Purification and characterization of interferon-γ-inducing molecule of OK-432, a penicillin-killed streptococcal preparation, by monoclonal antibody neutralizing interferon-γ-inducing activity of OK-432

An immunoglobulin M mouse monoclonal antibody (MAb) to streptococcal preparation OK-432, TS-2, was generated. The TS-2 MAb showed positive reaction with butanol extract of OK-432 and fungal, branched (1-->3)-gamma-glucans (lentinan and schizophyllan) as well as lipoteichoic acids. Moreover, the interferon (IFN)-gamma-inducing activity of OK-432 was neutralized by TS-2 MAb. The affinity column of butanol extract of OK-432 on CNBr-activated Sepharose 4B-bound TS-2 antibody was prepared and the fractions containing IFN-gamma-inducing activity were eluted. The polysaccharide sample carrying the IFN-gamma-inducing activity with a molecular weight of 700,000 was destroyed by treatment with acid or sodium metaperiodate, but was stable to treatment with heat, alkali, pronase, or neuraminidase. Survival time of human salivary adenocarcinoma-bearing animals given a combination of the polysaccharide sample purified from butanol extract of OK-432 and TS-2 MAb was significantly shorter compared with that of the tumor-bearing animals given only the purified polysaccharide sample of OK-432. Moreover, a high level of effector cell activity in natural killer (NK) and lymphokine-activated killer (LAK) assays and significant increase of IFN-gamma-positive cells or asialo-GM1-positive cells were detected in the spleen cells from the animals given the polysaccharide sample purified from butanol extract of OK-432. These findings indicate that the polysaccharide sample purified by the affinity chromatography of butanol extract of OK-432 on CNBr-activated Sepharose 4B-bound TS-2 MAb carries the IFN-gamma-inducing activity of OK-432 and marked antitumor activity.