Joe K. McIntosh

An Anti-Wnt-2 Monoclonal Antibody Induces Apoptosis in Malignant Melanoma Cells and Inhibits Tumor Growth

Activation of the Wnt/β-catenin signaling pathway has been associated with human cancers. To test whether Wnt-2 signal is a survival factor in human melanoma cells and thus represents a potential therapeutic target, we investigated the effects of inhibition of Wnt-2 signaling in human melanoma cell lines. We have developed a novel monoclonal antibody against the NH2 terminus of the human Wnt-2 ligand that induces apoptosis in human melanoma cells overexpressing Wnt-2. Whereas incubation of this antibody with normal cells lacking Wnt-2 expression does not induce apoptosis, Wnt-2 signaling blockade by the ligand-binding antibody is confirmed by down-regulation of Dishevelled and β-catenin. Wnt-2 small interfering RNA treatment in these cells yielded similar apoptotic effects and downstream changes. Down-regulation of an inhibitor of apoptosis family protein, survivin, was observed in both the Wnt-2 antibody-treated and small interfering RNA-treated melanoma cell lines, suggesting that the antibody induces apoptosis by inactivating survivin. In an in vivo study, this monoclonal anti-Wnt-2 antibody suppresses tumor growth in a xenograft model. These findings suggest that the anti-Wnt-2 monoclonal antibody may be useful for the treatment of patients with malignant melanoma.

Antitumor effect of recombinant tumor necrosis factor-α against murine sarcomas at visceral sites: tumor size influences the response to therapy

SummaryWe examined the antitumor efficacy of rTNF-α administration on established tumor at two visceral sites, lungs and liver. Treatment of B6 mice harboring multiple (>100 foci of ≤0.5 mm diameter) 10-day pulmonary macrometastases from the MCA-106 sarcoma, with dosages of rTNF-α (5–10 μg, single dose i. v.) that caused hemorrhagic necrosis and regression of a 6 mm MCA-106 s. c. tumor, had no impact on the number (or size) of lung nodules. Similarly, rTNF-α failed to show an antitumor effect in B6 mice with advanced day 8 or 10 multiple (>100 foci of ≤0.5 mm diameter) hepatic metastases at single i. v. doses up to 20 μg, as measured by either enumeration of residual liver nodules or survival. B6 mice injected s. c. with MCA-106 sarcoma and treated with rTNF-α as a single i. v. dose on day 0, 3, 5, or 7 experienced marked tumor regression only after the day 7 rTNF-α injection, when the tumor had achieved a size of 5–6 mm in diameter. Since tumor size appeared important for rTNF-α susceptibility in vivo, we next induced a single hepatic tumor of the MCA-106 sarcoma by the direct injection of cells into the left lobe of the liver and treated these mice at day 10 when the nodule had achieved a size of 5–6 mm in diameter. Increasing doses of rTNF-α (up to 8 μg) given as a single i. v. injection resulted in increasingly greater reductions in hepatic tumor as well as significant survival benefit of the treated mice. Sites of regressing hepatic tumor exhibited central necrosis accompanied by polymorphonuclear leukocytes and lymphocytes. Collectively, these results show that rTNF-α administration can mediate a significant antitumor effect on visceral tumor and suggest that tumor size is an important factor in rTNF-α susceptibility not only for tumors growing at s. c. sites but also for those established at visceral sites.

Studies of the mechanisms of toxicity of the administration of recombinant tumor necrosis factor α in normal and tumor-bearing mice

SummaryTumor-bearing mice have a greater sensitivity to the acute lethal effects of the administration of high-dose recombinant human tumor necrosis factor α (rhTNF-α) compared to normal, non-tumor-bearing mice. We studied whether or not the presence of tumor per se was responsible for the enhanced rhTNF-α toxicity. Tumor-bearing mice underwent tumor excision or sham operation before the systemic administration of rhTNFα at staged times (0.5–24 h) following surgery. There was little survival difference between sham-operated tumor-bearing mice and tumor-bearing mice undergoing tumor excision (at 24 h, treatment with 12 µg rhTNF-α, survival:sham-operated tumor bearers = 0/12, excised tumor-bearers = 0/12;P2 <0.01 compared to non-tumor-bearers). Mice without tumors receiving sham operation, had minimal toxicity (10 of 12 mice surviving). The injection of 3 ml Ringers lactate i.p. before i.v. rhTNF-α therapy increased survival in tumor-bearing animals; following pretreatment with Ringers lactate 30/42 mice survived 12 µg rhTNF-α compared to 6/42 surviving a similar rhTNF-α dose without hydration (P2 <0.001). Since the production of oxygen free-radical metabolites has been postulated to play a role in the acute toxicity of rhTNF-α, bismuth subnitrate was used to induce the enzyme metallothionein to act as a natural scavenger for these metabolites. Daily oral bismuth subnitrate treatments improved survival of mice with MCA-106 or MCA-102 sarcoma and of mice without tumors, with higher rhTNF-α doses (12–20 µg), without reducing the therapeutic effect of rhTNF-α against the weakly immunogenic MCA-106 sarcoma. These studies suggest methods for reducing the toxicity of rhTNF-α administration in clinical trials.

Effect of recombinant human tumor necrosis factor α on the induction of antibody-dependent cellular cytotoxicity in the treatment of established B16 melanoma liver nodules

SummaryIncubation of C3H/Hen thymocytes in the presence of recombinant human tumor necrosis factor α (TNF) and interleukin-2 (IL-2) augmented the generation of antibody-dependent cellular cytotoxicity (ADCC) when compared to cells cultured in TNF or IL-2 alone. This effect was optimal when 100–200 units/ml IL-2 was used together with 103–104 units/ml TNF. TNF alone at any concentration could not mediate the induction of ADCC. Similar to the results obtained in vitro, TNF, when given alone, had no effect on the generation of ADCC in vivo. The addition, however, of TNF to IL-2, given at 10 000 and 20 000 but not 40 000 units, enhanced the IL-2-induced ADCC on a per-cell basis. Furthermore, TNF enhanced the total ADCC activity in various organs including the liver, spleen and thymus as a result of an increase in the number of mononuclear cells isolated from these organs. The increase in total ADCC activity was optimal when 110 000–220 000 units (5–10 µg) TNF were employed together with IL-2. The combined treatment with TNF and IL-2 also increased the intracellular benzyloxycarbonyl-l-l-lysinethiobenzyl-ester esterase content in cells isolated from the livers of mice treated with these cytokines. On the basis of these results we treated mice bearing a single B 16 melanoma nodule with TNF and TNF + IL-2 given with or without anti-B 16 monoclonal antibody. We found that TNF administration augmented the anti-tumor effect of specific anti-B 16 antibodies, and the addition of IL-2 further increased this anti-tumor effect.

The Kinetics of Interleukin-6 Induction by the Systemic Administration of rhTNF-α in Mice

The production of IL-6 (interferon-&) from macrophages and fibroblasts has been previously detected in vitro after addition of recombinant human tumor necrosis factor (rhTNF-a).’ Recently, we have demonstrated that the in vivo administration of rhTNF-a to patients with metastatic cancer leads to the induction of circulating levels of IL-6.’ In the present study, we investigated the capacity of rhTNF-a (provided by Cetus, Emeryville, California) to induce IL-6 in normal, nontumor-bearing (NTB) mice and in mice bearing an established 14-day MCA-106 subcutaneous sarcoma (TB). IL-6 activity in serum samples was assayed by the B9 subclone hybridoma proliferation assay’ standardized by recombinant IL-6 (provided by Genetics Institute, Cambridge, Massachusetts). In addition, all activity was neutralized with a specific anti-murine IL-6 polyclonal antibody. TNF activity was detected by the L929 cytolytic colorimetric assay.‘ NTB and TB mice were treated with escalating doses of rhTNF-a (0-6 pg) and then their sera were extracted at 2 h after infusion and tested for IL-6 and TNF activity. A direct relationship existed between rhTNF-a administration and induction of serum IL-6 levels (TABLE 1). The kinetics of IL-6 induction then was determined in NTB and TB mice receiving a 4-pg dose of rhTNF-a iv. Pooled serum samples of six mice per group were collected at serial time points (0.25, 0.5, 1, 2, 4, 8, and 24 h) and they were tested for both IL-6 and TNF activity. As described previously, TNF levels peaked by 15 min after administration and were only minimally detectable by 4 h.5 In contrast, low levels of IL-6 were detectable within 15 min following rhTNF-a infusion, and consistent increases continued for 2-4 h after a single injection of rhTNF-a and waned by 8 h (TABLE 2). TB mice consistently produced more IL-6 in response to rhTNF-a than did NTB mice (at 2 h, 4 pg rhTNF-a, TB = 26,400, NTB = 2300 HGF U/mL IL-6). Serum