PURIFICATION AND CHARACTERIZATION OF INTERFERON-GAMMA-INDUCING MOLECULE OF OK-432, A PENICILLIN-KILLED STREPTOCOCCAL PREPARATION, BY MONOCLONAL-ANTIBODY NEUTRALIZING INTERFERON-GAMMA INDUCING ACTIVITY OF OK-432

An immunoglobulin M mouse monoclonal antibody (MAb) to streptococcal p reparation OK432, TS-2, was generated. The TS-2 MAb showed positive re action with butanol extract of OK432 and fungal, branched (1-->3)-gamm a-glucans (lentinan and schizophyllan) as well as lipoteichoic acids. Moreover, the interferon (IFN)-gamma-inducing activity of OK-432 was n eutralized by TS-2 MAb. The affinity column of butanol extract of OK-4 32 on CNBr-activated Sepharose 4B-bound TS-2 antibody was prepared and the fractions containing IFN-gamma-inducing activity were eluated. Th e 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, p ronase, or neuraminidase. Survival time of human salivary adenocarcino ma-bearing animals given a combination of the polysaccharide sample pu rified from butanol extract of OK-432 and TS-2 MAb was significantly s horter compared with that of the tumor-bearing animals given only the purified polysaccharide sample of OK-432. Moreover, a high level of ef fector cell activity in natural killer (NK) and lymphokine-activated k iller (LAK) assays and significant increase of IFN-gamma-positive cell s or asialo-GM1-positive cells were detected in the spleen cells from the animals given the polysaccharide sample purified from butanol extr act of OK-432. These findings indicate that the polysaccharide sample purified by the affinity chromatography of butanol extract of OK-432 o n CNBr-activated Sepharose 4B-bound TS-2 MAb carries the IFN-gamma-ind ucing activity of OK-432 and marked antitumor activity.