Interleukin 1-induced sequential myelorestoration: dynamic relation between granulopoiesis and progenitor cell recovery in myelosuppressed mice.

The myelorestorative effect of recombinant human interleukin 1 alpha (IL-1 alpha) was studied in mice treated with anticancer drugs. The treatment of mice with 5-fluorouracil (5-FU; 250 mg/kg body weight) or cyclophosphamide (CPA; 100 mg/kg) considerably decreased bone marrow or splenic colony-forming units in culture (CFU-C) or neutrophils in blood. The daily administration of IL-1 alpha (100 ng/mouse/day) after 5-FU treatment markedly accelerated the recovery of bone marrow CFU-C. This increase was followed by the recovery of splenic CFU-C and neutrophils in blood. In the CPA-treated mice the recovery of bone marrow CFU-C over the normal level was observed regardless of the administration of IL-1 alpha 3 days after CPA treatment. The daily administration of IL-1 alpha after CPA treatment markedly increased splenic CFU-C or neutrophils over the normal level following the increase of bone marrow CFU-C. Thus, in both 5-FU- and CPA-treated mice, the increase of bone marrow CFU-C after the administration of IL-1 alpha was observed several days earlier than the increase of splenic CFU-C and neutrophils in blood. The increase of splenic CFU-C and neutrophils in blood was observed concomitantly. The experiments, in which effective timing of IL-1 alpha injection was examined, indicated that the administration of IL-1 alpha within a few days after treatment with anticancer drugs was necessary for the accelerated recovery of bone marrow progenitor cells. On the other hand, the effective recovery of splenic progenitor cells and peripheral neutrophils required the administration of IL-1 alpha after the increase of bone marrow progenitor cells. Thus, the administration of IL-1 alpha daily or every other day was the most effective for recovery from myelosuppression induced by anticancer drugs.

Simple enzyme immunoassay methods for recombinant human tumor necrosis factor alpha and its antibodies using a bacterial cell wall carrier.

We have developed simple methods for measuring recombinant human tumor necrosis factor alpha (rHu-TNF alpha) and antibodies to rHu-TNF alpha in the sera of animals intravenously injected with rHu-TNF alpha. rHu-TNF alpha was measured by a competitive binding enzyme immunoassay (C-EIA) using standard rHu-TNF alpha, beta-galactosidase labeled rHu-TNF alpha as enzyme-labeled antigen (E-Ag) and anti-rabbit IgG goat immunoglobulins coupled to bacterial cell walls (insolubilized second antibody). In contrast, anti-rHu-TNF alpha antibodies were measured by a sandwich EIA (S-EIA) using purified anti-rHu-TNF alpha rabbit IgG as standard, beta-galactosidase labeled rHu-TNF alpha as E-Ag, and rHu-TNF alpha coupled to bacterial cell walls as insolubilized antigen. C-EIA permits the determination of serum rHu-TNF alpha within the range of 2-150 U/ml (about 0.7-52 ng/ml) with a CV of below 7.6% and 99% recovery. S-EIA permits the determination of anti-rHu-TNF alpha antibodies within the range of 70-1000 ng/ml with a CV of less than 4% and 94.8-106.9% recovery.

Production and characterization of recombinant human neutrophil chemotactic factor.

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.

[Combined effect of PT-050 (recombinant human TNF) and antitumor drugs on syngeneic murine tumors].

The combined effect of PT-050 (recombinant human TNF) and various antitumor drugs was investigated using murine colon 26 adenocarcinoma, Meth A sarcoma and B16 melanoma transplanted into syngeneic mice. When colon 26- or Meth A- bearing mice were intravenously given PT-050 in combination with mitomycin C (MMC), doxorubicin (DXR), cis-platinum (CDDP), 5-fluorouracil (5-FU) or cyclophosphamide (CPA), a significant synergistic effect was observed, that is, both the inhibition rate of tumor growth and the cured ratio were increased significantly when compared with those given each drug alone. Similarly, an augmentation of the antitumor effect was also observed in B16-bearing mice by a combined treatment with PT-050 and these antitumor drugs. These results suggest that the combination chemotherapy of PT-050 with various antitumor drugs may be useful for cancer therapy.

Recombinant human tumor necrosis factor--I. Cytotoxic activity in vitro.

Cytotoxic activity of recombinant human TNF (rHu-TNF) on various human cell lines was examined in vitro. rHu-TNF exerted a cytostatic effect on various types of human tumor cells such as carcinoma, sarcoma, leukemia, melanoma and other types. When the cytocidal effect was examined on the tumor cells which were cytostatically susceptible to rHu-TNF, the cytocidal effect of rHu-TNF was also noticed on many of these tumor cells. However, some tumor cells were affected cytostatically only. Human diploid cells were not affected cytostatically or cytocidally by rHu-TNF. WI-38 VA13 cells which are an SV-40-transformed derivative of WI-38 diploid cells, were affected both cytostatically and cytocidally by rHu-TNF. These results suggest that rHu-TNF exerts cytostatic and cytocidal effects against a broad spectrum of human tumor cells, and its cytotoxic activity is tumor-specific.

Interleukin-1 alpha: its possible roles in cancer therapy.

Our studies on recombinant human IL-1 alpha polypeptide were summarized with respect to molecular cloning, production, quantitative assay systems, antitumor activity, myelorestorative activity and augmentation of host resistance to infections. Recombinant human IL-1 alpha (18 kDa) was produced through the expression of the cloned human IL-1 alpha cDNA in Escherichia coli and purified to an endotoxin-free homogeneous polypeptide. The human IL-1 alpha inhibited dose-dependently the growth of syngeneic murine tumors transplanted in mice and completely regressed the tumors in some cases, and its antitumor activity was significantly enhanced in combination with indomethacin. The human IL-1 alpha accelerated the recovery of the numbers of peripheral leukocytes and neutrophils in a dose-dependent manner at a dose as low as 10 ng/mouse/day in myelosuppressed mouse model produced by administering anticancer chemotherapeutic drugs. The myelorestorative effect of IL-1 alpha was observed not only on leukocytes/neutrophils, but also on platelets in myelosuppressed mice. In addition, the human IL-1 alpha markedly augmented dose-dependently resistance of normal and leukopenic mice to various microbial infections. These results suggested that recombinant human IL-1 alpha might be useful for cancer therapy from the viewpoints of improving adverse effects such as myelosuppression caused by chemotherapy and/or radiation therapy and preventing infections. In addition, use of IL-1 alpha may permit more intensive chemo- and radiation therapies using higher doses. Finally, the antitumor activity of the IL-1 alpha itself may play an important role.

Accelerated recovery of antigen-presenting cell activity by the administration of interleukin 1 alpha in 5-fluorouracil-treated mice.

In this study, we investigated the effect of human recombinant interleukin-1 alpha (IL-1 alpha) on antigen-presenting cell (APC) activity of spleen cells in mice treated with 5-fluorouracil (5-FU). APC activity was determined by the antigen-specific proliferation of T cell clone D10.G4.1 cells. When mice were injected with 5-FU, APC activity of spleen cells was suppressed. The administration of IL-1 alpha accelerated the recovery from this suppression. The most accelerated recovery was observed when these mice were administered with IL-1 alpha both before and after the 5-FU treatment. The recovery was also accelerated when the mice were injected with IL-1 alpha after the 5-FU treatment, but not when injected before the 5-FU treatment. The injection of 5-FU also decreased the cell numbers of whole spleen cells, B cells, and non-T non-B cells (Ig- and Thy-1- cells). The administration of IL-1 alpha accelerated the recovery of the decreased cell numbers. Both B cells and non-T non-B cells possessed APC activity, but most APC activity of unseparated spleen cells was carried by non-T non-B cells. B cells possessed only 1/20 of the APC activity of non-T non-B cells. The injection of 5-FU decreased APC activity of both B cells and non-T non-B cells, but the administration of IL-1 alpha accelerated its recovery. Thus, the accelerated recovery of APC activity by IL-1 alpha was suggested to be due to the recovery in the numbers of APC activity-bearing cell subpopulations and also due to the recovery of the APC activity of each subpopulation. Possible mechanisms for the recovery were discussed.

Recombinant human tumor necrosis factor--II. Antitumor effect on murine and human tumors transplanted in mice.

Recombinant human tumor necrosis factor (rHu-TNF) was found to exhibit potent antitumor activities not only against murine tumors, i.e. Meth A sarcoma, B 16 melanoma, colon 26 adenocarcinoma, Lewis lung carcinoma and MH134 hepatoma, transplanted in syngeneic mice but also against human tumors, i.e. HMV-2 melanoma, PC-10 lung carcinoma and GOTO neuroblastoma, heterotransplanted in nude mice. rHu-TNF caused necrosis of all tumors tested and inhibited their growth in a dose dependent manner. Complete regression of tumors was observed in mice bearing Meth A, B16, colon 26, MH134, HMV-2 and PC-10 but not in mice bearing Lewis lung carcinoma and GOTO neuroblastoma. The prolongation of survival time was also observed in syngeneic mice transplanted with murine tumors except Lewis lung carcinoma. The antitumor effect of rHu-TNF was more evident when it was given intratumorally than when given intravenously. The feasibility of rHu-TNF as a drug for cancer therapy is discussed.

Augmentation of cytotoxic activity of recombinant human tumor necrosis factor (rHu-TNF) by recombinant human interferon-gamma (rHu-IFN-gamma).

The present study was undertaken to examine the effect of recombinant human interferon-gamma (rHu-IFN-gamma) on the in vitro antitumor activity of recombinant human tumor necrosis factor (rHu-TNF) against rHu-TNF-sensitive and resistant tumor cells. rHu-IFN-gamma augmented both cytostatic and cytocidal activity of rHu-TNF. When 14 tumor cells were tested, augmentation by rHu-IFN-gamma was observed in most of rHu-TNF-sensitive tumor cells and in few cases in rHu-TNF-resistant tumor cells. Among rHu-TNF-sensitive tumor cells, there was no relationship between the degree of augmentation and the susceptibility to rHu-TNF. Augmentation was marked when tumor cells were treated with rHu-IFN-gamma either before the exposure to rHu-TNF or during the early period of the exposure to rHu-TNF. On the other hand, augmentation was not observed when tumor cells were treated with rHu-IFN-gamma during a late period of the exposure to rHu-TNF. From these results, a combined treatment with both agents can be expected to provide an approach to get better results in clinical trials.

Synergistic enhancement of the antitumor activity of recombinant human TNF-alpha by recombinant human IFN-gamma.

The effect of recombinant human interferon-gamma (rHu-IFN-gamma) on the antitumor activity of recombinant human tumor necrosis factor (rHu-TNF-alpha) was examined in vitro and in vivo. rHu-IFN-gamma enhanced both cytostatic and cytocidal activity of rHu-TNF-alpha against most rHu-TNF-alpha-sensitive tumor cells in vitro. However, there was no correlation between the degree of enhancement by rHu-IFN-gamma and that of the susceptibility of tumor cells to rHu-TNF-alpha. The enhancing effect of rHu-IFN-gamma was most marked when tumor cells were treated with rHu-IFN-gamma either for 1 day before treatment with rHu-TNF-alpha or for the first day of the exposure to rHu-TNF-alpha. A marked enhancing effect of rHu-IFN-gamma was also observed in the in vivo antitumor activity of rHu-TNF-alpha against HMV-2 melanoma. A combined treatment with rHu-TNF-alpha and rHu-IFN-gamma in a patient with papillary adenocarcinomas was shown to be much more effective than treatment with rHu-TNF-alpha alone. These results suggest that combined treatment with both agents will have better results in clinical trials.

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor.

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.

Establishment of dye-uptake method (A375 assay) for quantitative measurement of IL-1: correlation with LAF assay.

The dye-uptake method (A375 assay) for quantitative measurement of biological activity or recombinant human interleukin-1 (IL-1) was established based on the cytostatic activity of IL-1 on human melanoma A375 cells. Although various factors, e.g., incubation temperature, inoculum cell size and cell growth phase, were found to influence the titration to some degree, the assay-to-assay fluctuation in the observed titers determined by this method could be made minimum after normalization with the observed titer of a standard IL-1. A good correlation was observed between A375 and LAF assays. Thus, A375 assay was shown to be a stable and reliable method and could be replaced with LAF assay for quantitative measurement of recombinant human IL-1.

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse.

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and 1 h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED50s of these antitumor effects after i.v. administration of rHu-TNF were about 50 times as high as ED50s after i.t. administration. ED50 of i.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED50 of i.v. rHu-TNF for vascular effect was only 2-3 times as high as that for cytotoxicity. The whole body autoradiographies with [125I]HSA given i.v. to see the blood influx into tumor tissue and [14C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues. In conclusion, the in vivo antitumor effect of rHu-TNF given i.t. or i.v. appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.