Cancer Immunotherapy: Current Status of Treatment With Interleukin 2 and Lymphokine-Activated Killer Cells

Subject Review Cancer Immunotherapy: Current Status of Treatment With Interleukin 2 and Lymphokine-Activated Killer Cells

CAROL M. van HAELST-PISANI, M.D., Department of Oncology; RICHARD J. PISANI, M.D., Division of Thoracic Diseases and Internal Medicine; JOHN S. KOVACH, M.D., Department of Oncology In recent years, the medical community has witnessed a growing interest in the use of adoptive immunotherapy in patients with malignant lesions refractory to standard treatments. Systemic administration of interleukin 2, in combination with the adoptive transfer of a patient's own activated immune cells, has resulted in objective regression of several types of advanced cancers. Pronounced regression of tumor has also been observed with use of systemic interleukin 2 alone. This ability to augment the immune defense system of the host against cancer has stimulated intense clinical and laboratory investigations.

During the past decade, interest in the role of the immune system in host defense against cancer has been renewed. One reason for the resurgence of tumor immunology is that major technologic advances in immunology and in molecular biol­ ogy have facilitated the identification, isolation, and large-scale production of substances that may enhance the immune response to tumor. These substances have been called "biologic re­ sponse modifiers." Among biologic response modifiers, interleukin 2 (IL 2) is distinguished by its unique capacity to generate a class of tumoricidal lymphocytes— lymphokine-activated killer.(LAK) cells. Interest in the potential value of LAK cells in the treat­ ment of human cancers was sparked by the ob­ servations by Rosenberg and associates 1,2 that IL 2 and LAK cells can cause objective regressions and occasionally complete remissions of advanced cancers. Since Rosenberg and colleagues published their preliminary results, enthusiasm for IL 2/LAK therapy has been tempered by an appreciation of

Address reprint requests to Dr. J. S. Kovach, Department of Oncology, Mayo Clinic, Rochester, MN 55905. Mayo Clin Proc 64:451-465, 1989

the pitfalls hindering widespread clinical appli­ cation: (1) a low rate of complete and long-lasting responses, (2) severe toxicity necessitating intensivecare monitoring, and (3) the labor-intensive nature of LAK cell preparation. Critics have argued that other investigational treatments for advanced malignant lesions 3,4 demonstrate response rates similar to that of IL 2/LAK and at lower mor­ bidity and cost.5'6 Nevertheless, the occasional complete remission of refractory cancers with biologic therapy is an important and exciting achievement. Intensive investigation of IL 2/LAK therapy as a model for studying mechanisms underlying host immune response to tumor will allow expansion of knowledge at a basic level and facilitate the development of more effective therapeutic regimens in the future. In this review, we summarize the immunologic and clinical aspects of IL 2/LAK therapy and discuss possible mechanisms that mediate tumor regression and IL 2 toxicity. BACKGROUND The body's defense against malignant processes seems to rely primarily on the cell-mediated arm of the immune system, composed of several classes of cytotoxic cells capable of recognizing

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and eliminating tumor cells. The mechanism of and can kill a variety of tumors without prior target recognition and the differential expression sensitization, although how they recognize their of specific surface molecules (surface markers) target is unknown. NK cells are characterized by help to distinguish among the different types of a molecule on their surface, termed "CD16," cytotoxic cells. Monoclonal antibodies have been which is the receptor for the Fc portion of IgG. produced that recognize and bind to these surface The IgG Fc receptor also enables the NK cell to molecules. Although a surface marker may not interact with IgG antibody-coated targets and to be unique to one class, dual staining with several mediate antibody-dependent cellular cytotoxicity. monoclonal antibodies to identify combinations A small subset of NK cells bear the CD8 molecule of two or more surface molecules can be quite spe­ but, unlike CTL, do not coexpress the CD3 mole­ cific in identifying a subset of cytotoxic lympho­ cule on their surface. cytes. A brief description of the various types of A third type of cytotoxic cell merits brief men­ cytotoxic effectors will help define the "LAK cell" tion because it may contribute to the "LAK func­ or, more appropriately, "LAK activity" (Table 1). tion" described in the subsequent material. A Target recognition, in the case of classic antigen- subset of T lymphocytes seem to mediate non11 specific cytotoxic T lymphocytes (CTL), relies on MHC-restricted cytotoxicity. Although they re­ a structure on the surface of each CTL called the arrange (or activate) the genes that encode the T-cell receptor (TCR). The TCR recognizes a target TCR and bear the CD3 molecule on their surface, cell only when it displays foreign antigen in asso­ they are not antigen-specific, and target recog­ ciation with a membrane-bound product of the nition is not limited to tumors that express selfmajor histocompatibility complex (MHC).7'8 The MHC molecules. Perhaps through a different re­ MHC is a large cluster of genes which encodes ceptor or through another mechanism altogether, molecules that are markers of individual identity they recognize a variety of autologous (self) and or self. Two classes of MHC proteins have been allogeneic (nonself) tumor cells. Leu-19, a surface identified. Class I MHC proteins are found on marker primarily restricted to peripheral blood virtually all cells, whereas class II MHC mole­ NK cells, is also expressed on these non-MHC12 cules appear primarily on cells that participate restricted T cells. in the immune response such as lymphocytes, Macrophages 13 and certain helper/inducer T macrophages, and specialized epithelial cells.9 lymphocytes 14,15 may mediate direct tumor cyto­ The requirement of dual stimuli (antigen and a toxicity; however, they are not thought to be LAK self-MHC protein) is called "MHC restriction." precursors or effectors. Most CTL are class I MHC-restricted, recognizing Description of the LAK Cell.—The LAK specific antigen only when associated with a phenomenon was first described in 1980.16"18 At class I MHC protein on the surface of the target that time, investigators at the National Cancer cell. Although all T lymphocytes are stained by Institute were studying a type of adoptive ima monoclonal antibody that recognizes the TCR- munotherapy in mice termed "specific adoptive associated complex, CD3, the antigen-specific immunotherapy." 19 Specific adoptive immunoCTL, is characterized by a second surface mole­ therapy relies on the adoptive transfer of tumorcule, CD8. specific lymphocytes (CTL) into a tumor-bearing A second class of cytotoxic cells, natural killer host. Although possible in mice, this type of (NK) cells, have a much broader target range adoptive immunotherapy is difficult to study in than do CTL. 10 NK cells are not MHC-restricted humans because of the weak immunogenicity of

Table 1.—Cytotoxic Lymphocytes Freshly Isolated From Peripheral Blood* % of peripheral Target recognition Phenotype Class blood lymphocytes Class I MHC-restricted CD3+, CD8+ CTL 20-30 CD3+, Leu-19+ Non-MHC-restricted 1-3 CTLt Non-MHC-restricted CD3", CD16+, Leu-19+, (CD8+) 10-15 NKt *CTL = cytotoxic T lymphocytes; MHC = major histocompatibility complex; NK = natural killer. tPredominant contributors to peripheral blood lymphokine-activated killer activity.

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most human tumors and the limitations to acquir­ ing large numbers of sensitized tumor-specific CTL. During the course of these studies, the investi­ gators found that nonsensitized lymphocytes, in­ cubated with the lymphokine IL 2, not only dem­ onstrated killing of the specific tumor cells but also lysed a range of autologous and allogeneic NK-cell-resistant fresh and cultured tumor targets. These IL 2-activated cells were originally termed "lymphokine-activated killer" (LAK) cells because they were thought not to belong to any previously described class of cytotoxic cells. Although this concept is still controversial, subsequent work by several laboratories suggests that LAK cells are activated NK cells with some component arising from non-MHC-restricted T cells.20"26 The term "LAK cell" continues to be used in a functional sense. Unlike sensitized CTL, cells demonstrating LAK activity are neither MHC-restricted nor restricted to a specific tumor type. The method commonly used to measure the tumoricidal activity of LAK cells is a 4-hour chromium-release assay in which the target is labeled with radioactive chromium (51Cr), and the effector (killer) cell is added at various effector:target ratios. Lysis of target cells results in release of 51 Cr. With use of such an assay system, investigators have shown that the LAK-mediated killing of a sensitive labeled tumor target can be inhibited by unlabeled (cold) target cells from a different LAK-sensitive tumor. 27 The cold-inhibition assay suggests the existence of a common mechanism by which different LAK-sensitive targets are rendered sus­ ceptible to LAK effectors. This effect could be mediated through a shared antigenic determi­ nant or through the differential expression of various adhesion molecules on the surface of susceptible targets. 28 Further study is necessary to define the interaction between the target cell and the cytolytic effector. Although the original definition of the LAK cell maintained that these cells recognized and lysed only transformed cells,17,18 several recent reports have demonstrated that LAK cells are capable of lysing normal endothelial cells and peripheral blood lympho­ cytes;29,30 thus, the tumor specificity of the LAK effector is open to question. Description of IL 2.—For the generation of LAK cells, IL 2 must be present. This glycoprotein, with a molecular weight of approximately 15,000 daltons, is secreted by activated T cells.

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Initially isolated from the culture supernatants of mitogen-stimulated T cell blasts, IL 2 was termed "T-cell growth factor" because of its abil­ ity to sustain the proliferation of activated T cells in long-term cultures. 31 Soon after its discovery, IL 2 was purified from conditioned media of a human lymphoblastoid leukemia cell line called the Jurkat line. 32 The cloning of the gene for IL 2 and its insertion and expression in Escherichia coli bacteria have made available large quantities of the recombinant product. 33,34 Several available commercial preparations differ slightly in amino acid composition 35 but behave similarly in in vitro functional assays when used at equivalent strengths. A unit of IL 2 is the amount that causes half-maximal proliferation of T cells in a standardized biologic assay. 36 IL 2 is pivotal in the immune response. Its prop­ erties have been reviewed in detail elsewhere. 37 In addition to its role in the proliferation of antigenstimulated T cells, 38 IL 2 increases the cytotoxicity of CTL and NK cells.39 IL 2 induces or enhances the secretion of other cytokines including interferon (IFN)-)', hematopoietic colony-stimulating factors, interleukin 1 (IL 1), tumor necrosis factor (TNF), and B-cell growth factors. 40 " 42 ANIMAL STUDIES With the recognition of the LAK cell and the availability of IL 2, a new type of "adoptive immunotherapy" arose, which has now been studied extensively in the mouse model by using several murine tumor lines, including a series of variably immunogenic methylcholanthrene (MCA)induced sarcomas. Inoculation of as few as 2 x 105 sarcoma cells into a mouse's tail vein results in pulmonary, hepatic, and peritoneal metastatic lesions at 3 days, which are grossly visible by 10 days. In such experiments, tumor-bearing mice (the murine MCA-105 sarcoma model) were treated with IL 2 at different doses for 4 to 5 consecutive days, beginning either on day 3 or on day 10 after tumor inoculation. In certain experiments, LAK cells were infused. LAK cells infused alone were largely ineffective against 3-day and 10-day metastatic lesions, but the coadministration of LAK cells and IL 2 re­ sulted in a dramatic reduction of metastatic tumor nodules in the lungs and livers of these animals. 43 " 45 Studies showed that the administra­ tion of IL 2 stimulated in vivo proliferation of the adoptively transferred lymphocytes. 46 The

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response was improved by increasing the number of LAK cell infusions or by increasing the dose of IL 2. The combination was equally effective in animals irradiated before treatment with a sublethal dose (500 rad) to induce lymphopenia, a finding that suggests that therapy with IL 2 and LAK cells is not dependent on host immune response and that this therapy may be effective in an immunocompromised host. The administration of IL 2 alone was not as effective as the combination of IL 2 and LAK cells. Higher doses of IL 2 were needed to achieve tumor regression, and in preirradiated mice, no antitumor effect was observed even when high doses of IL 2 were administered. 47 This lack of response confirms that IL 2 is not directly cytotoxic to neoplastic cells. The antitumor effects of IL 2 alone seem to be mediated through a radio­ sensitive endogenous component of the host's immune system—presumably LAK cells or other cytotoxic effector cells (or both) whose numbers or potency is augmented by IL 2. The effect of IL 2 on the host's immune sys­ tem was studied further. Administration of IL 2 was shown to cause proliferation of endogenous lymphocytes as well.48 Incorporation of [125I]5iododeoxyuridine, a radiolabeled thymidine ana­ logue, by lymphocytes was used as a measure of proliferation. Increased uptake was noted in the lung, liver, spleen, kidney, and mesenteric lymph nodes of the mice receiving IL 2 in comparison with untreated mice. This lymphoid proliferation was proportional to the dose of IL 2 administered and was largely eliminated by preirradiation of the host. In addition, lymphocytes isolated from these organs demonstrated considerable in vitro LAK activity, a confirmation that IL 2 can stim­ ulate generation of LAK cells in vivo. Finally, an intermediate dose of IL 2 caused minimal re­ gression of 3-day-old metastatic lesions but was effective in reducing the size and the number of metastatic nodes present at 10 days. 47,49 This result was puzzling because a lower tumor burden present on day 3 should have been more respon­ sive to therapy than a higher tumor burden on day 10. Further study revealed that the extent of the response of 10-day metastatic lesions de­ pended on the immunogenicity of the murine sarcoma. At an intermediate dose, IL 2 was in­ effective in reducing 10-day-old metastatic le­ sions of a nonimmunogenic sarcoma but was extremely effective at 10 days in the MCA-105

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sarcoma, which is weakly immunogenic (Table 2). This finding is consistent with the hypothesis that the antitumor effect of IL 2, given alone, is mediated through the host's immune response to the tumor. In tumors able to elicit an immune response (such as the MCA-105 sarcoma), this response would be better established by day 10 and would be enhanced by the administration of IL2. An important unanswered question is why, de­ spite pronounced tumor regression, a few meta­ static nodules persist in most animals. These nodules, which eventually cause death, do not seem to be inherently resistant to IL 2/LAK because the tumor cells from such nodules can be lysed by LAK cells in vitro. Furthermore, when these tumor cells are reinjected into a genetically identical mouse, the tumor nodules that regrow respond again to IL 2/LAK therapy. 50 Although the mechanisms by which tumor cells escape IL 2/LAK therapy remain unclear, the tumor regres­ sions and improved survival observed in animals treated with IL 2/LAK stimulated the introduc­ tion of this therapy into human studies.

Table 2.—Effect of Dose, Schedule, and Administration of Lymphokine-Activated Killer (LAK) Cells in the Murine MCA-105 Sarcoma Model 444749 Response to therapyt 3-dayJ Treatment* 10-dayt LAK cells only§ LAK cells plus ++ ++ Low-dose IL 2\\ +++ +++ Medium-dose IL 2^| +++ +++ High-dose IL 2# IL 2 only +/+/Low dose ++ Medium dose +/+++ +++ High dose *IL 2 = interleukin 2. t~ = no response (>200 pulmonary metastatic lesions found at autopsy 14 days after tumor inoculation, the same finding as in untreated control animals); +/- = minimal response (150-200 metastatic lesions); + = partial response (100-150 metastatic lesions); ++ = good response (20-100 metastatic lesions); +++ = excellent response (<20 metastatic lesions). fTherapy initiated 3 days or 10 days after inoculation of tumor. §Splenocytes (1 x 108) from a genetically identical mouse were incubated with IL 2 for 2-3 days and reinfused on days 3 and 6 or days 10 and 13. \\Low dose = 4,000-6,000 U/dose, administered intraperitoneally 3 times/day for 4-5 consecutive days. HMedium dose = 20,000-34,000 U/dose. #High dose = 100,000 U/dose.

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CLINICAL STUDIES: SYSTEMIC THERAPY In initial trials, LAK cells and IL 2 were eval­ uated separately for therapeutic effect and toxicity. Clinical Trials With LAK Cells Alone.—In early studies, the effects of phytohemagglutinin (PHA)-activated lymphocytes were analyzed. The cytotoxic activity of these lymphocytes is now thought to have originated from the generation of LAK cells, inasmuch as PHA stimulates T cells and causes them to release IL 2. In 1970 in one of the earliest studies of adoptive immunotherapy, Frenster and Rogoway51 treated five cancer patients (two with melanoma and one each with Ewing's sarcoma, teratocarcinoma, and embry­ onal cell carcinoma) with five daily intravenous injections of more than 108 PHA-activated autologous lymphocytes, for one to six courses. Mild chills and fever were the only toxic effects re­ ported. Three patients had objective regressions of pulmonary metastatic tumors of more than 50% for longer than a month after infusion. These observations, however, were not actively pursued until the early 1980s. In 1984, Mazumder and associates 52 reported on the propagation and use of PHA-stimulated lymphocytes for cancer therapy in humans. They had previously shown that, in vitro, these cells lyse autologous tumor but not normal lymphoid cells.53 Ten patients with advanced cancer under­ went between 7 and 15 leukaphereses. In vitro culture of their peripheral blood lymphocytes in PHA for 2 days generated a large number of activated cells that could be reinfused (up to 17 x 1010 cells for a period of 5 weeks) and caused tolerable side effects. By using 111-indium-labeled PHA-stimulated cells, this group studied the in vivo distribution of the infused cells. They dem­ onstrated preferential uptake in the spleen and liver, rapid clearance from the circulation, and little or no accumulation in the lymphatic system. Sequestration in the lungs was noted after re­ peated infusion of cells. Interestingly, activated peripheral blood lymphocytes were detected in the peripheral circulation of patients after mul­ tiple leukaphereses and infusions. These cells did not require ex vivo activation with PHA to lyse fresh tumor targets. These authors suggested that the accumulation of labeled infused cells in the lungs and the increased lytic activity detected in the peripheral circulation with repeated in­ fusion might result from saturation of reticulo-

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endothelial clearance mechanisms. No antitumor response was seen, but all patients had heavy tumor burdens. Clinical Trials With IL 2 Alone.—The avail­ ability of large amounts of recombinant IL 2 led to trials with IL 2 as a single agent in patients with advanced cancer. Initial phase I trials dem­ onstrated the short plasma half-life of IL 2 after administration of an intravenous bolus54,55 (approxi­ mately 7 minutes for the initial phase of distri­ bution and 70 minutes for the secondary phase of distribution). The rapid plasma elimination is due to metabolism by the renal tubular epithe­ lium. The mode of administration considerably influences the plasma half-life of IL 2. After administration of an intravenous bolus, IL 2 can be detected for 1 to 4 hours, depending on the dose given; levels of IL 2 are consistently detected throughout a 24-hour continuous infusion and are sustained for as long as 9 hours after subcutane­ ous injection. 56 These findings are important be­ cause the duration of exposure to detectable levels of IL 2 apparently influences the degree of immunomodulation. Nonneutralizing antibodies to recombinant IL 2 have been described in patients receiving the lymphokine, but they do not seem to be clinically significant in terms of pharmacokinetics or functional determinations. 57,58 No tumor regression was noted in several early phase I trials with IL 2 alone. Doses varied, however, and a variety of advanced tumors were treated. In the first trial with high-dose IL 2,59 10 patients were treated, and 3 of 6 patients with melanoma had a partial response, defined as a 50% or greater reduction in tumor burden. This result led to trials of high-dose IL 2 in combina­ tion with LAK cells. Clinical Trials With IL 2 Plus LAK Cells.— After the feasibility of infusing LAK cells in large quantity had been established and the tolerable doses of IL 2 had been determined, trials com­ bining IL 2 and LAK cells were initiated. We briefly outline two regimens of IL 2/LAK ther­ apy—one in which extremely high intermittent doses of IL 2 are administered and the other in which a low to moderate dose of IL 2 is given by 24-hour continuous infusion. Study Designs.—Rosenberg and associates 1 ' 2 conducted the initial trial of IL 2/LAK at the National Cancer Institute. IL 2 was administered by intravenous bolus at priming dosages of 100,000 U / k g every 8 hours (approximately 10

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million units/m 2 daily) for 5 consecutive days. these IL 2 regimens, with and without the addi­ Three days after termination of IL 2 injections, tion of LAK cells. 61-63 In order to proceed with the patients underwent a series of five daily large-scale clinical trials of IL 2/LAK therapy, leukaphereses. The collected cells were incubated techniques used for in vitro experiments have in high concentrations of IL 2 for 4 to 5 days. been expanded and automated to facilitate mass After culture, IL 2-activated cells were reinfused production of human LAK cells. Several recent into the patients in three separate injections. modifications in transfusion medicine have opti­ These cells demonstrated cytolytic activity against mized methods for collection, culture, and harvest a panel of tumor cell lines. IL 2 was administered of mononuclear cells.64,65 at the same dose as in the preceding week in Clinical Results.—The results from trials con­ conjunction with the LAK cell infusions and for ducted at the National Cancer Institute are sum­ 2 days thereafter. marized in Table 3. 62 In a nonrandomized trial, IL 2 has also been given by 24-hour continuous 139 patients with advanced cancer were treated infusion by West and colleagues. 60 They studied with IL 2/LAK and 79 patients were treated with 48 patients treated with IL 2 for 5 consecutive high-dose IL 2 alone. The overall response rates, days at dosages that ranged from 1 to 7 x 106 defined as complete and partial responses, were U/m 2 daily. Thirty -six hours after the end of the 21% and 16%, respectively. An additional 108 pa­ continuous infusion, patients underwent the first tients were randomized to receive either IL 2/LAK of four daily leukaphereses. Cells were cultured or IL 2 alone (Table 3). for 4 to 5 days in the presence of IL 2 and again Most responses occurred in patients with renal reinfused with IL 2 at the same dose and schedule cell carcinoma or melanoma. The seven patients as during the first week of therapy. with renal cell carcinoma who achieved a com­ Several centers throughout the United States plete response after IL 2/LAK therapy remained are treating patients with modified versions of free of disease at 2 to 14 months of follow-up, as

Table 3.—Results of Nonrandomized and Randomized Trials of Interleukin 2 (IL 2) Alone or in Combination With Lymphokine-Activated Killer (LAK) Cells in Patients With Various Types of Cancer 82 Response Overall response No. of Type of Complete Partial Type of cancer therapy* No. % patientsf Nonrandomized trial 10 17 IL 2/LAK 54 7 31 Renal carcinoma 4 3 7 18 IL2 38 3 3 6 18 Melanoma IL 2/LAK 34 0 6 6 26 IL2 23 1 2 3 11 27 Colorectal cancer IL 2/LAK 0 0 0 IL2 10 75 1 2 3 Non-Hodgkin's IL 2/LAK 4 0 0 0 lymphoma IL2 3 Randomized trial 10 29 6 4 IL 2/LAK 34 5 16 3 2 IL2 31 2 12 2 0 Melanoma IL 2/LAK 16 4 33 0 4 IL2 12 0 0 0 Colorectal cancer IL 2/LAK 5 0 0 0 IL2 7 1 50 0 1 Non-Hodgkin's IL 2/LAK 2 0 0 0 lymphoma IL2 1 *Patients receiving combination therapy underwent a 16-day treatment cycle that was divided into 3 phases: on days 1-5, IL 2 (100,000 U/kg) was administered every 8 hours; days 6 and 7 were rest days; on days 8-12, patients underwent leukapheresis daily. On days 12, 13, and 15, patients received LAK cells that had been harvested on days 8 and 9, 10, and 11 and 12, respectively. IL 2 (100,000 U/kg) was administered 3 times/day on days 12-16. fin the nonrandomized trial, an additional 20 patients with various types of tumors had no response to IL 2/LAK, and an additional 5 patients had no response to IL 2 alone. Renal carcinoma

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sors reappear 24 to 48 hours after bolus injection and 4 to 7 days after beginning a continuous infusion of IL 2. After the last dose of IL 2, a rebound lymphocytosis occurs that, after pro­ longed intravenous infusion, can result in as much as a 16-fold increase in circulating lympho­ cytes. 55 All phenotypes are affected. This phenom­ enon is not well understood but allows a high yield of cells when patients undergo leukaphere­ sis shortly after discontinuation of IL 2 therapy. The second effect of administration of IL 2 is an increase in plasma concentrations of IFN-y. Even though IL 1, TNF, and lymphotoxin can be easily detected in the culture supernatants of peripheral blood lymphocytes stimulated with IL 2, 66 to date the only cytokine consistently found in the plasma of these patients has been IFN-y. Lotze and asso­ ciates 55 reported up to 26 U of IFN-y in the plasma 2 hours after administration of an intravenous bolus of 106 U/kg of IL 2. Lower levels are ob­ In a study by West and associates 60 in which served with continuous infusion of IL 2. Whether patients were given IL 2 at low doses as a 24- IFN-y is secreted by the infused LAK cells or by hour continuous infusion with LAK cells, 13 of cells activated through IL 2 is unknown. 40 evaluable patients with a variety of tumors The third and fourth immunologic effects seem obtained partial remissions: 3 of 6 with renal cell to depend on sustained exposure of patient cells carcinoma, 5 of 10 with melanoma, 1 of 5 with to IL 2. After administration of IL 2 to humans, lung cancer, 1 of 2 with a parotid tumor, 1 with the number of circulating cells that express the Hodgkin's disease, 1 with non-Hodgkin's lympho- receptor for IL 2 on their surface increases. The ma, and 1 with an ovarian tumor. Although no IL 2 receptor is recognized by a monoclonal anti­ complete responses were observed, the study con­ body (Tac), and normally, less than 2% of periph­ firmed the sensitivity of renal cell carcinoma and eral blood lymphocytes are positive for Tac. After melanoma to this therapy. 1 to 3 weeks of continuous infusion of IL 2, 25 to 40% of the circulating cells are Tac positive. Immunomodulation.—Four major immunoTwo patients who received intravenous therapy logic effects have been observed in patients re­ and two patients who received intraperitoneal ceiving systemic IL 2: alteration in the in vivo therapy were studied in considerable detail with distribution of immune cells, production of "im­ two-color flow analysis of the Tac-positive periph­ mune interferon" (IFN-y), an increase in the 67 eral cells. Most of the Tac-positive cells costained number of circulating T lymphocytes bearing the receptor for IL 2, and an increase in circulating with monoclonal antibodies to CD4 or Leu-M3, markers for helper T cells and monocytes, respec­ LAK cells. The concept of a "priming dose" of IL 2, ad­ tively. These patients also had exceedingly high ministered to patients before leukapheresis, is levels of soluble IL 2 receptor in the serum. The based on the trafficking effect of IL 2 on immune significance of these findings remains unclear. cells. Within minutes after intravenous injection Finally, IL 2 promotes the generation of LAK of 105 or more units of IL 2/m 2 , NK cells and LAK cells in the body. Peripheral blood lymphocytes cell precursors disappear from the circulation, as obtained from patients after prolonged infusion evidenced by an inability to generate LAK activ­ of IL 2 demonstrate LAK activity when tested in ity with the incubation of peripheral blood mono- vitro. Kohler and associates, 68 who administered nuclear cells in IL 2. Within 4 hours, the numbers four consecutive 4-day cycles, noted stepwise in­ of all lymphocytes in the circulation are substan­ creases in the tumoricidal activity of peripheral tially decreased. The peripheral blood lympho­ blood lymphocytes with each successive week of cyte count returns to baseline and LAK precur­ IL 2 continuous infusion therapy. In a separate

did the four patients who achieved a complete response after IL 2 therapy alone (at 4+ to 13+ months). The complete responses in three pa­ tients with melanoma after IL 2/LAK therapy have lasted from more than 11 months to more than 31 months. In these trials at the National Cancer Institute, however, no responses occurred in seven patients with sarcoma, six with lung adenocarcinoma, three with breast carcinoma, and nine with other miscellaneous tumors. In the six extramural trials conducted to confirm these results, 35 patients with renal cell carcinoma have been treated with IL 2/LAK. 63 Of the 32 patients who received IL 2 and at least one infusion of LAK cells, 2 have had a complete response (and remain free of disease at 12+ and 9+ months) and 3 have had a partial response. Two of the patients with a partial response un­ derwent resection of residual disease and remain disease-free at 16+ and 15+ months.

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study, investigators demonstrated that the LAK effector cells, generated in vivo after 4 to 6 weeks of IL 2 therapy, expressed the Leu-19 surface marker. 69 Several ongoing clinical trials may help determine whether the in vivo generation of LAK cells obviates the need for LAK cell infusions. Toxicity.— The toxicity observed with IL 2 therapy is related to the cumulative dose admin­ istered. With the extremely high dosages described (more than 3 x 105 U / k g daily), substantial multi­ system toxicity is encountered. 70 All toxicities re­ verse after discontinuation of IL 2, although in some cases the symptoms of retention of fluids and toxicity of the central nervous system have persisted for 2 to 7 days after withdrawal of the drug. Other than fever, chills, and transient hypo­ tension in some patients, LAK cell infusions do not seem to cause appreciable additional toxicity. The major toxic effect of high-dose IL 2 therapy is a capillary leak syndrome associated with de­ creased intravascular volume, hypotension, oliguria, peripheral and pulmonary edema, and weight gain of up to 20 kg during a 3-week period. Within 24 hours after administration of IL 2, all patients who receive more than a 105 U / k g cumulative dose have evidence of fluid retention. The cause of this unexpected phenomenon is under investi­ gation. Skin biopsy specimens from patients who have received IL 2 have shown that endothelial activation occurs, as evidenced by the appearance of certain antigens or molecules on the endothelial cell surface after IL 2 therapy. 71 "Leaky" activated endothelial cells are similarly seen in hypersensitivity reactions in which localized edema oc­ curs. The endothelial activation is presumed to be indirectly mediated because IL 2 fails to induce these antigens in vitro on cultured endothelial cells. The vascular leak syndrome does not occur in nude mice or in mice immunosuppressed by irradiation and is considerably decreased by in­ jection of cyclophosphamide before administra­ tion of IL 2. 72 Therefore, both the therapeutic and the toxic effects of IL 2 are likely indirectly mediated. Another proposed explanation for the vascular leak syndrome is that LAK cells are directly toxic to endothelial cells. 30 The hypotension, although primarily related to diminished intravascular volume, may also be due in part to the vasodilatory effects of IL 2 therapy. In patients given IL 2 and monitored in an intensive-care unit, the cardiac index has increased in association with a profoundly low

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systemic peripheral resistance. 73 The clinical pic­ ture corresponds to that of septic shock, and the role of mediators such as TNF, IL 1, or activated complement components (C5a) is being evaluated. The blood pressure returns to baseline within 24 hours after IL 2 therapy has been discontinued. All excess weight gained during treatment is lost within 1 to 2 weeks after discontinuation. During therapy, however, serious toxicity may be encoun­ tered as a result of fluid extravasation and volume overload. In the National Cancer Institute trial, arrhythmias and myocardial infarctions occurred in this setting in 11% and 2% of patients, respec­ tively, respiratory distress necessitating intuba­ tion occurred in 8%, and death occurred in 2%.62 Hepatic dysfunction in association with jaundice and hepatomegaly has also been noted. In all patients who receive high-dose IL 2, im­ paired renal function develops, as evidenced by de­ creased urine output and increased concentrations of serum creatinine.74 This condition seems to be primarily prerenal in nature. During IL 2 therapy, the fractional excretion of sodium declines to ex­ tremely low levels, despite administration of fluids and infusions of low-dose dopamine, and elevated plasma renin levels have been noted.75 Almost immediately after discontinuation of IL 2 therapy, however, urinary output increases and, in most cases, the serum creatinine stabilizes with a return to baseline levels within a few days. In the Na­ tional Cancer Institute trial, 74 pretreatment creat­ inine and prior nephrectomy were found to be important risk factors in predicting the degree of renal insufficiency. Patients with a serum creati­ nine concentration between 1.5 and 2 mg/dl, es­ pecially those older than 60 years of age, tended to have a delayed recovery of renal function, regard­ less of the dose of IL 2. In all patients, renal function eventually returned to normal—although, in some, more than 30 days elapsed before complete recovery occurred. At lower doses of IL 2, renal dysfunction is less frequent; however, oliguria and peak serum creatinine levels between 2 and 4 mg/ dl have been noted even at doses of 3 x 106 U/m 2 daily.76 Other renal-related toxicities have been reported. Abnormal urinary sediments containing tubular epithelial cells or coarse granular casts (or both) have been found in some patients. 74 Increased urinary levels of AT-acetylglucosaminidase, a tubu­ lar enzyme suggestive of tubular damage, have been reported. 75 Renal tubular acidosis has also been noted. 41 Clearly, the pathophysiologic mech-

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anisms of the acute renal dysfunction seen with both high- and low-dose IL 2 are still not entirely understood. Fever and chills occur at doses that exceed 5 x 105 U/m 2 , approximately 4 hours after IL 2 is administered by intravenous bolus, continuous infusion, or subcutaneous injection. The cause of the fever is unclear; the late onset, unlike the early onset of fever associated with endotoxins, suggests an indirect mechanism. IL 2, however, is one of the few cytokines that do not induce synthesis of prostaglandin E2,77 a common path­ way for febrile responses. IFN-y, the production of which is increased by IL 2 treatment, is not believed to be responsible for associated fever because it is a relatively weak pyrogen. The febrile response may be due to IL 2-induced release of other pyrogenic cytokines that have a direct effect on the hypothalamus. Patients who receive IL 2 also experience malaise, anorexia, nausea, vomiting, and rash. 78 In some patients, an explosive diarrhea develops approxi­ mately 3 hours after injection of IL 2. Pain at the tumor site has been noted during therapy. 56 The cause of these adverse effects is unknown. Central nervous system toxicity, primarily disorientation, was seen in 35% of patients in the National Cancer Institute trial. 79 Somnolence, hallucinations, seizures, and coma have also oc­ curred in patients who have received IL 2. Whether these symptoms that involve the central nervous system result from cerebral edema or from neurohormones released in response to ad­ ministration of IL 2 is unknown. The occurrence of central nervous system symptoms is an indi­ cation that IL 2 therapy should be discontinued because, unlike other toxicities, these symptoms do not resolve during therapy and may persist for several days after IL 2 is discontinued. As with the other toxicities, the effects of IL 2 on the bone marrow seem to depend on the dose. In a study by Ettinghausen and associates, 80 pa­ tients had a profound hypoproliferative anemia and moderate to severe thrombocytopenia. In five patients, circulating erythroid and myeloid stem cells were monitored and found to be sub­ stantially decreased during IL 2 infusions. After withdrawal of IL 2 therapy, the hematopoietic precursors rebounded. At low doses of IL 2 ad­ ministered by continuous infusion, hematologic toxicity has been limited to mild anemia that only occasionally necessitates transfusion. The

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cause of the cytopenias is unclear, but a variety of cytokines (including IFN-y and TNF as well as IL 2) have been reported to exert inhibitory effects on hematopoiesis in colony culture. 81-83 Laboratory Abnormalities.—Regardless of the dose of IL 2 administered, eosinophilia has been a striking finding; eosinophils have consti­ tuted as much as 92% of the circulating leukocytes, and absolute counts of eosinophils as high as 35,000/mm 3 have been noted. Typically, a 1- to 2-week latency period elapses between the initi­ ation of IL 2 therapy and the appearance of eosinophils. Although an erythematous rash de­ velops in many patients and a few complain of urticaria, the occurrence of these side effects is seemingly unrelated to the degree of eosinophilia. It is thought that IL 2 triggers secretion of IL 3 or a more specific eosinophil-stimulating factor.84 Hypoalbuminemia develops within days after initiation of IL 2 therapy. This response is too rapid to be caused by decreased hepatic synthesis and probably is best explained by loss into the extravascular space. 75 Substantial increases in acute-phase proteins such as fibrinogen and Creactive protein have been noted and may be due to the effects of IL 1 and TNF. 41 The actions of IL 1 and TNF on the hypothalamic-pituitary axis are thought to be responsible for increased con­ centrations of circulating adrenocorticotropic hor­ mone, prolactin, cortisol, and growth hormone. Hypovitaminosis C has also been noted. A re­ cent study described 11 patients who had rapid severe decreases in plasma ascorbate concentra­ tions after the initiation of IL 2 therapy. 85 All 11 patients had normal nutritional states, and 10 of the 11 had normal pretreatment ascorbate levels. Interestingly, the three patients who responded to IL 2/LAK therapy had higher ascorbate con­ centrations before and during therapy than did the eight nonresponders. Ascorbic acid (vitamin C) may be needed for optimal cell-mediated immunity. CLINICAL STUDIES: REGIONAL THERAPY One approach that may reduce some of the side effects of IL 2/LAK therapy is regional admin­ istration. Distribution studies with radiolabeled LAK cells have failed to demonstrate preferential migration of infused IL 2-activated cells to tumor sites.16,51 Regional or local therapy with IL 2 or LAK cells (or both) would, in theory, deliver LAK cells to the site of the tumor and provide high

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local concentrations of IL 2 without necessitating the administration of high systemic doses of the lymphokine. Administration of IL 2 by direct splenic or hepatic artery infusion is also being explored, in an attempt to increase in vivo gener­ ation of LAK cells. 86 Several reports have described intralesional therapy. In 1972, Cheema and Hersh 87 reported that injection of autologous PHA-activated lymphocytes into 29 cutaneous nodules of metastatic melanoma in 15 patients yielded partial to complete regres­ sion or stabilization in 28 of the 29 nodules but in only 10 of 18 nodules injected with control lympho­ cytes. Injection of IL 2 alone into cutaneous tumor nodules in two patients with melanoma and in two patients with Kaposi's sarcoma related to acquired immunodeficiency syndrome produced no response. Pizza and colleagues, 88 however, reported complete tumor regression in three of six patients with bladder cancer who received intra­ lesional injections of high-dose IL 2 during a period of 7 to 45 days. In all patients, an increase in tumor-infiltrating lymphocytes was noted. No adverse clinical side effects were reported. Jacobs and associates 89,90 treated 15 patients with recurrent glioblastoma with a combination of IL 2 and autologous LAK cells injected directly into the brain tissue surrounding the glioblas­ toma resection site intraoperatively. They re­ ported an increase in the duration of survival from a median of 3 to 6 months to a mean of 14.7 months in 4 of 11 evaluable patients. No systemic or central nervous system toxicity was noted. More recently, IL 2/LAK cells were administered through an Ommaya reservoir in­ serted intraoperatively in seven patients under­ going resection of recurrent glioblastoma multiforme (N = 6) or oligoastrocytoma (N = l). 91 Although a partial response was reported for the patient with oligoastrocytoma, no objective re­ sponses were noted in the six patients with glio­ blastoma multiforme, and neurologic toxicity was significant. Intracavitary therapy with IL 2 or LAK cells (or both) is also being explored. Intraperitoneal administration of IL 2 has been studied at the National Cancer Institute, where IL 2 has been administered in increasing doses through a Tenckhoff catheter to patients with advanced cancer, not necessarily localized to the peritoneal cav­ ity. 92 At each dose level, systemic toxicity was equal to that seen with intravenous administra­

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tion of IL 2. Cytolytic cells were reported in the peritoneal cavity. More recently, Steis and asso­ ciates 93 reported objective responses in four of five patients with peritoneal carcinomatosis from ovarian and colorectal malignant lesions who were treated with a combination of IL 2 and LAK cells administered intraperitoneally. IL 2 or LAK cells (or both) have also been administered directly into the pleural cavity. West and colleagues 60 administered IL 2 plus LAK cells into the pleural cavity of a patient with lung cancer and a malignant pleural effusion who had obtained substantial regression of a large hilar mass with systemic IL 2/LAK therapy. The au­ thors reported disappearance of malignant cells from the effusion only after intrapleural therapy. In a study by Yasumoto and associates, 94 11 patients with malignant pleural effusions due to lung cancer were treated with daily intrapleural instillations of recombinant IL 2. In 9 of the 11 patients, the malignant cells and the pleural effusion reportedly disappeared within 4 to 10 days after initiation of therapy, and an increase in the number and the cytotoxicity of immunoblasts in the pleural effusion was noted. Similar cellular changes were not evident in the two nonresponders. In 8 of the 11 patients, a signif­ icant increase in pleural eosinophils was also observed. Finally, anecdotal reports have described intrathecal therapy with IL 2/LAK cells for patients with leptomeningeal malignant melanoma, 90 meningeal gliomatosis, and meningeal carcinomato­ sis. 95 Administration of LAK cells through an Ommaya reservoir of a ventriculoperitoneal shunt with concurrent administration of IL 2, though not curative, has reportedly resulted in clini­ cal improvement and a considerable reduction in or a disappearance of malignant cells from the cerebrospinal fluid. FUTURE DIRECTIONS A N D CONCLUSION Much remains unknown about the mechanisms by which IL 2 affects the immune response and causes end-organ toxicity. Several questions have become the focus of intense laboratory and clini­ cal research. How does treatment with IL 2 or IL 2/LAK mediate tumor regression? Are infusions of LAK cells necessary? Although the combination of IL 2/LAK is more effective than IL 2 alone in the

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mouse model, the ability of IL 2 to generate LAK cells in vivo suggests that determination of the optimal dose and schedule of IL 2 administration might eliminate the need for infusions of LAK cells. Currently, several randomized clinical trials are comparing the effectiveness of IL 2 alone versus IL 2/LAK. Is it, in fact, the LAK cell that mediates tumor lysis? Although the LAK cell (infused or endog­ enous) may directly mediate tumor regression, perhaps other classes of IL 2-activated cells are responsible. Dense infiltrates of CD3 + , CD8 + T lymphocytes have been observed in tumor biopsy specimens obtained from patients responding to IL 2 therapy. These cellular changes were not present before therapy and were not observed in the nonresponders. 96 The role of the various cytokines induced by IL 2 in mediating tumor regres­ sion remains unknown. In an attempt to answer this question, trials in which IL 2 is administered in conjunction with IFN or TNF are being initiated. LAK cell effectiveness may also depend in part on the source of the lymphocytes that are cultured in IL 2. Recent reports suggest that tumor-infiltrating lymphocytes may be prefer­ able to peripheral blood lymphocytes as a source of LAK effectors.97,98 Tumor-infiltrating lympho­ cytes have been isolated and characterized from many different tumors, but their in vivo physio­ logic role remains controversial. Various studies have differed about whether tumor-infiltrating lymphocytes are functionally suppressed99 or acti­ vated 100 and whether they enhance 101 or downregulate102 host antitumor response. One explana­ tion for these incongruous findings is that because different tumor types elicit distinct tumorinfiltrating lymphocyte phenotypes, 103-107 the func­ tional attributes of tumor-infiltrating lympho­ cytes may vary depending on the tumor from which they were extracted. In addition, altering the culture conditions (for example, the duration of exposure to IL 2 in vitro) of tumor-infiltrating lymphocytes may influence the phenotype, tar­ get specificity, and the cytotoxicity of cultured effectors.108 Clinical trials with use of adoptive transfer of tumor-infiltrating lymphocytes are underway. 109 ' 110

response are numerous but unsubstantiated. One possibility is that a suppressor cell population decreases responsiveness of tumor to immunologic attack. This possibility has led to clinical trials with a combination of IL 2 and low-dose cyclophosphamide in an attempt to eliminate suppressor T lymphocytes. 111 Another possibility is that excessive tumor burden limits the effect of immunotherapy. As a result, cytoreductive treatment with drugs and other modalities may improve the response to IL 2/LAK therapy in patients with advanced cancers. In the future, IL 2 may be found to be more effective in the ad­ juvant setting, when tumor burden is low. An additional area of exploration focuses on the absence of clinical response in most patients with malignant lesions. The expression of cer­ tain surface molecules by tumor cells may be important in determining their susceptibility to cytotoxic mechanisms. In a preliminary study, a striking correlation was noted between HLA-DR expression (a class II MHC molecule) on tumor cells of patients and subsequent clinical response.96 Targeting the LAK cells, by either selective ad­ ministration into the site of the tumor or coadministration with tumor-specific monoclonal antibodies, may increase the response. Finally, several unexpected side effects have been observed with IL 2 therapy. Virtually every organ in the body may be affected by the admin­ istration of high-dose IL 2. Investigation into the mechanisms underlying the toxicities may pro­ vide insight into the physiologic connection be­ tween the immune system and other organ systems. Although the clinical value of adoptive im­ munotherapy remains unclear, the IL 2/LAK phenomenon has definitely stimulated a wealth of basic, clinical, and interdisciplinary research. It seems reasonable to expect that one consequence of these investigations will be the development of a viable anticancer therapy that relies on aug­ mentation of immune responses already present in the host.

Although dramatic complete responses have occurred in patients in clinical trials and many of these patients remain in remission, most re­ sponses have been partial and of short duration. Explanations for a failure to achieve a complete

1. Rosenberg SA, Lotze MT, Muul LM, Leitman S, Chang AE, Ettinghausen SE, Matory YL, Skibber JM, Shiloni E, Vetto JT, Seipp CA, Simpson C, Reichert CM: Ob­ servations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 313:1485-1492,1985

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37. Mertelsmann R, Welte K: Human interleukin 2: molecular biology, physiology and clinical possibilities. Immunobiology 172:400-419,1986 38. Gillis S, Smith KA: Long term culture of tumour-specific T cells. Nature 268:154-156,1977 39. Henney CS, Kuribayashi K, Kern DE, Gillis S: Interleukin2 augments natural killer cell activity. Nature 291:335338,1981 40. Yamamoto JK, Farrar WL, Johnson HM: Interleukin 2 regulation of mitogen induction of immune interferon (IFNy) in spleen cells and thymocytes. Cell Immunol 66:333-341,1982 41. Mier JW: Therapeutic uses of recombinant interleukin 2 in patients with cancer. Cancer Bull 39:19-24,1987 42. Howard M, Matis L, Malek TR, Shevach E, Kell W, Cohen D, Nakanishi K, Paul WE: Interleukin 2 induces antigen-reactive T cell lines to secrete BCGF-I. J Exp Med 158:2024-2039, 1983 43. Mul§ J J , Shu S, Schwarz SL, Rosenberg SA: Adoptive immunotherapy of established pulmonary metastases with LAK cells and recombinant interleukin-2. Science 225:1487-1489,1984 44. Mul6 J J , Shu S, Rosenberg SA: The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo. J Immunol 135:646-652,1985 45. Lafreniere R, Rosenberg SA: Successful immunother­ apy of murine experimental hepatic metastases with lymphokine-activated killer cells and recombinant inter­ leukin 2. Cancer Res 45:3735-3741,1985 46. Ettinghausen SE, Lipford EH III, Mule J J , Rosenberg SA: Recombinant interleukin 2 stimulates in vivo pro­ liferation of adoptively transferred lymphokine-activated killer (LAK) cells. J Immunol 135:3623-3635, 1985 47. Rosenberg SA, Mule JJ, Spiess PJ, Reichert CM, Schwarz SL: Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic ad­ ministration of high-dose recombinant interleukin 2. J Exp Med 161:1169-1188,1985 48. Ettinghausen SE, Lipford EH III, Mule J J , Rosenberg SA: Systemic administration of recombinant interleukin 2 stimulates in vivo lymphoid cell proliferation in tissues. J Immunol 135:1488-1497,1985 49. Mul§ JJ, Yang JC, Lafreniere R, Shu S, Rosenberg SA: Identification of cellular mechanisms operational in vivo during the regression of established pulmonary metastases by the systemic administration of high-dose recombinant interleukin 2. J Immunol 139:285-294,1987 50. Mule J J , Ettinghausen SE, Spiess PJ, Shu S, Rosenberg SA: Antitumor efficacy of lymphokine-activated killer cells and recombinant interleukin-2 in vivo: survival benefit and mechanisms of tumor escape in mice un­ dergoing immunotherapy. Cancer Res 46:676-683, 1986 51. Frenster JH, Rogoway WM: Immunotherapy of human neoplasms with autologous lymphocytes activated invitro. Proc Leukocyte Culture Conf 5:359-371,1970 52. Mazumder A, Eberlein TJ, Grimm EA, Wilson DJ, Keenan AM, Aamodt R, Rosenberg SA: Phase I study of the adoptive immunotherapy of human cancer with lectin activated autologous mononuclear cells. Cancer 53:896905,1984 53. Mazumder A, Grimm EA, Rosenberg SA: Characteriza­ tion of the lysis of fresh human solid tumors by autol­ ogous lymphocytes activated in vitro with phytohemagglutinin. J Immunol 130:958-964,1983 54. Lotze MT, Frana LW, Sharrow SO, Robb RJ, Rosenberg SA: In vivo administration of purified human inter­ leukin 2.1. Half-life and immunologic effects of the Jurkat cell line-derived interleukin 2. J Immunol 134:157-166, 1985

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55. Lotze MT, Matory YL, Ettinghausen SE, Rayner AA, Sharrow SO, Seipp CA, Custer MC, Rosenberg SA: In vivo administration of purified human interleukin 2. II. Half life, immunologic effects, and expansion of periph­ eral lymphoid cells in vivo with recombinant IL-2. J Immunol 135:2865-2875,1986 56. Thompson JA, Lee DJ, Cox WW, Lindgren CG, Collins C, Neraas KA, Dennin RA, Fefer A: Recombinant inter­ leukin 2 toxicity, pharmacokinetics, and immunomodulatory effects in a phase I trial. Cancer Res 47:42024207,1987 57. Atkins MB, Gould JA, Allegretta M, Li J J , Dempsey RA, Rudders RA, Parkinson DR, Reichlin S, Mier JW: Phase I evaluation of recombinant interleukin-2 in pa­ tients with advanced malignant disease. J Clin Oncol 4:1380-1391,1986 58. Allegretta M, Atkins MB, Dempsey RA, Bradley EC, Konrad MW, Childs A, Wolfe SN, Mier JW: The develop­ ment of anti-interleukin-2 antibodies in patients treated with recombinant human interleukin-2 (IL-2). J Clin Immunol 6:481-490,1986 59. Lotze MT, Chang AE, Seipp CA, Simpson C, Vetto JT, Rosenberg SA: High-dose recombinant interleukin 2 in the treatment of patients with disseminated cancer: responses, treatment-related morbidity, and histologic findings. JAMA 256:3117-3124,1986 60. West WH, Tauer KW, Yannelli JR, Marshall GD, Orr DW, Thurman GB, Oldham RK: Constant-infusion re­ combinant interleukin-2 in adoptive immunotherapy of advanced cancer. N Engl J Med 316:898-905,1987 61. Paciucci PA, Konefal R, Ryder J, Odchimar R, Holland JF: Phase I-II study of adoptive immunotherapy with rIL-2 activated cells and escalating continuous infusion rIL-2 in patients with disseminated cancer (abstract). Proc Annu Meet Am Soc Clin Oncol 6:248, 1987 62. NCI to open more LAK/IL-2 cancer center programs. Clin Cancer Lett 10:2-5, October 1987 63. Fisher RI, Coltman CA Jr, Doroshow JH, Rayner AA, Hawkins MJ, Mier JW, Wiernik P, McMannis JD, Weiss GR, Margolin KA, Gemlo BT, Hoth DF, Parkinson DR, Paitta E: Metastatic renal cancer treated with interleukin2 and lymphokine-activated killer cells: a phase II clini­ cal trial. Ann Intern Med 108:518-523,1988 64. Muul LM, Director EP, Hyatt CL, Rosenberg SA: Large scale production of human lymphokine activated killer cells for use in adoptive immunotherapy. J Immunol Methods 88:265-275, 1986 65. Muul LM, Nason-Burchenal K, Carter CS, Cullis H, Slavin D, Hyatt C, Director EP, Leitman SF, Klein HG, Rosenberg SA: Development of an automated closed system for generation of human lymphokine-activated killer (LAK) cells for use in adoptive immunotherapy. J Immunol Methods 101:171-181,1987 66. Nedwin GE, Svedersky LP, Bringman TS, Palladino MA Jr, Goeddel DV: Effect of interleukin 2, interferon-y and mitogens on the production of tumor necrosis fac­ tors a and /3. J Immunol 135:2492-2497, 1985 67. Lotze MT, Custer MC, Sharrow SO, Rubin LA, Nelson DL, Rosenberg SA: In vivo administration of purified human interleukin-2 to patients with cancer: develop­ ment of interleukin-2 receptor positive cells and circulat­ ing soluble interleukin-2 receptors following interleukin2 administration. Cancer Res 47:2188-2195,1987 68. Kohler PC, Hank J, Hong R, Huseby-Moore K, Rosenthal N, Sondel PM: Enhanced in vivo generation of LAK cells by repetitive administration of IL-2: a phase 1 clinical study (abstract). Proc Annu Meet Am Soc Clin Oncol 6:249,1987

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