Immunotherapy of a plasmacytoma with attenuated salmonella.

An attenuated strain of Salmonella typhimurium, SL3235, developed as a prototypic typhoid vaccine, is shown to retard growth of a murine plasmacytoma, TEPC-183, and to prolong survival of tumor-bearing mice. Live salmonella, but not acetone-killed organisms, had antitumor activity. The immunotherapeutic effect was demonstrable when the tumor was injected intralesionally or intraperitoneally. Increased survival, longer mean time to death, and retardation of tumor growth were found when the salmonella were given intralesionally as late as the sixth day post-tumor injection. Timing of salmonella inoculation, as well as the salmonella dose, had an effect on treatment efficacy. Injection of salmonella intraperitoneally exerted a strong antitumor effect when given as late as the third day post-tumor inoculation. The highest dose (2 x 10(6)) of salmonella was less effective than doses 10- or 100-fold lower. TEPC-183 plasmacytoma is rapidly growing and highly immunosuppressive, so the ability of the salmonella to exert therapeutic activity against it is a measure of the potency of the vaccine. These observations are of interest, as they show that a genetically engineered, avirulent strain of Salmonella has immunotherapeutic properties similar to those of BCG and other biological response modifiers, and might have clinical potential as an antitumor agent.

Clinical results and immunologic effects of a mixed bacterial vaccine in cancer patients.

A biological response modifier, mixed bacterial vaccine (MBV), derived from Streptococcus pyogenes and Serratia marcescens was used as a single agent in the treatment of 11 patients with refractory malignancies. MBV's effect on interleukin-2 (IL-2) production, plasma interferon (IFN) and tumor necrosis factor (TNF) levels was monitored. Most patients' peripheral blood mononuclear cells continued to produce baseline to elevated levels of IL-2, in spite of age and disease status. Several patients maintained moderate to high IFN levels. In general there was little correlation between IL-2 and IFN levels or with the response to therapy. One of 11 patients had minor response, 1 of 11 had partial response, 4 of 11 had temporary stabilization of disease, and 5 of 11 had progressive disease. A patient with AIDS and Kaposi's sarcoma experienced a dramatic improvement in performance status and disease stabilization. In all patients side effects occurred only following i.v. and not i.m. administration and included fever and chills. No adverse hepatic, renal or hematologic effects were observed. MBV is a well-tolerated biological response modifier with modest activity in advanced human tumors.

Preliminary result of mixed bacterial vaccine as adjuvant treatment of hepatocellular carcinoma.

Mixed bacterial vaccine (MBV) was employed in the multi-modality treatment of hepatocellular carcinoma (HCC) during 1985-1988. Thirty eight patients undergoing palliative resection and cisplatin therapy (Series 1) and 48 patients with unresectable HCC who received hepatic artery ligation + intraarterial cisplatin infusion + radiotherapy (Series 2) were randomized to receive MBV or not. In series 1, the 1- and 2-year survival rates of MBV group and control were 75% vs 58% (P = 0.19) and 45% vs 39% (P = 0.23). In series 2, the 1-, 2- and 3-year survival rates were 59%, 41% and 41% for MBV group and 39%, 25% and 20% for the control, respectively (P1 = 0.07, P2 = 0.09, P3 = 0.07). In addition, MBV improved the "second look" resection rate to 40% as compared to 17% in the control (P greater than 0.05). MBV could also prevent such immunosuppression as decrease of macrophage activity caused by radiotherapy. We consider MBV a potential nonspecific immunostimulant in the multimodality treatment of HCC.

The effect of a bacterial vaccine on tumors and the immune response of ICR/Ha mice.

This study examined the effect of mixed bacterial vaccine (MBV), a biological response modifier prepared from Streptococcus pyogenes and Serratia marcescens, on the immune system of mice and on the regression of a transplantable mouse tumor sarcoma 37. The study examined MBV's biological properties and analyzed its chemical composition. The chemical composition varied with the growth media. A typical centrifuged, dialyzed supernate of the serum-containing preparation was found to consist mainly of protein and minimal amounts of carbohydrate and endotoxin, while MBV made with synthetic medium contained similar amounts of all three. MBV was nontoxic for mice, which gained weight following the injection of 0.5-1.0 ml of MBV. MBV caused regression of 20-100% of well-established mouse tumors without appreciable toxicity. MBV also had a striking effect on the immune response of mice to sheep red blood cells. When administered simultaneously with antigen injection, MBV increased the number of antibody-secreting splenocytes measured by the plaque-forming assay threefold. Serum antibody levels also increased two- to threefold. MBV did not enhance the immune response to pneumococcal polysaccharide type III, a B-cell-dependent response. However, the in vivo administration of MBV increased the in vitro response to MBV and the B-cell mitogen lipopolysaccharide. MBV compares favorably with other biological response modifiers because of its enhancing effect on the immune response and its oncolytic properties at nontoxic levels.

Effect of the mixed bacterial vaccine on the immune response of patients with non-small cell lung cancer and refractory malignancies.

Since 1984, 13 patients were entered into our study and 12 patients have completed one or more cycles of treatment with mixed bacterial vaccine (MBV), a natural biologic response modifier derived from Streptococcus pyogenes and Serratia marcescens. Eight patients with refractory malignancy were treated with MBV only (0.1 ml intravenously [IV]) twice weekly for 3-16 weeks (colorectal cancer, pancreatic cancer, chronic lymphatic leukemia, hepatoma [two patients], sarcoma [three patients]). Four patients with advanced non-small cell lung cancer were treated with MBV in combination with low-dose cyclophosphamide, day 1; cisplatin, day 15; and MBV, 0.1 ml IV, days 5, 7, and 9. Two patients in this study received cyclophosphamide and cisplatin alone. The cycle was repeated every 28 days. Plasma interferon levels, interleukin-2 production by peripheral lymphocytes, and lymphocyte subpopulations were monitored. Interferon levels and interleukin-2 production showed increased or sustained values in general. In some patients, B-cells and helper T-cell populations increased, whereas T-suppressor cell numbers declined. With one exception, side effects were mild and consisted of fever greater than 37.8 degrees C (nine of 13), chills (11 of 13), increased respiratory rate (nine of 13), minor changes in blood pressure (seven of 13), and nausea (three of 13). One patient with non-small cell lung cancer had a partial response. Two patients with non-small cell lung cancer and one patient with refractory malignancy had stable disease and performance status at the end of 8 weeks of treatment; one patient with refractory malignancy was stable at the end of 4 weeks of treatment. In this pilot study, cancer patients treated with MBV showed objective evidence of immune stimulation with acceptable toxicity.

Effect of cocaine on the immune response and host resistance in BALB/c mice.

This study focuses on the effect of varying regimens of cocaine administration on three parameters of the immune response: antibody production, resistance to infection by Streptococcus pneumoniae following immunization, and resistance to tumors. The effect of cocaine on antibody production of female and male BALB/c mice was investigated to both a T-independent (pneumococcal polysaccharide type III [SSS-III]) and a T-dependent antigen (the 2,4-dinitrophenyl ligand [DNP]). It was found that high doses of cocaine injected 3 times/day prior to SSS-III resulted in a small rise in antibody levels in male mice. Low doses given for 4 days prior to or subsequent to SSS-III injection had no effect on the antibody response nor on the susceptibility to infection by live S. pneumoniae. High dosages of cocaine administered 3-5 times/day had no effect on the anti-DNP immune response of male mice but resulted in an almost 2-fold increase of anti-DNP plaque-forming cells in female mice.

Cellular changes and antitumor responses in the plasmacytoma-bearing mouse following cyclophosphamide treatment.

Mice bearing the immunosuppressive plasmacytoma TEPC-183 exhibit a marked splenic hyperplasia. We have characterized these tumor-reactive splenocytes on the basis of cell surface marker expression, nonspecific esterase activity, and morphology. Although splenocyte numbers increased progressively throughout tumor growth, B- and T-lymphocytes, as defined by surface immunoglobulin and Thy-1 antigen expression, respectively, did not increase significantly. Fourteen days after tumor implantation, T-lymphocytes decreased from 70 million to 50 million per spleen. However, cells expressing Mac-1 antigen or nonspecific esterase activity increased from 10 to 65 million. This constituted a 6-fold increase in splenic macrophages. Further studies utilizing the expression of PC.2 antigen in conjunction with morphological examination indicated that metastatic TEPC-183 cells comprise approximately 5% of the tumor-host splenocyte population 14 days after implantation. Ablation of plasmacytoma by cyclophosphamide inhibited the tumor-associated splenocytosis and led to an increase in splenic T-cells (from 70 to 120 million). In addition, macrophage numbers returned to normal. This study also assessed the ability of splenocytes from animals with either actively growing tumors or those from cyclophosphamide-treated tumor-bearing mice to mediate an antitumor response. Splenocytes, when assessed 1 wk following tumor ablation by cyclophosphamide, demonstrated antitumor activity in Winn neutralization assays. This activity was not detectable in splenocytes from animals with progressively growing tumors. Additional studies revealed that the cell population involved in the antitumor effect was glass nonadherent, nylon-wool nonadherent, and expressed Thy-1 antigen. These observations were consistent with the expansion of the splenic T-lymphocyte population following cyclophosphamide treatment. However, the immune response directed against primary TEPC-183 tumor cells was not inhibitory to metastatic tumor cells.

Effect of TEPC-183 plasmacytoma on resistance of passively or actively immunized BALB/c mice to infection with Streptococcus pneumoniae.

This study focuses on the effect of TEPC-183 (tetramethylpentadecane)-induced plasmacytoma on resistance of mice to infection with Streptococcus pneumoniae type III. Vaccination of normal or of TEPC-183-bearing mice with either a polyvalent pneumococcal vaccine (Pnu-Imune) or purified polysaccharide type III protects both against challenge with live S. pneumoniae. However, a 38-fold increase in susceptibility of the tumor-bearing mice over that of controls was observed. Immunized splenectomized mice were ten times more susceptible to infection than were immunized normal mice. However, this increased susceptibility of splenectomized mice could be overcome by passively administered antibody. In passive protection experiments, antisera obtained from normal and TEPC-183-bearing or from cyclophosphamide-treated mice were almost equally protective. A 6-fold increase of antibody (133 ng of antibody), however, was required to protect TEPC-183-bearing mice against challenge with 500 organisms, as compared with the 22 ng of antibody required to protect normal mice, indicative of defective host defense mechanisms in addition to the lower production of antibody in the TEPC-183-bearing mice. In contrast, mice bearing an equal tumor load of Sarcoma 37 behaved similarly to normal mice, showing that the increased susceptibility to infection was due to TEPC-183, a particularly immunosuppressive tumor.

An immunosuppressive lipoprotein fraction from TEPC-183 bearing mice.

Lipoprotein (LP) fractions prepared from sera of normal and plasmacytoma (TEPC-183) bearing mice, were compared with respect to their effect on the in vitro anti-2,4,6-trinitrophenyl plaque forming cell response. Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions of sera from the plasmacytoma bearing mice contained immunosuppressive activity that was absent in VLDL and LDL fractions of normal serum LP fractions. The suppressive activity did not correlate with cholesterol or triglyceride content. Crossed-immunoelectrophoresis and polyacrylamide gel electrophoresis revealed components of the suppressive VLDL and LDL fractions which were not present in normal serum LP fractions, suggesting a modification of serum LPs by the plasmacytoma.

Effect of cyclophosphamide on the immune response of BALB/c mice bearing an immunoglobulin M plasmacytoma (TEPC-183).

Mice bearing TEPC-183, an immunoglobulin (M(kappa)-secreting plasmacytoma, exhibit severe suppression of their immune responses. Since chemotherapy with cyclophosphamide (CY) is used in the treatment of myeloma, the present study was undertaken to determine its effect on the immune response. CY was tested at different doses in order to establish whether a tumor-lytic dose could be established which would minimally impair the immune response. In addition, CY was injected at different time intervals in respect to antigen administration, and its short- and long-term effects on the immune response were determined. It was found that 1 mg CY suppressed the six-day primary immune response to the type 3 pneumococcal polysaccharide SSS-III, and even a secondary response 30 days later. Three mg CY were required to suppress the primary as well as the secondary immune response elicited by 2,4-dinitrophenyl-hemocyanin. One injection of 1 mg CY per mouse resulted in complete regression of all tumors. Splenectomy did not aid in the recovery of the immune response to SSS-III but accentuated the impairment. The time of CY administration played a crucial role in affecting the immune response. When CY was administered simultaneously with or two days after antigen injection, it was totally suppressive, whereas the immune response was enhanced when CY was given four days prior to antigen injection. Minimal effects were observed when CY was given at other time intervals.

Effect of splenectomy on the immune response of BALB/c mice bearing an immunoglobulin M plasmacytoma (TEPC-183).

Mice bearing TEPC-183, an immunoglobulin M(kappa)-secreting plasmacytoma, exhibit severe suppression of their immune responses to both thymus-dependent and thymus-independent antigens, 2,4-dinitrophenyl, and the type 3 pneumococcal polysaccharide SSS-III. This immunosuppression is not lifted by splenectomy of the tumor-bearing mice or prevented by removal of the spleen prior to tumor injection. On the contrary, splenectomy either before or after tumor implantation further accentuates the immunosuppressed state of tumor bearers and even depresses the immune response of normal mice. A secondary immune response of normal mice 34 to 51 days after splenectomy is still reduced. Thus, spleen cells may play a dual role. While splenectomy may remove a source of suppressor cells in tumor-bearing mice, it also eliminates a major source of antibody-producing cells and results in reduced immune responses of normal and TEPC-183-bearing mice. These findings have clinical relevance since splenectomy is used as a therapeutic and diagnostic procedure in neoplastic lymphoproliferative disorders.

Immunosupression of the primary and secondary immune response by an IgM plasmacytoma (TEPC-183).

Previously we had established that TEPC-183 IgM(K) suppressed the primary immune response (IR) to both the T-dependent antigens 2,4-dinitrophenyl-haemocyanin (DNP-HCY) and T-independent pneumococcal polysaccharides. In the current investigation, the effect of TEPC-183 on an ongoing immune response to SS-III and DNP-HCY was examined. It was found that when TEPC-183 was injected 6 days after the initial antigen injection, at the height of the primary IR, the response was significantly suppressed to SS-III and to the DNP ligand. In addition, suppression of the secondary IR occurred when mice were injected with tumour as late as 35 days after the first antigen injection. Tumour removal lifted the immunosuppression to DNP and the tumour-removed group had a similar number of both direct and indirect anti-DNP-PFC, although HA levels were still reduced. When mice were pretreated with serum from normal mice or serum or ascites from TEPC-183 bearing mice, one day prior to and on the day of antigen injection, the immune response to DNP was reduced by TEPC-183 serum but not by normal mouse serum (NMS), while the anti-SSS-III response was reduced by both NMS and TEPC-183 serum. Thus, NMS selectively suppressed the T-independent response, but only TEPC-183 serum suppressed both types of responses. The suppressive effect of serum on the IR of normal mice indicates a role for soluble regulatory suppressive factors present in the serum of normal and tumour-bearing mice. The data are consistent with the idea that the tumour exerts its effect on the inductive as well as the proliferative phase of the immune response.

The effect of plasmacytomas on serum immunoglobulin levels of BALB/c mice.

The effect of tumor growth on serum immunoglobulin levels and on the immune response to sheep erythrocytes (SRBC) was studied in BALB/c mice bearing MOPC-315 (IgA), MOPC-460 (IgA), MOPC-173 (IgG2a) and MOPC-104E (IgM) to gain insight into the immunologic competence of the plasmacytoma-bearing host. The initial increase of myeloma protein coincided with the first appearance of the tumor and increased as the tumor progressed. However, at the time of death there was little correlation between spleen weights, tumor size, and myeloma-protein levels. The mean serum concentration of the myeloma proteins reached a higher level in the mice bearing tumors transplanted i.p. compared to those with tumors transferred subcutaneously (s.c.). Non-myeloma immunoglobulin levels in the serum were reduced: IgM was significantly lowered in the presence of MOPC-315 injected i.p. and MOPC-460 injected s.c. and the IgG2 levels were depressed in mice injected i.p. with MOPC-315 and MOPC-104E. The only significant reduction of IgA levels was seen when MOPC-173 was transplanted i.p. The decreases observed in immunoglobulin levels correlated with plasmacytoma growth. They were specific for myeloma and were not due to tumor growth per se since the levels of all immunoglobulins tested increased in the presence of Sarcoma 37, a pleomorphic neoplasm. The primary plaque-forming cell (PFC) response of tumor-bearing animals after the injection of 0.5 ml of 10% SRBC was either similar or enhanced compared to the controls. However, with a lower SRBC dosage (0.5 ml of 2% SRBC) the indirect PFC were reduced with mice bearing MOPC-104E and MOPC-173. Tumor sizes did not seem to correlate with reduction of the PFC response. MOPC-460-bearing mice had a comparable number of PFC per spleen to those of the controls, but reduced numbers when calculated per 10 spleen cells. Consistently, hemagglutination titers were reduced in all tumor-bearing animals. The number of direct and indirect PFC per spleen was increased in mice bearing Sarcoma 37, compared to the controls. The possible implications of these findings are discussed.