自体LAK细胞和rIL－2治疗晚期肾癌的实验与临床研究

应用自体ＬＡＫ细胞和重组白细胞介素－２（ｒＩＬ－２）治疗２０例晚期肾癌患者。自患者周围血分离到的单个核细胞（ＰＢＭＣ）体外经ｒＩＬ－２短期培养，其ＮＫ、ＬＡＫ活性明显增强并于第５、７天达高峰。当这些ＬＡＫ细胞与ｒＩＬ－２过继回输给同一患者后，病人周围血ＮＫ、ＬＡＫ活性明显增加（Ｐ＜０．０ｌ），ＮＫ比率、ＩＬ－２受体表达明显增加（Ｐ＜０．０５），提示对肾癌患者的免疫调节作用。本组病人获部分缓解（ＰＲ）ｌ例，轻度缓解（ＭＲ）３例，平均缓解期５个月。毒副作用主要表现为发热、寒战，病人能耐受，表明ＬＡＫ／ｒＩＬ－２疗法是安全的方法。     

Immunotherapy of cancer with lymphokine-activated killer cells and recombinant interleukin-2

Lymphokine-activated killer (LAK) cells can be generated in vitro by incubation of normal murine or human lymphoid cells in recombinant interleukin-2 (RIL-2). These LAK cells are capable of mediating the lysis of fresh, noncultured tumor cells in 4-hour chromium release assays. The adoptive transfer of LAK cells plus RIL-2 is capable of mediating the inhibition of established pulmonary micrometastases from syngeneic tumors in mice. High-dose RIL-2 administered alone is also capable of mediating these antitumor effects, probably via the production of LAK cells in vivo. The immunotherapeutic effect of RIL-2 but not of LAK cells plus RIL-2 is abrogated in hosts that have received preirradiation with 500 rads. The administration of high-dose RIL-2 is also capable of reducing the growth of solid subdermal tumors as well. The use of LAK cells in conjunction with RIL-2 may be applicable to the treatment of cancer in humans, and clinical trials to evaluate this approach in humans have begun.     

An adoptive immunotherapy of patients with medulloblastoma by lymphokine-activated killer cells (LAK)

An adoptive immunotherapy of 6 patients with medulloblastoma by lymphokine-activated killer (LAK) cells is described. They were from 2 to 9 years in age and had cerebrospinal fluid (CSF) dissemination of the tumours. All patients underwent the whole-neuraxis irradiation and chemotherapy. After the usual treatments, they were submitted to an adoptive transfer of one-haplotype identical LAK cells. The LAK cells were induced from peripheral blood lymphocytes (PBL) of their relatives with human recombinant interleukin-2 (rIL-2). 3 - 15 x 10(9) LAK cells were transferred intrathecally in 2-3 months. In 3 of 6 patients, neurological signs were improved and malignant cells had never been detected on CSF cytology after the adoptive immunotherapy. One among these 3 patients showed complete response in 20 months. Thus, this is an attractive approach for the treatment of medulloblastoma with CSF dissemination of the tumour which current therapeutic intervention can not cure.     

Injection of interleukin-2 (IL-2) into the tumor-bearer's spleen augments lymphokine-activated killer (LAK) activity in vitro and inhibits tumor metastasis

The effect of interleukin-2 (IL-2), injected directly into the tumor-bearer's spleen, on inhibition of tumor metastasis was evaluated. C3H/HeN mice were inoculated intradermally with 2 X 10(6) X5563 syngeneic tumor cells on day 0, and the tumor was surgically resected on day 10. The operation failed to prevent tumor death of these mice within 3 weeks. Autopsy of these mice revealed that death was due to systemic metastasis of tumor cells to lymphoid organs including the liver although the tumors had been successfully removed without any visible local recurrence. In this model, we administered IL-2 by intrasplenic injection daily for 3 days after operation. Mice treated with an intrasplenic injection of IL-2 showed a significantly prolonged survival time. Histological findings after this treatment revealed lymphoid cell proliferation of the spleen, no metastatic foci were found in the liver. Lymphokine-activated killer (LAK) activity from IL-2 injected spleen was also augmented. Intravenous (i.v.) and subcutaneous (s.c.) administration of IL-2 were not effective. A major difficulty in achieving significant immunologic effect in vivo by IL-2 infusion is the relatively short half-life of IL-2. Therefore IL-2 administered directly into the responding lymphoid organ is theoretically reasonable. In fact, treatment with intrasplenic injection of IL-2 significantly augmented the antitumor activity. Splenic arterial infusion of IL-2 may be an appropriate route of administration for adjuvant immunotherapy in human cancer.     

Effects of serum immunosuppressive factors on the cytotoxicity of lymphokine-activated killer (LAK) cells induced by recombinant interleukin 2(R-IL2)

Fundamental studies were performed on adoptive immunotherapy, especially on effects on lymphocytic cytotoxic activity, of nonspecific immunosuppressive factors (ferritin, IAP, AFP) and of serum factors obtained from gastric cancer patients, and possible intervention of suppressor T cells in serum immunosuppressive activity on the cytotoxicity was also examined. The following results were obtained. 1) Cytotoxicity of LAK cells induced by culturing normal peripheral blood lymphocytes (PBL) with R-IL2 in the medium containing normal AB-type sera, was higher than that of PBL. 2) Effect of nonspecific immunosuppressive factors on cytotoxicity of LAK cells was lower than that on cytotoxicity of PBL. 3) Cytotoxicity of PBL was inhibited in a relatively specific fashion by sera from patients of the cancers which were of identical histological types with the target tumor cells, while that of LAK cells was hardly inhibited by patients' sera. 4) Cytotoxicity of Leu 15-PBL was inhibited by nonspecific immunosuppressive factors and also by cancer patients' sera in a relatively specific fashion in relation to histology. 5) Cytotoxicity of Leu 15-LAK cells was hardly inhibited by serum nonspecific and specific immunosuppressive factors. The above results showed that serum immunosuppressive factors might act on PBL cytotoxicity without intervention of suppressor T cells, and that LAK cells were hardly inhibited by such immunosuppressive factors. All these results suggested usefulness of adoptive immunotherapy with LAK.     

The intrapleural administration of recombinant interleukin-2 (rIL-2) to patients with malignant pleural effusion: Clinical trials

Recombinant IL-2 (rIL-2) was administered intra-pleurally according to an original protocol to 11 patients with malignant pleural effusion, 7 of whom suffered from breast cancer and 4 from esophageal cancer. The pleural effusions either disappeared or decreased roentgenographically, and malignant cells disappeared from all 13 pleural cavities in the 11 patients, confirming the validity of this therapy to be 100%. The mean survival time from the initial administration of rIL-2 was 15.9 months. We ensured that the concentration of IL-2 in the effusion was maintained at a high level for a sufficient period of time, and that the lymphokine-activated killer (LAK) activity of lymphocytes in the effusion was augmented. Fever, eosinophilia, and a transient increase in the pleural effusion were the main side effects, but the symptoms were temporary and not serious. The results of this study therefore suggest the efficacy of intrapleural rIL-2 for patients with malignant pleural effusion.     

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.     

Interstitial nephritis in a patient receiving adoptive immunotherapy with recombinant interleukin-2 and lymphokine-activated killer cells.

A patient receiving adoptive immunotherapy with recombinant interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells for metastatic melanoma developed acute renal insufficiency out of proportion to the decrease in her renal perfusion after 4 days of receiving IL-2. On the 8th day of IL-2 and the 3rd day of LAK cell infusion she expired suddenly from an acute intra-abdominal hemorrhage due to rupture of a metastasis. Postmortem examination of the kidneys showed an interstitial infiltrate consisting largely of lymphocytes and concomitant tubulitis. Immunoperoxidase staining of the infiltrating cells with a panel of lymphocyte-specific monoclonal antibodies showed the majority of cells to be T lymphocytes (70-75%), with a more focal infiltrate of B lymphocytes (25-30%) and rare monocytes, granulocytes and natural killer cells. This patient represents the first reported case of acute interstitial nephritis associated with IL-2 immunotherapy. Our finding is consistent with the hypothesis that the acute renal failure that accompanies this therapy may sometimes be due to intrinsic renal disease as well as the usual pre-renal failure. Nephritis may be caused by IL-2-mediated effects on lymphocytes, resulting in renal parenchymal infiltration.     

Laparotomy enhances intraperitoneal tumor growth and abrogates the antitumor effects of interleukin-2 and lymphokine-activated killer cells

The interrelationship between host resistance to cancer and the trauma of a surgical procedure is the subject of much speculation. Extensive study of animal models and human subjects is required to define these effects and to provide a theoretical model by which to interpret these data. We used a murine model of intraperitoneal cancer to demonstrate the augmentation of tumor growth by surgical trauma. In this intraperitoneal tumor model, a surgical procedure that included entry into the abdominal cavity resulted in augmented tumor growth; a surgical incision on the skin of the animal's back did not promote tumor growth. The immunotherapeutic effects of interleukin-2 and lymphokine-activated killer cells were significantly reduced by the performance of a laparotomy. This abrogation of the effects of the immunotherapeutic regimen was observed for up to 14 days after laparotomy but was lost by days 35 to 42. Healing tissue may promote tumor growth, and these effects are dominant over immunotherapy with interleukin-2 plus lymphokine-activated killer cells.     

Sterile abscesses in glioma patients treated by intraparenchymal injection of lymphokine-activated killer cells and recombinant interleukin-2: case reports

Earlier, we conducted Phase I clinical trials to determine any acute toxicity of adoptive immunotherapy with intralesional injections of autologous lymphocytes expressing lymphokine-activated killer (LAK) activity and recombinant interleukin-2 (rIL-2) in patients with malignant glioma. Within six weeks of craniotomy and intralesional injection of autologous LAK cells plus rIL-2, 3 of 29 patients demonstrated a decline in clinical status and evidence on computed tomographic and magnetic resonance imaging scans of edema and mass of unknown character at the site of previous surgery and immunotherapy. Craniotomy was performed to remove the tissue and reduce intracranial pressure. Microscopic examination of the excised material indicated no new tumor growth within the resected mass, but rather that the tissue had the histological characteristics of a chronic sterile abscess including necrosis, fibrosis, and influx of inflammatory cells. Factors that may have contributed to this reaction in the 3 patients were age, Karnofsky score, the extent of tumor excision, and immune status. All 3 had also been treated with greater than average numbers of rIL-2 activated lymphocytes that demonstrated significant in vitro LAK activity. The results suggest that in patients whose clinical status is good and who are not immunosuppressed by corticosteroids, the dose-limiting toxicity of intraparenchymal immunotherapy with LAK cells plus rIL-2 for glioma may be related to the total, absolute number of activated cells injected, and this toxicity develops over time and is manifested by development of a sterile abscess.     

Adoptive immunotherapy using lymphokine-activated killer cells and recombinant interleukin-2 in preventing and treating spontaneous pulmonary metastases of syngeneic Dunning rat prostate tumor

Lymphokine-activated killer (LAK) cells were generated from splenocytes of rats bearing a weakly immunogenic Dunning prostate tumor (R-3227 AT-3) and activated with recombinant interleukin-2 (rIL-2). The maximal LAK activity was obtained from splenocytes of rats bearing tumors for 10 to 14 days after incubation with 1000 U/ml./day of rIL-2 for five to eight days. The majority of these LAK cells expressed high levels of asialo GM1 (89%), laminin (83%), OX-19 (80%) and OX-8 (88%) surface markers. LAK cells exhibited higher cytotoxicity to rat prostate tumor cells and mouse lymphoma in vitro than to other non-prostate tumor cells or normal rat splenocytes and thymocytes. Splenocytes of rats bearing prostate tumors have higher LAK activity than normal splenocytes. The Winn type assay showed that Dunning prostate tumor growth was inhibited effectively by LAK cells at a tumor cell:LAK cell ratio of 1:50. The therapeutic efficacy of LAK cells in the treatment of primary solid prostate tumors and pulmonary metastases of Dunning rats was evaluated. LAK cells in combination with rIL-2 showed a greater therapeutic benefit in 1) prevention of prostate tumor metastases to lung, 2) retardation of the primary tumor growth, 3) regression of spontaneously established pulmonary metastases, and 4) prolongation of survival as compared to untreated controls or those groups treated with LAK cells or rIL-2 alone. The results of this study indicate that the conjunctive therapeutic approach of using surgical therapy to remove primary solid tumors followed by adoptive immunotherapy with LAK cells plus in vivo administration of IL-2 may be potentially valuable in the treatment of prostate tumors, particularly for the spontaneous pulmonary metastases.     

Interleukin-2 and autologous lymphokine-activated killer cells in the treatment of malignant glioma. Preliminary report

Nine patients with malignant glioma were treated with the lymphokine interleukin-2 (IL-2) or with lymphokine-activated killer (LAK) cells, and one patient received combination therapy with both LAK cells and IL-2. The LAK cells were generated by culturing recombinant IL-2 with peripheral blood lymphocytes obtained from brain-tumor patients. Escalating doses of LAK cells (10(8) to 10(10] or IL-2 (10(4) to 10(6) U) were administered intraoperatively by direct injection into the brain tissue surrounding the cavity left by debulking the tumor. There were no signs of systemic or neural toxicity following treatment. The selective killing of the tumor by LAK cells used for these treatments was demonstrated by a chromium release microcytotoxicity assay which showed in vitro the ability of the LAK cells to lyse glioma cells but not normal cells.     

Study of cytotoxicity of recombinant interleukin-2 (rIL-2) expanded tumor infiltrating lymphocytes (TIL) in renal cell cancer

We studied subsets and cytotoxicity of recombinant interleukin-2 (rIL-2) expanded tumor infiltrating lymphocytes (TIL) from renal cell cancer (RCC) patients. TIL were successfully expanded in 13 of 14 RCC cases using anti-CD3 during initial 48 hours of culture. Percentages of CD8 positive cells among rIL-2 expanded TIL at 1 tp 4 week(s) of culture were 56.2 +/- 15.1% (range 26.2 to 79.8%, N = 13) and not necessarily predominant over CD4 positive cells. NK and LAK activities of TIL at 3 to 6 weeks of culture were 31.6 +/- 15.8% (range 1.4 to 57.4%, N = 9) and 16.6 +/- 11.6% (range 3.8 to 35.6%, N = 6), respectively. Autologous and allogeneic RCC cytotoxicity of TIL at 3 to 4 weeks of culture were 17.9 +/- 19.7% (range 0 to 47.6%, N = 4) and 18.9 +/- 14.8% (range 0 to 47.3%, N = 12), respectively. Since there was no statistical difference between them, autologous specific cytotoxicity was not demonstrated. From these results of present study, it is unlikely that most of effector cells of rIL-2 expanded TIL in autologous RCC lysis are major histocompatibility complex restricted cytotoxic T cells. And we concluded that it is doubtful that TIL is significantly superior over LAK cells in immunotherapy of human RCC.     

Adoptive immunotherapy of human meningeal gliomatosis and carcinomatosis with LAK cells and recombinant interleukin-2

Previous in vitro studies have demonstrated that peripheral blood lymphocytes activated with recombinant interleukin-2 (rIL-2) generated cells that were lytic for fresh autologous tumor cells but not for normal lymphocytes or lymphoblasts. Adoptive transfer of autologous lymphokine-activated killer (LAK) cells induced in vitro with rIL-2 was used in two patients: one with meningeal gliomatosis and the other with meningeal carcinomatosis. The adoptive transfer of LAK cells was very effective in reducing the clinical symptoms and signs, and in eliminating the malignant cells from the cerebrospinal fluid. Thus, this therapy is an attractive approach for the treatment of malignant tumors that have poor immunogenicity and are insensitive to several anti-cancer agents, and for patients with severe immunosuppressive conditions induced by repeated radiation therapy or chemotherapy.     

A new approach to the therapy of cancer based on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2

A new approach to cancer therapy has been developed based on the adoptive transfer of autologous lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (IL-2). Forty-one patients with advanced cancer who have failed all standard treatments were treated in this experimental protocol. Fourteen patients experienced an objective regression of cancer, including one patient with metastatic melanoma who underwent a complete regression. Objective responses were seen in patients with colorectal cancer, renal cell cancer, melanoma, and lung adenocarcinoma. The sites of tumor regression included subcutaneous tissue, lung, and liver. The major side effect of therapy resulted from the administration of high-dose IL-2 and was manifested primarily as fluid retention, resulting in a generalized capillary permeability leak syndrome. This approach to adoptive immunotherapy represents a promising approach to the therapy of patients with metastatic cancer. Attempts to increase the potency and decrease the toxicity of therapy and extend this treatment to patients with smaller tumor burdens are in progress.     

Experimental studies on induction of various lymphokine-activated killer cells using recombinant interleukin 2

Fundamental studies were performed on adoptive immunotherapy, especially on promotion of cell proliferation and on augmentation of cytotoxicity of various lymphokine-activated killer cells, induced by recombinant interleukin 2(R-IL2) with (P-LAK) or without (LAK) PHA, from peripheral blood lymphocytes (PBL) of healthy volunteers. The following results were obtained. 1) In LAK induced by culturing normal PBL with R-IL2, a cell proliferation was observed in 14 days. Their cytotoxic activity against all the strain cells examined, was higher than PBL already on the third day of culture. 2) Culturing normal PBL with PHA (with or without R-IL2) for 3 days, followed by culturing with R-IL2 for 11 days, caused a marked increase in cell number by about 65 times in 14 days. Cytotoxicity of these cells against MKN-28, MKN-45 and KATO III was found to increase with lapse of culture time. 3) On the 14th day of culture, cytotoxicity of LAK was higher than that of P-LAK. 4) Surface phenotype analysis of LAK revealed that OKT3+ (cell ratio) tended to increase, OKT8+ increased significantly, OKT4+ and Leu7+ tended to decrease, and OKT4+/OKT8+ ratio decreased significantly. Analysis of P-LAK revealed that OKT3+ and OKT8+ increased significantly and OKT4+, Leu7+, and OKT4+/OKT8+ ratio decreased significantly. 5) The rate of total increase in cytotoxicity, calculated in multiplying the rate of cell number increase by the rate of increase in cytotoxicity, was higher in P-LAK than in LAK. The above results showed that P-LAK induced by addition of PHA for the first few days, could cause marked increase both in cell number and in total cytotoxicity.     

A study of perforin appearing in human lymphokine-activated killer (LAK) cells derived from spleen and peripheral blood mononuclear cells]

Perforin is regarded as one of the main cytotoxic factors in such cells. We succeeded in cloning human perforin cDNA and recently used it to assess perforin appearing in LAK cells. To derive the LAK cells, mononuclear cells separated from a human spleen were mixed with recombinant interleukin-2 and cultured. Almost no perforin mRNA appeared on day 0 but definitely accelerated on day 1 and tended to decrease on and after day 2. Peak of perforin mRNA observed on day 1 was found to precede cytotoxic activity on K562 and Daudi cells by one day. Similar results were found in LAK cells derived from peripheral blood mononuclear cells. Regarding the expression of perforin mRNA on day 0 as 1, the values on day 1 and 2 were calculated as 3.6 and 1.8, respectively. Immunological staining using an antiperforin antibody revealed perforin in the cytoplasm of large cells formed blasts, this also confirmed derivation of perforin on the protein level. Flow cytometry indicated that cells containing perforin included most CD16+ NK cells and some CD8+ T lymphocytes. The fact that TNF and IFN were simultaneously derived together with perforin suggested that the collective joint effect of these substances results in cytotoxic activity.     

肿瘤浸润淋巴细胞、重组白细胞介素2治疗对手术后原发性肝癌病人细胞免疫功能的影响

对５例经手术切除的原发性肝癌病人用肿瘤浸润淋巴细胞（ＴＩＬ）和重组白细胞介素２（ｒＩＬ－２）治疗。治疗前１周及治疗后１个月检测细胞免疫功能，治疗后外周血淋巴细胞的ＮＫ活性、白细胞介素２产生及活性均明显升高；植物血凝素（ＰＨＡ）皮试阴性转阳性。提示；ｒＩＬ－２和ＴＩＬ应用可明显提高原发性肝癌病人的细胞免疫功能，对杀灭残留癌细胞，抗肿瘤复发、转移，提高远期疗效有一定作用。可能成为肝癌术后抗肿瘤复发，转移的重要疗法。     

Isolation perfusion in extracorporeal circulation with interleukin-2 and lymphokine-activated killer cells in the treatment of in-transit metastases from limb cutaneous melanoma

Background: Therapies of advanced melanoma patients with interleukin-2 (IL-2) and cytotoxic lymphocytes have produced interesting results, but a larger diffusion of these treatments is limited by the severe side effects due to IL-2 systemic infusion. A strictly regional administration of IL-2 and cells by an isolation perfusion (IP) in extracorporeal circulation (ECC) for the treatment of regional melanoma metastases could improve tolerability and efficacy of this specific modality of immunotherapy. Methods: Ten patients were submitted to adoptive immunotherapy with IL-2 and lymphokine-activated killer (LAK) cells by IP in ECC. The schedule of treatment included the first course of a 5-day systemic administration of IL-2 (Proleukin, EuroCetus 9–12 × 10⁶ IU/M²/day continuous infusion); autologous LAK cells were obtained via leukapheresis and after in vitro activation were given (range 8–28 × 10⁹) along with IL-2 (120-2,400 IU/ml of perfusion priming) to the affected limb by IP; IL-2 (9–12×10⁶ IU/m²/day) was also administered by systemic continuous infusion for 5 days starting on the day after IP. Results: All patients concluded the treatment without any major local or systemic toxicities. Clinical responses included one complete and six partial remissions; three patients had stable disease. All patients are alive. Follow-up after IP ranged from 12 to 35 months (median: 22). The analysis of circulating lymphocytes revealed the rapid disappearance of LAK cells, suggesting their extravasation and/or endothelial adhesion in perfused tissues. Conclusions: IP with IL-2 and LAK cells is a new approach for the treatment of in-transit metastases due to cutaneous melanoma. The treatment appears to be feasible and reliable. Further biological and immunological studies should permit amelioration of the present modality of treatment.