Lymphokine-activated killer cell adoptive immunotherapy for cancer treatment and its significance]

New culture system, CDCS-T1, was developed for clinical conduction of lymphokine-activated killer (LAK) cell adoptive immunotherapy (AIT). Advanced or recurrent cancer patients of digestive tract were treated with AIT with LAK cells generated by CDCS-T1 in combination with plasma exchange. Partial responses were shown in 10 to 20% of patients treated. Long survival was found in some responders, indicating the significance of LAK therapy for cancer treatment. AIT with LAK cell transfer was also conducted in patients with esophageal cancer as postoperative adjuvant therapy. Better restoration of postoperative depression of immunological parameters was found in patients with postoperative LAK cell transfer. It is suggested that postoperative LAK cell transfer is a good candidate for adjuvant immunotherapy for cancer treatment.     

Preparing clinical grade Ag-specific T cells for adoptive immunotherapy trials

The production of clinical-grade T cells for adoptive immunotherapy has evolved from the ex vivo numerical expansion of tumor-infiltrating lymphocytes to sophisticated bioengineering processes often requiring cell selection, genetic modification and other extensive tissue culture manipulations, to produce desired cells with improved therapeutic potential. Advancements in understanding the biology of lymphocyte signaling, activation, homing and sustained in vivo proliferative potential have redefined the strategies used to produce T cells suitable for clinical investigation. When combined with new technical methods in cell processing and culturing, the therapeutic potential of T cells manufactured in academic centers has improved dramatically. Paralleling these technical achievements in cell manufacturing is the development of broadly applied regulatory standards that define the requirements for the clinical implementation of cell products with ever-increasing complexity. In concert with academic facilities operating in compliance with current good manufacturing practice, the prescribing physician can now infuse T cells with a highly selected or endowed phenotype that has been uniformly manufactured according to standard operating procedures and that meets federal guidelines for quality of investigational cell products. In this review we address salient issues related to the technical, immunologic, practical and regulatory aspects of manufacturing these advanced T-cell products for clinical use.     

Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies

Clinical effects and side effects were studied in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA)-mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of normal donors by means of initial stimulation with pokeweed mitogen (PWM). Six of 15 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastasis, pleural effusion and primary tumor. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Generally toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently except one patient who showed preshock like symptom accompanied with leukocytopenia and continuous hypotension immediately after infusion but was finally rescued. In the patients who received more than 10¹¹ of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies appeared in the patients. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells.Abbreviations PWM pokeweed mitogen - LAK lymphokine-activated killer - IL-2 interleukin 2 - PEL peripheral blood lymphocytes - TIL tumor-infiltrating lymphocytes - GVHD graft-versus-host disease - HLA human leukocyte antigen     

Cancer immunotherapy with interleukin-2-activated natural killer cells

Natural killer (NK) cells are a subset of lymphocytes with a distinct morphologic appearance (large granular lymphocytes [LGL]) and the ability to kill virally infected and tumor targets but to spare most normal cells. NK cells respond to a variety of biologic agents, such as interleukin-2 (IL-2), or interferons, by upregulation of cytolytic, secretory, and proliferative functions. In cancer-bearing hosts, NK cells have been considered to be the major component of antitumor immunity responsible for rapid elimination of malignant cells from the blood. More recently, however, studies have demonstrated the ability of adoptively transferred, IL-2-activated NK cells to selectively localize into solid tumors tissue and to eliminate established tumors. While these findings indicate a role for NK cells in cancer immunotherapy, additional studies are needed in both animal models and in humans to optimize clinical protocols of cancer therapy based on these cells.     

Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies.

Clinical effects and side effects were investigated in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA)-mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of healthy donors with the same blood types as those of patients. Recently we succeeded in increasing the proliferation rate and enhancing the cytotoxic activity of LAK cells by means of initial stimulation with pokeweed mitogen (PWM, PWM-LAK cells). Five of 12 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastases and pleural effusion. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently. In the patients who received more than 10(11) of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies existed in the patients; this phenomenon suggests the safety of the adoptive immunotherapy using allogeneic LAK cells. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells. Recently, LAK cells, particularly PWM-LAK cells were found to obtain significantly potent and prompt lectin-dependent cell-mediated cytotoxicity (LDCC). All tumor cells confluent in microtest plate well could be annihilated by PWM-LAK cells plus PWM less than 8 hours. New immunotherapy using PWM-LAK cells or lectin-stimulated LAK cells with PWM or other lectins is discussed.     

Lymphokine-activated killer cells with interleukin-2: dose toxicity and localization in isolated perfused rat lungs

Lymphokine-activated killer (LAK) cells combined with recombinant interleukin-2 (rIL-2) can produce tumor regression in murine models and in patients with pulmonary metastatic disease. However, the dose escalations of rIL-2 required for optimal therapeutic effect often result in increased vascular permeability ("vascular leak syndrome") and other toxic systemic consequences. To avoid systemic distribution, lung perfusion was used to administer LAK and rIL-2 locally. Preliminary to using these agents to treat tumor-bearing lungs, we used a nonblood-perfused isolated rat lung model to study the localization of radiolabeled rIL-2 and LAK and to characterize effects on normal lung tissue of increasing dosages and exposure times of rIL-2 and LAK cells, individually and combined. Lung function or permeability was assessed by measuring lung weight gain and pulmonary arterial pressure during the perfusion, extravascular lung water by double indicator dilution techniques, and wet weight to dry weight ratio. After perfusion for 1 hour using 200,000 U (1,300 U/ml) rIL-2, injury was detected as visible pulmonary edema, weight gain and increases in wet to dry weight ratio, and extravascular lung water; no injury was detected at lower, clinically appropriate dosages. When 1 X 10(8) LAK cells combined with 100,000 U rIL-2 (666 U/ml) were perfused for up to 2 hours, no injury was ascertained. Uptake and distribution of the radiolabeled rIL-2 or LAK was uniform to all lung lobes and corresponded to the decrease of 12% of the rIL-2 or 50% of the LAK from the perfusate after 1-hour perfusion.     

Homing of radiolabelled recombinant interleukin-2 activated natural killer cells and their efficacy in adoptive immunotherapy against murine fibrosarcoma

Natural killer (NK) cells are spontaneously cytotoxic against tumour target cells. Their number was found to be four times more in the spleen of tumour-bearing Swiss albino mice. After activation with recombinant interleukin-2 (rIL-2), NK cells were tested and found to seek out the tumour site when injected intravenously in tumour-bearing mice. Their potential for fighting tumours in vivo was further seen following adoptive transfer of rIL-2 activated NK (A-NK) cells in tumour-bearing mice. After surgical removal of tumour load, adoptive transfer of A-NK cells inhibited tumour recurrence in 92.3%cases, thereby suggesting the use of this protocol for therapeutic purposes to obtain a better outcome.     

Homing of radiolabelled recombinant interleukin-2 activated natural killer cells and their efficacy in adoptive immunotherapy against murine fibrosarcoma

Natural killer (NK) cells are spontaneously cytotoxic against tumour target cells. Their number was found to be four times more in the spleen of tumour-bearing Swiss albino mice. After activation with recombinant interleukin-2 (rIL-2), NK cells were tested and found to seek out the tumour site when injected intravenously in tumour-bearing mice. Their potential for fighting tumours in vivo was further seen following adoptive transfer of rIL-2 activated NK (A-NK) cells in tumour-bearing mice. After surgical removal of tumour load, adoptive transfer of A-NK cells inhibited tumour recurrence in 92.3% cases, thereby suggesting the use of this protocol for therapeutic purposes to obtain a better outcome.     

Tumor-derived interleukin-2-dependent lymphocytes in adoptive immunotherapy of lung cancer

Summary A trial of adoptive immunotherapy was performed in which long-term cultured, interleukin-2 (IL2)-dependent T-lymphocytes were administered to patients with metastatic adenocarcinoma of the lung. Lymphocytes were isolated from explants of cancer tissues that were cultured in medium with recombinant IL-2. These T-cells expressed surface markers of activation, and killed a broad panel of tumor targets. Intravenously injected ¹¹¹indium-labeled T-cell blasts distributed primarily to lungs, liver, and spleen. Despite a paucity of infused lymphocytes detected by external imaging at sites of tumor, five of seven patients showed reduction of their cancers. However, in no case was greater than 50% reduction of total tumor burden achieved. Evidence of increased delayed cutaneous hypersensitivity to protein antigens was observed in three patients following therapy. We conclude that long-term cultured tumor-derived T-cells can be transferred safely into humans and that these cells may be capable of enhancing immune responses and mediating tumor reduction in vivo.     

Clinical-grade,large-scale,feeder-free expansion of highly active human natural killer cells for adoptive immunotherapy using an automated bioreactor

Background aimsNatural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. Ex vivo expansion and activation of NK cells under good manufacturing practice (GMP) conditions are crucial for facilitating large clinical trials. The goal of this study was to optimize a large-scale, feeder-free, closed system for efficient NK cell expansion.MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors and myeloma patients were cultured for 21 days using flasks, cell culture bags and bioreactors. Final products from different expansions were evaluated comparatively for phenotype and functionality.ResultsSignificant NK cell expansions were obtained in all systems. The bioreactor yielded a final product rich in NK cells (mean 38%) ensuring that a clinically relevant cell dose was reached (mean 9.8 × 10⁹ NK cells). Moreover, we observed that NK cells expanded in the bioreactor displayed significantly higher cytotoxic capacity. It was possible to attribute this partially to a higher expression level of NKp44 compared with NK cells expanded in flasks.ConclusionsThese results demonstrate that large amounts of highly active NK cells for adoptive immunotherapy can be produced in a closed, automated, large-scale bioreactor under feeder-free current GMP conditions, facilitating clinical trials for the use of these cells.     

A new human pancreatic carcinoma cell line developed for adoptive immunotherapy studies with lymphokine-activated killer cells in nude mice

Summary A human tumor cell line designated SU.86 has been established from a moderate-to-poorly differentiated pancreatic carcinoma of ductal origin specifically for adoptive immunotherapy studies. This line was characterized as to its ability to be lysed in vitro by autologous and allogeneic lymphokine-activated killer (LAK) and natural killer cells and to grow in nude mice. SU.86 has been growing continuously in cell culture for more than 100 passages since 22 September 1986. Transplantation orthotopically and heterotopically into athymic Swiss nude mice showed that tumor take was 100% in the orthotopic position when young (4 to 6 wk old) mice were used and 0% when adult (8 wk old) mice were used (P=0.004). In the heterotopic position (subcutaneous), tumor take was 100% in neonate (2 to 3 wk old) and young mice and 50% in adults. The rate of tumor growth was inversely correlated with age (P<0.001). The histologic pattern is similar to that observed in most human pancreatic carcinomas with pseudoglandular structures and frequent mitotic figures. SU.86 has a doubling time of 77 h in vitro and produces carcinoembryonic antigen, 594 ng/10⁶ cells in 3 d. Chromosomal analysis shows heterogeneity with two notable cell subpopulations. The cell line is moderately sensitive to lysis by LAK cells in a standard, 4-h chromium-51 release assay (35.4±4.0%). When grown together with LAK cells in vitro, it is lysed completely in culture in 8 to 15 d, depending on the serum concentration.     

Lessons from the clinical trials of interleukin-2

The initial results of interleukin-2 (IL-2) therapy in man are reviewed from the perspective of how they conform to predictions from preclinical studies. These preclinical models predict that tumors will vary in their susceptibility to IL-2 therapy and will be most successfully treated at lower tumor burdens. In addition, the dose and schedule of IL-2 are important for successful therapy. Host-related factors, including the presence of suppressor activities, may also be important. In these models, the addition of other cytokines, including interferon-alpha or tumor necrosis factor, to IL-2 can enhance antitumor activity. The concomitant administration of ex vivo IL-2-activated lymphokine-activated killer cells or tumor-infiltrating lymphocytes also enhances the IL-2 antitumor effect. Clinical trials addressing all of these issues have been completed or are underway; the results suggest overall that the preclinical models are predictive, with both host- and tumor-related factors as well as such IL-2-therapy-related factors as dose, schedule, route and the use of additional agents all playing a role in the success of therapy. A more complete understanding of the mechanisms of response and resistance involved in this therapy will facilitate the rational development of more effective and less toxic IL-2-based therapy of human malignancy.     

Rapid expansion of tumor-reactive cells from HLA-matched siblings for adoptive immunotherapy of melanoma

BACKGROUND: Administration of expanded tumor-infiltrating lymphocytes in association with lymphodepleting chemotherapy is effective in some patients with advanced malignant melanoma. However, obtaining lymphocytes and subsequent expansion is labor intensive, making it impractical for broad clinical application. Allogeneic transplantation may have anti-melanoma efficacy because of a graft vs. tumor effect. The disappointing tumor control observed post-transplant suggests that adoptive immunotherapy using melanoma-reactive cells will be essential for sustained responses. METHODS: Melanoma cell lines were grown from two patients with advanced disease. High-level CD80 and CD86 expression was obtained in the tumor lines using a retroviral vector for gene transfer. Transduced melanoma and controls were cultured with mononuclear cells from HLA-identical sibling donors. RESULTS: Expression of CD80 and CD86, particularly the former, promoted marked expansion of lymphocytes from HLA-matched sibling donors. Proliferation of up to 300-fold after 4 weeks of culture was observed. Lymphocytes from cultures stimulated with CD80 demonstrated cytotoxicity against recipient-untransfected melanoma (45-75% specific lysis at an E:T ratio of 50:1). Although expanded lymphocytes were predominantly CD4(+), cytotoxicity was greatest in the numerically smaller CD8(+) subpopulation. Both CD4(+) and CD8(+) cells secreted IFN-gamma (but not IL-4) on exposure to untransduced stimulator melanoma cells. DISCUSSION: Our strategy generates a large number of melanoma-reactive lymphocytes from HLA-identical siblings using a 4-week culture strategy. Lymphocytes expanded in this way offer an alternative to tumor-infiltrating lymphocytes for allogeneic cellular immunotherapy after stem cell transplantation in young patients.     

Ex vivo expansion of non-MHC-restricted cytotoxic effector cells as adoptive immunotherapy for myeloma

BACKGROUND: PBMC can be expanded ex vivo into aggressive cytotoxic effector cells (CEC) comprising T, NK and NKT cells. We identified the phenotype, cytotoxicity and mechanisms of killing of these CEC. METHODS: CY- and G-CSF-mobilized PBMC from myeloma patients were placed in Aim-V serum-free medium, IL-2 (50 IU/mL) and OKT-3 (50 ng/mL). Cytotoxicity was evaluated by selectively blocking the TCR, MHC class I or NKG2D receptor. RESULTS: The CEC expanded three-fold by day 7 and aggressively lysed myeloma cells (41.9%) compared with day 0 (4%; P=0.012). CD8+ CD56+ NKT cells performed the majority of lysis. The CD8+ cells greatly increased NKG2D expression during culture (P=0.005). Cytotoxicity correlated with target NKG2D ligand expression (P=0.0002). Blocking the TCR or MHC class I did not affect cytotoxicity (P>0.22). CD8+ cell-mediated lysis dropped 48% when the NKG2D receptor was blocked. Day 7 CEC aggressively lysed myeloma cells in an MHC- and non-MHC-restricted fashion, through the NKG2D receptor. DISCUSSION: Because MHC expression is often down-regulated on tumor cells and the NKG2D ligands are generally specific to malignant cells, the adoptive transfer of CEC that kill through different pathways may circumvent tumor-resistant mechanisms and improve outcomes.     

Rise of iPSCs as a cell source for adoptive immunotherapy

Adoptive T cell transfer is a potentially effective strategy for treating cancer and viral infections. However, previous studies of cancer immunotherapy have shown that T cells expanded in vitro fall into an exhausted state and, consequently, have limited therapeutic effect. One way to overcome this obstacle is to use induced pluripotent stem cells (iPSCs) as a cell source for making effector T cells. In recent years, there have been several reports on generating effector T cells suitable for adoptive immunotherapy. The reported findings suggest that using iPSC technology, it may be possible to stably derive large numbers of juvenile memory T cells targeted to cancers or viruses. In this review, we describe a strategy for applying iPSC technology to immunotherapy and the characteristics of T cells derived from iPSCs. We also discuss how these technologies can be applied clinically in the future.     

Evaluation of natural killer cell expansion and activation in vivo with daily subcutaneous low-dose interleukin-2 plus periodic intermediate-dose pulsing

 Natural killer (NK) cells may be expanded in vivo with a prolonged course of daily subcutaneous interleukin-2 (IL-2). However, cellular activation requires higher concentrations of IL-2 than are achieved with low-dose therapy. The objective of the current trial was to determine the toxicity and immunological effects of periodic subcutaneous intermediate-dose IL-2 pulses in patients receiving daily low-dose therapy. A group of 19 patients were treated with daily subcutaneous low-dose IL-2 at 1.25×10⁶ International Units (1.25 MIU) m^–2 day^–1. After 4–6 weeks, patients received escalating 3-day intermediate-dose IL-2 pulses administered as single daily subcutaneous injections, repeated at 2-week intervals. The maximum tolerated pulse dose was 15 MIU m^–2 day^–1, with transient hypotension, fatigue, and nausea/vomiting dose-limiting. Subcutaneous IL-2 resulted in in vivo expansion of CD56⁺ NK cells (796±210%) and CD56^bright natural killer (NK) cells (3247±1382%). Expanded NK cells coexpressed CD16, and showed lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity in vitro. Intermediate-dose pulsing resulted in serum IL-2 concentrations above 100 pM. Cellular activation was suggested by rapid margination of NK cells following pulsing, coincident with peak IL-2 levels, with return to baseline by 24 h. In addition, interferon γ production in response to lipopolysaccharide was augmented. Subcutaneous daily low-dose IL-2 with intermediate-dose pulsing is a well-tolerated outpatient regimen that results in in vivo expansion and potential activation of NK cells, with possible application in the treatment of malignancy and immunodeficiency. Received: 31 December 1997 / Accepted: 20 April 1998     

The use of BRM-activated killer cells in adoptive immunotherapy: A pilot study with nine advanced cancer patients

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood T cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 107) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN- and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood T cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 109 BRM-activated killer (BAK) cells composed of CD56+ T cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90–100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated T cells producing IFN- were assayed as an indication marker of BAK therapy. The normal range of IFN- producing T cells comprised 6.9 ± 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN- producing T cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood T cells of Patients Nos. 1–7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN- producing T cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial T cell counts, a low frequency of these cells may contraindicate BAK therapy.