Antitumor efficacy of lymphokine-activated killer cells and recombinant interleukin-2 in vivo: survival benefit and mechanisms of tumor escape in mice undergoing immunotherapy

The studies described in this paper showed that the combination of i.v.-transferred lymphokine-activated killer (LAK) cells and i.p. injections of recombinant interleukin-2 (RIL-2) was highly effective in vivo in reducing established pulmonary metastases of natural killer cell-resistant, MCA-105 sarcoma and B16 melanoma in mice. A 3-day in vitro incubation of normal C57BL/6 splenocytes in medium containing pure RIL-2 generated LAK cells that, when combined with RIL-2, reduced the mean number of established pulmonary micrometastases of the B16 melanoma and of the MCA-105 sarcoma from 179 and 140, respectively (in groups treated with Hanks' balanced salt solution alone), to 12 (P = 0.01) and 6 (P = 0.01), respectively. This combined immunotherapy also consistently resulted in significant prolongation of survival in mice with established, 3-day or 10-day pulmonary metastases of the MCA-105 sarcoma. Mice autopsied at time of death revealed a massive involvement of tumor in the lungs and liver in the group receiving Hanks' balanced salt solution alone compared to a small number of residual large lung or liver metastases in the group receiving LAK cells plus RIL-2. Experiments were designed to test whether variants existed in the original tumor cell inoculum that were resistant to killing by LAK cells and thus could account for the metastases that "escaped" the combined immunotherapy of LAK cells plus RIL-2 in vivo. Metastases of the MCA-105 sarcoma that escaped the combined therapy of LAK cells plus RIL-2 were dissected from the organs of mice upon autopsy and directly tested for susceptibility in vitro to lysis by LAK cells in 4-h and 18-h 51Cr release assays. Target cells derived from the metastases were lysed to an equivalent extent as those prepared from a fresh MCA-105 sarcoma that was growing s.c. In addition, successful reduction of pulmonary metastases established by the i.v. infusion of MCA-105 sarcoma cells obtained from metastases that escaped a prior round of therapy with LAK cells and RIL-2 could be achieved in vivo by the combined immunotherapy as well as by high doses of RIL-2 alone. Culture adapted, natural killer cell-resistant B16 melanoma cells surviving two successive treatments with LAK cells in vitro remained as susceptible to LAK cell lysis as untreated B16 melanoma cells in 18-h 51Cr release assays.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antitumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: successful immunotherapy of established pulmonary metastases from weakly immunogenic and nonimmunogenic murine tumors of three district histological types

We have recently shown that the systemic administration of lymphokine activated killer cells (LAK cells) plus relatively low doses of recombinant interleukin 2 (RIL-2) or the administration of high doses of RIL-2 alone can reduce the number of established pulmonary metastases from the weakly immunogenic MCA-105 sarcoma in mice. We have now analyzed the therapeutic efficacy of these treatments on both weakly and nonimmunogenic tumors of three distinct histological types in two different mouse strains. In all experiments, LAK cells were administered i.v. on days 3 and 6 and RIL-2 was injected i.p. from days 3 through 8 after tumor induction. The MCA-101 sarcoma was completely nonimmunogenic as defined by its inability to successfully immunize C57BL/6 mice. Nevertheless, administration of LAK cells plus 7,500-10,000 units RIL-2 was highly effective in reducing the number of established 3-day pulmonary metastases from this sarcoma [at 7,500 units RIL-2, mean number of metastases 37 +/- 11 (SE); P less than 0.05; at 100,000 units, 2 +/- 1; P less than 0.05] when compared to Hanks' balanced salt solution treated control animals (116 +/- 9). Likewise, RIL-2 alone at doses of 20,000 units/injection or greater had significant antimetastatic effects (77 +/- 12; P less than 0.05). Established 3-day pulmonary metastases from the MCA-38 adenocarcinoma in C57BL/6 mice and the M-3 melanoma in C3H mice were also susceptible to adoptive immunotherapy with LAK cells plus RIL-2 and with high dose RIL-2 alone. Treatment of mice with LAK cells alone or with low doses of RIL-2 alone (less than or equal to 20,000 units/injection) had little if any antitumor effects. LAK cells were tested for cytolytic activity in vitro against tumor target cells of a variety of histological types; there was no discernible relationship between susceptibility to lysis by LAK cells in vitro and therapeutic efficacy in vivo. These findings have thus demonstrated that the successful immunotherapy of established pulmonary metastases with LAK cells plus RIL-2 or with high dose RIL-2 alone includes: tumors that are immunogenic and nonimmunogenic; tumors of distinct histological types such as sarcoma, adenocarcinoma, and melanoma; and tumors in at least two different mouse strains, C57BL/6 and C3H, and that there is little correlation between the in vitro lysability of tumor cells by LAK effectors and the susceptibility of these same tumors to successful immunotherapy in vivo.     

Successful immunotherapy of murine experimental hepatic metastases with lymphokine-activated killer cells and recombinant interleukin 2

Lymphokine-activated killer (LAK) cells are generated in vitro by the incubation of normal murine splenocytes in interleukin 2. We have shown previously that the systemic injection of LAK cells in conjunction with recombinant interleukin 2 can reduce the number of established pulmonary metastases in mice. In an attempt to study this approach in the treatment of hepatic metastases, we developed a technique for the induction of hepatic metastases in mice based on the intrasplenic injection of tumor cells and have tested the effects of LAK cells and recombinant interleukin 2 produced in Escherichia coli (RIL-2) therapy on these metastases. Treatment with LAK cells alone in 14 consecutive experiments rarely produced significant reduction in metastases over control (mean percentage reduction, 12%). Therapy with RIL-2 alone produced a dose-dependent reduction in the number of liver metastases. In 20 consecutive experiments when RIL-2 was administered i.p. three times a day at doses varying from 1,000 to 5,000, 10,000 to 15,000, and 25,000 units, a statistically significant (P less than 0.05) reduction in liver metastases was seen in 2 of 12, 2 of 4, and 8 of 12 determinations, respectively (percentage reduction, 0 to 97; mean, 42%). At doses greater than 25,000 units, the reduction in metastases was highly reproducible (percentage reduction, 66 to 95; mean, 83%) and was statistically significant in 14 of 14 determinations. When LAK cells were given i.v. in addition to RIL-2 administration in 16 consecutive experiments, the percentage reduction in liver metastases was markedly increased over that seen with RIL-2 alone (mean percentage reduction, 77% at doses of 5,000 to 25,000 units of RIL-2 and mean reduction, 97% for doses greater than 25,000 units of RIL-2). At doses of 5,000, 10,000, 25,000, and greater than 25,000 units of RIL-2 plus LAK cells, significant reduction of liver metastases (P less than 0.05) was achieved in 3 of 7, 2 of 2, 8 of 8, and 6 of 6 determinations, respectively. When animals were given fresh splenocytes or splenocytes cultured in complete medium without RIL-2 instead of LAK cells, no reduction in liver metastases was seen except for that attributable to the administration of RIL-2 alone. Sublethal total body irradiation of the mice prior to therapy abrogated the therapeutic effects of RIL-2, but the effects of treatment with LAK cells plus RIL-2 were maintained. Thus, treatment with RIL-2 alone or in combination with LAK cells is effective in reducing the number of established hepatic micrometastases in a murine model.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunotherapy of murine hepatic metastases with lymphokine-activated killer cells expanded in serum-free media and recombinant interleukin 2

It has been shown that the systemic administration of lymphokine-activated killer (LAK) cells with recombinant interleukin 2 (RIL-2) is effective in reducing the number of established pulmonary and hepatic metastases in murine models. Similarly, this modality of therapy has been proven effective against certain selected human tumors as well. In view of the rising concern with transmission of virally related communicable diseases such as hepatitis and AIDS, we have undertaken the evaluation of a serum-free medium (AIM V) for the generation and expansion of murine LAK cells for use in in vivo tumor immunotherapy against murine hepatic metastases. Day 3 LAK cells generated in AIM V medium demonstrated a greater percentage of viable cells than cells generated in serum containing complete medium (CM) (mean percentage of yield, 59 versus 25%, AIM V medium versus CM, respectively, P less than 0.001, N = 6 consecutive experiments). When day 3 LAK cells were transferred to new medium (CM to CM and AIM V to AIM V), a highly reproducible expansion of these cells was demonstrated which was significantly better for cells expanded in AIM V medium versus cells expanded in CM (mean fold expansion on day 21 of culture; 201 versus 54, AIM V medium versus CM, respectively, P less than 0.005, N = 4 consecutive experiments). When day 3 LAK cells, day 5 expanded LAK cells, and day 13 expanded LAK cells grown in CM or in AIM V medium were given in vivo with RIL-2 to mice harboring hepatic metastases, cells grown in AIM V medium demonstrated an increased antitumor activity compared to cells grown in CM. As an example in experiment 1, the mean number of metastases with day 5 expanded LAK cells grown in CM and given with RIL-2 was 47 while the mean number of metastases with day 5 expanded LAK cells grown in AIM V medium and given with RIL-2 was 5 (P less than 0.002). These experiments demonstrate that AIM V medium can be utilized to generate greater numbers of murine LAK cells with enhanced in vivo antitumor activity compared to cells generated in CM. These findings could be applied to the expansion of cytotoxic cells for human antitumor therapy.     

Augmentation of interleukin-2 immunotherapeutic effects by lymphokine-activated killer cells and allogeneic stimulation in murine tumor cells

Interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells were used in intraperitoneal and pulmonary tumor models in C57BL/6 mice. To maintain the immunotherapeutic effects of IL-2 plus LAK treatment but reduce its toxicity, ways were sought to augment IL-2 effects. The investigation showed that the adoptive transfer of LAK cells was a prerequisite for successful therapy of intraperitoneal cancer. When LAK cells were given on consecutive days within one course of immunotherapy, antitumor efficacy was augmented with additional doses of LAK cells. However, with the reduction of 1 complete cycle of IL-2 + LAK cells, no further reduction in intraperitoneal tumor was observed as compared to the reduction after 2 or 4 cycles. LAK cells generated from splenocytes of mice that had received an allogeneic tumor challenge 1 week earlier exerted a highly increased cytotoxicity as compared to normal LAK cells. Furthermore, the potentiation effect of an allogeneic response of the host at the tumor site was demonstrated by decreased numbers of lung implants and improved survival in mice given mixtures of syngeneic and allogeneic tumor cell suspensions. An alloimmune response within the microenvironment of tumor tissue markedly enhanced the antitumor effect of IL-2 against the syngeneic tumor. It was concluded that there is a fundamental need to improve the recruitment of adoptively transferred LAK cells or LAK precursors into tumor tissue. This may be the next step required in the further development of IL-2 and LAK immunotherapy.     

Comparison of cellular immunotherapies and anti-CD3 in the treatment of MCA-38-LD experimental hepatic metastases in C57BL/6 mice

An experimental model of hepatic metastases in C57BL/6 mice was used to compare the antitumor effects of lymphokine-activated killer (LAK) cells, anti-CD3-activated T-cells (ATC), and anti-CD3 alone. Liver metastases were produced by in vivo passage of MCA-38-LD adenocarcinoma via the ileocolic vein. LAK cells and ATC were generated by 3-day in vitro incubation of spleen cells in interleukin 2 and anti-CD3, respectively. Percentage of tumor volume in livers was determined with a morphometric technique. With less than therapeutic LAK cell doses (0.5-1.0 x 10(7) cells), no effect was seen in mean (+SE, -SE) percentage of tumor volume of control [23.3 (29.3, 18.5)] compared to LAK cell-treated [21.6 (29.3, 15.9)] animals. The same number of ATC significantly reduced the mean percentage of tumor volume [2.7 (4.7, 1.4)] (P less than 0.005). High dose interleukin 2 also significantly decreased tumor volume. More strikingly, a single dose of anti-CD3 alone had a beneficial effect on mean percentage of tumor volume when given i.p. [1.0 (1.9, 0.4)] or i.v. [1.2 (1.7, 0.7)] (P less than 0.0003). A total of 33% of anti-CD3-treated mice had no detectable liver metastases. In 51Cr release assays, the cytotoxicity of ATC was shown to be partially mediated by nylon wool-adherent accessory cells. The effectiveness of anti-CD3 in this immunotherapy model suggests that a similar approach may be taken to immunotherapy of human malignancies, without the requirements for in vitro-generated killer cells or exogenously administered interleukin 2.     

Reduction of pulmonary metastases of B16 melanoma by human recombinant interleukin 2 and lymphokine-activated killer cells in immunosuppressed C57BL/6 mice receiving anticancer agent

The immunosuppressive effect of a water-soluble nitrosourea derivative, 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), was evaluated in terms of the cytotoxicity of spleen lymphocytes, and the restoring effect of lymphokine-activated killer (LAK) cells and/or human recombinant interleukin-2 (rIL-2) on the cytotoxicities of spleen lymphocytes was examined in ACNU-treated C57BL/6 mice. In addition, we tested whether the administration of LAK cells and/or rIL-2 could reduce the increased numbers of pulmonary metastases in ACNU-treated mice. The maximum effective dose of ACNU suppressed the cytotoxicity of spleen lymphocytes and pretreatment with ACNU enhanced the induction of artificial pulmonary metastases. The administration of LAK cells and/or human rIL-2 restored the cytotoxicity of spleen lymphocytes against YAC-1 and syngeneic B-16 melanoma cells in ACNU-treated mice, and these treatments partially suppressed the increased numbers of artificial pulmonary metastases of B-16 melanoma cells in ACNU-treated mice. These results are extremely important in providing a rationale for the introduction of adoptive immunotherapy using LAK cells and rIL-2 in patients with advanced cancer who are being treated with anticancer agent(s).     

Increased local antitumor effects of interleukin 2 liposomes in mice with MCA-106 sarcoma pulmonary metastases

The effects of liposome formulations of interleukin 2 (IL-2) and local route were studied in C57BL/6 mice with MCA-106 sarcoma pulmonary metastases. IL-2 liposomes made by hydration of powdered dimyristoyl-phosphatidylcholine with aqueous recombinant IL-2 had 95% of the IL-2 associated with the lipid fraction. When mice with pulmonary micrometastases were treated once daily with free cytokine on days 5, 6, and 7 after tumor inoculation, the intrathoracic route was superior to the i.p. or s.c. routes. When IL-2 liposomes were administered by the local intrathoracic route, significantly better antitumor effects (P less than 0.01) were seen compared to empty liposomes or free IL-2 as determined by (a) increased survival and (b) reduced numbers of pulmonary metastases. Minimal toxicity was observed. Results indicate that local route and incorporation of IL-2 in liposomes may enhance therapeutic efficacy and facilitate more practical daily dosing regimens.     

Enhancement of therapeutic effect of interleukin-2 on spontaneous pulmonary metastases of Lewis lung carcinoma by killer helper factor associated with increased induction of killer activity

Killer helper factor (KHF) was previously found to be produced by a human T cell hybridoma, 24A . CA2. We studied the therapeutic effects of interleukin-2 (IL-2) and KHF on the inhibition of pulmonary metastases of syngeneic Lewis lung carcinoma (3LL) in C57BL/6N mice. Multiple subcutaneous (sc) injections of IL-2 plus KHF had significantly more effect than injections of IL-2 alone in inhibiting spontaneous pulmonary metastases and prolonging survival of the mice. The effect of KHF with IL-2 on induction of lymphokine (IL-2)-activated killer (LAK) activity against P-29 cells was examined in the murine system. Spleen cells generated LAK activity after incubation for 4 days with more than 500 U/ml of IL-2. In contrast, KHF alone did not render spleen cells cytotoxic. The combination of these lymphokines at subthreshold concentrations, however, resulted in significant in vitro induction of LAK activity. The LAK activity of splenocytes incubated with IL-2 plus KHF was maximal after 4 days, and persisted for longer than that of cells treated with IL-2 alone. The LAK cells induced by KHF plus IL-2 were also cytotoxic to FBL and YAC-1 cells. Moreover, spleen cells of mice bearing lung metastases could be induced to the cytotoxic state by sc injections of IL-2 plus KHF. These results indicate that combination treatment with IL-2 and the new lymphokine KHF should be useful clinically in inducing LAK activity for inhibition of pulmonary metastases.     

Immunogenicity of the tumor determines the outcome of immunotherapy with interleukin-2, ABPP, and cyclophosphamide of micro- and macrometastatic intraperitoneal tumor

We have shown previously that interleukin-2 (IL-2) and the interferon inducer ABPP can induce lymphokine activated killer (LAK) cell activity in vivo after intraperitoneal (i.p.) administration. The antitumor effects of various immunotherapy regimens with IL-2, LAK cells, ABPP, and cyclophosphamide (CY) on microscopic (day 3) and on macroscopic (day 8) i.p. tumors, differing in histology and immunogenicity, were studied in C57BL6 mice. The immunogenic sarcomas MCA-105, -106, and the colon adenocarcinoma MCA-38, and the nonimmunogenic sarcomas MCA-101, -102 were used. After i.p. inoculation of 1 X 10(5) tumor cells i.p. on day 0, therapy with IL-2 +/- LAK cells consisted of 1 X 10(8) LAK cells, i.p., on day 3 and IL-2, 10k to 25k U, i.p., b.i.d., on days 3 to 7. Treatment with ABPP +/- CY consisted of CY, 50 mg/kg, i.p., on day 3 and/or 8 ABPP, 250 mg/kg on days 3, 4 and/or 8, 9. In the treatment of micrometastases, IL-2 + LAK cell therapy was effective against all tumors. Therapy with low dose IL-2 alone was effective only against immunogenic tumors. Combined therapy with CY was very effective against the immunogenic tumors and prolonged survival significantly. Only marginal antitumor effects were seen against nonimmunogenic tumors. In the setting of advanced tumor, chemoimmunotherapy was only successful against immunogenic tumors. These observations demonstrate that the immunogenicity of the tumor is of major importance in the outcome of immunotherapy, especially in the setting of advanced disease. This indicates that, apart from LAK cells, the in vivo activation of other cytotoxic effector cells is important in the rejection of immunogenic tumors.     

Local conditions in the host influence immunotherapy with interleukin-2 and LAK cells

Production of biological response modifiers through recombinant techniques has stimulated interest in immunotherapy of cancer. One of these, interleukin-2 (IL-2), will induce in vivo as well as in vitro proliferation of noncommitted T lymphocytes into lymphokine-activated killer (LAK) cells: cells cytolytic for a broad range of tumor cells. We have demonstrated earlier that immunotherapy with IL-2 and LAK cells will reduce tumor load and prolong survival in a significant way in an intraperitoneal (ip) tumor model as well as in other models. Nevertheless, mice die of one or two metastases escaping immunotherapy. Activation of the host immune system might boost endogenous IL-2 production. Activation might also enhance immunotherapy by increasing the necessary cofactors. Loco-regional allogeneic pretreatment ip 14 days prior to syngeneic tumor challenge did not enhance, but completely abrogated, ip IL-2 and LAK cell therapy (peritoneal cancer index, 0.6 +/- 0.3 vs 2.6 +/- 0.2, P2 = 0.003). Tumor bulk is not the reason for escape of immunotherapy either. One week after intracutaneous (ic) tumor inoculation a noncurative or sham tumor resection was performed, followed by IL-2 and LAK cell therapy either ip or in and around the tumor nodule. No significant difference in tumor diameter or survival of mice was seen. Allogenic tumor cells admixed with syngeneic tumor cells will induce an inflammatory reaction locally and regionally. This inflammatory reaction in the syngeneic host will enhance the treatment with IL-2. The allogeneic (P815) and syngeneic (MCA-105) tumor cell mixture was injected ic. Growth rate was retarded and survival prolonged in a significant way when the cell mixture was treated with ip IL-2 injections; no difference was seen when the admixture was not treated or the syngeneic ic tumor alone was treated with IL-2. We conclude that host immune status and recruitment of immunocompetent cells locally to the tumor site determine the outcome of immunotherapy with IL-2 and LAK cells.     

Inhibition of pulmonary metastasis of Lewis lung carcinoma by a glucan, Schizophyllan

The effect of a glucan, Schizophyllan (SPG), on pulmonary metastases in syngeneic mice bearing Lewis lung carcinoma (3LL) was examined. As a model of pulmonary metastases, 3LL cells were implanted into the footpads of C57BL/6 mice, the resulting primary tumor was removed 9-10 days later. The inhibitory effect of SPG was evaluated from the number of pulmonary surface nodules on the lungs about 3 weeks after tumor implantation. SPG was found to have antimetastatic activity, which depended on its dose and time of injection. A single injection of 100 or 200 mg/kg or daily injections of 20 or 50 mg/kg of SPG after removal of the primary tumor markedly inhibited pulmonary metastases. Combined therapy with cyclophosphamide and SPG significantly prolonged the survival of mice with pulmonary metastases. Enhancement of the in vitro cytotoxic activity of peritoneal macrophages and bronchoalveolar or whole lung cells against 3LL cells was noted in SPG-treated mice on day 7 after a single intraperitoneal injection of 100 mg/kg SPG. Intravenous transfer of peritoneal macrophages activated with SPG inhibited the development of pulmonary micrometastases.     

Enhanced antimetastatic activity of lymphokine-activated killer cells purified and expanded by their adherence to plastic

We have recently reported a simple and reproducible technique for the purification and rapid expansion of homogeneous populations of large granular lymphocytes expressing a natural killer cell phenotype and high levels of broad antitumor cytotoxic activity [lymphokine-activated killer (LAK) activity]. This technique exploits the observation that, in the presence of recombinant interleukin 2 (rIL-2), large granular lymphocytes/natural killer cells become adherent to plastic surfaces, actively proliferate, and acquire high levels of LAK activity. Because of their adherent properties these cells have been termed adherent LAK or A-LAK cells. The present studies investigate the antimetastatic effects of A-LAK cells in a syngeneic rat model of experimental pulmonary and hepatic metastases. For pulmonary metastases, F344 rats received i.v. injections with a natural killer-resistant mammary adenocarcinoma, MADB106, and, for hepatic metastases, animals received an intrasplenic injection of MADB106 tumor cells followed by surgical splenectomy. Three days later, the animals were treated with A-LAK cells alone, A-LAK cells plus rIL-2, or rIL-2 alone. These treatments were compared to immunotherapy using standard cultures of LAK cells (unfractionated spleen cells) and rIL-2. The results indicate that the administration of unfractionated LAK cells plus interleukin 2 (IL-2) was effective in reducing established lung or liver metastases in this rat model. However, the results also indicate that purified populations of A-LAK cells in combination with rIL-2 demonstrate dramatic and superior antimetastatic effects when compared to LAK cells cultured under standard conditions. The antimetastatic effects of standard LAK cells or A-LAK cells plus IL-2 translated into significant survival benefits compared to animals receiving no therapy or IL-2 therapy alone. Survival after therapy with A-LAK cells plus IL-2 was significantly prolonged compared to treatment with standard LAK cells. These data suggest that purified populations of LAK cells (derived from natural killer cells) may prove superior for adoptive immunotherapy in the clinical setting.     

Studies of effects of recombinant human tumor necrosis factor on autochthonous tumor and transplanted normal tissue in mice

The acute hemorrhagic necrosis of tumor nodules caused by the systemic administration of recombinant human tumor necrosis factor alpha (rhTNF-alpha) has been partially attributed to changes in tumor neovascularity. In this study, the effects of rhTNF-alpha were tested on primary autochthonous sarcomas induced in C57BL/6 mice by 3-methylcholanthrene, on spontaneous mammary tumors in C3H/HEN mammary tumor virus positive mice, and on the rejection of normal tissue transplants at different stages of maturity in C57BL/6 mice. Primary i.m. tumors induced by injection of 3-methylcholanthrene grew slowly over a 3-month period and became acutely necrotic after i.v. injection of rhTNF-alpha (2-6 micrograms). In addition, rhTNF-alpha caused a reduction in tumor area of 24% over 10 days compared to a 43% increase in tumor area in control mice receiving excipient (P2 less than 0.01). Histopathologically, tumors underwent central necrosis with a neutrophilic infiltration as was observed previously for serially transplanted tumors following rhTNF-alpha administration. Spontaneous, virally induced mammary tumors underwent a 11% regression on administration of rhTNF-alpha (4-6 micrograms) compared to a 24% growth in mice receiving excipient (P2 less than 0.05). Normal mice were grafted with syngeneic (C57BL/6) or partially allogeneic (C57BL/10 to C57BL/6) skin and were treated with a single dose of rhTNF-alpha (5-20 micrograms) i.v. at either 5, 10, or 15 days posttransplantation. rhTNF-alpha administration had no effect on the integrity of the skin grafts at any maturation point tested (syngeneic graft survival at 60 days: excipient, 35 of 36 versus 20 micrograms rhTNF-alpha, 35 of 36; allogeneic graft survival: excipient, 46 +/- 8 days versus 20 micrograms rhTNF-alpha, 48 +/- 10 days). In addition, rhTNF-alpha had no effect on the integrity of a syngeneic neonatal s.c. heart graft (graft survival at 60 days, excipient, 35 of 36 versus rhTNF-alpha, 30 of 33). Thus, although rhTNF-alpha administration led to marked necrosis and growth inhibition of vascularized tumor, no effect was observed on vascularized normal tissue transplants. To evaluate possible systemic effects of the tumor bearing state on the maturing neovascularity of normal tissue grafts, the three transplant models were studied in mice bearing a 9-day established MCA-106 s.c. sarcoma. After treatment with rhTNF-alpha (2-6 micrograms), acute necrosis and tumor size reduction was apparent in the s.c. tumors; however, no effect was seen in any of the normal tissue transplants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biological and antitumor effects of recombinant human macrophage colony-stimulating factor in mice

The administration of recombinant human macrophage colony-stimulating factor (M-CSF) i.p. in doses of 25 or 100 micrograms twice daily for 5 consecutive days to non-tumor-bearing C57BL/6 mice resulted in a dose-dependent infiltration of mononuclear cells in the livers but not the lungs of these treated animals. Immunohistochemical examination of fixed liver tissue with the murine macrophage-specific monoclonal antibody, F4/80, revealed a greater than 5-fold increase in the number of hepatic macrophages. Quantification of F4/80-positive cells in a mononuclear single cell suspension derived from liver revealed a greater than 25-fold expansion in the number of hepatic macrophages compared to control mice. These cells were then tested in 18-h 51Cr release assays for tumoricidal activity, after an 18-h incubation with or without gamma-interferon, against cultured P815 targets. Significant tumor cell lysis by these liver-associated mononuclear cells occurred, which was enhanced by gamma-interferon preincubation. The systemic administration of M-CSF alone at high dose had no antitumor impact in vivo against 3-day pulmonary metastases from the MCA-203 sarcoma and B16 melanoma or hepatic metastases from the B16 melanoma or MCA-105, -203, or -207 sarcomas. Although the systemic administration of M-CSF in combination with tumor-specific monoclonal antibody had no effect on 3-day pulmonary metastases from the B16 melanoma, significant reductions in liver metastases were seen. These murine studies demonstrate the biological activity of recombinant human M-CSF in vivo and suggest that the administration of this cytokine in combination with specific monoclonal antibody may be useful in the treatment of patients with metastatic disease at sites of monocyte/macrophage accumulation.     

Enhancement of Therapeutic Effect of Interleukin-2 on Spontaneous Pulmonary Metastases of Lewis Lung Carcinoma by Killer Helper Factor Associated with Increased Induction of Killer Activity

Killer helper factor (KHF) was previously found to be produced by a human T cell hybridoma, 24A CA2. We studied the therapeutic effects of interleukin-2 (IL-2) and KHF on the inhibition of pulmonary mctastascs of syngeneic Lewis lung carcinoma (3LL) in C57BL/6N mice. Multiple subcutaneous (sc) injections of IL-2 plus KHF had significantly more effect than injections of IL-2 alone in inhibiting spontaneous pulmonary mctastascs and prolonging survival of the mice. The effect of KHF with IL-2 on induction of lymphokine(IL-2)-activated killer (LAK) activity against P-29 cells was examined in the murine system. Spleen cells generated LAK activity after incubation for 4 days with more than 500 U/ml of IL-2. In contrast, KHF alone did not render spleen cells cytotoxic. The combination of these lymphokines at subthreshold concentrations, however, resulted in significant in vitro induction of LAK activity. The LAK activity of splenocytes incubated with IL-2 plus KHF was maximal after 4 days, and persisted for longer than that of cells treated with IL-2 alone. The LAK cells induced by KHF plus IL-2 were also cytotoxic to FBL and YAC-1 cells. Moreover, spleen cells of mice bearing lung metastases could be induced to the cytotoxic state by sc injections of IL-2 plus KHF. These results indicate that combination treatment with IL-2 and the new lymphokine KHF should he useful clinically in inducing LAK activity for inhibition of pulmonary metastases.     

Immunotherapy of murine sarcomas using lymphokine activated killer cells: optimization of the schedule and route of administration of recombinant interleukin-2

Interleukin-2 (IL-2) at high doses or at low doses in concert with lymphokine-activated killer (LAK) cells can produce regression of established pulmonary and hepatic metastases from a variety of tumors in mice. IL-2 appears to mediate its antitumor effect through the generation of LAK cells in vivo from endogenous lymphocytes and by the stimulation of host and transferred LAK cell proliferation in tissues. In this paper we have investigated different strategies for IL-2 administration to determine which regimen produced maximal in vivo proliferation and optimal immunotherapeutic efficacy of LAK cells. Tissue expansion of lymphoid cells was assessed using an assay of in vivo labeling of dividing cells by the thymidine analogue, 5-[125I]iododeoxyuridine. The therapeutic effect of the different IL-2 administration protocols was determined by evaluating their efficacy in the treatment of established, 3-day pulmonary metastases from sarcomas in mice. The selection of IL-2 injection regimens for evaluation was based upon pharmacokinetic studies of IL-2 in mice. A single i.v. or i.p. dose yielded high peak IL-2 levels that could be measured for only a few hours after injection, while IL-2 given i.p. thrice daily produced titers that were detectable throughout the study periods (greater than or equal to 6 units/ml of serum after 100,000 units of IL-2 i.p. thrice daily). Using the proliferation and therapy models, we tested the same cumulative daily doses of IL-2 administered by i.v. or i.p. once daily, or i.p. thrice daily regimens. The i.p. thrice daily protocol stimulated greater lymphoid cell proliferation in the lungs, for example, than did the other regimens. Similarly, 300,000 units of IL-2 divided i.p. thrice daily were more successful in reducing metastases (n = 16) than was the entire dose given i.v. once daily (n = 190; P less than 0.05) or i.p. once daily (n = 71; P less than 0.05). When compared to the i.p. or i.v. once daily protocols, the i.p. thrice daily regimen for IL-2 also produced greater proliferation of exogenous LAK cells, as well as a more effective therapeutic outcome when IL-2 was combined with transferred LAK cells. Thus, sustained, lower levels of IL-2 were more effective than brief, high peak titers for stimulation of proliferation and antitumor activity. We then evaluated the effect of duration of IL-2 treatment as well as the number of LAK cell injections in the two models.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differences in the effects of host suppression on the adoptive immunotherapy of subcutaneous and visceral tumors

A syngeneic transplantable sarcoma induced in C57BL/6 mice, MCA 105, was used in studies to examine host suppression on the adoptive immunotherapy of established intradermal and experimentally induced pulmonary and hepatic metastases. Fresh immune splenocytes were generated from mice immunized to the MCA 105 tumor by a mixture of viable tumor cells and Corynebacterium parvum. The adoptive immunotherapy of intradermal MCA 105 tumor with immune cells required prior immunosuppression of the recipient by sublethal irradiation with 500 R or T-cell depletion. The effect of whole-body sublethal irradiation appeared to eliminate a systemic host suppression mechanism, since partialbody irradiation involving the tumor-bearing area did not permit successful immunotherapy. Host irradiation was not required to achieve successful immunotherapy of experimentally induced pulmonary or hepatic metastases. In nonirradiated recipients bearing both intradermal and pulmonary tumors, host suppression did not affect the function of transferred immune cells to induce regression of pulmonary metastases. Thus, suppression of adoptive immunotherapy appears to be relevant to tumors confined to the skin and subcutaneous tissue but not to tumor in visceral sites, such as the lung and liver.     

Effects of the immunomodulator LS 2616 on growth and metastasis of the murine B16-F10 melanoma

The carboxamide-quinoline LS 2616 is a novel immunomodulator augmenting natural killer (NK) cell activity and T-lymphocyte related effector functions. To investigate the possible usefulness of LS 2616 in immunotherapy of tumors, the effect of the substance on growth and metastasis of the B16-F10 melanoma in syngeneic C57BL/6 mice was investigated. Treatment with LS 2616 from the time of s.c. inoculation of B16-F10 cells significantly reduced tumor take. Continuous treatment of mice with LS 2616 initiated 4 days prior to i.v. injection of tumor cells reduced the number of pulmonary metastases by 85%. When treatment with LS 2616 was started 4 days after i.v. injection of tumor cells, a time when established tumor foci were readily detectable in the lungs, a significant reduction in the number of pulmonary metastases resulted. LS 2616 significantly reduced the number of spontaneous pulmonary metastases developing from a B16-F10 tumor growing in the footpad. When treatment with LS 2616 was initiated after the establishment of grossly visible spontaneous pulmonary metastases, no significant effect on the number of metastases was found after 2 weeks of treatment. However, combined treatment with a dose of cyclophosphamide which in itself was ineffective resulted in a statistically significant 70% reduction in the number of remaining pulmonary metastases. Injection of antibodies to asialomonoganglioside which strongly reduce NK cell activity in various organs was used as a probe for the involvement of NK cells in the effects of LS 2616 on the B16-F10 tumor. The therapeutic efficiency of LS 2616 on tumor take when given from the time of s.c. inoculation, on the number of i.v. induced pulmonary metastases when treatment was started before tumor cell injection, as well as the spontaneous development of pulmonary metastases during exposure to the substance was abrogated by simultaneous injection with antibodies to asialomonoganglioside. In contrast, the beneficial effects of LS 2616 on already established i.v. produced or spontaneous pulmonary metastases were unaltered in mice made NK cell deficient by injection of anti-asialomonoganglioside antibodies. In conclusion, LS 2616 has potent antitumor activities mediated by NK cells as well as non-NK cell related defense mechanisms.     

Experiments evaluating antitumor immunity induced by cholesterol hemisuccinate-treated syngeneic cell vaccines

Evaluation of the efficiency of immunizations with syngeneic tumor vaccines prepared from cells treated with cholesterol hemisuccinate (CHS) was performed in five animal models: P815 mastocytoma in DBA/2 mice, MCA-103 fibrosarcoma in C57BL/6N mice, L1210 leukemia in DBA/2 mice, Ehrlich ascites carcinoma in C57BL/6N mice, and CBP pancreatic cancer in CB/SsLak hamsters. Animals received two to four weekly intraperitoneal immunizations with 10(6) or 10(7) tumor cells, followed by challenges with syngeneic viable tumor cells. Survival and tumor growth rates were observed. No significant differences were observed among animals immunized with CHS-treated irradiated tumor vaccines, nontreated irradiated tumor vaccines, and nonimmunized controls in the P815, MCA-103, L1210, and CBP models. Mice immunized with nontreated irradiated Ehrlich ascites cell vaccines showed longer survival than those immunized with CHS-treated irradiated cell vaccines and nonimmunized controls. Results indicated that CHS-treated tumor cell vaccines were not effective in protecting against tumor challenges in five different syngeneic tumor models.