Peptides as requirement for immunotherapy of the guinea-pig line-10 tumor with endotoxins

Summary The transplantable line-10 hepatocellular carcinoma of guinea-pigs has been used as a model for the study of immunotherapy of malignant tumors. Cure rates of up to 100% have been obtained with ReGl-CM from 0 antigen-deficient (Re) mutant strains of Enterobacteriaceae, provided they were combined with mycobacterial trehalose dimycolate (cord factor, P3). Whereas highly endotoxic LPS extracts from all wild-type strains so far tested have failed to cause tumor regression, acid hydrolysis of such LPS samples led to residual fractions (RESI) that cross-reacted serologically with ReGl-CM samples (Chang and Nowotny, 1975) and provided cure rates up to 100%. RESI from Serratia marcescens was essentially nonpyrogenic and 100 times less lethal for chick embryos than potent endotoxins. Antigenic material associated with endotoxic extracts appears to be cryptic or sterically hindered from being effective in wild-type LPS but is exposed in ReGl and RESI samples.Reduction of the aminoacid content of ReGl-CM by microparticulate silica gel chromatography or by treatment with Triton X-100 significantly lowered the ability to bring about tumor regression without affecting endotoxicity. Antitumor activity could be restored by the addition of synthetic N-acetyl-muramyl-l-seryl-d-isoglutamine (MDP) or a nontoxic lipoid side fraction recovered during the isolation of ReGl-CM, which contained a small amount of peptidic substances. It is concluded that the addition of peptidic material, which may act as an adjuvant, to endotoxins is required to make them useful for immunotherapy of the weakly immunogenic line-10 tumor.Chemical procedures known to detoxify endotoxins while retaining adjuvanticity, such as succinylation and phthalylation, resulted in complete loss of endotoxicity and tumor-regressive potency of ReGl-CM. Transesterification with sodium methoxide led to a water-soluble phase, which cured 50% of tumor-bearing animals even though lethality and pyrogenicity were reduced by 100 times and 50 times, respectively. Thus there was no direct correlation between endotoxic potency and tumor-regressive activity. In addition, our findings indicate that a low level of toxicity may be required to obtain optimal levels of tumoricidal action.Abbreviations P3 trehalose dimycolate isolated by microparticulate silica gel chromatography (Ribi et al., 1974) - LPS lipopolysaccharide from wild-type gram-negative bacteria - ReGl endotoxic glycolipids from Re mutant gram-negative bacteria - CM chloroformmethanol - PW phenol-water - PCP phenol-chloroform-petroleum ether - ReGl-CM ReGl-PW, ReGl-PCP refer to ReGl extracted with CM, PW, or PCP, respectively - ACP acetone-precipitated by-product of ReGl-CM - B1, B2, B4 chromatographic fractions of ReGl-CM - lipid A hydrochloric acid hydrolyzate of LPS or ReGl - RESI organic solvent-insoluble fraction of lipid A (Chang and Nowotny, 1975) - KDO keto-3-deoxyoctonate - BSA bovine serum albumin - CWS cell wall skeleton of BCG (Bacillus Calmette-Guérin) - PPD purified protein derivative (tuberculoprotein) - TAP tuberculin-active peptide - MDP N-acetyl-muramyl-l-seryl-d-isoglutamine