Repetitive weekly cycles of interleukin-2. II. Clinical and immunologic effects of dose, schedule, and addition of indomethacin.

Clinical trials with high doses of interleukin 2 (IL-2) have shown antitumor responses, but many of the patients have experienced severe and occasionally life-threatening toxic effects. Preclinical studies indicate that modifications in IL-2 dose, route, and schedule can influence both immune activation and antitumor effects. This study evaluated the clinical tolerance to and immunologic modifications induced by four repetitive weekly cycles of IL-2, with two dose levels (1 X 10(6) and 3 X 10(6) U/m2 per day) of IL-2 and three different daily administration schedules [bolus, continuous, or combined (bolus and continuous)], with and without indomethacin treatment. Patients in all treatment groups experienced acceptable, non-life-threatening toxic effects and immune system stimulation characterized by rebound lymphocytosis with increased numbers of natural killer and lymphokine-activated killer cells and enhanced direct cytolytic function. These immune changes were significantly enhanced by the repetition of IL-2 cycles beyond the first week of therapy. At an IL-2 dose of 3 X 10(6) U/m2 per day, bolus IL-2 was less immunostimulatory than continuous-infusion IL-2. The combined regimen (with half of each daily dose given as a bolus and half as a 24-hr infusion) was as stimulatory as continuous-infusion IL-2 and also induced antitumor effects. Finally, the addition of indomethacin to this regimen did not significantly modify in vitro or in vivo immune response parameters but appeared to worsen the systemic toxic effects of renal dysfunction and capillary leakage. These results suggest that continuous or combined infusion of IL-2 at 3 X 10(6) U/m2 per day on this schedule should be considered for further testing in phase II trials or in combination with other therapeutic modalities.

Repetitive weekly cycles of recombinant human interleukin-2: responses of renal carcinoma with acceptable toxicity.

A phase I trial of repetitive weekly cycles of human recombinant interleukin-2 (IL-2) was performed in 23 patients with metastatic carcinoma. Patients received 4 days of IL-2 each week, followed by 3 days of observation, for 4 consecutive weeks. IL-2 was administered iv at 1.0 or 3.0 X 10(6) U/m2/day by one of three schedules involving continuous or bolus infusions. All treatment was carried out in a general hospital ward without intensive care unit monitoring or support. Seventeen patients had metastatic renal cell carcinoma; three of these demonstrated measurable (greater than 50% shrinkage) partial responses. This study demonstrates that IL-2 given alone without lymphokine-activated killer cells in this manner can induce antitumor effects with acceptable toxicity.

In vitro analysis of donor bone marrow following monoclonal antibody treatment for the prevention of acute graft versus host disease.

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality following bone marrow transplantation. The in vitro removal of the GVHD-causing T-lymphocytes from donor marrow is one approach which could control this complication. Treatment of the donor bone marrow with lectins and erythrocyte-forming rosette depletion, anti-T-cell antisera or monoclonal antibodies are methods currently being tested to accomplish this. CT-2 is an immunoglobulin monoclonal antibody specific for the T-cell erythrocyte-forming rosette receptor. Bone marrow from 23 consecutive donors was treated in vitro with CT-2 and complement, prior to infusion, as a potential means of controlling GVHD. Surface marker analysis using erythrocyte-forming rosetting, and OKT-3 and OKT-11 monoclonal antibodies on paired samples of treated and untreated marrow demonstrated a mean depletion to 1% of the original number of T-cells. Proliferative responses to alloantigens and mitogens as well as cytotoxic and natural killer cell function were tested and found to be markedly reduced. Despite these effects on T-lymphocytes, viable hematopoietic stem cell colonies were retained. Clinical results following the in vitro T-lymphocyte depletion of donor bone marrow for the 8 histocompatible and 15 nonhistocompatible bone marrow transplantation are reported. Prompt engraftment with minimal GVHD, despite no posttransplant GVHD prophylaxis, was seen in seven of the matched patients. In the nonhistocompatible bone marrow transplantation, failure of engraftment occurred in 11 patients. Grades III-IV GVHD were seen in two of the four patients that engrafted despite good T-lymphocyte depletion. No predictive correlation could be found between the in vitro analysis of marrow following CT-2 treatment and clinical outcome.

Repetitive weekly cycles of interleukin 2: effect of outpatient treatment with a lower dose of interleukin 2 on non-major histocompatibility complex-restricted killer activity.

Fifteen patients with advanced malignancy who had failed conventional therapy were entered into a protocol consisting of 1 inpatient mo of repetitive weekly cycles of interleukin 2 (IL-2) at 3 x 10(6) units/m2/day by constant infusion for the first 4 days of each week. This was followed by IL-2 administered on an outpatient basis at the same schedule but at a dose of 1 x 10(6) units/m2/day for the next 1 to 6 mo. Nine patients had renal carcinoma, four had melanoma, and two had lymphoma. Thirteen patients completed the induction month, and ten patients completed greater than or equal to 1 mo of outpatient therapy. Only one patient had therapy discontinued because of toxicity due to IL-2. No major toxicities occurred during outpatient therapy. After 1 mo of IL-2 at 3 x 10(6) units/m2/day, profound changes similar to those previously documented were seen in peripheral blood lymphocyte (PBL) counts (4.7-fold increase), lymphokine-activated killer activity (16-fold increase), and the percentage of PBL with natural killer-associated markers including a 3.6-fold increase in the percentage of PBL expressing the Leu 19 (NKH-1) marker, a 3.7-fold increase in Leu 11 (FcIgGR), and a 3.0-fold increase in Leu 17 (OKT10). These indicators of IL-2 effect all remained elevated relative to the baseline at the end of 1 and 2 mo of outpatient therapy at the lower dose. However, lymphokine-activated killer activity and Leu 17 percentage were significantly reduced relative to the effect of the higher induction dose. PBL taken from patients while receiving maintenance therapy showed strong and rapid responses to IL-2 in vitro, confirming the in vivo effects of prolonged IL-2 treatment. Nevertheless, there were no complete or partial antitumor responses seen. This study demonstrates that an immunologically active dose of IL-2 can be given long term as outpatient therapy with tolerable toxicity and results in highly IL-2-responsive "primed" lymphokine-activated killer cells.

In vivo induction of the lymphokine-activated killer phenomenon: interleukin 2-dependent human non-major histocompatibility complex-restricted cytotoxicity generated in vivo during administration of human recombinant interleukin 2.

The availability of purified human recombinant interleukin 2 (IL-2) has enabled clinical trials to test its in vivo effects. We report here the immunological effects of 7 consecutive days of IL-2 treatment given to 25 patients with cancer in a clinical Phase I study. Peripheral blood lymphocytes obtained from patients following therapy with IL-2 had enhanced proliferative responses to IL-2 and enhanced direct cytotoxic activity on K562 target cells. This lytic activity was further augmented by the addition of IL-2 during the 51Cr release assay. Fresh peripheral blood lymphocytes from some patients who had just completed treatment at the higher IL-2 dose levels were able to kill both the natural killer-resistant Daudi cell line and fresh tumor cells while pretreatment samples and peripheral blood lymphocytes from healthy controls were not. This lytic activity was best detected when IL-2 was present in the in vitro effector assay. These results demonstrate that the administration of IL-2 to patients with cancer induces a population of effector cells able to directly destroy natural killer-resistant target cells, when assayed in the presence of IL-2.

Clinical and immunological effects of recombinant interleukin 2 given by repetitive weekly cycles to patients with cancer.

Eleven patients received four consecutive weekly cycles of human recombinant interleukin 2 (IL-2) by continuous infusion for 4 days/week. Two dose levels were tested, 1 and 3 X 10(6) units/m2/day. Toxicities experienced by most patients included fever, rigors, fatigue, anemia, eosinophilia, and liver function abnormalities. All side effects from treatment reversed and no severe or life-threatening problems occurred. A marked lymphocytosis was seen following the 4 weeks of therapy. Fresh lymphocytes obtained during this lymphocytosis mediated enhanced destruction in vitro of a natural killer cell-resistant tumor cell line (Daudi). The increase in the absolute number of circulating lymphocytes and their enhanced ability to mediate direct lysis of Daudi targets resulted in a greater than 100-fold mean increase in cytotoxic potential by the end of IL-2 treatment. One patient, with renal carcinoma, who was treated at 3 X 10(6) units/m2/day experienced a sustained measurable response with greater than 50% regression of pulmonary and hepatic metastases. Five patients were retreated with a second course of IL-2, lasting 4 weeks. This therapy was well tolerated in four of these five patients, with similar immunological changes occurring. No further antitumor responses were seen in these patients. Thus, a relatively well tolerated immunotherapy regimen using IL-2 can induce dramatic increases in lymphocyte number and augment their in vitro antitumor reactivity.

Altered tryptophan and neopterin metabolism in cancer patients treated with recombinant interleukin 2.

Immune stimulation or interferon administration induces indoleamine 2,3-dioxygenase and GTP cyclohydrolase activity in humans, resulting, respectively, in tryptophan degradation to kynurenine and in neopterin production. To determine if similar effects result from interleukin 2 (IL-2) administration, plasma tryptophan and urinary kynurenine and neopterin were measured in patients undergoing a phase 1 toxicity trial of recombinant IL-2 given by daily bolus or continuous i.v. administration for 7 days at doses of 1 x 10(5) to 1 x 10(7) units/m2/day. Significant dose-dependent decreases in plasma tryptophan levels and corresponding increases in urinary kynurenine and neopterin were observed. These metabolic effects of IL-2 are probably mediated by induction of gamma-interferon production, although elevated levels of gamma-interferon were not found in the sera of these patients. In view of the indispensable role of tryptophan in synthesis of protein, niacin, and serotonin, we suggest that some of the toxic side effects may be the result of this loss of tryptophan. Since these metabolic changes were detected at relatively low doses of IL-2, these assays provide a highly sensitive means for monitoring in vivo metabolic responses to IL-2 therapy.

Phase I study of toremifene in patients with advanced cancer.

A phase I multicenter evaluation of a novel antiestrogen, toremifene, was undertaken in postmenopausal women with various advanced difficult-to-treat malignancies. One hundred and seven women were treated at one of six dosage levels (10, 20, 40, 60, 200, or 400 mg/d orally) for at least 8 weeks. Weekly evaluations for toxicity were conducted. The most common side effects were nausea (31%), vomiting (12%), and hot flashes (29%). Five patients were removed from the study for possible adverse reactions: three patients experienced hypercalcemia; one experienced tremulousness, fatigue, and inability to think clearly; and one had vaginal bleeding. Twelve patients died while on study, 11 with disease progression and one with a pulmonary embolus. Sex hormone-binding globulin (SHBG) levels increased and there was a modest decline in serum antithrombin III levels. Four of 48 assessable patients had partial responses: three with breast cancer and one with endometrial cancer. Toremifene was generally well tolerated at the doses tested.

Transplantation of HLA-haploidentical T-cell-depleted marrow for leukemia: autologous marrow recovery with specific immune sensitization to donor antigens.

Autologous marrow recovery without engraftment of donor marrow was observed after bone marrow transplantation (BMT) for two patients with acute lymphoblastic leukemia. Each had received marrow from a haploidentical mixed lymphocyte culture (MLC) reactive donor after pretransplant conditioning with total body irradiation and high-dose cyclophosphamide. To minimize graft-vs-host disease, the marrow was depleted of T cells in vitro by treatment with a monoclonal anti-T-cell antibody and complement. Two weeks after each transplant, reactive lymphocytes were noted transiently in the blood of each patient. Analysis of karyotype, HLA type, and in vitro MLC responsiveness proved the lymphocytes to be of host, not donor, origin. MLC studies showed rapid proliferative responses specifically to stimulating cells from the BMT donor, indicating in vivo sensitization to donor antigens. Return of hematopoietic function was markedly delayed, but it eventually normalized after several months, without evidence of chimerism. These studies confirm that some immune and hematopoietic stem cells of host origin survive the high-dose chemoradiotherapy used as transplant conditioning. Because these immune cells are specifically reactive to donor alloantigens, more potent suppression of host immunity may be needed to prevent nonengraftment of T-cell-depleted, HLA-mismatched bone marrow.

Transplantation of HLA-haploidentical T-cell-depleted marrow for leukemia: addition of cytosine arabinoside to the pretransplant conditioning prevents rejection.

A total of 41 patients with hematologic malignancies (other than acute leukemia in relapse) received allogeneic bone marrow transplants at the University of Wisconsin from 1 April 1980 through 31 March 1984. In an effort to minimize graft-versus-host disease, marrow was depleted of T-lymphocytes in vitro with monoclonal anti-T-cell antibody and complement prior to infusion for seven of 19 recipients of marrow from HLA-identical, MLC-nonreactive siblings, and for all 22 recipients of marrow from MLC-reactive HLA-haploidentical donors. The recipients of HLA-identical T-depleted marrow all showed excellent engraftment following standard pre-BMT conditioning with cyclophosphamide and total body irradiation. In contrast, five of five recipients of T-depleted haploidentical marrow failed to engraft following this same conditioning regimen. The addition of cytosine arabinoside to the pretransplant conditioning appeared to correct this problem, allowing engraftment in 14 of 17 subsequent patients. These clinical results, coupled with prior in vitro data, demonstrate the need to adequately suppress residual host-versus-graft immunity in order to prevent the rejection of T-cell-depleted HLA-haploidentical bone marrow.

The cellular immunotherapy of cancer: current and potential uses of interleukin-2.

The potential for immune-mediated destruction of neoplasms was suggested nearly one century ago. Despite this, no "magic bullet" has yet been identified. Nevertheless, the physiology of cell-mediated immune reactions has been well characterized in molecular, cellular, and clinical studies of allograft and microbial immunity. Extensive studies performed in laboratory animal models have documented the in vitro and in vivo destruction of various neoplastic tissues by immune cells. This destruction can be directed against autologous, syngeneic, or allogeneic tumors in several systems with varying degrees of "tumor specificity". Two approaches exist towards utilizing these immune reaction in vivo. The first involves providing the tumor bearer with immunostimulatory agents, either specific or nonspecific, designed to activate and amplify the destructive potential of the individual's endogenous immune cells able to recognize and destroy autologous tumor. The second approach provides immune cells with antitumor capacity to a tumor-bearing individual, these cells having been activated exogenously. A number of successful regimens involving these two approaches, and combinations of them, have been delineated in animal tumor models. These experimental studies lay a strong foundation for initiating clinical trials of these concepts for patients with cancer. This review summarizes the diverse experimental studies in animals leading to clinical trials, presents recent data from ongoing clinical trials directly testing the potential for cellular immunotherapy, and then presents some of the major challenges facing further development and application of this potential therapeutic approach.

The in vivo distribution of indium-111 labeled in vitro alloactivated syngeneic lymphocytes in a patient with relapsed acute myelomonocytic leukemia: implications for adoptive chemoimmunotherapy.

A single patient who had a leukemic relapse six months after receiving a syngeneic bone marrow transplant was given "adoptive chemoimmunotherapy." Lymphocytes from the patients identical twin were alloactivated and grown in T cell growth factor in vitro. After receiving chemotherapy to reduce the number of relapsed leukemia cells, he received 1.1 X 10(10) in vitro alloactivated twin lymphocytes via intravenous and intraperitoneal injections. Although progressive Candidal pneumonia was fatal and prevented analysis of either efficacy or delayed toxicity of this potential form of therapy for this patient, radioactive 111In labelling and scanning showed dissemination of these lymphocytes following either injection route, with no clinical evidence of immediate toxicity.

Status and potential of interleukin-2 for the treatment of neoplastic disease.

Interest in interleukin-2 results from the fact that it works in vivo on the cell-mediated arm of the immune response and in vitro it is effective on naive lymphocytes. Although it has no direct cytotoxic effect on neoplastic cells, it does help mediate the destruction of tumor cells. Current clinical trials are attempting to determine optimum dose and duration of treatment, since the toxicity of IL-2 appears to increase in proportion to dosage. The authors summarize our knowledge to date.

The role of interleukin-2 in cancer therapy.

Despite the abundance of evidence from murine models suggesting a powerful immunological approach to the treatment of cancer, the available data on clinical response have not been as dramatic. The use of interleukin-2 (IL-2) either alone or in combination with lymphokine activated killer (LAK) cells clearly is therapeutic for some patients, albeit a minority. It does not appear from the available evidence that the regimens tested show major differences in antitumour activity, although there is a sense that higher doses may be slightly more effective. Nor is it clear that the addition of LAK cells significantly or sufficiently enhances clinical responses to warrant their widespread use. It must therefore be concluded that other patient and tumour related factors must have an undefined role in the ability to attain meaningful responses. Immunological response to IL-2 and tumour burden are factors which can be examined given the available clinical data. While animal studies have shown that antitumor effects are related to the dose and number of LAK cells given, this is not as clear in patient studies. Some reports have suggested a correlation between clinical response and the in vivo induction of LAK activity or the magnitude of the rebound lymphocytosis. Clinical trials at the University of Wisconsin have shown striking increases in both the number of peripheral blood lymphocytes and LAK induction in patients who showed no clinical response. This in vivo LAK induction, as expected, is not the sole determinant in achieving a measurable response. Whether it is a necessary biological response needed to achieve antitumour effects remains uncertain. The role of tumour burden in response to IL-2 remains elusive. In many trials, including our own, patients with bulky disease have responded to therapy that was ineffective in other patients with what appeared to be a minimal tumour burden. Despite the disappointment that initial expectations have not quite been met, something extremely important has occurred in the treatment of cancer. For the first time, the controlled activation of a patient's endogenous immune system has been shown to have some promise as an antitumour treatment. Clearly, if the response rates reported to date are the best attainable its role will be limited. This remains doubtful as investigators are actively exploring combinations of IL-2 with other biologicals, cytokines, monoclonal antibodies and chemotherapeutic agents. Preclinical data suggest enhanced antitumour effects will be mediated by these combinations. While the magic bullet has not yet been found, there has been a major step forward since IL-2 was first described.

Clinical adoptive chemoimmunotherapy with allogeneic alloactivated HLA-haploidentical lymphocytes: controlled induction of graft-versus-host-reactions.

A total of 13 cancer patients were treated with Adoptive Chemoimmunotherapy (ACIT) using alloactivated HLA haploidentical lymphocytes. Donor lymphocytes were activated in vitro using a pool of irradiated allogeneic lymphocytes (MLC-cells) and some further expanded by culturing in T-cell growth factor (TCGF-cells). The first 6 patients received i.v. cyclophosphamide (CPM) followed 24 h later by escalating doses of MLC-cells, then 7 days later they received an infusion of TCGF-cells. Minimal toxicity was seen. The next 7 patients received CPM (800 mg/m2) and a combined MLC and TCGF-cell infusion (total cell dose ranged from 0.79 x 10(10) to 2.26 x 10(10)). Of these 7 patients, 3 developed mild graft-versus-host reaction (GVHR) which resolved without treatment, and 2 patients had progressive GVHR which was arrested by methylprednisolone (2 mg/kg). Peripheral blood lymphocytes from these 2 patients, during the GVHR, had increased activated T-cells (OKT-10+ and OK-Ia+). In vitro expansion, in TCGF, of these activated T-cells enabled HLA typing to prove they were of donor origin. Only 1 clinical antitumor response was observed in the first 6 patients. The results of this study indicate that this form of ACIT can be given to patients with acceptable toxicity. Self-limited or easily controlled GVHR may be induced and primed donor cells persisting in the circulation are probably responsible. Further testing is required to determine whether the immune response induced by this form of ACIT may be therapeutically effective.

Destruction of autologous human lymphocytes by interleukin 2-activated cytotoxic cells.

Human and murine lymphocyte populations differentiate into lymphokine activated killer (LAK) cells after in vitro or in vivo exposure to interleukin 2 (IL 2). LAK cells mediate destruction of neoplastic tissue in vitro and have been reported to spare normal tissue. However, systemic toxicity is observed in mice and patients receiving IL 2 infusions. Some aspects of this toxicity are similar to that seen in graft-vs-host disease, suggesting that IL 2 may cause an immune-mediated destruction of normal tissues. We have evaluated this issue by examining the destructive potential of fresh human lymphocytes cultured in media containing highly purified recombinant human IL 2. In the absence of any exogenous antigen or allogeneic stimulating cells, strong proliferative responses were induced after 6 days of exposure to IL 2. Lymphocytes harvested from these 6-day cultures were highly cytotoxic to K562 and Daudi target cells. These IL 2-activated cells were also cytotoxic against autologous and allogeneic normal lymphocyte target cells. This autologous lymphocyte destruction was detected in media containing autologous serum and was directly dependent on the concentration of IL 2 added to the cultures. These studies demonstrate that populations of IL 2-activated lymphocytes, containing LAK activity, can mediate low-level but significant destruction of normal lymphocytes in vitro.

Clinical response of a patient with diffuse histiocytic lymphoma to adoptive chemoimmunotherapy using cyclophosphamide and alloactivated haploidentical lymphocytes. A case report and phase I trial.

Adoptive chemoimmunotherapy has cured experimentally induced tumors in animals, but its clinical use has been limited. Six patients were treated with refractory neoplasms in a Phase I study with cyclophosphamide (CPM) and alloactivated haploidentical lymphocytes. Patients received an immunosuppressive dose of CPM (800 mg/m2) followed by haploidentical lymphocytes primed in vitro with alloantigens in mixed lymphocyte culture (MLC). One week later patients received a second infusion of alloactivated lymphocytes expanded in T-cell growth factor (TCGF). The total number of cells given to each patient progressively increased, with a single patient receiving 35.5 X 10(9) cells. Transient febrile responses and delayed-type hypersensitivity reactions at the intravenous sites were the only toxicities noted. A complete clinical response lasting 12 weeks was seen in a single patient with diffuse histiocytic lymphoma. Our experience indicates that adoptive chemoimmunotherapy can be given to patients safely and merits further clinical testing.

Phase 1 clinical evaluation of recombinant interleukin-2.

The availability of large quantities of pure recombinant IL-2 has permitted clinical studies of its effects in patients with cancer. Based on the rapid half-life (3-5 minutes) and the need for prolonged exposure to IL-2 to maximally stimulate responsive cells we conducted a Phase 1 clinical trial designed to compare the clinical tolerance, immunologic effects and antitumor activity of IL-2 given by CI and daily BI for 7 consecutive days. The MTD of IL-2 based on completion of 7 days of treatment was found to be 3 X 10(6) U/m2/day for BI and 10(6) U/m2/day for CI. These results are approximately equivalent to those reported by others (Lotze, 1985 and Atkins, 1986), provided corrections are made for activity (2.3 Cetus IL-2 units = 1 BRMP IL-2 Unit). Although dose limiting toxicity was observed at the higher doses of IL-2 tested, these doses were tolerated for a shorter period of time (3-4 days) with striking immunologic effects (Sondel et al. 1987). As such, shorter treatment schedules utilizing higher doses of IL-2 warrant further investigation. The toxicity associated with the administration of IL-2 is considerable and clearly dose related. Of particular clinical importance is the fever, hypotension, fluid accumulation and decrease in PS. The etiology of these toxicities appears to be related to the release of endogenous cytokines following activation of the patient's immune system. It is also possible that the in vivo activation by IL-2 of cells mediating NRC may play some role in the toxicity associated with IL-2 (Sondel, 1986). It remains unclear whether immunosuppressive methods such as the use of cyclophosphamide or steroids will enable blockade of the immune mediated toxicity without also diminishing its therapeutic value. Further studies examining methods to control the severe toxicity associated with high doses of IL-2 are needed. The future role that IL-2 may play in treating cancer is unclear. Whether IL-2 alone can have significant antitumor activity may depend on factors such as tumor bulk, the patient's inherent ability to generate cytotoxic cells or the susceptibility of a particular malignancy. As such further studies will need to examine the importance of dose, timing, schedule, method of IL-2 administration as well as tumor burden in a wide variety of cancers.(ABSTRACT TRUNCATED AT 400 WORDS)