Charles A. McLaughlin

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

SummaryThe 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.

Further studies on the structural requirements of agents for immunotherapy of the guinea pig line-10 tumor

SummaryIn this laboratory and elsewhere, cord factor or some less complex analogue has been found to be an important constituent of agents for suppression and immunotherapy of cancer. In further attempts to delineate structural requirements, we have tested several such analogues, in combination with the optimum quantity (150 μg) of glycolipid from an Re mutant salmonella (Re glycolipid), for ability to produce regression of transplantable one-week-old line-10 tumors in guinea pigs. The synthetic diester, trehalose 6,6′-dipalmitate, has been reported to be a useful antibacterial prophylactic and a tumor-suppressive agent, but neither alone nor in combination with Re glycolipid was it effective in therapy of established line-10 tumors, even in doses up to 1500 μg. Trehalose myocolates (cord factor, P3) were also ineffective when given alone, but as little as 15 μg of P3, combined with Re glycolipid and oil droplets, produced a high rate of regression. Some analogues of higher molecular weight were effective but, within the limits of these experiments, only when the fatty acid residues contained side chains at the alpha carbon atom. It was striking that a naturally-occurring 6,6′-trehalose diester of a branched chain fatty acid(s) containing the carbon equivalent of two condensed palmitic acid residues was as effective as any of the higher-molecular weight compounds, including the mycolates. Thus, it appears that there may be requirements for a certain molecular size and/or for a particular molecular conformation. Only such a compound, in conjunction with Re glycolipid or other suitable immunogen, has been found to bring about complete cures, including regression of the primary tumor, elimination of metastases in the regional lymph node, and specific systemic immunity to rechallenge with the line-10 tumor.

Regression of line-10 hepatocellular carcinomas following treatment with water-soluble, microbial extracts combined with trehalose or arabinose mycolates

SummaryIntratumor injections of the aqueous phase of phenol-water extracts of Re mutant Salmonella typhimurium (Re glycolipid) in combination with trehalose dimycolate at dose levels of 150 to 15 μg were consistently and highly effective (65–93%) in producing regression of line-10 tumors in strain-2 guinea pigs. We observed that the rate of regression was more rapid than that seen after treatment with cell walls from Mycobacterium bovis strain Bacillus of Calmette and Guèrin (BCG). Arabinose mycolate could be substituted for trehalose dimycolate in the Re glycolipid-mycolate mixture without appreciably compromising antitumor activity, providing that the level of arabinose mycolate was not reduced below 15 μg. In addition to the Re glycolipid preparation, similarly prepared aqueous extracts from Mycobacterium bovis strain BCG and strain AN5 in combination with trehalose dimycolate also possessed tumor-regressive activity. The activity of these last extracts was reduced when the arabinose mycolate was substituted for the trehalose dimycolate. The aqueous extract of a rickettsia, Coxiella burnetii, in combination with either trehalose dimycolate or arabinose mycolate was also active (50 and 80% tumor regression rates, respectively). Intracutaneous administration of Re glycolipid or aqueous extracts from BCG in combination with trehalose or arabinose mycolates did not produce life-threatening, clinical signs of toxicity in young mice. If additional toxicity studies demonstrate that adverse side effects can be satisfactorily controlled, these watersoluble extracts may prove beneficial in the treatment of spontaneous tumors of humans and other animals.

Tumor regression induced by defined microbial components in an oil-in-water emulsion is mediated through their binding to oil droplets

SummaryOil-in-water emulsions containing aqueous-soluble glycolipids combined with synthetic or naturally occurring fatty acid esters derived from mycobacteria were found to be potent tumor regressive preparations. An apparent mode of action of these fatty acid esters in regression of treated tumors was shown to be that of binding glycolipids to oil droplets. A model is presented.

Synergistic tumor regressive activity observed following treatment of line-10 hepatocellular carcinomas with deproteinized BCG cell walls and mutant Salmonella typhimurium glycolipid

SummarySynergistic tumor-regressive activity was observed when the water-soluble portion of a phenol-water extract from mutant Salmonella typhimurium whole cells was combined with deproteinized cell walls from Mycobacterium bovis strain BCG. As little as 50 μg deproteinized cell walls combined with 50 μg water-soluble extract from the mutant salmonella produced 89–100% cures of line-10 dermal tumors in treated strain 2 guinea-pigs. However, none of the animals was cured following treatment with 50 μg of deproteinized cell walls alone. Only 17% of treated animals were cured following treatment with 50 μg of the water-soluble extract from the mutant salmonella. The deproteinized cell walls and water-soluble extract were suspended in oil-in-water emulsions and injected directly into 10 mm tumors. The deproteinized cell walls were prepared by treating BCG cell walls with proteolytic enzymes and denaturing agents (KCl, urea, Triton X-100, and guanidine hydrochloride). Urea or a combination of denaturing agents reduced the protein content of protease-treated cell walls from approximately 2% (w/w) protein to 0.7% protein. The antigenicity of the effectively deproteinized cell walls, as measured by skin testing in presensitized guinea-pigs, was reduced approximately ten-fold compared with untreated cell walls. Injection to mice of 500 μg deproteinized cell walls in combination with 500 μg water-soluble extract from the mutant salmonella produced transient, clinical signs of toxicity (malaise, conjunctivitis, diarrhea, and rough hair coats) lasting approximately 5 days. However, no deaths were observed. The synergistic antitumor effect of combining deproteinized BCG cell walls with the water-soluble extract from mutant salmonella may be useful for treatment of certain cases of spontaneous neoplastic disease.

Eradication of microscopic lymph node metastases of the guinea pig line-10 tumor after intradermal injection of endotoxin plus mycobacterial components

SummaryNon-viable microbial agents were used to treat lymph node metastases of the line-10 hepatocarcinoma in strain two guinea pigs. Oil droplet vaccines were administered by intradermal injection adjacent to the site of dermal tumors. The primary tumors were removed surgically before or after immunotherapy. Control animals, treated with surgery alone, died of metastatic tumor growth. The mycobacterial glycolipid, P3, plus polysaccharide deficient endotoxin (Re Et) eliminated lymph node metastases when the primary tumors were excised 7 days or 1 day after immunotherapy. The combination of P3, BCG cell wall skeleton and Re Et was also effective when there was an interval of 1 or 7 days between immunotherapy and surgery. In addition, this combination retarded, and in some experiments, eliminated metastatic tumor growth in animals given immunotherapy immediately prior to surgery and in animals given immunotherapy 2 days after surgery.

TUMOR REGRESSION CAUSED BY ENDOTOXINS COMBINED WITH TREHALOSE DIMYCOLATE

ABSTRACT The synergistic antitumor activity of two adjuvants, endotoxic glycolipid extracted from Re mutants of gram-negative bacteria and trehalose mycolate isolated from mycobacteria, against guinea pig syngeneic line-10 tumor was abrogated after peptidic substances accompanying the endotoxic extracts had been removed. This activity could be restored by combining peptide-free endotoxin with either cell wall skeleton from Bacillus Calmette-Guerin, a polymeric mycolic acid-arabinogalactan-mucopeptide, or with a combination of two separate components, trehalose dimycolate and synthetic N-acetyl-muramyl-L-alanyl-(L-seryl)-D-isoglutamine (MDP). The tumor regressive activity could also be restored to the refined endotoxin trehalose mycolate mixture by the addition of an essentially nontoxic lipoid side fraction recovered during the isolation of endotoxic glycolipids, which contained a small amount of peptidic substances, or by the addition of nontoxic Brauns lipoprotein known to contain covalently bound MDP moieties. Highly endotoxic lipopolysaccharide (LPS) extracted from wild type Enterobacteriaceae so far tested failed to cause tumor regression. However, acid-hydrolysis of wild type Serratia marcescens LPS led to a residual peptide-containing fraction, designated RESI, which serologically cross-reacted with endotoxins from Re mutant Salmonellae and which, in combination with trehalose dimycolate, provided a cure rate of 90%. This RESI was essentially nonpyrogenic and was about 100 times less toxic than a typical potent endotoxin, suggesting that there was no correlation between the antitumor property and endotoxicity.

Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

SummaryThe purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 106 line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 105 viable line-10 cells. The results showed that as few as 107 immune spleen cells completely inhibited the growth of 105 tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 107 immune cells admixed with 105 tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.