Experimental models of immunochemotherapeutic synergism: study of the influence of the treatment schedule

Graded doses of LSTPA or L1210 leukemia cells were injected ip or iv into fully compatible hosts or mice incompatible for Multiple Minor Histocompatibility Loci (MMHL). Three days later the animals were treated with single doses of BCNU, NM, DTIC and VCR. The results showed that NM and VCR could synergize with the weak anti-tumor immune responses of MMHL-histocompatible mice only upon ip injection of the tumor. If the same tumor has been injected iv, only BCNU could synergize with the host's antitumor response. On DTIC treatment, no synergistic effects were detectable for either route of tumor's challenge.     

Disruption of DNA synthesis and structure of mouse L1210 leukemia cells, sensitive and resistant to 1-methyl-1 nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in vivo

Leukemia L1210 cells with acquired resistance to 1-methyl-1-nitrosourea (MNU) (L1210/MNU) and 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (L1210/BCNU) were developed from leukemia L1210 cells sensitive to these drugs (L1210/0). The modal chromosome number of leukemia L1210/MNU and L1210/BCNU cells increases from 40 (L1210/0) to 41. It was shown that in leukemia L1210/MNU cells the inhibition of DNA synthesis after MNU administration in a therapeutic dose (80 mg/kg) is lasted within 24 hours, while that in leukemia L1210/0 cell--within 96 hours. After administration of BCNU (20 mg/kg) inhibition of DNA synthesis in leukemia L1210/BCNU cells reached of 50% of control in comparison with practically complete inhibition of DNA synthesis in leukemia L1210/0 cells. Centrifugation on alkaline sucrose density gradients revealed no differences in the rate of sedimentation of leukemia L1210/0, L1210/MNU and L1210/BCNU cell lysates. After 1 hour treatment with MNU of mice bearing L1210/MNU and L1210/0 leukemia cells single-strand breaks in DNA were determined. After 4 hours these strand-breaks retained in leukemia L1210/0 cells, but were eliminated in leukemia L1210/MNU cells. Administration of BCNU to mice with leukemia L1210/0 and L1210/BCNU cells resulted in both cases in the production of DNA aggregates. There is no complete cross-resistance between MNU and BCNU which allows a substitution of these drugs providing for the increase in their therapeutic efficiency.     

Rejection of reovirus-treated L1210 leukemia cells by mice

Summary L1210 leukemia cells were treated in vitro with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and reovirus to determine their interactive effects on rejection of these tumor cells by mice. The cells were treated with BCNU at concentrations of 0, 3, or 10 M, incubated for 48 h, then treated with reovirus at a multiplicity of infection of 0, 10, 30, or 100 for 2, 6, or 12 h. The survival of mice injected with cells treated with any amount of reovirus, regardless of BCNU treatment, was greater than that of mice injected with untreated cells. Exposure of the cells to reovirus for 6 or 12 h increased the survival of mice injected with these cells as compared with that of mice injected with cells exposed to reovirus for 2 h. Of the survivors, 76% were resistant to subsequent challenge with untreated L1210 cells. These results suggest that activities associated with reovirus replication may cause modifications of L1210 cells that enable them to induce an immune response, thus facilitating their rejection. A lack of correlation between differences in DNA synthesis (measured by ³H-thymidine uptake) by treated cells and the ability of those cells to kill recipient mice indicates that rejection of cells treated with reovirus or BCNU is not due to a decrease in their ability to proliferate or, presumably, to generate lethal tumors. The survival of mice injected with treated L1210 cell preparations containing as few as 2.9% reovirus-infected cells was enhanced to the same degree as that of mice injected with those containing as many as 14.6% infected cells, indicating that modification of only a minor component of the tumor cell population is sufficient to alter the ability of the cells to generate a lethal tumor.This work was supported by a research grant from the Miami University Faculty Research Committee and a Sigma Xi Grant-in-Aid of Research     

Postsurgical adjuvant chemoimmunotherapy with recombinant interleukin-2 and 1,3-bis-(2-chloroethyl)-1-nitrosurea on spontaneous metastases of a non-immunogenic murine tumour

Summary The efficacy of the association of recombinant interleukin-2 (rIL-2) with chemotherapy has been investigated on an experimental model representative of clinical tumours, i.e. on post-surgical spontaneous metastases of a non-immunogenic tumour. We used the M5076 ovarian reticulum cell sarcoma, which metastatizes to the liver after intra-footpad implantation. Such a tumour appeared to be non-immunogenic by a variety of commonly used in vivo assays. Four clinically widely employed drugs, i.e. doxorubicin,cis-diamminedichloroplatinum II, cyclophosphamide and 1,3-bis-(2-chloroethyl)-1-nitrosurea (BCNU), were tested and BCNU proved to be the most effective one when administered as single injection at the maximum tolerated dose (33 mg/kg i.p.) 1 day after tumour excision. When moderate doses of rIL-2 (6 × 10⁵ IU in three injections per day for 5 days) were administered at three different intervals after BCNU, namely before the nadir of white blood cells (1 day after BCNU), at the nadir (3 days after BCNU) or at recovery (6 days after BCNU), no increase in BCNU antitumour activity was observed. The same results were obtained by administering rIL-2 for 5 days before BCNU. Higher doses of rIL-2 (1.2 × 10⁶ IU in three injections per day for 5 days), which were always well tolerated in sham-excised non-tumour-bearing mice, proved lethal in two out of four experiments in tumour-bearing animals. In the two experiments in which no lethality was observed, the administration of high doses of rIL-2 1 or 6 days after BCNU significantly increased the antitumour activity of BCNU alone. rIL-2 alone was not active even when administered at high doses. These results indicate that high but not moderate doses of rIL-2 may increase the activity of BCNU against a non-immunogenic tumour. Moreover, they suggest that rIL-2 tolerability is reduced in tumour-bearing mice.     

Relation between the changes in the structure and synthesis of DNA in the cells of mouse leukemia L1210 induced by 1-methyl-1-nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea

The damage of DNA structure and synthesis in murine leukemia L1210 cells upon single administration in therapeutic doses of antitumour agents of N-nitrosourea type, such as 1-methyl-1-nitrosourea (MNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied. MNU and BCNU were characterized by stronger inhibitory effects on de novo DNA synthesis compared to additional pathway of DNA synthesis in leukemia L1210 cells in vivo. Centrifugation in alkaline sucrose density gradients of L1210 cell lysates has revealed persistent single-strand breaks and alkaline-labile sites in newly replicated DNA. Parental DNA structure was more stable to damaging drug effects than that of newly replicated DNA. The results are consistent with our previous data on the differences in the mechanisms of MNU and BCNU action and the absence of complete cross resistance between the drugs.     

The role of N-nitrosourea-induced changes in the nucleoid structure and activity of repair enzymes in the development of drug resistance in mice with leukemia L1210

Using centrifugation of the nucleoid in a neutral sucrose gradient, the damages in the secondary structure of DNA and the activity of repair enzymes, such as DNA-polymerases alpha and beta and poly(ADP-riboso) polymerase, induced by 1-methyl-nitrosourea (MNU) and 1.3-bis (2-chloroethyl)-1-nitrosourea (BCNU) injected at maximal nonlethal single doses to mice bearing parent leukemia cells (L1210/0) and resistant to MNU and BCNU leukemia L1210 cells (L1210/MNU and L1210/BCNU), were studied. The MNU-induced production of single-strand breaks in L1210/0 and L1210/MNU cells was more conspicuous in newly replicated DNA than in those in preexisting DNA. A more fast repair of the damages in newly replicated DNA was detected in L1210/BCNU and especially in L1210/MNU leukemia cells as compared with L1210/0 cells. The data obtained suggest that there are prone errors in the repair of DNA template, since most of the single-strand breaks were revealed in the newly replicated DNA synthesized on the repaired DNA. The repair of DNA damages in L1210/BCNU and especially in L1210/MNU cells was accompanied by the activation of DNA-polymerases alpha and beta and poly(ADP-riboso)polymerase. Both DNA-polymerases--alpha and beta--were shown to be involved in repair of DNA damages induced by MNU and only DNA-polymerase beta was involved in the repair of damages induced by BCNU.     

Immunological resistance to L1210 leukemia induced by viable L1210/DTIC cells

Summary Permanent, drug-induced antigenic alterations, not detectable in parental cells and transmissible after the withdrawal of treatment with the drug, have been obtained in mouse lymphoma. Viable L1210/DTIC cells, because they are rejected by syngeneic animals and carry L1210-associated TAA, can elicit host resistance to a subsequent inoculum of parental L1210. Mice challenged with viable L1210/DTIC cells, following rejection, were more resistant than mice immunized with inactivated parental cells. Resistance was specific and related to the immunogenicity of the TAA of the original tumor line employed.Active immunization was potentiated by adoptive transfer of immune lymphocytes, as evidenced by marked improvement in animal survival. Also, the treatment of tumor-bearing animals with anticancer compounds in conjunction with immunological alteration may result in an improved therapeutic response. BCNU administered to immunized animals 6 days after challenge with parental tumor cells resulted in augmented host survival, possibly attributable to partial resistance of a secondary immune response to the drug and a late nadir of immunosuppression, occurring after the completion of therapeutic action. Cyclophosphamide given before immunization enhanced host survival to a subsequent challenge of L1210 leukemia, conceivably as the result of preferential inhibition of T suppressor cells.     

Tumor target-derived soluble factor synergizes with IFN-gamma and IL-2 to activate macrophages for tumor necrosis factor and nitric oxide production to mediate cytotoxicity of the same target.

Inflammatory mouse peritoneal macrophages were activated by IFN-gamma in synergy with IL-2 or Lipid A to mediate TNF production for autocrine generation of cytotoxic nitric oxide (NO) to kill P815 or L1210 tumor targets. It was determined that for IL-2, but not Lipid A, to effectively trigger activation of IFN-gamma-primed macrophages, the tumor targets must be also present for interaction with effector macrophages to mediate the production of TNF and NO. IFN-gamma- and IL-2-activated macrophages from syngeneic DBA/2 and allogeneic C3H mice had identical MHC-unrestricted requirements for interaction with DBA/2 mouse-derived P815 and L1210 targets to mediate production of TNF and NO for tumor cytotoxicity. To further define the mechanistic requirements for macrophage-tumor target interaction, IFN-gamma- and IL-2-activated macrophages were separated from P815 targets in culture by a semipermeable membrane. Under these conditions, both TNF and NO were produced by the macrophage, which indicated that the requirement for tumor target-macrophage interaction may be due to a soluble factor produced by the target rather than to direct physical contact. This was confirmed by experiments in which 24-h cell-free culture fluids, derived from either P815 or L1210 tumor targets, substituted for the intact tumor cells in the stimulation of TNF mRNA synthesis and secretion with NO generation of TNF mRNA synthesis and secretion with NO generation by IFN-gamma- and IL-2-activated C3H or DBA/2 macrophages. The activity in 24-h culture fluids derived from P815 and L1210 tumor targets was tentatively designated as tumor-derived recognition factor(s) (TDRF) since it was produced constitutively by the tumor targets and synergized with IFN-gamma and IL-2 to induce macrophage production of TNF and NO for death of the same targets. A variety of nontransformed human and mouse fibroblasts, mouse spleen lymphocytes, and two adherent mouse fibrosarcomas did not produce detectable TDRF activity, whereas two mouse T lymphomas, EL4 and EL4.IL-2, produced TDRF activity similar to L1210 mouse leukemia and P815 mastocytoma. The C3H/MCA, a TDRF-nonproducing mouse fibrosarcoma, was susceptible to cytotoxicity mediated by macrophages activated by IFN-gamma and Lipid A, but not by IL-2 triggering. Exogenous TDRF derived from L1210 targets reconstituted the cytotoxic activity for C3H/MCA MCA targets mediated by IFN-gamma- and IL-2-activated macrophages accompanied by the production of TNF and cytotoxic NO.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of X rays on BCNU-induced DNA crosslinking

We have used the technique of alkaline elution to study DNA interstrand crosslinking in 9L rat brain tumor cells treated with combinations of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and X rays. Irradiation with doses as low as 50 rad of X rays immediately or 6 hr after a 1-hr treatment with 60, 80, or 100 microM BCNU produced a significant increase in BCNU-induced DNA interstrand crosslinking. If cells were irradiated before BCNU treatment, the amount of crosslinking was not affected compared with BCNU alone. Cell survival experiments using 600 rad of X rays and 1-hr treatments with 0-30 microM BCNU were also performed. As found in the crosslinking studies, irradiation immediately or 6 hr after the BCNU treatment produced enhanced cell kill, but irradiation 6 hr before BCNU treatment did not produce enhanced cell kill. Therefore, the X-ray-mediated increase in BCNU-induced DNA interstrand crosslinking may be the mechanism through which cell kill is increased by combination treatment with the agents.     

Possible Novel Therapy for Malignant Gliomas with Secretable Trimeric TRAIL

Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and many novel therapeutic approaches, average survival for the patients with malignant gliomas is only about 1 year. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown potent and cancer-selective killing activity and drawn considerable attention as a promising therapy for cancers, but concerns over delivery and toxicity have limited progress. We have developed a secretable trimeric TRAIL (stTRAIL) and here evaluated the therapeutic potential of this stTRAIL-based gene therapy in brain tumors. An adenovirus (Ad-stTRAIL) delivering stTRAIL was injected into intra-cranial human glioma tumors established in nude mice and tumor growth monitored using the magnetic resonance imaging (MRI). Ad-stTRAIL gene therapy showed potent tumor suppressor activity with no toxic side effects at therapeutically effective doses. When compared with 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a conventional therapy for malignant gliomas, Ad-stTRAIL suppressed tumor growth more potently. The combination of Ad-stTRAIL and BCNU significantly increased survival compared to the control mice or mice receiving Ad-stTRAIL alone. Our data indicate that Ad-stTRAIL, either alone or combined with BCNU, has promise as a novel therapy for malignant gliomas.     

Immunotherapy of EL4 lymphoma with reovirus

Summary EL4 lymphoma was grown as an ascitic tumor in the peritoneal cavity of C57Bl/6 mice. Animals with different tumor burdens (either 10⁷ or 10⁹ cells) were treated with a single intraperitoneal injection of BCNU using doses from 20–40 mg/kg. Response as measured by mean survival time and percent survival was dependent on tumor burden and dose of drug. The objective of chemotherapy was to increase the mean survival time, but not the percent survival, in order to evaluate the therapeutic effect of reovirus. Mice were given 10⁸, 10⁹, or 10¹⁰ Pfu of reovirus at various times with respect to chemotherapy. The number of mice cured after treatment with both BCNU and reovirus was significantly greater compared to mice treated with BCNU only. Mice cured with combination therapy developed tumor-specific immunity as measured by cytotoxic lymphocytes and serum, and resistance to a lethal tumor challenge. The Abbreviations used are: BCNU: 1,3-bis-(2-chloroethyl)-1-nitrosourea; Saline: 0.9% NaCl solution; MEM: minimal essential medium; Pfu: plaque-forming units; FCS: fetal calf serum; BME: basal eagle's medium; SSC: sodium citrate-sodium chloride     

Increased oxidative stress created by adenoviral MnSOD or CuZnSOD plus BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) inhibits breast cancer cell growth

Superoxide dismutases (SODs) have been found to decrease tumor formation and angiogenesis. SOD gene therapy, as with many other gene transfer strategies, may not completely inhibit tumor growth on its own. Thus, concomitant therapies are necessary to completely control the spread of this disease. We hypothesized that intratumoral injection of AdSOD in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy would synergistically inhibit breast cancer growth. Our data indicate that BCNU when combined with SOD overexpression increased oxidative stress as suggested by elevated glutathione disulfide (GSSG) production in one of three breast cancer cell lines tested, at least in part due to glutathione reductase (GR) inactivation. The increased oxidative stress caused by BCNU combined with adenovirally expressed SODs, manganese or copper zinc SOD, decreased growth and survival in the three cell lines tested in vitro, but had the largest effect in the MDA-MB231 cell line, which showed the largest amount of oxidative stress. Delivery of MnSOD and BCNU intratumorally completely inhibited MDA-MB231 xenograft growth and increased nude mouse survival in vivo. Intravenous (iv) BCNU, recapitulating clinical usage, and intratumoral AdMnSOD delivery, to provide tumor specificity, provided similar decreased growth and survival in our nude mouse model. This cancer therapy produced impressive results, suggesting the potential use of oxidative stress-induced growth inhibitory treatments for breast cancer patients.     

Development of host-dependent high-grade tumor-specific immunity through a novel mechanism triggered by the Lyt-2+ tumor-specific T cell clone (K7L) that induces temporal growth of L1210 leukemia-K7L-variant

When a murine leukemia L1210-specific Lyt-2+ T cell clone, K7L, was injected i.p. into CD2F1 mice together with L1210, the normal growth of L1210 in the peritoneal cavity of the mice at the early stage (days 0 to 5) was strongly inhibited, but L1210 grew progressively at the middle-stage (days 5 to 10), and then was rejected at the late stage (days 10 to 20). The mice thus survived for long times (more than 60 days), whereas the normal control injected with L1210 alone died within 14 days. The L1210 that grew at the middle stage in mice initially inoculated with L1210 together with K7L was a K7L-insensitive (K7L-) variant. All of eight tumor clones established from L1210-K7L- by limiting dilution was insensitive to the antitumor activity of K7L, and this property of tumor clones was stable after repeated in vitro passage. The initial depression of the L1210 growth by K7L followed by growth and rejection of the variant L1210-K7L- by the host T cell activity was then found to prepare a strong, long-lasting (more than 3 mo) immunity to protect mice against the high-dose (10(7) cells per mouse) challenge of original L1210. Corresponding to this result, definite tumor (L1210)-specific cytotoxic T lymphocyte (CTL) activity against both variant and original L1210 targets was developed by antigen (L1210) restimulation in the culture of spleen cells from these mice, but was not increased to a detectable level before L1210-K7L- variant started to grow. It was suggested that the 1210-K7L- variant and the original L1210 should have the common tumor-specific antigen that was independent of the K7L-reactive antigen, and that original L1210, whose growth was retarded by K7L, primed the host with the common antigen to be enormously boosted by the subsequently growing L1210-K7L- variant.     

Tumorigenicity of interleukin-2 (IL-2)-cDNA-transfected L1210 lymphoma and its in vivo variants is modulated by changes in IL-2 expression; potential therapeutic implications

Summary To study parameters that affect the tumorigenicity of L1210 lymphoma we have analyzed the structure of MHC class I antigens of this tumor. In addition this tumor was transfected with interleukin-2 (IL-2) cDNA in order to determine the effects of high concentrations of IL-2 within the tumor environment. The nucleotide sequence of the class I K^d, D^d and L^d mRNAs from this tumor showed that the encoded amino acid sequence of the corresponding antigens is normal, thus suggesting that the tumorigenicity of L1210 lymphoma is not due to defective antigen presentation to tumor-specific cytotoxic T cells. In contrast, induction of IL-2 expression by cDNA transfection led to loss of tumorigenicity of the IL-2-secreting tumor cells. However, a fraction of long-term-surviving mice developed progressively growing variant tumors that showed substantial decrease or loss of IL-2 expression. These results suggest that IL-2 secretion by tumors is suicidal but, because of tumor heterogeneity, IL-2-loss-variant tumors may arise that are able to escape the immune defenses of the host. The observed consistent loss of IL-2 expression in variant tumors implies that specific targeting of large quantities of IL-2 to tumor cells may be a valuable approach to immunotherapy of cancer. In addition we find that under specific gamma ray irradiation IL-2-secreting tumor cells lose their ability to multiply yet continue to secrete IL-2 at levels equivalent to those secreted by unirradiated cells. Such IL-2-secreting irradiated tumor cells were found to be superior immunogens in comparison to the irradiated parental tumor cells, suggesting their use as tumor vaccines.     

Priming of the antitumor response promotes efficacy of interleukin-2 therapy

 We have studied the effect of active specific immunization (ASI) on the antitumor response induced by locoregional, low-dose interleukin-2 (IL-2) therapy. On day 0, mice were injected i.p. with viable, syngeneic tumor cells and with irradiated tumor cells (ASI). Low-dose IL-2 treatment was given i.p. for 5 consecutive days. ASI led to extended survival in two out of seven models tested. In these two models, enhanced efficacy was observed when both ASI and IL-2 were administered. In the five models in which ASI had no therapeutic value, ASI+IL-2 treatment was no more effective than IL-2 therapy. In the SL2 lymphoma model, use of ASI prior to IL-2 therapy given as early as days 1–5 led to at least 60% cure, whereas IL-2 therapy without ASI was only effective when administered after day 9. In the P815 mastocytoma model, however, ASI, IL-2, and the combination caused negative (suppressive) effects when administered on days 6–10. IL-2 administered on days 6–10 was therapeutically effective in this model when mice were treated with cyclophosphamide on day 6. In both the SL2 and the P815 tumor models, cured mice were specifically immune. The positive and negative effects observed were not due to the increased number of cells injected (non-specific inflammation) nor to possible antigenic alteration of the ASI cells by irradiation, as ASI with fragmented tumor cells was also effective in inducing synergy. Investigations into the underlying mechanism indicated that CD4⁺ cells play an important role. In total, the results indicate that ASI may be a good supplement to locoregional IL-2 treatment if care is taken to alleviate immunosuppressive activities. Received: 6 February 1997 / Accepted: 6 March 1997     

Evaluation of time and dose in treating mammary adenocarcinoma with immunostimulants

Summary BCG, C. parvum, and reovirus were used as immunostimulants in treating murine mammary adenocarcinoma (A-10) after tumor burden had been minimized with BCNU. Immunostimulants were administered at different times with respect to chemotherapy. Different doses were used to determine the optimal response as measured by survival. BCG induced the best response when 6.67×10⁵ organisms were given 2 days after chemotherapy. The optimal response with C. parvum was observed after a dose of 0.35 mg was given 1 or 2 days after chemotherapy. Similarly, reovirus produced the best response when 10¹⁰ plaque-forming units were given 2 days after chemotherapy. These data are consistent with previous findings and support the notion that immunostimulants require an appropriate lymphoid substrate in order to induce an adequate anti-tumor response.Tge abbreviations used are: BCNU: 1,3-bis-(2-chloroethyl)-1-nitrosourea Saline: 0.9% NaCl solution; BCG: Bacillus Calmette-Guerin; C. parvum: Corynebacterium parvum; pfu: plaque-forming unitsThis study was supported, in part, by Contract No. N01-CB-43864 and Grant No. CA 14460 from The National Cancer Institute     

Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.     

Potentiation of the efficacy of murine L1210 leukemia vaccine by a novel immunostimulator 7-thia-8-oxoguanosine: Increased survival after immunization with vaccine plus 7-thia-8-oxoguanosine

Summary We have investigated the ability of a novel immunopotentiator, 7-thia-8-oxoguanosine (7T8OG) to increase the efficacy of a weakly immunogenic murine L1210 leukemia vaccine. The vaccine was prepared by irradiating L1210 leukemia cells in a cesium source with a total of 6000-R dose. DBA/2 mice were treated with 150 mg/kg 7T8OG and/or with vaccine consisting of 10⁷ irradiated cells. In combination therapy, mice first received the vaccine and then were injected with 75 mg/kg 7T8OG 2 h and 4 h after vaccination. One week after the last treatment all mice were inoculated with 10⁴ live leukemia cells intraperitoneally. Control, untreated mice (n = 66) injected with 10⁴ live leukemia cells had a mean survival time ± standard error of 10.5±0.2 days. Treating mice (n = 66) with one, two or three doses of 7T8OG administered i.p. 1 week apart did not increase survival (mean survival time = 10.7 days). Mice immunized with one, two or three doses of vaccine had 14.5±1.1, 45.4±6.2 and 68.3±10.6 days mean survival, respectively. 7T8OG-stimulated vaccination increased the survival dramatically. The best survival was noted when the mice were treated with 2× (vaccine + 7T8OG). Immunization of mice (n = 30) with this treatment regimen increased the mean survival to 156±10.0 days. Over 90% of mice that were treated this way had a cumulative survival time greater than 160 days. In contrast, only 12% of the mice immunized twice with the leukemia vaccine alone survived over 160 days. These results suggest a rationale for the use of this immuno-potentiator with various vaccines for a more effective immunization.     

Therapeutic efficacy of human recombinant interleukin-2 (TGP-3) alone or in combination with cyclophosphamide and immunocompetent cells in allogeneic,semi-syngeneic,and syngeneic murine tumors

Summary The potential for a recombinant human interleukin-2 (rIL-2, TGP-3) alone, in combination with cyclophosphamide, and in combination with cyclophosphamide and normal immunocompetent cells to manifest biological activity in vivo was tested using allogeneic, semi-syngeneic, and syngeneic tumor-host systems in mice. The biological activity of rIL-2 was evaluated by the inhibition of the growth of tumors and the inhibition of metastases in short-term assays and, in long-term assays, the prolongation of the survival time of mice bearing subcutaneously (s.c.) or intradermally transplanted tumors. rIL-2 was injected s. c. daily continuously for up to 40 days or intermittently two to four times into mice bearing established tumors. In the short-term assays, the dose and schedule dependence of activity of rIL-2 alone was significantly manifested against sarcoma 180 in ICR mice (allogeneic) by the regression of the tumor, and was confirmed against Meth-A fibrosarcoma in BALB/c mice (syngeneic) by retarding the growth of the tumor. When assessed using these tumors, it was found that the antitumor activity of rIL-2 was scheduledependent: the growth of tumors was more significantly suppressed when rIL-2 was injected every day for 10 days, starting on the 7th day after tumor transplantation, than when rIL-2 was injected five times every other day or twice every 5th day, even if the total amounts of rIL-2 injected were same. The continuous injection for 10 days was considered to be a standard regimen and the daily effective doses of rIL-2 were 5, 10, and 25 µg/mouse. Using the standard regimen and the effective doses, the activity of rIL-2 alone was also observed against two other syngeneic tumors: Colon carcinoma 26 in BALB/c mice, by retarding the growth of the tumor, and Lewis lung carcinoma in C57BL/6 mice by reducing the formation of lung metastases. When assessed using M5076 reticulum cell sarcoma, in a long-term assay, the activity of rIL-2 alone was not manifested in C57BL/6 mice (syngeneic) even when rIL-2 was injected for a long period (20 days) but it was observed in BDF1 (semi-syngeneic) mice. On the other hand, it was found that rIL-2 was effective in combination with cyclophosphamide in prolonging the survival time of C57BL/6 mice bearing the tumor. After cyclophosphamide (2.0 mg) had been administered orally to mice on the 6th day after tumor transplantation, the tumor regressed temporarily but regrew; however, when rIL-2 at a dose of 10 µg was also injected daily for a long period (40 days), the regrowth was retarded and the survival time of the mice was significantly prolonged. Moreover, when normal immunocompetent cells were transferred at the tumor sites, the regrowth of the tumors was retarded more significantly even at a daily dose of 1 µg or 3 µg rIL-2, and mice were observed to be cured by daily doses over 3 µg. The results obtained in the syngeneic tumor-host systems indicate that the continuous injection of rIL-2 is necessary and important for its activity to be manifest in vivo, and that, when combined with cytotoxic drugs and/or with immunocompetent cells, the potential efficacy of rIL-2 is valuable in cancer therapy.