Toyama

Comparison of the Chase Effects of Avidin, Streptavidin, Neutravidin, and Avidin‐Ferritin on a Radiolabeled Biotinylated Anti‐tumor Monoclonal Antibody

Injection of avidin can decrease the background radioactivity due to a radiolabeled biotinylated monoclonal antibody. We compared the chase effects of avidin, streptavidin, neutravidin, and avidin‐conjugated ferritin on a radiolabeled antitumor monoclonal antibody in tumor‐bearing nude mice. A radioiodine‐labeled biotinylated monoclonal antibody (OST7) was administered to athymic mice bearing osteogenic sarcomas. After 24 h, an avidin, streptavidin, neutravidin or avidin‐conjugated ferritin chaser was intravenously injected into the mice. At 2 h after the chase, the biodistribution of the radiolabeled monoclonal antibody was determined. Clearance from the blood was dose‐dependently accelerated by avidin and its effect was 10‐fold stronger than that of neutravidin or avidinferritin. Streptavidin did not promote clearance of the biotinylated antibody. Avidin was the most effective chasing agent for improving the biodistribution of the radiolabeled biotinylated monoclonal antibody among the four avidin derivatives tested.

Membrane receptor mobility changes by sendai virus

Treatment with Sendai virus does affect the lateral mobility of cell surface components on cultured human cells (KB) and cultured mouse cells (3T3). Diffusion coefficients and mobile fractions of a fluorescent lipid analog, tetramethyl rhodamine-succinyl concanavalin A (ConA) and tetramethyl rhodamine-anti human β2 microglobulin antibodies on the cell surface were measured by fluorescence photobleaching recovery (FPR). Diffusion coefficients of receptors for ConA and for antibodies to β2 microglobulin (presumably histocompatibility antigens) were increased 2- to 3-fold by exposure to the ultraviolet inactivated virus at virus doses from 250 to 2 500 HAU/ml, regardless of whether the particular cells measured had been fused. No mobility enhancement was induced with trypsinized Sendai virus, nor by influenza virus, indicating a probable requirement for the Sendai virus F protein. Virus treatment reduced the diffusion coefficient of a lipid analog in 3T3 cells. The results are compatible with the hypothesis that the Sendai virus disrupts a mobility restraint system, perhaps cytoskeleton-membrane connections.

In vivo instability of reduction-mediated 99mTc-labeled monoclonal antibody

A murine monoclonal antibody that reacts with human osteogenic sarcoma (OST7) was reduced and directly labeled with 99mTc without any loss of immunoreactivity. No fragmentation of the antibody was detected by high performance liquid chromatography after the labeling. However, SDS-PAGE analysis of the labeled antibody demonstrated the presence of low molecular weight species. Although more than 95% of the radioactivity remained bound at the antibody after incubation with human serum for 24 h, 99mTc-labeled OST7 was cleared faster from the circulation than 125I-labeled OST7 or 111In-labeled OST7 in mice. Urinary and fecal excretion of 99mTc were higher than those of 125I. When the 125I-labeled antibody was dual-labeled with 99mTc, the blood clearance of 99mTc was faster than that of 125I, suggesting release of 99mTc from the antibody in vivo. 99mTc-labeled OST7, however, gave a higher tumor-to-blood ratio than 125I- or 111In-labeled OST7 in mice bearing human osteogenic sarcoma. The 99mTc-labeled antibody prepared by the direct method was unstable in vivo, but retained a good tumor targeting ability.

Altered cell-fusion capacity in lines of KB cells resistant to Sendai virus-induced cytolysis.

Lines of KB cells resistant to Sendai virus-induced cytolysis (sil cells) have been isolated and characterized. Acquisition of resistance to the cytolysis was found to be associated with changes in the cell fusion capacity. The ability of sil cells to adsorb Sendai virus was essentially the same as that of KB cells, although they were found to have a decreased amount of sialic acid compared to KB cells. The elution of adsorbed virus from sil cells was increased over that from KB cells. The penetration of the virus was markedly reduced at high multiplicities of infection, while it proceeded normally at low multiplicities of infection. The consequences of these results are discussed.

Increased streptavidin uptake in tumors pretargeted with biotinylated antibody using a conjugate of streptavidin-fab fragment

Radiolabeled streptavidin accumulated in tumors pretargeted with biotinylated antibody. However, the absolute delivery of radioactivity was limited. To increase the tumor uptake of radioactivity further, we conjugated streptavidin with a mouse monoclonal antibody (MAb) fragment, OST6Fab, which recognizes antigen on human osteosarcoma. Another mouse MAb, OST7, which also reacts with the same tumor but recognizes an epitope different from the OST6 epitope, was biotinylated. The radioiodinated streptavidin-OST6Fab conjugate was administered to tumor-bearing mice after the biotinylated OST7 pretargeting. The uptake of the conjugate in tumors pretargeted with the biotinylated antibody was significantly higher than that of streptavidin and that of the conjugate of streptavidin and irrelevant Fab fragment. Renal uptake of radioactivity was decreased markedly, and the blood clearance was retarded by the conjugation with Fab fragment. In conclusion, the conjugate of streptavidin with specific Fab fragment increased the accumulation of radioactivity in tumors pretargeted with biotinylated antibody.

Okadaic acid gives concentration-dependent reciprocal effects on the fluid phase endocytosis activated by Ca2+ and phorbol 12-myristate 13-acetate

Incubation of a human fibrosarcoma cell line HT‐1080 in increasing concentration of Ca2+ was found to enhance endocytic internalization of a fluid phase marker, horseradish peroxidase. At 16.8 mM Ca2+, generation of the effect required incubation for more than 45 min. The effect was reversed by removal of the excess ion for 30 min. Monitoring the intracellular concentration showed that the incubation induced a transient large Ca2+ influx followed by a recovery to 230 ± 50 nM instead of the normal level of 83 ± 5 nM. The activation was not inhibited by inhibitors of protein kinases nor a cAMP antagonist. In contrast, the effect was prevented by okadaic acid (OKA) at 100 nM without detectable effect on the basal activity. Fluid phase uptake by HT‐1080 cells was also enhanced by phorbol 12‐myristate 13‐acetate (PMA). In contrast to the case with Ca2+, OKA at 100 nM did not prevent the PMA effect but further enhanced the endocytosis. The effect of OKA was concentration‐dependent, as the reagent at 1 μM inhibited not only both the activation but also the basal activity. In Ca2+‐or PMA‐stimulated cells, FITC‐dextran was delivered to endosomes that had been labeled with TRITC‐transferrin. In contrast, following treatment with a combination of PMA and 100 nM OKA, fluid phase was internalized in vesicular compartments devoid of transferrin labeling. These results suggest that, through differential modifications of protein phosphorylation, endocytosis can be enhanced distinctively either by employing conventional receptor‐bearing compartments or generating a new endosomal population.