Augmentation of organ-associated natural killer activity by biological response modifiers. Isolation and characterization of large granular lymphocytes from the liver.

Natural killer (NK) activity in the rat and human has been attributed to cells having the morphology of large granular lymphocytes (LGL). However, this association has been less clear in the mouse, largely because of difficulties in obtaining highly enriched populations of LGL from normal spleen and blood. We have previously observed that the administration of the biological response modifier (BRM) maleic anhydride divinyl ether (MVE-2) strongly augmented NK activity in lung and liver, and the augmented NK activity coincided with increased resistance to the formation of experimental metastases in these organs. The degree of NK augmentation was most striking in the liver, an unexpected and previously unreported observation. In the present study, both MVE-2 or Corynebacterium parvum induced a dramatic augmentation of liver NK activity, which reached maximum levels 3-5 d after treatment. This augmentation of NK activity in the liver coincided with a large increase in the number of lymphoid cells with the morphological characteristics of LGL that could be isolated from enzymatically digested suspensions of perfused liver. The yield of LGL per liver following BRM treatment corresponded to a 10-50-fold increase as compared to normal mice. LGL were purified from these enzymatically digested suspensions of perfused liver by depletion of adherent cells on nylon wool columns and subsequent enrichment for low-density lymphoid cells by fractionation on Percoll density gradients. The enrichment of LGL correlated with greatly increased NK activity against YAC-1. Conversely, the higher-density fractions were depleted of both LGL and NK activity. This increase in NK activity in the liver was suppressed by in vivo treatment with anti-asialo GM1 (asGM1) serum. This treatment also resulted in a corresponding reduction in both the total number and percentage of LGL. By flow cytometry analysis, the phenotype of the majority of these highly cytolytic LGL isolated from the livers of BRM-treated mice were asGM1+, Thy-1+, Ly-5+, Qa-5+, Mac-1+, and Gma-1+, whereas these LGL were Ly-1-, Lyt-2-, L3T4-, and surface Ig-. We conclude that the livers of BRM-treated mice can provide a rich source of highly active mouse LGL that could be used for further characterization of this lymphocyte subset. Further, these studies imply a potential for BRM therapy of neoplastic or viral diseases through augmentation of organ-associated immune responses.

Mobilization of long-term reconstituting hematopoietic stem cells in mice by recombinant human interleukin 7.

Administration of recombinant human interleukin 7 (rh)IL-7 to mice has been reported by our group to increase the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakarocyte macrophage) from the bone marrow to peripheral organs (blood, spleen[s], and liver). We now report that IL-7 also stimulates a sixfold increase in the number of more primitive CFU-S day 8 (CFU-S8) and day 12 (CFU-S12) in the peripheral blood leukocytes (PBL) of mice treated with rhIL-7 for 7 d. Moreover, > 90% of lethally irradiated recipient mice that received PBL from rhIL-7-treated donor mice have survived for > 6 mo whereas none of the recipient mice that received an equal number of PBL from diluent-treated donors survived. Flow cytometry analysis at 3 and 6 mo after transplantation revealed complete trilineage (T, B, and myelomonocytic cell) repopulation of bone marrow, thymus, and spleen by blood-borne stem/progenitor cells obtained from rhIL-7-treated donor mice. Thus, IL-7 may prove valuable for mobilizing pluripotent stem cells with long-term repopulating activity from the bone marrow to the peripheral blood for the purpose of gene modification and/or autologous or allogeneic stem cell transplantation.

Recombinant transforming growth factor beta 1 and beta 2 protect mice from acutely lethal doses of 5-fluorouracil and doxorubicin.

Transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2 can reversibly inhibit the proliferation of hematopoietic progenitor cells in vivo, leading us to hypothesize that such quiescent progenitors might be more resistant to high doses of cell cycle active chemotherapeutic drugs, thereby allowing dose intensification of such agents. Initial studies showed that whereas administration of TGF-beta 1 or TGF-beta 2 did not prevent death in normal mice treated with high doses of 5-fluorouracil (5-FU), those mice that received TGF-beta 2 did exhibit the beginning of a hematologic recovery by day 11 after administration of 5-FU, and were preferentially rescued by a suboptimal number of transplanted bone marrow cells. Subsequently, it was found that the administration of TGF-beta 2 protected recovering progenitor cells from high concentrations of 5-FU in vitro. This protection coincided with the finding that significantly more progenitors for colony-forming unit-culture (CFU-c) and CFU-granulocyte, erythroid, megakaryocyte, macrophage (GEMM) were removed from S-phase by TGF-beta in mice undergoing hematopoietic recovery than in normal mice. Further studies showed that the administration of TGF-beta protected up to 90% of these mice undergoing hematologic recovery from a rechallenge in vivo with high dose 5-FU, while survival in mice not given TGF-beta was < 40%. Pretreatment of mice with TGF-beta 1 or TGF-beta 2 also protected 70-80% of mice from lethal doses of the noncycle active chemotherapeutic drug, doxorubicin hydrochloride (DXR). These results demonstrate that TGF-beta can protect mice from both the lethal hematopoietic toxicity of 5-FU, as well as the nonhematopoietic toxicity of DXR. This report thus shows that a negative regulator of hematopoiesis can be successfully used systemically to mediate chemoprotection in vivo.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contributes to interferon gamma-dependent natural killer cell protection from tumor metastasis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed by in vitro activated natural killer (NK) cells, but the relevance of this observation to the biological function of NK cells has been unclear. Herein, we have demonstrated the in vivo induction of mouse TRAIL expression on various tissue NK cells and correlated NK cell activation with TRAIL-mediated antimetastatic function in vivo. Expression of TRAIL was only constitutive on a subset of liver NK cells, and innate NK cell control of Renca carcinoma hepatic metastases in the liver was partially TRAIL dependent. Administration of therapeutic doses of interleukin (IL)-12, a powerful inducer of interferon (IFN)-gamma production by NK cells and NKT cells, upregulated TRAIL expression on liver, spleen, and lung NK cells, and IL-12 suppressed metastases in both liver and lung in a TRAIL-dependent fashion. By contrast, alpha-galactosylceramide (alpha-GalCer), a powerful inducer of NKT cell IFN-gamma and IL-4 secretion, suppressed both liver and lung metastases but only stimulated NK cell TRAIL-mediated function in the liver. TRAIL expression was not detected on NK cells from IFN-gamma-deficient mice and TRAIL-mediated antimetastatic effects of IL-12 and alpha-GalCer were strictly IFN-gamma dependent. These results indicated that TRAIL induction on NK cells plays a critical role in IFN-gamma-mediated antimetastatic effects of IL-12 and alpha-GalCer.

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice.

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.

Antitumor effects of interleukin-7 and adoptive immunotherapy on human colon carcinoma xenografts.

The antitumor properties of recombinant human IL-7 (rhIL-7) on a human tumor was evaluated by engrafting a human colon carcinoma into immunodeficient mice and then treating the mice with rhIL-7 and adoptively transferred human peripheral blood T cells. It was found that rhIL-7 alone had no effect on the survival of the tumor-bearing recipients. However, the combination of rhIL-7 and human T cells significantly promoted the survival of the recipients compared with mice receiving either treatment by itself. When the surviving mice were analyzed 6 mo later for the degree of human cell engraftment, the recipients receiving both rhIL-7 and human T cells had greater numbers of human CD8+ T cells in the spleens. However, the human T cells recovered from the surviving mice showed low lytic activity against the tumor in vitro. Supernatants from human T cells cultured with the tumor and rhIL-7 in vitro were found to inhibit tumor growth and were demonstrated to contain high levels of IFN-gamma. Antibodies to IFN-gamma neutralized the growth inhibition of the tumor both in vitro and in vivo demonstrating that the in vivo mechanism underlying the antitumor effects of this regimen was partly dependent on the production of IFN-gamma by the T cells and not their cytolytic capability. Interestingly, systemic administration of rhIFN-gamma to tumor-bearing mice yielded little antitumor effect suggesting that adoptive immunotherapy with rhIL-7 was superior possibly because of the continuous local release of the cytokines. Therefore, rhIL-7 may be of clinical use as an antineoplastic agent and the human/mouse model is a potentially important preclinical model for in vivo evaluation of the efficacy of this and other immunotherapies.

IFN-gamma and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy.

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8(+) T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8(+) T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-gamma-dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-gamma production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-gamma and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8(+) T cell-mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.

Hydrodynamics-based gene delivery of naked DNA encoding fetal liver kinase-1 gene effectively suppresses the growth of pre-existing tumors.

Antiangiogenic gene therapy is a promising strategy for cancer treatment, which generally requires highly efficient delivery systems. To date, success of this strategy has depended almost exclusively on the delivery of high titers of viral vectors, which can result in effective transgene expression. However, their cytotoxicity and immunogenicity are a major concern for clinical applications. Recent advances in delivery efficiency of naked DNA could potentially meet the requirement for both high transgene expression and minimal side effects. To investigate whether naked DNA can be used for antiangiogenic cancer therapy, an expression plasmid was generated that encodes a soluble form of fetal liver kinase-1 (Flk-1) gene, a receptor for vascular endothelial growth factor (VEGF). Hydrodynamic injection of this plasmid resulted in close to 0.1 mg/ml of soluble Flk-1 protein in mouse serum and blocked VEGF-driven angiogenesis in matrigel in vivo. The same delivery significantly suppressed the growth of two different pre-existing subcutaneous tumors, Renca renal cell carcinoma and 3LL lung carcinoma. CD31 immunohistochemistry revealed that the tumor-associated angiogenesis was also highly attenuated in soluble Flk-1-treated mice. Thus, expression of genes by hydrodynamics-based gene delivery of naked DNA appears to be a promising approach for antiangiogenic cancer gene therapy.

Isotope-release cytotoxicity assay with the use of indium-111: advantage over chromium-51 in long-term assays.

The adaptation of indium-111-oxine (also known as 8-hydroxyquinoline) (111In Ox) chelate for long-term (18-48 hr) isotope-release assays of cell-mediated cytotoxicity (CMC) and its advantages over the use of 51 Cr are described. Labeling of DBA/2 P815 mastocytoma cells with 111InOx resulted in the incorporation of as many as a million counts per minute in 10(5) cells with no reduction in cell viability. 111InOx labeled both mouse and human tumor cells. 111InOx, like 51Cr, primarily labeled cytoplasmic constituents; up to 80% of the label existed in a releasable form. 111InOx was quantitatively released from labeled P815 in response to specifically sensitized C57BL/6 lymphocytes. The high labelling efficiency of 111InOx offered a significant advantage over 51Cr in 18- to 48-hour assays for CMC by reducing the counting error and thus making the assay more precise. Because of its higher labeling efficiency, 111InOx can be used in microcytotoxicity assays. 111InOx has the added advantage of a lower spontaneous release in culture than 51Cr. This feature of 111InOx also makes the calculation of specific isotope release more accurate than that achieved with 51Cr in long-term cytotoxic assays.

Alterations in sensitivity to nonspecific cell-mediated lysis associated with tumor progression: characterization of activated macrophage- and natural killer cell-resistant tumor variants.

Certain "membrane-mutant," lectin-resistant (Lecr) variants derived from the highly metastatic and poorly immunogenic DBA/2 mouse tumor MDAY-D2 previously were found to differ substantially in their ability to grow and to metastasize. In the present study, the parental MDAY-D2 tumor and several wheat germ agglutinin-resistant (WGAr) variants were examined for alterations in sensitivity to activated macrophage (M phi)- and natural killer cell (NK)-mediated lysis. The results indicated that selection in WGA after mutagenic treatment of a metastatic parental tumor cell line (MDAY-D2), which was M phi-sensitive (M phi S) and NK-resistant (NKR), can result in the isolation of a significantly M phi-resistant (M phi R) and NK-sensitive (NKS) tumor variant, MDW4. The in vivo hybridization of the M phi R, NKS, Lecr MDW4 variant with a normal host-derived cell within a primary subcutaneous tumor, previously demonstrated to result in the progressive and selective outgrowth and metastasis of hybrid products, was found to be associated directly with reversion to the M phi S, NKR phenotype of the metastatic parental MDAY-D2 cell line. DMA/2 mice given iv injections of 10(5) M phi R, NKS cells (MDW4 or MDW4-110c1, a cloned line isolated from a subcutaneous primary tumor of an MDW4-injected animal) survived for a significantly prolonged period as compared to animals given injections of either the parental tumor or M phi S, NKR hybrid products isolated from a MDW4 subcutaneous primary tumor (MDW4-110c2) or visceral metastases (MDW4-24a, MDW4-24b, and MDW4-24c). The results clearly indicate an inverse relationship among the tumor variants in their ability to be lysed by either M phi or NK and suggest a central role for NK rather than M phi surveillance in this tumor system.

Immunomodulation of natural killer cell activity by flavone acetic acid: occurrence via induction of interferon alpha/beta.

The investigational drug flavone acetic acid (FAA) systemically augments natural killer (NK) cell activity in normal and tumor-bearing mice and in human cancer patients. The results from the present investigation demonstrate that in vivo administration of FAA induces in a dose-dependent manner high levels of serum interferon (IFN) within 4 hours in BALB/c, C57BL/6, and BALB/c nude mice. Antibody neutralization studies indicated that FAA induced IFN of the alpha/beta type, while molecular hybridization studies demonstrated that FAA rapidly stimulated the production of IFN alpha mRNA in splenic leukocytes. In vivo administration of anti-IFN alpha/beta antibodies to FAA-treated mice inhibited the FAA-induced augmentation of splenic NK cell activity at 4 hours. These results suggest that FAA mediates its anti-tumor effects indirectly by immunomodulation as well as directly by antiproliferative or cytotoxic activity.

Reversal of drug resistance in a human colon cancer xenograft expressing MDR1 complementary DNA by in vivo administration of MRK-16 monoclonal antibody.

One strategy to overcome multidrug resistance in neoplasia is to inhibit the gp170 glycoprotein (relative molecular mass, 170,000) that functions as a plasma membrane, energy-dependent, drug-efflux pump. The human colon cancer cell line HT-29, which grows as an ascitic tumor in athymic NCr-nu/nu nude mice, was made multidrug resistant by infection with an MDR1 (also known as PGY1) retrovirus. Referred to as HT-29mdr1, it was used to study reversal of drug resistance in vivo by the anti-P-glycoprotein monoclonal antibody MRK-16. Flow cytometry and radioimmunoassay demonstrated a marked increase in MRK-16 reactivity on HT-29mdr1 cells as compared with its reactivity on the parental, uninfected cell line (HT-29par). The 50% inhibitory concentrations (IC50) of vincristine on HT-29par and HT-29mdr1 cells were 2.5 and 15 ng/mL, respectively. The MRK-16 monoclonal antibody did not affect the vincristine sensitivity of the HT-29par cells. Pretreatment of HT-29mdr1 cells with 10 micrograms/mL MRK-16 in tissue culture partially restored the vincristine sensitivity (IC50 = 7 ng/mL). This modulation of vincristine sensitivity by MRK-16 was then tested in vivo. The median survival times of mice given intraperitoneal transplants of 5 x 10(6) HT-29par or HT-29mdr1 were 37 and 39 days, respectively. Treatment of mice with 1 mg/kg vincristine weekly for 3 weeks, beginning 10 days after tumor injection, resulted in a significant increase in the median survival time of the HT-29par tumor-bearing mice (68 days, P less than .0001), but it had no effect on the HT-29mdr1 tumor-bearing mice. However, treatment of mice bearing the HT-29mdr1 tumor with MRK-16 before vincristine therapy reversed the resistance to the drug (median survival time = 64 days, P less than .0001). The MRK-16 monoclonal antibody alone had no effect on the median survival time of mice given an injection of either HT-29par or HT-29mdr1 cells. These results suggest that strategies employing monoclonal antibody against gp170 may be clinically useful to reverse multidrug resistance.

Enhancement by recombinant human interferon alfa of the reversal of multidrug resistance by MRK-16 monoclonal antibody.

BACKGROUND: The anti-P-glycoprotein monoclonal antibody MRK-16 mediates the reversal of multidrug resistance. Recombinant human interferon alfa (rHuIFN alpha) enhances the cytotoxic activity of diverse chemotherapeutics and may modulate multidrug resistance. PURPOSE: Our purpose was to determine the outcome of combination treatment with MRK-16, rHuIFN alpha-2a, and cytotoxic agents on tumor cells that express P-glycoprotein (Pgp). METHODS: Three Pgp-expressing, multidrug-resistant human tumor cell lines were used: the MDR1 retrovirus-infected HT-29 colon adenocarcinoma (HT-29mdr1), the doxorubicin (Adriamycin)-resistant MCF-7 (AdrR MCF-7) breast carcinoma, and the de novo Pgp-acquired, HCT-15 colon carcinoma. The parental cell lines HT-29par and MCF-7 were used as controls. The in vitro effects of MRK-16 and rHuIFN alpha-2a were studied on: (a) chemosensitivity of parental and multidrug-resistant cell lines to vincristine, doxorubicin, or paclitaxel (Taxol); (b) intracellular drug concentrations; and (c) Pgp expression. The efficacy of vincristine alone or in combination with MRK-16 and/or rHuIFN alpha-2a was assessed against HT-29mdr1 cells in female, athymic NCr-nu/nu mice. RESULTS: For vincristine, the IC50 (i.e., the concentration that causes 50% inhibition of cell growth) was 7.0 ng/mL in HT-29mdr1 cells. Pretreatment of HT-29mdr1 cells with MRK-16 partially restored vincristine sensitivity (IC50 = 4.8 ng/mL), which was enhanced by noncytotoxic concentrations of rHuIFN alpha-2a (IC50 = 2.9 ng/mL) via a mechanism independent of Pgp modulation or [3H]vincristine efflux. rHuIFN alpha-2a potentiated MRK-16 reversal of multidrug resistance with both doxorubicin and paclitaxel on HT-29mdr1 cells and with vincristine on AdrR MCF-7 and HCT-15 tumor cells. Treatment of mice with 1 mg/kg vincristine weekly for 3 weeks, beginning 10 days after tumor injection, significantly increased the median survival times of the HT-29par tumor-bearing mice (60 days versus 35 days; P < .0001) but was only marginally therapeutic for HT-29mdr1 tumor-bearing mice (52 days versus 46 days). Pretreatment with MRK-16 (500 micrograms) and rHuIFN alpha-2a (5 x 10(4) U), alone or in combination, 24 hours before vincristine therapy did not affect the survival of HT-29par tumor-bearing mice. In contrast, the survival of mice bearing HT-29mdr1 tumors was significantly increased following treatment with MRK-16 before vincristine (80 days; P < .0001). Administration of a nontherapeutic dose of rHuIFN alpha-2a (5 x 10(4) U) with MRK-16 before vincristine treatment further increased the median survival times of HT-29mdr1 tumor-bearing mice (116 days; P < .0001). CONCLUSIONS: MRK-16 used in combination with rHuIFN alpha-2a was significantly more effective than MRK-16 in overcoming multidrug resistance.

Gradual loss of T-helper 1 populations in spleen of mice during progressive tumor growth.

BACKGROUND: The carefully orchestrated events that result in a protective immune response are coordinated to a large extent by cytokines produced by T helper 1 (Th1) and T helper 2 (Th2) T-cell subsets, which are two arms of the immune system. Th1 cells preferentially produce interleukin 2 (IL-2), interferon gamma (IFN gamma), and tumor necrosis factor (TNF), resulting in a cellular response that helps to eliminate infected cells. In contrast, Th2 cells produce IL-4, IL-5, IL-6, and IL-10 and stimulate an antibody response that helps to prevent the cells from becoming infected. The clinical progression of several infectious diseases, including human immunodeficiency virus, some types of parasitoses, and tuberculosis, is thought to be associated with the predominance of a Th2-type T-cell response. Recent reports have demonstrated the presence of T cells producing Th2 lymphokines (IL-4, IL-6, and IL-10) in tumor-infiltrating lymphocytes of renal cell carcinoma. PURPOSE: The purpose of this study was to investigate at the molecular level whether there was any change in the splenic T cells of mice with progressively growing tumors from a Th1 to a Th2 DNA-binding pattern or phenotype. METHODS: Splenic T cells from mice bearing renal cell carcinoma or MCA-38 colon carcinoma were tested for cytokine production after in vitro activation. Nuclear extracts of splenic T cells were used for the DNA-binding assay using IFN-gamma core promoter region, the kappa B (kappa B) site from immunoglobulin gene, and the nuclear factor of activated T-cell (NFAT) site from IL-2 gene. RESULTS: Splenic T cells from mice bearing renal cell carcinoma or MCA-38 colon carcinoma preferentially produced Th2 cytokines (i.e., IL-4) upon activation and showed a marked decrease in Th1 cytokine (particularly IFN gamma) production compared with the production observed in normal splenic T cells. The DNA-binding assay with the IFN-gamma core promoter region confirmed the gradual decline in the nuclear transcription factors associated with the Th1 phenotype during tumor progression in both tumor models. Renal cell carcinoma-bearing mice, successfully treated with flavone-8-acetic acid and recombinant human IL-2, showed a reversion to a Th1-like pattern. In addition, nuclear extracts of T cells from tumor-bearing animals showed a Th2-type kappa B-binding pattern. Moreover, the NFAT complex present in the normal splenic T cells was lost at the later stages of tumor progression; instead, a new complex was present in mice bearing long-term tumors. CONCLUSION: T cells from tumor-bearing mice lose the Th1 phenotype with progressive tumor growth.

Administration of interleukin 12 with pulse interleukin 2 and the rapid and complete eradication of murine renal carcinoma.

BACKGROUND: Interleukin 2 (IL-2) and interleukin 12 (IL-12) are potent immunoregulatory cytokines that exhibit antitumor activity. Preliminary evidence suggests that combined administration of IL-2 and IL-12 may yield greater antitumor activity than that observed with either agent alone. PURPOSE: We evaluated the ability of combination regimens of IL-2 and IL-12 to induce regression of established primary and metastatic murine renal carcinoma (Renca) tumors. METHODS: BALB/c mice were given either subcutaneous or intrarenal injections of 10(5) Renca cells; tumor cell injections were given to 10-12 mice for each treatment group. Mice bearing subcutaneous primary tumors were treated with chronic IL-2 (300,000 IU given on a daily basis) or pulse IL-2 (300,000 IU given twice daily one day per week) alone, IL-12 alone (0.5 micrograms given on a daily basis), or IL-12 in combination with either chronic or pulse IL-2. Mice with metastatic tumors (arising from intrarenal implants; animals were nephrectomized to remove the primary tumors) were treated with IL-12 plus or minus pulse IL-2; in these experiments, IL-12 was given at doses of either 0.5 or 1.0 micrograms. In most experiments, treatment was continued for at least 3 weeks. Two-sided statistical tests were used to evaluate the data. RESULTS: Most mice with subcutaneous Renca tumors treated with the combination of IL-12 and chronic IL-2 died of treatment-related toxic effects within 7-14 days. In contrast, treatment with IL-12 plus pulse IL-2 was well tolerated, and six of 10 mice experienced complete tumor regression; none of the mice treated with either IL-12 alone or pulse Il-2 alone experienced a curative response. Seven of eight and nine of nine mice with metastatic tumors experienced complete tumor regression after treatment with 0.5 micrograms IL-12 plus pulse IL-2 or 1.0 microgram IL-12 plus pulse IL-2, respectively; two of 12 mice treated with pulse IL-2 alone and 10% or less of mice treated with IL-12 alone were cured of metastatic tumors (with 0.5 micrograms IL-12, none of 10 mice; with 1.0 micrograms IL-12, one of 10 mice). Five of 10 mice with metastatic tumors treated with a short-course regimen of IL-12 and pulse IL-2 (two pulses of IL-2 flanking 5 days of 0.5 micrograms IL-12) experienced complete tumor regression, while only one of the 12 mice treated with IL-2 alone and none of the mice treated with IL-12 alone experienced complete tumor regression. Virtually all curative response frequencies obtained with IL-12 and pulse IL-2 combination regimens differed significantly (P < .05) from those obtained with corresponding single-agent treatments. CONCLUSIONS: IL-12 administered in combination with pulse IL-2 induced rapid and complete regression of primary and metastatic Renca tumors and displayed greater antitumor activity than that observed with either IL-12 or IL-2 alone.

Restoration of interferon alpha potentiation of a recombinant ricin A chain immunotoxin following cytoreduction of xenografts of advanced ovarian tumors.

BACKGROUND: We have demonstrated that, in the human ovarian carcinoma cell line (OVCAR-3), recombinant human interferon alpha (rHuIFN-alpha) potentiated in vitro inhibition of protein synthesis by immunotoxins. The antitumor activity of intracavitary immunotoxin administered to nude mice 5 days after tumor cell injection was enhanced by a nontherapeutic dose of rHuIFN-alpha, as evidenced by increased survival time. PURPOSE: Our purpose was to determine the outcome of treatment with immunotoxin and rHuIFN-alpha in xenografts of more advanced tumors. METHODS: At 10 or 15 days after tumor cell injection, nude mice with peritoneal OVCAR-3 xenografts were treated intraperitoneally with immunotoxin or with 454A12 monoclonal antibody (MAb) recombinant ricin A chain (rRA), alone or combined with a nontherapeutic dose of rHuIFN-alpha. The immunotoxin was composed of rRA covalently bound to an anti-CD71 (transferrin receptor) MAb. In other experiments, mice were treated intraperitoneally with cyclophosphamide and cisplatin to reduce tumor size on days 20 and 27 after tumor cell inoculation and then, beginning on day 40, with immunotoxin alone or combined with rHuIFN-alpha. RESULTS: Initiation of treatment 10 days after OVCAR-3 transplantation significantly increased median survival from 41 to 89 days (10% survivors on day 120) with 454A12 MAb rRA alone and to more than 120 days (70% survivors) with 454A12 MAb rRA combined with rHuIFN-alpha (P < .0001). The increase in survival time between tumor-bearing mice treated with immunotoxin combined with rHuIFN-alpha and those treated with immunotoxin alone was statistically significant (P = .017). In contrast, the 15-day transplant tumors were not curable with immunotoxin therapy (survival, 72 days; 0% survivors) and were refractory to rHuIFN-alpha potentiation (survival, 75 days; 0% survivors). After the second course of chemotherapy to reduce the size of the advanced tumors (day 40), during the ascites cell count nadir, initiation of treatment with 454A12 MAb rRA alone or combined with rHuIFN-alpha resulted in significantly different survival times of 129 and 162 days, respectively (P = .0037). Pathologic examination of surviving mice treated with chemotherapy and 454A12 MAb rRA alone or in combination with rHuIFN-alpha revealed that one (17%) of six mice and 11 (65%) of 17 were tumor free, respectively. CONCLUSIONS: The synergy between immunotoxins and IFN-alpha is dependent on tumor burden. These agents are less effective against large tumor burdens (i.e., advanced stage disease), but their beneficial effects re-emerge after cytoreduction by combination chemotherapy. IMPLICATIONS: The ideal setting for testing the efficacy of intracavitary immunotoxin combined with rHuIFN-alpha after front-line chemotherapy is in patients with residual tumor refractory to additional chemotherapy or in those with toxic effects that prevent delivery of effective doses.

Adjuvant immunotherapy of established murine renal cancer by interleukin 2-stimulated cytotoxic lymphocytes.

We have used a transplantable experimental murine renal carcinoma (Renca) to evaluate the adjuvant immunotherapeutic potential of cytotoxic lymphocytes stimulated by in vitro incubation for 24 h with human recombinant interleukin 2 (rIL-2). The Renca tumor model is therapeutically challenging since, following intrarenal implant, it grows progressively with local invasion and development of spontaneous metastases in abdominal lymph nodes, lungs, and liver. Therefore, successful treatment requires control of both primary tumor, local and regional invasive growth, and systemic metastases. Our studies have shown that rIL-2-stimulated cytotoxic lymphocytes efficiently lyse Renca in vitro. Further, both doxorubicin hydrochloride and an active metabolite of cyclophosphamide also inhibited the growth of Renca in vitro. In vivo administration of doxorubicin hydrochloride, cyclophosphamide or rIL-2-stimulated cytotoxic lymphocytes and rIL-2 to mice bearing established Renca tumors (7-day disease) resulted in a significant (P less than 0.01) increase in short-term survivors (at 45 days), but only 17% cures. However, combination chemoimmunotherapy consisting of doxorubicin hydrochloride and rIL-2-stimulated cytotoxic lymphocytes plus rIL-2 results in the cure of 67% of mice bearing established Renca. These results demonstrate that chemotherapy and immunotherapy with adoptively transferred cytotoxic lymphocytes can function synergistically in the treatment of established murine renal carcinoma.

Correlation between in vivo induction of cytokine gene expression by flavone acetic acid and strict dose dependency and therapeutic efficacy against murine renal cancer.

The investigational chemotherapeutic drug flavone acetic acid (FAA) acts as an immunomodulator by augmenting natural killer activity in both humans and rodents after in vivo administration. The accumulated data derived from a series of experiments also demonstrates that FAA synergizes with interleukin 2 (IL-2) for the treatment of murine renal cancer. The immunomodulatory and immunotherapeutic effects of FAA are strictly dose dependent with doses of FAA greater than 150 mg/kg effectively synergizing with IL-2, and doses less than 150 mg/kg exhibiting very little therapeutic effect. The antitumor and immunomodulatory effects of FAA are more pronounced in vivo than in vitro. Collectively, these results suggested that cytokines induced by FAA may contribute to these effects, and that the induction of such cytokines may also be very dose dependent. Studies were therefore initiated to investigate whether the in vivo administration of FAA would alter the expression of cytokine mRNA in leukocytes. Splenic leukocytes or liver nonparenchymal cells from untreated and FAA-treated mice were used as a source of RNA for Northern blot analysis. Interferon alpha and interferon gamma mRNA in the spleen was upregulated within 1.5 h after FAA administration, with peak induction occurring by about 2 h. An upregulation of tumor necrosis factor alpha mRNA was detected in the spleen by 0.5-1 h after treatment with peak induction occurring by 1-1.5 h. Induction of tumor necrosis factor alpha mRNA was also detected in hepatic nonparenchymal cells. No up-regulation of splenic mRNA for tumor necrosis factor beta, IL-1 alpha or beta, or IL-2 was detected after FAA administration. IFN and TNF activities were detectable in the serum by bioassay immediately following the appearance of mRNA in FAA mice. The observed up-regulation by FAA of cytokine mRNA and the corresponding serum protein was strictly dose dependent with substantial induction of both mRNA and proteins occurring only at FAA doses greater than or equal to 150 mg/kg, a dose range also shown to be the minimum required for immunomodulatory and immunotherapeutic effects. In summary, these results demonstrate that FAA acts as a potent inducer of at least three cytokines in vivo, and suggest that the immunomodulatory and immunotherapeutic effects of FAA may be partially mediated by these induced cytokines.

Immune-mediated arrest and reversal of established visceral metastases in athymic mice.

The metastasizing MDAY-D2 tumor of DBA/2 mice disseminates in BALB/c allogeneic athymic nude (nu/nu) mice in a manner identical to that observed in the syngeneic host. Both the kinetics and organ distribution pattern of metastases from s.c. implants of MDAY-D2 are routinely predictable at any given tumor dose. BALB/c heterozygote (nu/+) litter-mates reject MDAY-D2 grafts on the basis of the multiple minor histocompatibility differences that exist between DBA/2 and BALB/c mice. The in vitro cell-mediated cytotoxic response detected in tumor-bearing BALB/c nu/+ mice is "low grade" (isotope release is approximately 40 to 50% by 24-hr 111-indium-8-hydroxyquinoline assay and approximately 6 to 8% by 6-hr 51Cr assay) and yet correlates directly with tumor rejection. BALB/c nu/nu mice can be protected against MDAY-D2 by previous reconstitution with lymphoid cells from normal or MDAY-D2-sensitized BALB/c nu/+ mice. In addition, surgically documented, established visceral metastases in BALB/c nu/nu mice can be arrested and regressed by the adoptive transfer of MDAY-D2-sensitized BALB/c nu/+ spleen cells. This represents one of the few models where established metastases have been immunotherapeutically regressed. As such, the MDAY-D2 BALB/c nu/nu mouse model offers unique advantages for studying the role of the immune system in regulating the metastatic process.

Antitumor effects of alpha-interferon and gamma-interferon on a murine renal cancer (Renca) in vitro and in vivo.

Previous studies have shown that established murine renal cancer (Renca) can be successfully treated with the investigational drug flavone acetic acid (FAA) used in combination with recombinant interleukin 2 (IL-2). Additional experiments demonstrated that the in vivo administration of FAA rapidly induced the expression of the genes, as well as the biologically active proteins, for alpha- and beta-interferons (IFNs) as well as tumor necrosis factor alpha. Both IFN-alpha and IFN-gamma have been shown to have direct antiproliferative effects against some tumors, as well as being potent immunodulators for the induction of antitumor effector cells. Thus, the present study was designed to investigate the ability of IFN-alpha and/or IFN-gamma to mediate direct antiproliferative effects against Renca in vitro as well as to cause regression of Renca in vivo. The present study confirms that RAA and/or IL-2 are inactive against Renca in vitro, further suggesting an indirect mechanism for FAA-induced antitumor effects in vivo. However, the exposure of Renca in vitro to recombinant human IFN-alpha A/D, murine IFN-alpha or murine IFN-beta resulted in a dose dependent growth inhibition of Renca as assessed by the microculture tetrazolium dye incorporation assay. Very little growth inhibition was induced by recombinant murine IFN-gamma. Interestingly, IFN-alpha (100-1000 units/ml) when combined with very low doses of recombinant murine IFN-gamma (1-10 units/ml) yielded significantly more pronounced growth inhibition than either cytokine alone. This effect was most evident by 5 days of culture where combinations of 100-1000 units/ml recombinant human IFN-alpha A/D with 1-5 units/ml recombinant murine IFN-gamma yielded growth inhibition in the range of 45-99%. In order to determine whether the mechanisms for the antitumor activity of recombinant human IFN-alpha A/D and recombinant murine IFN-gamma was due to their direct antiproliferative effects, we also studied the efficacy of these combinations against i.p. Renca in athymic mice. In contrast to the potent antitumor effects observed in euthymic mice, the combination of IFN-alpha and IFN-gamma only slightly increased mean survival times in athymic mice and no long term survivors were obtained. Subsequent studies demonstrated that most mice (77%) cured of peritoneal Renca by recombinant human IFN-alpha A/D plus recombinant murine IFN-gamma were immune to rechallenge. Therefore the combination of IFN-alpha and IFN-gamma may directly inhibit the growth of Renca, but a major effect of IFNs in vivo must be to contribute to the induction of an anti-Renca immune response.