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Background: The rising incidence of malignant melanoma and the lack of curative therapies for metastatic disease represent a therapeutic challenge. New agents effective in treating this disease are needed., Purpose: Because of the additive antitumor effects of interleukin 1 alpha (IL-1 alpha) and indomethacin in vivo, we conducted a phase II trial of this combination in patients with melanoma. We used the recommended dose determined from our phase I trial to ascertain the antitumor activity of the combination., Methods: From August 1, 1990, through July 28, 1992, 49 patients entered the study. They were stratified into two groups based on the presence of visceral (n = 14) and nonvisceral (n = 35) metastases. The patients received 7 days of both IL-1 alpha (O.1 micrograms/kg per day by intravenous bolus) infusion) and indomethacin (50 mg orally every 8 hours). At least two cycles of therapy, repeated at 21-day intervals, were planned. Additional treatment was given to those patients who had stable or responding lesions. A chi-squared test for homogeneity of proportions was used to compare groups on several measures. All P values resulted from two-sided tests., Results: Fever, chills, and hypotension were among the most common side effects. None of the 14 patients with visceral metastases responded to the treatment. Of the 35 patients with non-visceral metastases, three showed a partial response for 6 months each and one showed a complete response for more than 34 months; the response rate was 11% (95% confidence interval [CI] = 5%-26%). All responding patients required phenylephrine for treatment of IL-1 alpha-induced hypotension, whereas six (19%) of 31 of the nonresponding patients with nonvisceral metastases required phenylephrine (P = .0008). The response rate in women was higher; three of 10 women (30%; 95% CI = 11%-60%) responded, whereas one of 25 men (4%; 95% CI = 0%-20%) responded (P = .029). All three women were positive for human leukocyte antigen (HLA) B7 expression (P = .011)., Conclusions: The combination of IL-1 alpha and indomethacin has minimal antitumor activity in melanoma patients. All responses were confined to patients with nonvisceral metastases. IL-1 alpha-induced hypotension, gender, and HLA B7 expression were positively associated with response., Implications: Administration of higher doses of IL-1 alpha alone has been shown to produce hypotension in a large proportion of patients but can be given safely with phenylephrine support. Because of the association of hypotension with antitumor activity, treatment with higher IL-1 alpha doses alone may be a strategy for attaining better response rates.

Background: Point mutations in the ras proto-oncogene that activate its oncogenic potential occur in approximately 30% of human cancers. Previous studies have demonstrated that T-cell immunity against some forms of mutant Ras proteins could be elicited, and some effectiveness against tumors expressing activated Ras has been reported., Purpose: The goal of this study was to determine if immunization of mice with two forms of mutant Ras protein can induce high levels of Ras mutation-specific T-cell immunity in vitro and tumor regression in vivo., Methods: Mice (BALB/c or C3H/HeJ) were immunized subcutaneously at 2-week intervals with purified Ras oncoproteins mixed with the immunologic adjuvants Antigen Formulation or QS-21, both of which have been shown to enhance the induction of T-cell-mediated immunity when included as components of soluble protein vaccines. In some experiments, mice were immunized directly with heat-killed Escherichia coli that had been induced to express one of the mutant Ras proteins. Spleen cells plus lymph node cells from Ras-immunized mice were tested in vitro for lysis of syngeneic Ras-expressing tumor cells and proliferation in response to mutant Ras peptides. For some of the cytolytic activity experiments, the spleen cells were grown under TH1 conditions (growth in presence of interleukin 2, interferon gamma, and an antibody directed against interleukin 4 to stimulate a cell-mediated immune response) or TH2 conditions (growth in presence of interleukins 2 and 4 to stimulate a humoral immune response). The specificity of immunity was examined in vivo by challenge of Ras-immunized mice with syngeneic tumor cells expressing mutant Ras oncoproteins (HaBalb, i.e., BALB/c mouse cells expressing Ras with arginine substituted at amino acid position 12 [Arg 12 Ras]; C3HL61, i.e., C3H/HeJ mouse cells expressing Ras with leucine substituted at position 61 [Leu 61 Ras]). Ten mice per group were used in each experiment., Results: Proliferative and cytolytic T-cell responses directed against the Arg 12 Ras protein were generated in BALB/c mice, resulting in protection against challenge with cells expressing Arg 12 Ras and therapeutic benefit in mice bearing established tumors expressing this protein. In C3H/HeJ mice, high levels of cytolytic and proliferative responses were induced against Leu 61 Ras. Immunization with heat-killed E. coli genetically engineered to express Leu 61 Ras also led to the induction of anti-Ras T-cell immunity. T cells grown under TH1 conditions were cytolytic against Ras-transformed tumor cells, whereas those grown under TH2 conditions were not., Conclusions: Immunization as described here leads to Ras mutation-specific antitumor immunity in vitro and in vivo, with therapeutic efficacy in an established tumor model.

Background: Activated forms of the ras proto-oncogene have been found in approximately 30% of human malignancies, including pancreatic, colon, and lung adenocarcinomas. Ras oncoproteins arise by somatic mutation and contain amino acid changes at residues 12, 13, or 61, thus generating unique tumor-specific proteins that are attractive targets for cancer therapy., Purpose: The goal of this study was to determine whether vaccination with mutant Ras protein could lead to the generation of cytotoxic T lymphocytes (CTLs) specific for the mutant epitope and to protection against challenge with tumor cells expressing the mutant oncoprotein., Methods: To determine a methodology for generating CTL responses following immunization with soluble protein, ovalbumin was used as a model tumor antigen. C57BL/6 mice were immunized with soluble ovalbumin administered intraperitoneally at 2-week intervals or with intravenous injection of ovalbumin or osmotically loaded splenocytes. Immunized mice were challenged with E.G7 cells (which express a transfected ovalbumin gene), and tumor growth was monitored. Generation of ovalbumin-specific CTLs was determined by 51Cr release assays. Purified wild-type or mutant H-Ras proteins (containing single amino acid substitutions at position 12 converting Gly to Arg or Val) were used to immunize BALB/c mice intraperitoneally. Ras-immunized mice were challenged with tumor cells containing Arg 12 or Val 12 mutations or not harboring mutant forms of Ras. Cytolytic and proliferative responses to mutant forms of Ras were studied, and the effects of in vivo depletion of CD4+ or CD8+ T lymphocytes were determined., Results: In vivo challenge with E.G7 showed that intraperitoneal immunization with soluble ovalbumin was as effective as osmotic loading, resulting in long-term disease-free survival of some mice and the development of ovalbumin-specific CTLs. Immunization with Arg 12 Ras led to disease-free survival in nine of 10 animals challenged with tumor cells containing an Arg 12 mutation, while no protection was afforded against tumors expressing other forms of Ras or other oncogenes. Splenocytes from BALB/c mice immunized with Arg 12 Ras demonstrated cytolytic activity specific against tumor cells expressing Arg 12 Ras, with most of this activity residing in the CD8+ subset. Mutation-specific proliferation to Arg 12 Ras peptides was also observed. Immunization with Val 12 Ras did not elicit detectable Val 12-specific immunity., Conclusions: Antigen-specific CTLs can be induced following intraperitoneal immunization of mice with purified, soluble proteins. For both ovalbumin and Arg 12 Ras, specific in vivo protection against tumor cell challenge was observed.

Background: Experiments in animal tumor models suggest that the antitumor effects of interleukin-2 (IL-2) or IL-2 in combination with lymphokine-activated killer (LAK) cells can be enhanced by chemotherapy agents such as cyclophosphamide or doxorubicin or by the biologic agent interferon alpha., Purpose: We determined the toxicity and clinical response rate of an IL-2-LAK cell regimen modified by the addition of moderate, immunomodulatory doses of chemotherapy and sequenced with interferon alfa-2a (IFN alpha-2a) in patients with metastatic melanoma and renal cell carcinoma., Methods: IL-2 (3-6 million units/m2 per day) was administered by continuous infusion on days 0-5 and days 11-16. LAK cells were infused on days 11 and 12 or on days 11, 12, and 14. Low doses of cyclophosphamide (300 mg/m2) and doxorubicin (25 mg/m2) were given on day 9 before the LAK cell infusions. Following the IL-2-LAK cell infusion, IFN alpha-2a (12 million units/m2) was administered for a total of nine doses to complete a cycle of treatment. A total of 89 patients were enrolled in the study., Results: For each histology, there were eight partial responses in 40 assessable patients, for an overall response rate of 20% (90% confidence interval = 10%-33%). The median response duration was 5 months, although two patients with renal cell carcinoma and one patient with metastatic melanoma had almost complete disappearance of tumor and are still responding after 26+, 22+, and 26+ months, respectively. Toxic effects were severe in patients receiving the highest dose of IL-2 administered in this study and similar to those reported with other high-dose IL-2-LAK cell regimens. Although toxic effects were completely reversible in most patients, there were four treatment-related deaths., Conclusions: This regimen is active in patients with metastatic melanoma and renal cell carcinoma and produces meaningful responses in a small percentage of these patients; however, it is not clear whether cyclophosphamide, doxorubicin, and IFN alpha-2a as used in this protocol appreciably augmented the antitumor activity of the IL-2-LAK cell regimen.