Lymphokine-activated Killer Research Articles

Abstract 4403: Antitumor properties of ouabain in lipid double emulsion integrated with tumor cell membrane fractions

Abstract Cell membrane proteins such as integrins are crucial for cell-cell recognition, binding and communication, cell-stromal interaction and adhesion, tumor invasion and metastasis. It was reported that cell-size hydrophobic beads coated with tumor membrane molecules could stimulate the lymphokine activated killer (LAK) cells to release IFN-alpha and TNF-alpha, and interrupted the recognition between LAK cells and tumor cells (Chong AS, 1988, 1991). Lipid double emulsion is a convenient formulation method to integrate lipid materials, hydrophilic and hydrophobic drugs in uniformed particles to achieve combinational chemotherapy. However the nonspecific uptake of double emulsion particles in vivo limits its targeting therapeutic potential. In current study, we sought to coat double emulsion particles with tumor cell membrane fraction to mimic the tumor cell surface in order to enhance the interaction and uptake of emulsion particles into tumor cells. Canine osteosarcoma cells and lymphoma cells were chosen for this study because canine cancers develop in immunocompetent environments and share many similar features observed in human cancers, such as long periods of latency for initiation, progression and development, metastasis, acquired drug resistance and recurrence. We use ouabain as therapeutic warhead in the emulsion particles because ouabain is known to have extremely high anticancer potential at nanomolar concentration level from our previous studies. The major obstacle of applying ouabain in vivo therapy is that the therapeutic dose window of ouabain is very narrow. Therefore in an attempt to mitigate such narrow therapeutic margin of safety, ouabain was formulated in lipid double emulsion by using microfluidic synthesis method and membrane emulsification method. The double emulsion particles were further coated with osteosarcoma cell membrane fraction. Our preliminary results showed enhanced antitumor activities of ouabain in tumor cell mimic-double emulsion or nanoemulsion against canine osteosarcoma cells and lymphoma cells in vitro and in orthotopic xenograft models in vivo, suggesting a promising therapeutic benefit in canine cancer targeting therapy. Citation Format: Jun Wu, Mariele Mondala, Meng-Yin Hsieh, Eugene Roberts, Richard Ermel. Antitumor properties of ouabain in lipid double emulsion integrated with tumor cell membrane fractions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4403. doi:10.1158/1538-7445.AM2015-4403

Immunotherapy of breast cancer by single delivery with rAAV2-mediated interleukin-15 expression.

Recombinant adenovirus-associated vector serotype 2 (rAAV2) is one of the most promising gene transfer vectors due to its advantage of causing non-pathogenic infection, low immunogenicity, and long-term gene expression in human clinical trials. Human interleukin 15 (hIL15) has been implicated in modulation of antitumor activity of lymphokine-activated killer (LAK) cells, including T cells and NK cells. In this study, the rAAV2-hIL15 vector was produced and subjected for treatment with xenograft JC breast cancer model. Results showed that tumor onset was significantly delayed, the tumor growth was suppressed, and the lifespan of tumor-bearing mice were prolonged by rAAV2-hIL15. In addition, rAAV2-hIL15 was able to produce a substantial expression of IL15 protein that ultimately activated the cytotoxic activity of LAK cells. Furthermore, prominent apoptosis was observed in tumor lesions following injection of rAAV2-hIL15. Taken together, our results suggested that rAAV2-hIL15 appears as a new potential therapeutic tool for breast cancer immunotherapy.

Present and Future of Allogeneic Natural Killer Cell Therapy.

Natural killer (NK) cells are innate lymphocytes that are capable of eliminating tumor cells and are therefore used for cancer therapy. Although many early investigators used autologous NK cells, including lymphokine-activated killer cells, the clinical efficacies were not satisfactory. Meanwhile, human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation revealed the antitumor effect of allogeneic NK cells, and HLA-haploidentical, killer cell immunoglobulin-like receptor ligand-mismatched allogeneic NK cells are currently used for many protocols requiring NK cells. Moreover, allogeneic NK cells from non-HLA-related healthy donors have been recently used in cancer therapy. The use of allogeneic NK cells from non-HLA-related healthy donors allows the selection of donor NK cells with higher flexibility and to prepare expanded, cryopreserved NK cells for instant administration without delay for ex vivo expansion. In cancer therapy with allogeneic NK cells, optimal matching of donors and recipients is important to maximize the efficacy of the therapy. In this review, we summarize the present state of allogeneic NK cell therapy and its future directions.

Armed hunter killers: discerning the role of adoptive T-cell transfer for glioblastoma.

Glioblastoma (GBM) is the most malignant primary brain cancer with an incidence around 3.19 per 100,000/year in the USA [1]. Poor prognosis is due to its infiltrative nature in the brain, genetic heterogeneity and the belligerent GBM stem cells. Despite the advance in new generation alkylating agents, such as temozolomide, along with radiation and tumor vasculature-targeting monoclonal antibodies, such as bevacuzimab, the overall survival has only accented by a few months above the natural history of the untreated tumor [2]. The Cancer Genome Atlas Research Network recently unraveled a genetic alteration which pinpointed potential antigenic targets in GBM [3]. Hence, targeted gene immunotherapy could be a vital hope for finding an efficient GBM cure. Immunotherapy is an intriguing biologic which harnesses the power of the immune system to selectively kill tumor cells [4]. Active immunization with dendritic cell vaccines and immune recovery with cytokines, are encouraging approaches however, they are limited by their inability to expand tumor antigen-specific T cells, which are often anergized by the tumor hostile milieu [4]. One of the means to overcome this limitation is passive immunization with adoptively-transferred T cells [5]. Adoptivelytransferred T cells are tumor-specific T cells prepared ex vivo, either de novo or by enriching a pre-existent tumor specific T-cell population, then retransferred back to patients, often after lymphodepletion [6]. Two tactics were scrutinized to generate tumor affinitymatured T cells by transducing T cells either with high-affinity tumor reactive T-cell receptor (TCR) genes or genes encoding chimeric antigen receptors (CARs) [7]. Broader adoptive therapy concept includes natural killer cells [8], lymphokine-activated killer cells [4]; as well as neural or mesenchymal stem-cells [9,10]. This review will highlight the use of T-cell adoptive therapy potential for GBM while embarking upon some challenges that need to be overcome to pave the way to an excelled GBM cure in the near future.

Therapeutic effect of lymphokine-activated killer cells treated with low-dose ionizing radiation on osteosarcoma

The aim of the present study was to investigate the effect of lymphokine-activated killer (LAK) cells, which received low-dose ionizing radiation, on the treatment of osteosarcoma in rats. The cultured UMR-106 cells were inoculated under the anterior chest skin of 24 rats to establish an osteosarcoma model. In addition, the LAK cells from 24 mice were exposed to doses of 0 (control group), 0.65 or 3.25 mGy X-ray radiation. The tritiated thymidine (3H-TdR) release method and Winn assay were performed to determine the antitumor effects of the LAK cells. The proliferation of the mouse LAK cells treated with 3.25 mGy radiation was significantly higher than that for those treated with 0 or 0.65 mGy radiation, which suggested that low-dose ionizing radiation stimulates the proliferation of LAK cells. The tumor-bearing rats were divided into three groups and injected with LAK cells that had already received 0, 0.65 or 3.25 mGy radiation. The mean survival time of the 3.25-mGy group was longer than that of the 0- and 0.65-mGy groups. After 30 days, tumors with weights of ~6.25 and 2.0 g were identified in the rats of the 0- and 0.65-mGy groups, respectively. However, tumor proliferation was not detectable in the rats of the 3.25-mGy radiation group. Therefore, low-dose ionizing radiation effectively kills osteosarcoma cells in rats by stimulating the proliferation and enhancing the cytotoxicity of LAK cells.

Hepatitis C viral infection is associated with activated cytolytic natural killer cells expressing high levels of T cell immunoglobulin- and mucin-domain-containing molecule-3.

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) is an inhibitory receptor implicated in T cell exhaustion characteristic of chronic viral infection. Limited data exist on NK cell Tim-3 expression and functional consequences. In chronic hepatitis C virus (HCV)-infected subjects, we found increased Tim-3 on NKs, which was associated with an activated phenotype. The high level of Tim-3 was not reversed by successful IFN-alpha-based antiviral therapy. Tim-3high NK cells up-regulated TRAIL in response to IFN-alpha to a greater extent and demonstrated greater lymphokine-activated killing activity, viral control, and degranulation but similar cytokine production than their Tim-3low counterparts. Our results suggest that Tim-3 on NKs is associated with activation of this innate lymphocyte population that is polarized towards cytotoxicity in chronic HCV. These findings reveal roles for Tim-3 in the regulation of NKs that might represent targets for treatment of chronic viral infections.

T-cell-associated cellular immunotherapy for lung cancer.

The aim of the present study was to discuss recent findings on the role of T cells in lung cancer to provide information on their potential application, especially in cellular immunotherapy. Data on the different types of T cells that are currently used for the treatment of lung cancer were obtained by searching the PUBMED database. Cytotoxic T lymphocytes, natural killer T cells, γδ T cells, lymphokine-activated killer cells, tumor-infiltrating lymphocytes, cytokine-induced killer cells and gene-modified T cells were analyzed to determine the benefits and drawbacks of their application in the treatment of lung cancer. Advances in the study of their antitumor mechanisms and directions for future research were discussed. T cells are critical for tumorigenesis and therefore important targets for the treatment of lung cancer. T-cell-associated cellular immunotherapy opens up a window of opportunity for the development of complementary methods to traditional lung cancer treatments, which warrants further investigation to improve the clinical outcomes of lung cancer patients.

왕느릅나무 껍질 열수 추출물의 마우스에서의 in vivo 면역조절 효과

Traditional medicinal plants are widely used to treat many diseases, such as inflammation, infections, and even cancer. Ulmus macrocarpa Hance, a Chinese elm species, is distributed in Korea, China, and Japan. The stem bark is widely employed in Korean traditional medicine to treat dermatitis, mastitis, and edema. The aim of this study was to investigate whether water extract of U. macrocarpa Hance bark (Ulmus cortex) has a immune-modulating function in a mouse model. Three different concentrations (30 ㎎/㎏, 100 mg/kg, and 300 ㎎/㎏) of Ulmus cortex water extract (UCWE) were orally administered to mice for 14 days, and their immune responses were analyzed. Cytokines, such as interleukin (IL)-2, IL-12, and IFN-γ, increased in the blood of UCWE-fed groups when compared with a control group. In contrast, the IL-4 level did not change in any of the UCWE-fed groups Cell-mediated cytotoxicity was also assayed using lymphokine-activated killer cells (LAK). LAK showed greater cytotoxicity in the UCWE-fed groups than LAK in the control group. Internal organ indices, such as liver, kidney, spleen, and thymus, were similar in all the groups, including the control group, indicating that UCWE may have been nontoxic in the experimental animals. These data suggest that UCWE has an immune-modulating function in a mouse model.

Impact of IL-2 and IL-2R SNPs on proliferation and tumor- killing activity of lymphokine-activated killer cells from healthy chinese blood donors.

One of the goals of tumor immunotherapy is to generate immune cells with potent anti-tumor activity through in vitro techniques using peripheral blood collected from patients. However, cancer patients generally have poor immunological function. Thus using patient T cells, which have reduced in vitro proliferative capabilities and less tumor cell killing activity to generate lymphokine-activated killer (LAK) cells, fails to achieve optimal clinical efficacy. Interleukin-2 (IL-2) is a potent activating cytokine for both T cells and natural killer cells. Thus, this study aimed to identify optimal donors for allogeneic LAK cell immunotherapy based on single nucleotide polymorphisms (SNP) in the IL-2 and IL-2R genes. IL-2 and IL-2R SNPs were analyzed using HRM- PCR. LAK cells were derived from peripheral blood mononuclear cells by culturing with IL-2. The frequency and tumor-killing activity of LAK cells in each group were analyzed by flow cytometry and tumor cell killing assays, respectively. Regarding polymorphisms at IL-2-330 (rs2069762) T/G, LAK cells from GG donors had significantly greater proliferation, tumor-killing activity, and IFN-γ production than LAK cells from TT donors (P<0.05). Regarding polymorphisms at IL-2R rs2104286 A/G, LAK cell proliferation and tumor cell killing were significantly greater in LAK cells from AA donors than GG donors (P<0.05). These data suggest that either IL- 2-330(rs2069762)T/G GG donors or IL-2R rs2104286 A/G AA donors are excellent candidates for allogeneic LAK cell immunotherapy.

Cannabinoids increase lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1

Cannabinoids have been shown to promote the expression of the intercellular adhesion molecule 1 (ICAM-1) on lung cancer cells as part of their anti-invasive and antimetastatic action. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study addressed the impact of cannabinoid-induced ICAM-1 on cancer cell adhesion to lymphokine-activated killer (LAK) cells and LAK cell-mediated cytotoxicity. Cannabidiol (CBD), a non-psychoactive cannabinoid, enhanced the susceptibility of cancer cells to adhere to and subsequently be lysed by LAK cells, with both effects being reversed by a neutralizing ICAM-1 antibody. Increased cancer cell lysis by CBD was likewise abrogated when CBD-induced ICAM-1 expression was blocked by specific siRNA or by antagonists to cannabinoid receptors (CB1, CB2) and to transient receptor potential vanilloid 1. In addition, enhanced killing of CBD-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen-1 (LFA-1) suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. ICAM-1-dependent pro-killing effects were further confirmed for the phytocannabinoid Δ9-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), a hydrolysis-stable endocannabinoid analogue. Finally, each cannabinoid elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less pronounced (CBD, THC) or absent (MA) ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate cannabinoid-induced upregulation of ICAM-1 on lung cancer cells to be responsible for increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of cannabinoids.

Association of Changes in Fitness and Body Composition with Cancer Mortality in Men

Both baseline cardiorespiratory fitness and adiposity predict the risk of cancer mortality. However, the effects of changes in these two factors over time have not been evaluated thoroughly. The aim of this study was to examine the independent and joint associations of changes in cardiorespiratory fitness and body composition on cancer mortality. The cohort consisted of 13,930 men (initially cancer-free) with two or more medical examinations from 1974 to 2002. Cardiorespiratory fitness was assessed by a maximal treadmill exercise test, and body composition was expressed by body mass index (BMI) and percent body fat. Changes in cardiorespiratory fitness and body composition between the baseline and the last examination were classified into loss, stable, and gain groups. There were 386 deaths from cancer during an average of 12.5 yr of follow-up. After adjusting for possible confounders and BMI, change hazard ratios (95% confidence intervals) of cancer mortality were 0.74 (0.57-0.96) for stable fitness and 0.74 (0.56-0.98) for fitness gain. Inverse dose-response relationships were observed between changes in maximal METs and cancer mortality (P for linear trend = 0.05). Neither BMI change nor percent body fat change was associated with cancer mortality after adjusting for possible confounders and maximal METs change. In the joint analyses, men who became less fit had a higher risk of cancer mortality (P for linear trend = 0.03) compared with those who became more fit, regardless of BMI change levels. Being unfit or losing cardiorespiratory fitness over time was found to predict cancer mortality in men. Improving or maintaining adequate levels of cardiorespiratory fitness appears to be important for decreasing cancer mortality in men.

Lymphocyte recovery is impaired in patients with chronic lymphocytic leukemia and indolent non-Hodgkin lymphomas treated with bendamustine plus rituximab.

The immune system has the potential to either attenuate tumor growth or to promote tumor progression. The goal of cancer immunotherapy is to shift the balance in favor of tumor immunosurveillance, so that the immune system can recognize the tumor, eliminate it, and prevent its recurrence. Bendamustine plus rituximab is generally considered effective and safe in patients with previously untreated chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin lymphomas. To evaluate the effects of bendamustine-rituximab and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) on the recuperation of immune system, we analyze the distribution of CD4+ and CD8+ T cells, B cells, and NK cells in peripheral blood of 18 patients who received 4-6 cycles of rituximab-bendamustine (BR) or six R-CHOP before therapy and 6 months after completing treatment. Our results indicate that lymphocyte recovery is impaired in patients with chronic lymphocytic leukemia and indolent lymphomas treated with bendamustine plus rituximab. Low CD4 T cells (<200 cells/μl) induced by bendamustine (BR) suggest prophylaxis should be applied against opportunistic infections. Asymptomatic EBV and CMV reactivations support a negative effect of BR on the immune system. If cellular immune therapy such as lymphokine-activated killer (LAK) or effector lymphocytes infusion is planned, regimes other than BR should be the first choice.

COMPARISON OF EFFICACY AND TOLERANCE OF THE INTRAPERITONEAL CELL-BASED AND IL-2 IMMUNOTHERAPY OF PATIENTS WITH CHEMOTHERAPY-RESISTANT GASTRIC CANCER AND MALIGNANT ASCITES

Most often gastric cancer is accompanied with abdominal carcinomatosis tumor (metastases) ascites (25 %), which is a life-threatening complications of tumor process, often recurring after systemic drug therapy which not always leads to a longer remission, but is also accompanied by severe side effects. Intraperitoneal immunotherapy of metastatic ascites interleukin-2 (IL-2) in combination with allogeneic (donor) lymphokine - activated killer (LAK) and IL-2 monotherapy is highly effective and well tolerated in patients with gastric cancer. Objective effect of the treatment was 50.0 % and 42.8 %, respectively. Proposed form of immunotherapy can be considered as one of the stages combined drug treatment, has good tolerance and improves their quality of life.

Purification, initial characterization and immune activities of polysaccharides from the fungus, Polyporus umbellatus

Polyporus umbellatus (formerly Grifola umbellatus), an edible fungus, has been used as traditional Chinese medicine. The present investigation aimed to uncover the active ingredients in this fungal species, with the emphasis on its polysaccharides. Two polysaccharides, one from fermented mycelium and the other from the fruiting body, were chromatographically purified. Molecular weights, anti-microbial and immune activities of the polysaccharides were determined. The mycelium polysaccharide was found to have a molecular weight of 857kDa and consist of glucose and galactose at a molar ratio of 1.57:1, while the one from the fruiting body, 679kDa in molecular weight, was also made up of glucose and galactose but at a molar ratio of 5.42:1. In immune assays, these fungal polysaccharides increased the killing potency of natural killer (NK) and lymphokine-activated killer (LAK) cells from mouse spleen and promoted proliferation of mouse B and T cells. Additionally, both P. umbellatus mycelium and its extract were found to inhibit Escherichia coli and Staphylococcus aureus, but had no effects on Aspergillus niger or Aspergillus nidulans. These results indicated that fermented P. umbellatus mycelium is indeed immune active, most probably due to its ingredients of polysaccharides. The consumption of this fungus may be beneficial for human health.

Benefits of a continuous therapy for cancer patients with a novel adoptive cell therapy by cascade priming (CAPRI).

A growing body of evidence shows that immune cells are pivotal in the fight against cancer. First, association studies have identified immune-protective immune response genes against cancer. Second, the presence of immune cells in the respective malignant tumor correlated with a better prognosis for the patients. Third, adoptive cell therapy (ACT) showed, in recent reports, an efficient reduction or even cure for malignant tumors. The focus of this review is a novel invitro ACT technique, using the patient's cascade-primed immune cells. The cascade-priming procedure stimulates APCs from the peripheral blood. Stimulated APCs digest and present tumor material better and differentiate naive cytotoxic T-lymphocyte effector cells against the patient's cancer. The principle and the impressive results of the cascade-primed immune cell treatment in patient case series is compared with the ACT concepts of lymphokine-activated killer, macrophage-activated killer, macrophage-activated killer-dendritic cell, cytokine-induced killer and tumor-infiltrating lymphocyte methods.

Gemcitabine sensitizes lung cancer cells to Fas/FasL system‐mediated killing

Gemcitabine is a chemotherapy agent commonly used in the treatment of non-small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine-induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real-time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL-expressing cells was evaluated by flow cytometry, and caspase-8 and caspase-3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine-activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti-Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane-bound FasL (mFasL) and of mFasL-positive apoptotic H292 cells, as well as caspase-8 and caspase-3 cleavage. Moreover, gemcitabine increased CH11-induced caspase-8 and caspase-3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine-treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up-regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas-dependent apoptosis mediated by caspase-8 and caspase-3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine-induced lung cancer cell killing.

Pancreatic Cancer Treatment

Pancreatic canceris one of the most aggressive and lethal human cancers, with more than 200, 000 deaths worldwide every year. Despite recent efforts, conventional treatment approaches, such as surgery, classic chemotherapy, radiotherapy, biologic therapy and palliative care have only slightly improved patient outcomes. Among the conventional chemotherapies the combination of abraxane (nab-paclitaxel) with gemcitabine and FOLFIRINOX (5-FU/leucovorin, irinotecan, and oxaliplatin) with gemcitabine in metastatic pancreatic cancer patients are promising. Among the targeted therapies the combination of gemcitabine, erlotinib and capecitabine are likely to form the base for future treatment. Under biologic or immunotherapy KRAS-targeting vaccines, telomerase-targeting vaccine (GV1001), gastrin-based vaccine, dendritic cell (DC)-based vaccine alone or combined with lymphokineactivated killer (LAK) cells and allogeneic GM-CSF-secreting vaccine (GVAX) are well tolerated by patients and could represent a new therapeutic option for pancreatic cancer. Moreover a novel strategy for using mesenchymal stem cells (MSCs) as means of delivering anticancer genes to the site of pancreas is also encouraging. Current and future clinical trials using natural compounds such as vitamin E compound delta-tocotrienol and curcumin in combination with standard chemotherapy are ongoing to discover more effective ways of treating advanced pancreatic cancer.

Immunotherapy of Cancer—A Historical Note

We examined the possibility that the anti-estrogens, tamoxifen (TX) and toremifen (TO) interacted with the immune system. Indeed, both TX and TO stimulated cells mediated cytotoxicity reactions by various killer cells: killer T (TK), natural killer (NK), lymphokine activated killer (LAK) cells. Both TX and TO inhibited the growth of tumors that express estrogen receptors. Thus these antiestrogens inhibited tumor growth and stimulated killer cells for cytotoxicty on such tumors. Therefore these agents were presumed to stimulate tumor immunity. We tested the P815 mouse mastcytoma with TK, LK, and TX or TO. A therapeutic effect was observed in both experiments. The SL2-5 murine lymphoma was tested with NK and TX cells or TO cells and successful immunotherapy was observed. We digested human breast carcinomas and lung tumors with collagenase. The killer cells were separated from tumor cells on Ficoll gradients. TX and TO enhanced the cytotoxic effect of autologous killer cells on the corresponding tumor cells. This experiment indicates that the results obtained in animals are also valid for human malignant disease.

IL-7 enhancement of antigen-driven activation/expansion of HIV-1-specific cytotoxic T lymphocyte precursors (CTLp).

CD8+ cytotoxic T lymphocytes are an important component in the immunologic control of human viral diseases. IL-7, a stromal cell-derived cytokine, has been demonstrated to enhance both anti-tumour and anti-viral CTL as well as lymphokine-activated killer (LAK) activity. We studied the ability of IL-7 to support the activation and the growth of in vitro antigen-specific CTL precursors (CTLp) present in peripheral blood mononuclear cells (PBMC) from HIV-infected patients. Results from these studies demonstrate that inclusion of IL-7 in a vaccinia/HIV-1 vector-based stimulation strategy greatly augmented overall CTL reactivities, whereas addition of IL-7 to unstimulated cultures failed to induce any significant anti-viral cytolytic activity. In four of six patients, HIV-specific lytic activities were significantly higher in cultures stimulated with antigen plus IL-7 compared with in vitro stimulation (IVS) with antigen alone. Cytotoxic activity was principally mediated by CD8+ effector cells, and CD3+/CD8+ cell expansion was increased by 2.7-fold in the presence of IL-7. In PBMC from seronegative donors, IL-7 enhanced anti-vaccinia CTL activities with less effect on cell proliferation. Furthermore, anti-gag CTL frequencies determined by limiting dilution analysis were increased by 2- and 10-fold in two asymptomatic patients following IVS plus IL-7 compared with antigen stimulation alone. Cytofluorimetric analysis revealed that IL-7 preferentially expanded CD8 memory cells (CD45RO+) and CD8+ lymphocytes expressing activation molecules. IL-7 was also able to support the growth of CD4+ lymphocytes, while having no effect on natural killer (NK)/K lymphocytes. Taken together, these data suggest that IL-7 acts cooperatively with the antigen supporting in vitro maturation of CTLp into functional cytotoxic effectors. Thus IL-7 may be an important biologic entity to consider as part of future immune-based therapies in which ex vivo expansion of antigen-driven CTL is an important determinant.

Activity of Outpatient Intravenous Interleukin-2 and Famotidine in Metastatic Clear Cell Kidney Cancer

Outpatient daily intravenous infusions of interleukin-2 (IL-2) have been developed to maintain anticancer activity and decrease toxicity of this agent against kidney cancer. Lymphokine activated killer cell (LAK) numbers are increased with these IL-2 schedules. Famotidine may enhance the LAK activity by increasing IL-2 internalization by the IL-2 receptor on lymphocytes. Fifteen patients with metastatic clear cell kidney cancer received IL-2 18 million IU/M² intravenously over 15-30 minutes preceded by famotidine 20 mg IV daily for 3 days for 6 consecutive weeks as outpatients. Cycles were repeated every 8 weeks. Patient characteristics were seven males/eight females, median age 59 (range: 28-70), median Eastern Cooperative Oncology Group (ECOG) performance status-1; common metastatic sites were lungs (14), lymph nodes (9), liver (4), bone (4), and pancreas (4). Prior systemic therapies were oral tyrosine kinase inhibitor (8), IL-2 (6), and mTor inhibitor (2). Most common toxicities were rigors, arthralgia/myalgia, nausea/emesis, fever, and hypotension. All episodes of hypotension were reversible with intravenous fluid. No patients required hospitalization due to toxicity. One complete response (7%) and four partial responses (26%) were seen (total response rate=33%; 95% confidence interval: 15%-59%). Responses occurred in the lungs, liver, lymph nodes, and bone. Outpatient intravenous IL-2 with famotidine has activity in metastatic clear cell kidney cancer.