Isolation of a nonendotoxic antitumor preparation from Serratia marcescens

White-type polysaccharide preparation (WPS) obtained from Serratia marcescens bacteria by hot 0.2 N acetic acid extraction was shown to have antitumor effects. These were manifested by enhanced resistance to the take of TA3 transplantable murine adenocarcinoma and by the induction of regression of Meth A sarcoma in mice. Optimal conditions for the liberation and isolation of these substances were sought to achieve the highest antitumor activity and the lowest endotoxin (ET) content. Simultaneously, the activities of the WPS preparations were tested in various tests which are frequently used as in vitro correlates of in vivo antitumor effects, such as the activation of macrophage cytotoxicity, activation of natural killer (NK) cells, and tumor necrosis factor (TNF) generation. We found that the enhanced resistance to the take of TA3 tumor correlated with ET content of the WPS preparations. Preparations with reduced or no ET content showed diminishing activity in this assay or were without any measurable effect. The induction of TNF production and NK activation did not show such close relationship with the ET content. This was particularly evident if testing WPS samples obtained after 60 or 120 min hydrolysis at 90°C. The greatest discrepancy was found between ET content and the Meth A regression induction. Samples with no detectable ET content and no activity in the macrophage, NK, or TNF tests were potent inducers of Meth A regression. Partial purification of such WPS samples could be achieved and a preparation was obtained with high Meth A regression capacity. Preliminary chemical analysis of this preparation showed 25.5% amino acid, 53.7% neutral carbohydrate, <0.4% KDO, 0.8% hexosamine, <0.1% phosphorous, and <1.0% long-chain carboxylic acid content. The above chemical analytical data are not consistent with designating such preparations as ET or ET derivatives, such as Lipid A or its split products. This conclusion was confirmed by the lack of endotoxic properties as determined by biological assays on this preparation.