Reno Parker

Biologically active components from mycobacterial cell walls: IV. protection of mice against aerosol infection with virulent Mycobacterium tuberculosis

Abstract Cell walls prepared from Bacillus Calmette-Guerin (BCG) and associated with oil droplets have been shown to protect mice from aerosol infection with the virulent H37Rv strain of Mycobacterium tuberculosis. A combination of the peptidoglycolipid cell wall skeleton (CWS-I), prepared by exhaustive organic solvent extraction of BCG cell walls, and P3, isolated by centrifugal microparticulate bed chromatography from the free lipids of the mycobacterial cell wall, gave protection comparable to that provided by whole BCG cell wall. Neither component by itself was capable of protecting mice. The mechanism of protection based upon the chemistry of the mycobacterial components is discussed.

Preparation and antitumor activity of nontoxic lipid A

SummaryHighly refined, disaggregated endotoxic glycolipids (B5) from heptose-less (Re) mutant Salmonella typhimurium quantitatively converted to nontoxic (lethality for chick embryos) and nonpyrogenic (fever in rabbits) lipid A by treatment with boiling 0.1 N HCl (B5-HC1). Nontoxic B5-HCl, like toxic B5, caused regression of line-10 tumors and elimination of lymph node metastasis in 27 of 32 (84%) syngeneic strain 2 guinea pigs at a dosage of 150 μg. At this dosage, toxic B5 led to a cure in 54 of 67 (81%) tumor-bearing animals. All cured animals rejected a second line-10 tumor cell transplant. This activity depended on combining the toxic or nontoxic endotoxins with mycobacterial trehalose mycolate (P3) and an essentially nontoxic peptide-containing side-fraction (ACP) recovered during the isolation of B5. In contrast to toxic B5 or endotoxins in general, nontoxic B5-HCl did not cause endotoxic shock when combined with adjuvant dipeptide (MDP) and injected IV into guinea pigs. Chemical analysis showed that the phosphate content of nontoxic B5-HCl was about one-half that observed in toxic B5 or in toxic KDO-free lipid A, which was obtained by treating toxic B5 with sodium acetate at pH 4.5 at 100° C (B5-pH 4.5). The molar ratio of glucosamine: phosphorus: fatty acids was 2:1:4 for nontoxic B5-HCl and was 2:2:4 for toxic B5-pH 4.5. These results demonstrate that endotoxic extracts could be selectively detoxified while retaining antitumor properties. Thus, nontoxic B5-HCl may be a potential candidate for immunotherapy of human cancer.

IMMUNOTHERAPY WITH NONVIABLE MICROBIAL COMPONENTS

Structural components of microorganisms have been studied for immunopotentiating effect with the aid of transplantable (line 10) tumors in syngeneic guinea pigs. Microbial components were associated with oil droplets, suspended in Tween-saline, and injected intralesionally. BCG cell walls, given in this way, produced regression and cure of 50-60% of established tumors, as did viable BCG. Lipid extraction markedly reduced the tumor-regressing potency of cell walls, but P3, a trehalose mycolate present in the extract, restored full activity to the cell wall residue. P3 alone was nonsensitizing and had no antitumor activity, but it enhanced the latter property of various other microbial products. For example, the cure rates produced by cell walls of M. tuberculosis, M. bovis, M. phlei, or M. smegmatis were enhanced from 20-60% to as much as 90% by addition of P3. P3 also conferred antitumor activity on products from unrelated microbes, such as cell walls of E. coli, and in combination with endotoxins from rough Re mutant salmonellae, it produced cure rates of up to 93%. These results suggest that P3 is essential to the immunopotentiating activity of mycobacteria and that it may be broadly applicable in immunotherapy of cancer with microbial agents.

Separation of the mixture of trehalose 6,6′-dimycolates comprising the mycobacterial glycolipid fraction, “P3”

Summary Ultra-purified trehalose dimycolate, “P3”, an agent with potent host-reactive and immunological properties, including the ability to enhance tumor immune responses, was isolated from various strains of Mycobacterium tuberculosis, M. bovis, M. avium , or M. phlei and subjected to per-trimethylsilylation to permit chromatographic resolution of trehalose dimycolates containing different pairs of mycolic acids. Samples of trimethylsilylated P3 from virulent strains of human and bovine tubercle bacilli were resolved into six different components based on pairs of α, β, and γ-mycolic acids, whereas P3 from avirulent or attenuated strains contained fewer components due to the absence of detectable β-mycolic acid-containing diesters. The virulence of pathogenic mycobacteria may depend to a significant extent upon the presence or absence of a given component.

Intralesional immunotherapy of malignant melanoma with mycobacterium smegmatis cell wall skeleton combined with trehalose dimycolate (P3).

The clinical efficacy of intralesional immunotherapy utilizing Mycobacterium smegmatis cell wall skeleton (CWS) and trehalose dimycolate attached to oil droplets was investigated in 15 patients with advanced malignant melanoma. Patients received 300 μg to 1050 μg of the CWS combined with one‐half that amount of trehalose dimycolate every 1 to 2 weeks for a total of 8 treatments. Therapy was continued if regression of injected lesions only occurred. Therapy was discontinued if regression of noninjected disease also occurred. Six of the 15 patients had regressionof at least one injected lesion. Four of these 6 patients also had regression of noninjected disease lasting 4+, 6, 16 and 18+ months. Response was highly related to immune status. Six (83%) of 7 patients who reacted to one of a battery of skin tests responded. All 8 patients who did not react to skin tests failed to respond to therapy. There was no correlation of response with sex, prior therapy, disease‐free interval or presence of visceral disease. Mycobacterial CWS and trehalose dimycolate is an effective immunotherapeutic agent. Additional studies of purified immunoadjuvants are warranted. Cancer 44:495‐503, 1979.

Phase I study of intradermal immunotherapy with oil-attached Mycobacterium smegmatis cell wall skeleton and trehalose dimycolate

SummaryThe clinical toxicity of intradermal immunotherapy with a nonviable mycobacterial vaccine consisting of oil-attached Mycobacterium smegmatis cell wall skeleton (CWS) and trehalose dimycolate (P3) was evaluated. Fifteen patients with advanced hypernephroma, lung cancer, or malignant melanoma were evaluated. Patients received one to ten separate intradermal injections in the subclavicular areas weekly for up to 8 weeks. Each separate injection usually contained 75 μg CWS and 37.5 μg P3.There were few systemic side effects. Mild fever occurred in 30% of 69 treatments. Severe local toxicity with ulceration and/or abscess formation occurred in seven patients. Regression of disease was observed in one patient to occur on two separate occasions following separate courses of therapy.Although intradermal CWS/P3 can be locally toxic, treatment with up to four separate injections of 75 μg CWS combined with 37.5 μg P3 every 1–2 weeks appears appropriate, from this study, for additional clinical trials.

Moieties of mycobacterial mycolates required for inducing granulomatous reactions.

Abstract Trehalose dimycolates and monomycolates isolated from a variety of Mycobacteria species as well as synthetic trehalose mycolates and trehalose behenylbehenate produced granulomatous responses in the lungs of mice. Trehalose alone or mycolic acids or their methyl esters, however, did not. These data suggest that the sugar moiety of these defined fatty acid esters is required for the production of this cellular inflammatory reaction. When mice were challenged with virulent Mycobactorium tuberculosis they showed increased resistance against infection during the time when the granulomatous response was greatest.

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.