SummaryONO-4641 is a next-generation sphingosine 1-phosphate (S1P) receptor agonist selective for S1P receptors 1 and 5. The objective of the study was to characterize the immunomodulatory effects of ONO-4641 using preclinical data. ONO-4641 was tested in both in-vitro pharmacological studies as well as in-vivo models of transient or relapsing-remitting experimental autoimmune encephalomyelitis (EAE). In vitro, ONO-4641 showed highly potent agonistic activities versus S1P receptors 1 and 5 [half maximal effective concentration (EC50) values of 0·0273 and 0·334 nM, respectively], and had profound S1P receptor 1 down-regulating effects on the cell membrane. ONO-4641 decreased peripheral blood lymphocyte counts in rats by inhibiting lymphocyte egress from secondary lymphoid tissues. In a rat experimental autoimmune encephalomyelitis (EAE) model, ONO-4641 suppressed the onset of disease and inhibited lymphocyte infiltration into the spinal cord in a dose-dependent manner at doses of 0·03 and 0·1 mg/kg. Furthermore, ONO-4641 prevented relapse of disease in a non-obese diabetic mouse model of relapsing-remitting EAE. These observations suggest that ONO-4641 may provide therapeutic benefits in the treatment of multiple sclerosis.

We have investigated the in vivo antitumor mechanism of natural human tumor necrosis factor (n‐TNF) isolated from a culture of human leukemic B cell line (BALL‐1), especially its action as an immunomodulator, and found that the in vivo antitumor effect of n‐TNF on Meth A sarcoma implanted in BALB/c mice pretreated with monoclonal antibody against T cell‐specific surface antigen (Thy‐1) was significantly diminished. Furthermore, when BALB/c mice were treated with T cell subset‐specific monoclonal antibodies, anti‐L3T4 or anti‐Lyt‐2.2, the antitumor effect of n‐TNF on Meth A sarcoma was significantly reduced. Therefore, it was suggested that the in vivo antitumor mechanism of n‐TNF might involve a T cell‐mediated immunological route.

Dihydrof olate reductase (DHFR) deficient Chinese hamster ovary (CHO) cells were transf ected with expression plasmid constructed from human tissue-type plasminogen activator (t-PA) and DHFR gene. The transf ected cells were selected with a medium containing methotrexate (MTX, 0-5NM), followed by isolation of several DHFR+ clones. It was definitely obserbed that the expression levels of t-PA were dose dependently elevated on the MTX resistance and the gene copy number. This recombinant t-PA had a similar specific activity and the same N-terminal amino acid sequence to human melanoma cell derived t-PA, and also showed similar characteristics in a neutralization test with a human t-PA, SDS-PAGE, PAS staining, zymography and human plasma clot lysis time assay. The MTX-selected high expression clone could keep constant production of t-PA without MTX for at shortest 5 months. These results strongly suggestted that CHO cells-DHFR gene amplification system was useful for producing a large amount of human glycoprotein with a complicated structure such as t-PA.

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