Jonathan E. Kolitz

A phase-II study of low-dose cyclophosphamide and recombinant human interleukin-2 in metastatic renal cell carcinoma and malignant melanoma

SummaryRecent preclinical and clinical studies that have demonstrated antitumor activity of high-dose recombinant interleukin-2 (rIL-2), and animal models that demonstrated a synergistic effect of low-dose cyclophosphamide, led us to study rIL-2 (Cetus Corp., Emeryville, Calif) in a phase II clinical trial in combination with low-dose cyclophosphamide in 32 patients, 18 with malignant melanoma and 14 with renal cell carcinoma. rIL-2 was given once daily at 3×106 U/m2, as a 30-min infusion for 14 days in cycle I and for 2×5 days in cycles II and III respectively; if tolerated, the dose was increased to a maximum of 6×106 U m−2 day−1; the cycles, separated by 1 week treatment-free intervals, were preceded each by a single i.v. bolus of cyclophosphamide at 350 mg/m2. The most prominent side-effects encountered in this trial consisted of a capillary leak syndrome, myalgia and fever that required dose reduction during the first cycle in one-half of the patients. Given the limit of tolerable toxicities in a standard care unit, the regimen employed achieved minor antitumor activity. No remission was achieved in patients with renal cell carcinoma, and 15% of melanoma patients showed objective responses (partial response + minor response).

T-cell activation defect in common variable immunodeficiency: restoration by phorbol myristate acetate (PMA) or allogeneic macrophages.

Common variable immunodeficiency (CVI) represents a group of familial and sporadic diseases characterized by a range of B-cell, T-cell, and macrophage defects. A defect in T-cell activation, involving reduced proliferation and IL-2 production after stimulation with OKT3 antibody, has been described previously. In the present study we found that these defects could be corrected in vitro by adding phorbol myristate acetate (PMA) to OKT3-stimulated peripheral blood mononuclear cells (PBMC) of 14 patients with CVI. PBMC of 6 out of 7 patients with CVI studied also exhibited a profound defect in IL-2 receptor expression when incubated with OKT3 antibody. IL-2 receptor expression after stimulation with PMA alone was normal, indicating that the OKT3- but not the PMA-induced pathway of IL-2 receptor expression was defective. On the RNA level, the genes for IL-2 and IL-2 receptor were expressed after stimulation with OKT3 antibody. IL-2 and IL-2 receptor gene expression were normal, indicating a possible post-transcriptional defect. To investigate whether the defect in T-cell activation was at the macrophage or the T-cell level, we prepared adherent cells and monocyte-depleted T cells (E+) from 3 patients with CVI and from normal blood donors. Incubating CVI E+ cells with normal adherent cells resulted in normal proliferation and IL-2 production in the presence of OKT3, whereas incubation of normal E+ cells with adherent cells from patients with CVI under the same conditions showed reduced IL-2 production and proliferation, suggesting the macrophage as the origin of the failure in T-cell activation in the patients with CVI studied. Inhibition by macrophage-secreted prostaglandins was excluded by failure to correct the IL-2 production and proliferation defects in the presence of indomethacin.

A phase I trial of intraperitoneal recombinant interleukin 2 in patients with ovarian carcinoma

SummarySeven patients with refractory stage III ovarian carcinoma were treated with escalating doses of human recombinant interleukin 2 (rIL-2) administered via the intraperitoneal (IP) route in an attempt to establish a dose and schedule of rIL-2 suitable for prolonged outpatient IP administration. Three patients went on to receive outpatient maintenance treatment twice weekly for 2–3 months. Doses ranged from 105 to 5 × 107 U/m2. The dose found most suitable for twice weekly outpatient IP administration was 106 U/m2. Dose-limiting toxicities consisted of diarrhea resulting in hypovolemia (5 patients) fever and chills (4 patients), nausea and vomiting (1 patient), mental status changes (2 patients), and azotemia (1 patient). These side effects were not prevented by indomethacin. Significant hypotension was not observed. Pharmacokinetic studies revealed extremely high IP concentrations of IL-2 which persisted for more than 24 hours. After a dose of 106 U/m2, the IP concentrations ranged from 670 to 760 U/ml. In one patient in whom concurrent serum concentrations were determined, the IP concentrations were over 100-fold higher than serum levels. After a dose of 107 U/m2, the IP concentrations of IL-2 ranged from 8700 to 14000. Concurrent serum levels in one patient revealed IP concentrations over 500-fold higher than serum levels. There were no consistent changes in T cell surface and activation markers on mononuclear cells from peripheral blood in 3 patients tested. Natural killer cell (NK) activity in peripheral blood increased in the three patients in whom it was measured. Four of the 7 patients progressed on treatment; 3 patients remained stable. We conclude that 106 U/m2 of rIL-2 is well-tolerated when administered by the IP route and that concentrations of IL-2 well in excess of that required to enhance cell-mediated cytotoxicity in vitro persist in the IP fluid for at least 24 hours.

Cell-mediated toxicity of interleukin-2-activated lymphocytes against autologous and allogeneic human myeloma cells

We studied the sensitivity of human myeloma (plasma cell leukemia) toward autologous and allogeneic lymphokine-activated killer (LAK) cells. Fresh plasma cell leukemia (PCL)-derived peripheral blood mononuclear cells (PBMC) and PBMC from 3 normal donors were cultured in the presence of recombinant interleukin-2 (rIL2; 1,000 U/ml) for subsequent use as cytotoxic effectors against fresh and continuously cultured myeloma cells. Target cell lysis was measured in a 4-hour 51Cr radioisotope release assay. At an effector to target (E:T) ratio of 50:1, rIL2-induced PCL-PBMC lysed 48 +/- 19% (mean +/- 1 SD) of autologous myeloma targets, as compared to 89 +/- 5, 95 +/- 15, and 100 +/- 9% lysis of standard LAK-sensitive Daudi cells and allogeneic myeloma cell lines SKO-007, and RPMI-8226, respectively. Normal PBMC-derived rIL2-induced (LAK) cells exhibited a slightly lower cytotoxic reactivity against allogeneic targets (61 +/- 9, 60 +/- 6, and 81 +/- 8% cytolysis of SKO-007, RPMI-8226, and Daudi cells, respectively, at a 50:1 E:T ratio). Cytotoxicity against myeloma (PCL) of autologous PCL-derived killer cells could be significantly (at least 2-fold) enhanced when rIL-2-induced effector cells were preincubated for 18 h in the presence of recombinant Interferon-alpha rIFN-alpha; 1,000 U/ml). In summary, our results indicate the potential antitumor efficacy of rIL2- and rIL2 + rIFN-alpha-activated killer cells in human myeloma (PCL).

Ewing’s Sarcoma: ex vivo Sensitivity towards Natural and Lymphokine-Activated Killing

We studied the ex vivo cell-mediated cytotoxicity of natural killer (NK) and lymphokine-activated killer (LAK) cells against continuously cultured Ewings sarcoma cells from 3 different patients. Target cell lysis was measured in a 4-hour 51Cr radioisotope release assay. At an effector to target (E:T) ratio of 50:1, the mean (+/- 1 SD) cytolysis by fresh purified large granular lymphocytes (NK cells) was 20 +/- 8, 25 +/- 2, and 21 +/- 3% in Ewings sarcoma cell lines 6647, 5838, and A4573, respectively. Under identical conditions, NK cells lysed 56 +/- 7% of K562 (a standard NK target), and 3 +/- 3% of Daudi (a standard NK-resistant LAK target). When compared to fresh unseparated peripheral blood mononuclear cells (PBMC), purified NK cells did not exhibit an enhanced cytotoxic reactivity against either Ewings sarcoma target. In contrast, LAK cells (i.e., PBMC that were preincubated for 4 days in the presence of rIL-2) were highly cytotoxic against all three Ewings sarcoma targets. LAK activity was dependent on the concentration of rIL-2 used in PBMC cultures. Optimum cell-mediated toxicity against the standard LAK target Daudi (99 +/- 10% cytolysis at 50:1 E:T ratio) was achieved at rIL-2 concentrations of 1,000 u/ml. LAK cells grown under these conditions were also effective against Ewings sarcoma cells. At an E:T ratio of 50:1, 86 +/- 16, 85 +/- 16, and 67 +/- 13% inhibition was observed in 6647, 5838, and A4573 cells, respectively, as compared to 17 +/- 10, 19 +/- 15, and 29 +/- 11% cytolysis by fresh uninduced PBMC. In summary, our results suggest that rIL-2-induced LAK-type immune effector cells may be of some therapeutic value in the treatment of poor prognosis Ewings sarcoma.

Human Interleukin 2 Gene is Located on Chromosome 4

The interleukin-2 (IL2) gene is assigned to human chromosome 4. A synthetic oligonucleotide representing bases 285 to 324 of the IL2 cDNA clone described by Taniguchi et al. [Nature (London) 302:305-310, 1983] was used as hybridization probe in Southern blots. Eco RI digests of DNA derived from 11 somatic mouse-human cell hybrid clones were used. The IL2 gene was localized to human chromosome 4 based on the observed combinations of segregating chromosomes and bands. Under the conditions of stringency utilized, a single 3.6 kilobase Eco RI fragment hybridized to the oligonucleotide. Molecular weight standards were provided by rehybridizing the blots with an actin cDNA clone, pAct 1, which identified actin gene Eco RI fragments of defined size.

Anti-Tumor Efficacy of Interleukin-2-Activated Killer Cells in Human Neuroblastoma ex vivo

We studied the ex vivo sensitivity of continuously cultured neuroblastoma cells from 3 different patients towards interleukin-2-induced cell-mediated cytotoxicity. A mean (+/- SD) target cell lysis (4 h 51Cr release) of 49 +/- 11, 46 +/- 8, and 32 +/- 11% in SMS-SAN, LA-N-1, and SK-N-BE2 cell lines, respectively, was achieved when neuroblastoma cells were co-cultured at an effector-to-target (E:T) ratio of 50:1 with peripheral blood mononuclear cells (PBMC) that had been preincubated for 4 days in the presence of recombinant interleukin-2 (rIL-2; 100 U/ml). Under identical conditions, 93 +/- 9% of Daudi cells (a standard target for rIL-2-activated killer cells) were lysed. Preincubation of rIL-2-induced PBMC cultures in the presence of irradiated neuroblastoma targets (LA-N-1, SK-N-BE2) resulted in a significant cytolytic augmentation. At E:T ratios of 50:1 and 10:1, day-4 rIL-2/LA-N-1-stimulated PBMC produced 69 +/- 7 and 41 +/- 11% lysis of LA-N-1 cells, as compared to 46 +/- 8 and 22 +/- (mean +/- SD) 7% lysis by untargeted PBMC that were preincubated with rIL-2 (100 U/ml) in the absence of LA-N-1 target cells (p less than 0.05). Co-incubation of rIL-2-induced PBMC preparations with irradiated LA-N-1 and SK-N-BE2 cells, respectively, did not significantly enhance the cytolytic activity against other neuroblastoma targets and the standard Daudi cell line (p greater than or equal to 0.3).(ABSTRACT TRUNCATED AT 250 WORDS)

Mitoxantrone, etoposide, mitoguazone and vinblastine chemotherapy (MV2) in relapsed and refractory lymphomas

SummaryAs part of a program to develop less leukemogenic chemotherapy regimens for the treatment of favorable prognosis Hodgkins disease, a phase I–II trial of mitoxantrone, etoposide, mitoguazone, and vinblastine was used to treat patients with relapsed and refractory malignant lymphoma and Hodgkins disease. An over-all partial response rate of 41% was observed. Although useful responses were seen, the absence of complete remissions is disappointing.