Lymphokine-activated Killer Therapy Research Articles

Postoperative immunosuppression cascade and immunotherapy using lymphokine-activated killer cells for patients with esophageal cancer: Possible application for compensatory anti-inflammatory response syndrome

Immunological parameters were measured in order to elucidate a postoperative immunosuppression mechanism in transthoracic esophagectomy for patients with esophageal cancer. Moreover, lymphokine-activated killer (LAK) cells were transferred just after the surgery to overcome the postoperative immunosuppression. Fifteen consecutive patients who underwent transthoracic esophagectomy were subjected to the postoperative measurement of immunological parameters. Ten patients who underwent open cholecystectomy served as controls. Heparinized venous blood was obtained pre- and postoperatively, and serum levels of cytokines IL-6 and IL-10 and immunosuppressive acidic protein (IAP) were measured. Peripheral blood lymphocytes were harvested and analyzed by flow cytometry for phenotype detection and by a mixed lymphocyte reaction for detecting concanavalin (Con)-A-induced or -non-induced suppressor activity. Another 29 consecutive patients who underwent transthoracic esophagectomy were randomly enrolled in a postoperative immunotherapy trial either with or without lymphokine-activated killer cells. It was found that, in the esophagectomy group, IL-6 and IL-10 increased postoperatively and peaked on day 1, followed by an increase in IAP, peaked again on day 4, with a profound decrease in helper and cytotoxic T-cell subsets, followed by increases in Con-A-induced (on day 7 or later) and spontaneous (on day 10) suppressor activities. These changes were minimal in the cholecystectomy group. LAK cell transfer restored the postoperative decrease in the helper and cytotoxic T-cell population, and there was a trend of reduction for postoperative remote infection such as pneumonia and surgical site infection in the LAK therapy group. Taken together, we would like to propose the existence of a postoperative immunosuppression cascade consisting of increases in cytokines and immunosuppressive proteins, decreases in helper and cytotoxic T-cell populations, and the development of suppressor T-cell activities in surgery for esophageal cancer. Postoperative adoptive transfer of LAK cells may be a novel clinical application in surgery for esophageal cancer as a means of treating this postoperative immunosuppressive condition that may be identical to the status of compensatory anti-inflammatory response syndrome (CARS).

Effect of intraportal adoptive immunotherapy on liver metastases after resection of pancreatic cancer

The prognosis of patients with resected pancreatic cancer remains poor. This study evaluated the effect of adoptive immunotherapy (AIT) using intraportal infusion of lymphokine-activated killer (LAK) cells after curative resection and intraoperative radiation therapy (IORT) on advanced pancreatic cancer. Twenty-nine consecutive patients with advanced pancreatic cancer (Japan Pancreas Society stage III or IV) were divided into two groups. The control group (n = 17) underwent tumour resection and IORT. The treatment group (n = 12) underwent resection, IORT and intraportal infusion of LAK cells combined with recombinant interleukin 2 (rIL-2). The incidence of liver metastasis and the survival rate of these two groups were compared. Although the overall survival between groups was not statistically different (P = 0.082), there were more patients (four) alive 3 years after operation in the test group (36 per cent versus zero), and the incidence of liver metastases in the treatment group was significantly lower (three of 12 versus ten of 15; P < 0.05). LAK therapy influenced survival positively in multivariate analysis. These preliminary observations suggest that AIT warrants further study as a possible adjuvant for patients undergoing curative resection and IORT for pancreatic cancer.

Clinical Study of Brain Metastasis of Renal Cell Carcinoma

Objectives: To evaluate the natural history and the efficacy of treatments for renal cell carcinoma (RCC) with brain metastasis, we reviewed 18 patients with this disease. Methods: Out of 325 cases with RCC treated at Osaka University Hospital from 1957 to 1993, 18 (5.5%, male:female ratio 16:2) cases developed brain metastases. Median follow-up was 44 months after the initial treatment of the primary lesion. Twelve patients had surgical resection of brain metastases (surgical group), and 7 of them received adjuvant radiotherapy. Six patients with poor performance status were treated with supportive therapy alone (nonsurgical group). Results: Of 18 RCC patients with brain metastasis, 16 were male and 2 female. All brain metastases except for 1 case were symptomatic. Median interval between the initial treatment of the primary lesion and the diagnosis of brain metastasis was 19 months. The most frequent metastatic site prior to brain was the lung, which was detected in 7 cases (38.9%). Median survival of the entire group, measured from the onset of brain metastasis, was 9.5 months. One-year survival rate after the diagnosis of brain metastasis was 43.2% (64.8% in surgical group, 0% in nonsurgical group), 3-year 18.5% and 5-year 0%. Among 109 metastatic RCC, 14 patients were treated by lymphokine-activated killer (LAK) therapy. Out of 14 metastatic RCC patients treated by LAK therapy, 3 (21.4%) developed brain metastases. On the other hand, out of 95 metastatic RCC patients without LAK therapy, 15 (15.8%) had brain metastases. There was no significant difference in the rate of brain metastases between these two groups. Conclusion: There was a trend for prognosis of the surgical group to be better compared to that of the nonsurgical group, although it is not statistically significant. The optimum treatment for brain metastasis of RCC remains undefined, but our data suggested surgical resection in selected patients might contribute to prolonged survival of patients with brain metastasis. LAK therapy was not necessarily the risk factor of the brain metastasis.

Effect of lymphokine-activated killer cells with or without radiation therapy against malignant brain tumors.

The use of autologous lymphokine-activated killer (LAK) cells to treat malignant brain tumors was evaluated in 10 patients, one with metastatic malignant melanoma and nine with malignant glioma. LAK cells were obtained by culturing autologous peripheral blood lymphocytes with human recombinant interleukin-2 (rIL-2) for 7-28 days. All patients underwent surgery to remove as much tumor as possible and an Ommaya reservoir was implaced in the tumor cavity. Two of the 10 patients had received radiotherapy elsewhere, so were treated with LAK cells alone. Eight patients were treated with a combination of LAK cells and radiotherapy, using 1.8-2.0 Gy fractions given five times a week with a total dosage between 54 and 65 Gy. LAK cells and rIL-2 were injected to the tumor cavity via the Ommaya reservoir once a week for inpatients and once a month for outpatients. The duration of the LAK therapy ranged from 3 to 23 months (mean 13.7 mos). Neuroimaging evaluation revealed two complete responses, three partial responses, four no changes, and one progressive disease. In one patient with pontine glioma, the Karnofsky performance score was raised from 20 to 60. There were no side effects after the injection of LAK cells and rIL-2. The results suggest low-dose LAK therapy is a useful and safe treatment modality for malignant brain tumors.

Targeting therapy for glioma by LAK cells coupled with bispecific antibodies

Targeting of T cells or natural killer (NK) cells to tumours by using bispecific antibodies has attracted increasing interest in the past few years. We treated 31 patients with malignant glioma using adoptive transfer of lymphokine-activated killer (LAK) cells coupled to a bispecific antibody (anti-CD3+anti-glioma) as post-operative adjuvant therapy. Although this study excluded patients with deeply-seated tumours or a poor performance status, approximately 50% of the patients remained alive after 3 years, and 40% were free of recurrence. Serial CT scans revealed disappearance of remnant tumours in some patients. In addition, CT-guided stereotactic biopsy of the tumour in 3 patients showed extensive necrosis and degeneration after specific targeting therapy (STT). Five patients developed acute infection, and one of them died of bacterial meningitis. Our results suggest that antibody-targeted LAK therapy can achieve a higher response rate in patients with standard LAK therapy or any type of conventional treatment.

Lysis of endothelial cells by autologous lymphokine-activated killer cells.

The mechanisms of lysis of endothelial cells derived from human umbilical vein (HUVEC) by autologous lymphokine-activated killer (LAK) cells, generated from cord blood lymphocytes of the same donor, were investigated. Freshly isolated HUVEC as well as HUVEC cultured for several passages were efficiently lysed by autologous LAK cells, and their susceptibility to the LAK cells was almost the some as that of allogenic HUVEC. Complement-depletion experiments revealed that the lysis was mainly dependent on CD16+ natural killer (NK) LAK cells. pretreatment of HUVEC with recombinant interferon gamma (rIFN gamma) for 24 h made them resistant to lysis by autologous LAK cells, while pretreatment with either rIL-1 beta. rTNF alpha, or acidic or basic fibroblast growth factor did not alter the lytic sensitivity of HUVEC. The resistance of rIFN gamma-treated HUVEC was specific to lysis by CD16+ NK LAK cells, and their lysis by CD3+ T-LAK cells was not significantly altered. Moreover, in comparison with control HUVEC or rIL-1 beta-treated HUVEC, rIFN gamma-treated HUVEC had a significantly less potent inhibitory effect on the lysis of untreated HUVEC, when used as an unlabeled target. This suggests that rIFN gamma treatment may down-regulate the recognition of some molecules on HUVEC by rIL-2-activated NK cells. These data suggest that damage of the endothelium during LAK therapy is mainly dependent on LAK cells with a NK phenotype that can specifically recognize a certain molecule on autologous endothelial cells.

Use of N-acetyl cysteine to increase intracellular glutathione during the induction of antitumor responses by IL-2.

IL-2 therapy can induce marked oxidative stress via reactive oxygen and nitrogen intermediates. Glutathione, the major intracellular reductant, may become rate limiting to cytotoxic lymphocyte activation and proliferation under these circumstances. N-Acetyl cysteine (NAc-cys) was used to increase intracellular glutathione levels during lymphokine-activated killer (LAK) cell activation by IL-2. Incubation of splenocytes with NAc-cys (0.6 to 1.0 mM) resulted in significant changes in intracellular reduced and total glutathione (92% and 58% increase, respectively) at 96 h. These levels correlated with markedly enhanced cell proliferation (threefold) and cytolytic effector cell generation (> fivefold increase in LU/10(6) cells) induced by the combination of NAc-cys with IL-2. IL-2 exposure by itself unexpectedly increased intracellular reduced glutathione by 43%. IL-2 and NAc-cys were synergistic in increasing glutathione levels (reduced glutathione: 292% increase; total: 251% increase). Inhibition of glutathione synthesis, using L-buthionine-(S,R)-sulfoximine reversed the effects of NAc-cys on intracellular glutathione, as well as cellular proliferation and cytotoxicity. This experiment established that the effects of NAc-cys required de novo glutathione synthesis. In conjunction with IL-2/LAK treatment, oral NAc-cys administration (260 to 900 mg/kg/day for 7 days) significantly decreased tumor progression in a refractory s.c. tumor model. A small fraction of mice (11 to 17%) had complete tumor regressions. NAc-cys may be useful as an adjunct to increase the antitumor activity of IL-2/LAK therapy.

Colon adenocarcinoma cells inhibit anti-CD3-activated killer cell induction.

Adoptive immunotherapy with lymphokine-activated killer (LAK) cells has shown some promise in the treatment of certain cancers that are unresponsive to conventional treatment approaches. However, colon adenocarcinomas tend to respond poorly to LAK therapy, possibly as a result of tumor-induced immunosuppression. Recently, in vivo administration of anti-CD3 antibody has been shown to induce mouse T lymphocytes to mediate major-histocompatibility-complex(MHC)-unrestricted tumoricidal activity which is distinct from natural-killer-cell-derived LAK activity. It has therefore been suggested that anti-CD3 therapy may find application in tumor immunotherapy in humans. However, the effectiveness of anti-CD3-activated killer cell induction within the environment found in the vicinity of colon adenocarcinoma cells has not been evaluated. The present report demonstrates that colon cancer cells of human (HT-29) and mouse (MCA-38) origin markedly inhibit the generation of activated killer cells in murine spleen cell cultures. DNA synthesis and interleukin-2 production by spleen cells following stimulation with anti-CD3 antibody are also profoundly depressed in the presence of MCA-38 and HT-29 adenocarcinoma cells. MCA-38- and HT-29-mediated inhibition of activated killer cell development is exerted through the production of a tumor-associated soluble factor that is distinct from transforming growth factor beta or prostaglandins. Local immunosuppression associated with sites of tumor growth may therefore represent a major obstacle to successful anti-CD3 immunotherapy of certain colon adenocarcinomas.

Combination of lymphokine‐activated killer cells and interleukin‐2 in treating metastatic renal cell carcinoma

To study the properties of lymphokine-activated killer (LAK) cells and the effect of immunotherapy with a combination of autologous LAK cells and interleukin-2 (IL-2) [LAK therapy] in 10 patients with metastatic renal cell carcinoma (RCC). The LAK cells were generated from peripheral blood lymphocytes (PBL) by incubation in a serum-free medium (AIM-V) supplemented with IL-2 for 4 days and killer cells were administered intravenously twice a week. The LAK cells showed cytotoxicity against allogenic RCC cell lines and augmented NK and LAK activities. Their phenotypes were CD25+, HLA-DR+, CD3+, and CD16+. Furthermore, LAK cells released IFN-gamma, IL-1 beta, and TNF-alpha. The total number of LAK cells administered ranged from 3.8 x 10(9) to 52.6 x 10(9) cells and the total amount of IL-2 ranged from 150 x 10(5) to 900 x 10(5) U. The effect on pulmonary metastasis in response to LAK therapy was studied. The outcome was complete response (1), partial response (1), minor response (2), no change (4) and disease progression (2). Toxic effects were transient and no serious side-effects occurred. Evaluation of host immune parameters indicated that a clinical response was expected in patients with increasing proportions of CD16+, CD25+, CD57+, HLA-DR+ and CD3+DR+ cells among PBL and with augmentation of NK and LAK activities. Brain metastases were detected in three patients during or after treatment. LAK therapy appears to be effective in treating some patients with RCC and pulmonary metastasis. The potential for inducing brain metastasis, however, should be taken into account.

Ｌｙｍｐｈｏｋｉｎｅ−ａｃｔｉｖａｔｅｄ ｋｉｌｌｅｒ（ＬＡＫ）療法が奏効した肺転移を伴った無色素性悪性黒色腫の１例

An amelanotic malignant melanoma successfully responded to lymphokine-activated killer (LAK) therapy is reported. A 62-year-old male was referred to our department complaining of tumor on the upper right gingiva (on October 23, 1990). Clinical examination revealed tumor formation on the upper right alveolar gum and multiple lung metastases. He was diagnosed as an amelanotic malignant melanoma histopathologically. He was treated with chemotherapy (DTIC, ACNU, and VCR) and adoptive immunotherapy with LAK cells which were generated in serum-free RD5F medium. After 2 course of chemotherapy and 5 course of LAK therapy, the size of lung metastases was reduced significantly.

Immunological reconstitution after high-dose chemotherapy and autologous blood stem cell transplantation for advanced ovarian cancer

We evaluated the immunological reconstitution of patients who underwent high-dose chemotherapy and autologous blood stem cell transplantation (ABSCT) for advanced ovarian cancer. Sixty days after transplantation a complete reconstitution of lymphocytes and of the CD3, CD4, CD8, CD19, and CD16/56 subsets was observed in this series. A significant increase in the count of interleukin-2 receptor expressing lymphocyte (CD25) was found on day +60 after transplantation compared to that obtained at diagnosis and before transplantation. A significantly higher lymphokine-activated killer (LAK) precursor activity was seen on day +60 compared to the values obtained at diagnosis and before transplantation while natural killer activity did not show any significant variation. We conclude that ABSCT gives prompt and complete immunohaematopoietic reconstitution after high-dose treatment. Moreover, our data support the feasibility of interleukin-2/LAK therapy as consolidative therapy after ABSCT.

Lnterleukin-2 ± Lymphocytes as Consolidative Immunotherapy after Autologous Bone Marrow Transplantation for Hematologic Malignancies

Patients who undergo autologous bone marrow transplantation (ABMT) for advanced hematologic malignancies experience a high relapse rate. Therapy with interleukin-2 (IL-2) +/- lymphokine-activated killer (LAK) cells has induced clinical responses in some patients with advanced malignant lymphoma (ML) or acute myelogenous leukemia (AML). It is postulated that IL-2 +/- LAK cells represents a potentially non-cross-resistant therapeutic modality which might prevent or delay relapses if used as consolidative immunotherapy after ABMT, at a time of minimal residual disease. Therefore, we first studied the reconstitution of IL-2-responsive LAK precursor cells after ABMT and found them in the circulation as early as 3 weeks after ABMT. A phase Ib clinical trial was then performed which identified a tolerable IL-2 regimen which could be administered early after ABMT and which could induce immunomodulatory effects. We then initiated a clinical trial to determine the feasibility of generating and administering autologous LAK cells using this IL-2 regimen after ABMT for 16 patients with ML. The results show that IL-2+LAK therapy early after ABMT is feasible but is more toxic than IL-2 alone. Patients with AML on the phase I IL-2 trial and with ML on the IL-2+LAK protocol were evaluated for tumor status. Of 8 patients with AML in first relapse or at a later stage who underwent ABMT and received IL-2, 2 have relapsed, while 6 remain in complete remission 26+ to 40+ (median 28+) months after ABMT. Of 16 patients with ML considered at high risk for relapse who were treated with ABMT+IL-2+LAK, 5 have relapsed, while 11 remain in complete remission at 6+ to 21+ (median 10+) months after ABMT. The results in both trials are quite encouraging and appear to be better than those in nonrandomized historical controls at our institution. Prospectively randomized trials of IL-2 versus no IL-2 after ABMT in such patients are being initiated to assess definitively the effect, if any, on the relapse rate.

Lymphokine-Activated Killer Traffic Assay and Our Preliminary Clinical Results of Regional Arterial Infusion of Lymphokine-Activated Killer Cells for Renal Cell Carcinoma

In vivo distribution of radiolabeled lymphokine-activated killer (LAK) cells that were administered either through the caudal vein or the abdominal aorta of Lewis rats was studied. The LAK traffic study showed remarkable increase in radioactivity in several organs peripheral to the arterial injection site. We began treating the patients with metastatic renal cancer by means of regional arterial administration of LAK cells. This treatment was supported by the results of the traffic assay. Our regional LAK therapy, which involves a weekly leukapheresis in combination with daily systemic low dose interleukin-2 administration, has been in practice for over 2.5 years. Seven out of 12 metastatic lesions in the 7 patients treated showed regression. Two responding cases have been alive for over 2 years. Responding lesions were bone, muscle, para-aortic lymph node, and/or retroperitoneum. The regional arterial administration of LAK cells is local therapy. However, this mode of therapy could be useful for those patients with extrapulmonary nonresectable metastasis and at least one injectable feeding artery. This study shows our laboratory data concerning the localization of LAK cells transferred via arterial route and our clinical cases treated with regional arterial administration of LAK cells.

Treatment of malignant ascites with allogeneic and autologous lymphokine-activated killer cells

Two cases of peritonitis carcinomatosa with gynecological cancer, which did not respond to conventional treatment, were treated by intraperitoneal adoptive immunotherapy with allogeneic or autologous lymphokine-activated killer (LAK) cells plus interleukin-2. In case 1, reduction of acute ascites and disappearance of malignant cells from the peritoneal fluid and decreased levels of tumor markers (CA12-5 and CA19-9) were demonstrated during the treatment. In case 2, which also received additional treatment for pleuritis carcinomatosa, reduction of ascites and pleural effusion and disappearance of malignant cells from the pleural fluid were noted. In these cases allogeneic LAK therapy was well tolerated. This therapy did not prolong the patients' lives, but the findings indicate that it had appreciable local antitumor effects on peritonitis and pleuritis carcinomatosa in these patients with gynecological cancer.

Treatment of malignant ascites with allogeneic and autologous lymphokine-activated killer cells

Two cases of peritonitis carcinomatosa with gynecological cancer, which did not respond to conventional treatment, were treated by intraperitoneal adoptive immunotherapy with allogeneic or autologous lymphokine-activated killer (LAK) cells plus interleukin-2. In case 1, reduction of acute ascites and disappearance of malignant cells from the peritoneal fluid and decreased levels of tumor markers (CA12-5 and CA19-9) were demonstrated during the treatment. In case 2, which also received additional treatment for pleuritis carcinomatosa, reduction of ascites and pleural effusion and disappearance of malignant cells from the pleural fluid were noted. In these cases allogeneic LAK therapy was well tolerated. This therapy did not prolong the patients' lives, but the findings indicate that it had appreciable local antitumor effects on peritonitis and pleuritis carcinomatosa in these patients with gynecological cancer.

Histological evidence of natural killer cell aggregation against malignant melanoma induced by adoptive immunotherapy with lymphokine-activated killer cells.

Adoptive immunotherapy with lymphokine-activated killer (LAK) cells and systemic administration of recombinant Interleukin-2 (RIL-2) was carried out in a case of malignant melanoma with lung metastases. Histological specimens from the lung showed a metastatic melanoma heavily invaded by atypical lymphoid cells with convoluted nuclei of varying size. Immunohistochemistry revealed that these cells had the characteristic exclusively of natural killer cell (Leu-7+). Nodules of these cells mimicked the appearance of non-Hodgkin's lymphoma of pleomorphic type. Molecular cytogenetic analysis, however, showed the absence of rearranged bands for the T-cell receptor beta-chain gene, indicating the absence of T-cell clones. At autopsy, 1 month after the LAK therapy, the heavy invasion of convoluted cells had disappeared. These findings clearly indicate that the LAK cell plus RIL-2 therapy induced Leu-7+ lymphoid cells, phenotypically suggestive of natural killer cell aggregation in the tumours.

Generation and expansion of lymphokine-activated killer cells from lymph node lymphocytes in human lung cancer

We cultured lymph node lymphocytes (LNL) from lung cancer patients in the presence of recombinant interleukin 2 (rIL2). The ability of LNL to respond to rIL2 was not affected by the advance of cancer stage when tested for proliferation and for lymphokine-activated killer (LAK) activity. The LAK activity of LNL was comparable to that of the corresponding peripheral blood lymphocytes. The rIL2-induced proliferation of macrophage-depleted LNL was augmented by the reconstitution with autologous alveolar macrophages while the LAK activity was not affected. However, macrophage-reconstituted LNL expanded rapidly and reached higher cell densities and exhibited a significantly lower LAK activity than macrophage-depleted LNL. The diminished LAK activity in macrophage-reconstituted LNL were markedly augmented by the subculture at a low cell density. From these results, we conclude that LNL can be a good material for the postoperative LAK therapy and that macrophage is useful in culture of LAK cells.

Regional transarterial infusion of autologous lymphokine-activated killer (LAK) cells for advanced renal cell carcinoma and its preliminary clinical result

We herein report the preliminary but appreciable results of regional transarterial infusion of 2 gravity subtypes of autologous lymphokine-activated killer (LAK) cells into the metastatic sites in combination with systemic recombinant interleukin-2 (rIL-2) administration in 3 patients with advanced renal cell carcinoma. Leukapheresis was performed once a week and peripheral blood lymphocytes were separated into 2 different subtypes by Percoll gradient centrifugation. These lymphocytes were incubated with rIL-2 for a few days to induce LAK cells. LAK cells were transferred to the metastatic lesions through cannula twice a week. A large iliac bone metastasis disappeared 3 months after the initial LAK cell therapy via a superior gluteal artery. A case of complete disappearance of psoas muscle and para-aortic lymphnode metastasis as well as partial regression of a lumbar bone metastasis was seen after lumbar arterial infusion treatment. Another case with brain metastasis showed a rapid exacerbation of brain edema after one week's LAK therapy. Our treatment modality seems to be worthwhile and promising for treatment of the advanced renal cell carcinoma.