Preparing clinical-grade myeloid dendritic cells by electroporation-mediated transfection of in vitro amplified tumor-derived mRNA and safety testing in stage IV malignant melanoma

BACKGROUND: Dendritic cells (DCs) have been used as vaccines in clinical trials of immunotherapy of cancer and other diseases. Nonetheless, progress towards the use of DCs in the clinic has been slow due in part to the absence of standard methods for DC preparation and exposure to disease-associated antigens. Because different ex vivo exposure methods can affect DC phenotype and function differently, we studied whether electroporation-mediated transfection (electrotransfection) of myeloid DCs with in vitro expanded RNA isolated from tumor tissue might be feasible as a standard physical method in the preparation of clinical-grade DC vaccines. METHODS: We prepared immature DCs (IDCs) from CD14(+ )cells isolated from leukapheresis products and extracted total RNA from freshly resected melanoma tissue. We reversely transcribed the RNA while attaching a T7 promoter to the products that we subsequently amplified by PCR. We transcribed the amplified cDNA in vitro and introduced the expanded RNA into IDCs by electroporation followed by DC maturation and cryopreservation. Isolated and expanded mRNA was analyzed for the presence of melanoma-associated tumor antigens gp100, tyrosinase or MART1. To test product safety, we injected five million DCs subcutaneously at three-week intervals for up to four injections into six patients suffering from stage IV malignant melanoma. RESULTS: Three preparations contained all three transcripts, one isolate contained tyrosinase and gp100 and one contained none. Electrotransfection of DCs did not affect viability and phenotype of fresh mature DCs. However, post-thaw viability was lower (69 ± 12 percent) in comparison to non-electroporated cells (82 ± 12 percent; p = 0.001). No patient exhibited grade 3 or 4 toxicity upon DC injections. CONCLUSION: Standardized preparation of viable clinical-grade DCs transfected with tumor-derived and in vitro amplified mRNA is feasible and their administration is safe

Pilot Study of Human Recombinant Interferon Gamma and Accelerated Hyperfractionated Thoracic Radiation Therapy in Patients with Unresectable Stage IIIA/B Nonsmall Cell Lung Cancer.

PURPOSE: Gamma interferon has a wide range of properties, including the ability to sensitize solid tumor cells to the effects of ionizing radiation. The North Central Cancer Treatment Group has previously completed pilot studies of accelerated hyperfractionated thoracic radiation therapy (AHTRT) in patients with unresectable Stage IIIA/B nonsmall cell lung cancer (NSCLC). This Phase I study was designed to assess the toxicity of concomitant gamma interferon and AHTRT in a similar patient population. METHODS AND MATERIALS: Between December 1991 and May 1992, 18 patients with unresectable Stage IIIA/B NSCLC were treated with daily gamma interferon (0.2 mg subcutaneously) concomitant with AHTRT (60 Gy given in 1.5 Gy twice daily fractions). All patients had an Eastern Cooperative Oncology Group performance status of 0 or 1 with weight loss \u3c 5%. Eight patients had Stage IIIA and 10 had Stage IIIB disease. RESULTS: Nine patients (50%) experienced severe, life-threatening, or fatal toxicities. Eight of the patients (44%) developed significant radiation pneumonitis, which was severe in six patients and fatal in two patients (11% treatment-related mortality). Two patients (11%) developed severe radiation esophagitis. With follow-up of 15-21 months, 2 patients are alive, and 16 have died. The median survival time and 1-year survival rate is 7.8 months and 38%, respectively. CONCLUSION: Gamma interferon appeared to sensitize normal lung tissue to the effects of radiation, as demonstrated by the high incidence of severe or fatal radiation pneumonitis. We do not recommend pursuing gamma interferon as a radiosensitizer in this setting

Phase II Study of Methotrexate, Vinblastine, Doxorubicin, and Cisplatin in Patients with Squamous Cell Carcinoma of the Upper Respiratory or Alimentary Passages of the Head and Neck.

BACKGROUND: The chemotherapy drugs methotrexate, vinblastine, doxorubicin, and cisplatin have shown activity in patients with recurrent or metastatic squamous cell carcinoma arising from the upper respiratory or alimentary passages of the head and neck. This study was undertaken to assess the antitumor activity and toxicity profile of the drug combination methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) in this patient population. METHODS: Patients with histologically confirmed unresectable, recurrent, or metastatic squamous cell carcinoma arising from the upper respiratory or alimentary passages of the head and neck were treated with MVAC over a 4-week cycle. The doses were as follows: 30 mg/m2 of methotrexate on Days 1, 15, and 22; 3 mg/m2 of vinblastine on Days 2, 15, and 22; 30 mg/m2 of doxorubicin on Day 2; and 70 mg/m2 of cisplatin on Day 2. The total cumulative dose of doxorubicin was not to exceed 450 mg/m2. Treatment was discontinued after four cycles for those whose disease remained stable. Patients were evaluated for chemotherapy response, progression free survival, and survival. RESULTS: Thirty-six patients were accrued onto this study between April 1993 and February 1996. One patient (3%) with a history of cardiac heart failure was declared ineligible. Severe leukopenia (leukocyte count \u3c 2000 cells/m3) was observed in 55% of the patients during the first cycle of treatment and in 81% of the patients during the entire course of their treatment. The overall objective response rate over the first 4 cycles of treatment was 46% (90% confidence interval [CI], 33-60%). Two of the 18 patients who responded had a complete response. The median time to progression was 19 weeks, and 1-year progression free survival rate was 17% (95% CI, 8-36%). The median survival was 49 weeks, and the 1-year survival rate was 43% (95% CI, 29-63%). Among the 22 patients with unresected residual or recurrent disease, the median time to progression was 11 weeks, and 1-year progression free survival rate was 14% (95% CI, 5-39%), and median survival was 24 weeks, and the 1-year survival rate was 36% (95% CI, 21-63%). Among the 13 patients with metastatic disease, the median time to progression was 26 weeks, and the 1-year progression free survival rate was 23% (95% CI, 9-62%), the median survival was 54 weeks, and the 1-year survival rate was 54% (95% CI, 33-89%). CONCLUSIONS: Methotrexate, vinblastine, doxorubicin, and cisplatin is an active chemotherapy regimen in patients with recurrent or metastatic squamous cell cancer arising from the upper respiratory or alimentary passages of the head and neck

Combined Levamisole with Recombinant Interleukin-2 (IL-2) in Patients with Advanced Renal Cell Carcinoma: A Phase II Study.

Adoptive immunotherapy (AI) with interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells is an antineoplastic modality in which immune-activated cells are administered to a host having cancer in an attempt to mediate tumor regression. Levamisole (LEV), an immune stimulant, has been suggested as having therapeutic effectiveness in a variety of cancers. After a phase I trial of recombinant IL-2 plus LEV, a phase II trial of this combination was conducted in patients who had advanced renal cell carcinoma. The regimen was IL-2 at 3 x 10(6) U/m2 daily x 5 plus LEV at 50 mg/m2 perorally three times a day x 5. Only one of the 22 eligible patients had a regression. It was a partial regression, 85 days in duration. The median time to treatment failure (refusal, progression, or off study because of toxicity) was 36 days. The only grade 4 toxicity reported was lethargy. This regimen is not recommended for further testing in patients who have advanced renal cell carcinoma