Intralesional infusion of lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2) for the treatment of patients with malignant brain tumor

Twenty patients with supratentorial, intracerebral lesions defined by computed tomographic scan or magnetic resonance imaging were treated by surgery and adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant Interleukin-2 (rIL-2, Cetus). Seventeen patients had glioblastoma, two had high-grade oligodendroglioma, and one patient had two metastatic sarcoma lesions. LAK cells were produced from blood mononuclear cells (MNC) obtained by 2 to 3 leukapheresis procedures and cultured (2.5 x 10(6) MNC/ml) 3 to 5 days with 1000 units rIL-2/ml. Although LAK cells could be produced from MNC of all patients, those taking steroids or with a low Karnofsky functional status generated, on average, suboptimal LAK cell activity. Age, sex, and serum anticonvulsant levels do not seem to influence a patient's ability to produce LAK cells in vitro. For therapy, cultured MNC (1-15 x 10(9] containing LAK cells were suspended in saline containing 10(6) units rIL-2 and injected into tissue surrounding the tumor cavity during craniotomy. For 3 days after their operations, patients received 10(6) units rIL-2 into the tumor cavity through an Ommaya reservoir. The treatment protocol was tolerated well by all patients, although they all experienced some degree of headache, fever, or lethargy that cleared within a few days of the last rIL-2 injection. When computed tomographic (CT) scans were obtained soon after treatment, areas of low density suggested a greater-than-normal extent of edema around the operative site. At the present time, CT scans indicate that the tumors of seven patients have recurred with an average disease-free interval of 25 +/- 6 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunomagnetic separation of infiltrating T lymphocytes from brain tumors

Tumor-infiltrating lymphocytes (TIL's) were isolated from human glioma biopsy specimens by immunomagnetic separation using T cell-specific monoclonal antibodies coupled to paramagnetic beads, and were expanded in culture with feeder cells and interleukin-2 (IL-2). The infiltrating cells from five of seven patients proliferated in culture. When tested after 2 to 3 weeks of culture, virtually all of the cells stained with antibodies against the CD2 and CD3 antigens. Most cells also expressed human leukocyte antigen class II molecules, while varying percentages of cells stained with antibodies against the IL-2 receptor and the CD4 and CD8 antigens. The cytotoxicity of the cultured TIL's against autologous and allogeneic glioma cells and the K562 and Daudi cell lines was measured and compared with that of lymphokine-activated killer (LAK) cells from the same patients. None of the TIL's showed significant cytotoxicity against these targets, whereas LAK cells lysed all of the targets.     

Augmentation of the lymphokine-activated killer cell response in head and neck cancer patients by combination interleukin-2 and interferon-alpha

The majority of patients with head and neck cancer present with advanced disease (stage III and IV), for which current chemotherapeutic regimens offer dismal results. Although known to have defects in their cell-mediated immunity, their poor performance status makes them unlikely candidates for aggressive immunotherapeutic protocols because of associated severe toxicities. This study evaluates the effect of subthreshold recombinant interferon-alpha (rIFN-alpha) and interleukin-2 (rIL-2) on the generation of lymphokine-activated killer (LAK) cells from the peripheral blood of patients with head and neck cancers. In vitro treatment of patients' lymphocytes consisted of incubation in 1,000 U/mL rIL-2, 100 U/mL rIL-2, 100 U/mL rIFN-alpha, and 100 U/mL rIFN-alpha plus 100 U/mL rIL-2 for 4 to 5 days. Cytotoxicity was measured using a standard 4-hour chromium-51 (51Cr)-release assay with Raji (B lymphoblastoid) tumor target cells. LAK activity was arbitrarily defined as greater than 20% cytolysis of Raji target cells. LAK activity was generated in a smaller percentage of the head and neck cancer patients by 1,000 U/mL rIL-2 compared with normal adult donors: 54% versus 100%, p less than 0.05; IFN-alpha (100 U/mL) induced LAK activity in approximately 50% of the cancer patients. The addition of rIFN-alpha (100 U/mL) to rIL-2 (100 U/mL) resulted in LAK generation in a higher percentage of patients (83% versus 54%), as well as increased levels of cytotoxicity, p = 0.05. This combination also resulted in cytotoxicity levels equivalent to high-dose (1,000 rIL-2 U/mL). These in vitro data support a clinical trial to assess the therapeutic efficacy of combined low-dose rIL-2 and rIFN-alpha in vivo in head and neck cancer patients.     

Improved in vivo efficacy of tumor-infiltrating lymphocytes after restimulation with irradiated tumor cells in vitro

Background: We investigated different culture conditions for tumor-infiltrating lymphocytes (TILs) with regard to proliferation, phenotypic changes, in vitro cytotoxicity, and in vivo therapeutic efficacy. Methods: After enzymatic digestion of the murine fibrosarcoma, MCA-105, TIL cultures were initiated as pure lymphocyte (groups 1 and 2) or mixed lymphocyte/tumor suspensions (groups 3 and 4). Group 1 TILs were grown in culture medium containing 100 IU/ml recombinant interleukin-2 (rIL-2). Group 2 TILs were stimulated with solid-phase anti-CD3 monoclonal antibody (mAb) for 48 h and cultured in rIL-2 (100 IU/ml)-containing medium. Group 3, which consisted initially of a surplus of tumor cells, received the same treatment as group 2. Group 4 was also activated with anti-CD3 mAb and rIL-2 but was additionally restimulated weekly with irradiated tumor cells (TILs to tumor, 20:1). Results: Groups 1 and 2 showed up to twofold higher increases in TIL numbers compared with groups 3 and 4 by the end of culture week 5. Although the original lymphocyte/tumor cell suspension consisted of 12.0 ± 3.8% CD4⁺ T cells and 5.3 ± 3.3% CD8⁺ T cells, all four TIL cultures showed 80% CD8⁺ TILs and no CD4⁺ TILs by the end of culture week 4. In vitro cytotoxicity did not correlate with in vivo efficacy of the examined TIL cultures. By using the MCA-105 pulmonary metastases model in C57BL/6 mice, only suboptimal doses of TILs (2 × 10⁶) from group 4, which had been restimulated weekly with irradiated tumor, showed significant tumor eradication compared with all other treatment groups (p<0.01). Conclusions: We conclude that in vitro tumor restimulation of TILs improves in vivo efficacy, most likely through the education of tumor-reactive T cells.Presented at the 48th Annual Meeting of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.     

Anti-tumor reactivity of human lymphokine activated killer (LAK) cells against fresh and cultured preparations of renal cell cancer

Immunotherapy utilizing the adoptive transfer of lymphokine activated killer (LAK) cells in conjunction with recombinant interleukin-2 (IL-2) is capable of mediating the regression of established cancer in a variety of animal tumor models as well as advanced metastatic cancers in humans. We have thus examined the variability of the anti-tumor lytic reactivity of LAK cells obtained from patients with metastatic renal cell cancer (RCC). Tumor cell suspensions were prepared by enzymatic digestion from 37 consecutive renal cell tumors. The mean (+/- SEM) total number of cells recovered was 1.5 +/- 2.2 X 10(9) cells per tumor. The percentage of tumor cells in the suspension was 39.1 +/- 3.3% (range: 6 to 75%). Thirteen of 13 different fresh renal tumor cell preparations tested in 57 experiments and tow of two renal tumor lines tested in 10 experiments were all lysed by LAK cells. RCC patients, like normal donors, generated good LAK effectors with broad antitumor activity against autologous as well as allogenic tumors. Both renal and nonrenal tumors were equally lysed by LAK cells. LAK killing of the erythroleukemic tumor lines K562 and Daudi was significantly better than the lysis of fresh autologous and allogeneic tumor targets or cultured RCC tumor lines. Short term tumor cultures derived from renal cancer preparations proved to be sensitive and reliable tumor targets for studying the in vitro killing by LAK cells. Clinical trials testing the therapeutic role of LAK cells and IL-2 in patients with advanced renal cell cancer are currently in progress.     

膀胱癌TIL的分离培养及其表型分析

对３例膀胱移行细胞病患者手术切除的肿瘤组织中肿瘤浸浸润性淋巴细胞进行了体外分离与培养，结果２例获得成功，ＴＩＬ体外扩增培数分别达５８－１７０，在培养２４ｄ时对自体肿瘤靶细胞的最高杀伤活性达４１％以上，且呈现一定的靶细胞特异性。     

Tumor-infiltrating lymphocytes derived from human renal cell carcinoma: Clonal analysis of its characteristics

Aim:   To assess the characteristics of activated tumor-infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4^- CD8⁺ TIL-clone derived from human renal cell carcinoma (RCC).
Methods:   Bulk TILs were expanded from a human RCC and the lymphocytes were separated into a CD8⁺ enriched population. Subsequently, using the limiting dilution technique, a TIL clone was established and its growth response, phenotype and cytotoxic activity were analyzed.
Results:   A clone, T16-13, by day 94 numbering 1 × 10⁷ cells, was harvested and characterized as a CD4^- CD8⁺ clone. On day 144, the cytotoxic activity of this clone against the autologous tumor was relatively high (2.3 ± 0.7 LU₃₀/10⁶ cells). Meanwhile, against allogeneic renal tumors, there was no cytotoxic activity (−0.1 LU₃₀/10⁶ cells).
Conclusions:   A TIL clone possessing modest autologous tumor-specific cytotoxicity can be isolated from human RCC. The characteristics analysis of various TIL clones may provide a better understanding of an RCC tumor microenvironment and may help to establish new modalities for the treatment of patients with metastatic kidney cancer.     

肾癌TIL的分离培养和其表型分析

对两例原发性肾癌患者手术切除肿瘤组织中肿瘤浸润性淋巴细胞（ＴＩＬ）进行了体外分离与培养。结果表明：ＴＩＬ体外扩增倍数分别达３２～２０３倍，对自体肿瘤靶细胞的最高杀伤活性达５３％和６４％，且呈现一定的靶细胞特异性。免疫组化分析结果：未经激活的ＴＩＬ细胞其膜抗原（ＣＤ３，ＣＤ４，ＣＤ８）的表达动态变化不大，但经ＩＬ－２激活的ＴＩＬ细胞随着培养无数的增加，其ＣＤ３细胞数比例及ＣＤ４／ＣＤ８比值上升明显，在培养至３２天时分别达９５％和１．６５。     

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.     

Substance P increases in vitro lymphokine-activated-killer (LAK) cell cytotoxicity against fresh colorectal cancer cells.

Gut-associated lymphoid cells are modulated by several gut hormones. We postulated that lymphokine-associated-killer (LAK) cell cytotoxicity of lymphocytes isolated from the gut mucosa may be increased by substance P (SP). Intestinal lamina propria mononuclear cells (LPMC) and colonic cancer cells were isolated from operative specimens by successive mechanical and enzymatic dissociation methods. Effector LAK cells were induced by culturing LPMC with recombinant interleukin-2 at a concentration of 250 U/ml. Substance P (10(-5) M) was added to the culture medium. Targets consisted of fresh colon cancer cells, HT-29 (cultured human colon cancer cell line), and control cell lines. After 4 days of incubation, cytotoxicity was measured using a 4-h 51Cr release assay. LAK cells alone showed moderate cytotoxicity against HT-29 and none against fresh colon cancer cells. LAK cells generated in the presence of substance P showed moderate cytotoxicity against HT-29 and strong cytotoxicity against fresh colorectal cancer cells. The percentage of cytotoxicity +/- SEM at various effector to target ratios was [(*) denotes P < 0.05 compared with above]: [table: see text] We conclude that substance P significantly increases LAK cell cytotoxicity against fresh colon cancer cells, but not against cultured cells.     

Induction of LAK cells and CTL in patients with brain tumor and research of its clinical application

We studied whether lymphokine-activated killer (LAK) cells and cytotoxic T lymphocytes (CTL) were capable of being induced in vitro from peripheral blood lymphocytes of patients with brain tumor. The LAK cells were generated by culturing recombinant IL-2 with peripheral blood lymphocytes. The culture was continued for 72 hours; then cytotoxicity to Hela cell was examined by 4-hr 51Cr release assay. LAK cells were induced from lymphocytes of patients with brain tumor, but the cytotoxicity was rather less than that of healthy subjects, and it was accompanied by clinical deterioration. CTL was generated by co-culture of patient's peripheral blood lymphocytes and autologous brain tumor cells with addition of rIL-2. The cytotoxicity to autologous and allogenic brain tumor cells was examined by 16-hr51Cr release assay. The cytotoxicity to autologous tumor was approximately 30-40%, and there was cross reaction to allogenic tumor cells. The adoptive transfer of CTL to four patients was performed. One patient improved clinically, and on CT scan, growth of the tumor appeared to have been reduced.     

Long-term follow-up of patients with recurrent malignant gliomas treated with adjuvant adoptive immunotherapy

Between August 1986 and October 1987, the Denver Brain Tumor Research Group conducted a clinical trial using autologous human recombinant interleukin-2 (rIL-2)-activated lymphocytes to treat 20 patients with recurrent high-grade gliomas. The trial involved surgical resection and/or decompression followed by intracavitary implantation of lymphokine-activated killer (LAK) cells and autologous stimulated lymphocytes (ASL) along with rIL-2 in a plasma clot. One month later, stimulated lymphocytes and rIL-2 were infused through a Rickham reservoir attached to a catheter directed into the tumor bed. The LAK cells were rIL-2-activated peripheral blood lymphocytes cultured for 4 days; the ASL were lectin- and rIL-2-activated peripheral blood lymphocytes cultured for 10 days. Of the 20 patients treated, 11 were evaluated as a group (mean age, 44 years, range, 15-61 years; mean Karnofsky rating, 69, range, 50-100; mean Decadron dose at entry, 14 mg/d, range, 0-32). The average number of lymphocytes implanted was 7.6 x 10(9) (range, 1.9-27.5 x 10(9], together with 1 to 4 x 10(6) U of rIL-2. To date, 10 of the 11 patients died, all from recurrent tumor growth. The median overall survival time was 63 weeks (range, 36-201; mean, 86). The median survival time after immunotherapy was 18 weeks (range, 11-151; mean, 39). No significant difference in survival after immunotherapy was found between those patients who had received previous chemotherapy and those who had not. The use of steroids or prior chemotherapy did not influence the in vitro generation of ASL or LAK cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

肿瘤浸润淋巴细胞治疗消化道肿瘤的初步探讨

本文初步探讨了肿瘤浸润淋巴细胞（ＴＩＬ）治疗消化系癌肿的临床疗效。从肿瘤组织中分离出力ＴＩＬ，体外经重组白细胞介素－２（ｒＩＬ－２）培养扩增，扩增后ＴＩＬ的ＣＤ细胞亚群比例升高，细胞毒活性增强。ＴＩＬ输注总量１．５×１０９～３×１０９，治疗有效者外周血Ｔ细胞亚群比例明显升高，自然杀伤细胞（ＮＫ）、淋巴因子激活的杀伤细胞（ＬＡＫ）细胞活性明显增强。５例行根治切除者随访１１～１９个月无复发，４例姑息切除者复发２例，９例未切除者总缓解率达６６．７％。证明ＴＩＬ治疗消化系肿瘤有一定价值。     

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.     

白细胞介素(IL)-2与IL-4对小鼠膀胱癌肿瘤浸润性淋巴细胞增殖及细胞毒性的协同调节作用

目的 探讨白细胞介素（ＩＬ）－２与ＩＬ－４对膀胱癌肿瘤浸润性淋巴细胞（ＴＩＬ）体外增殖及细胞毒性免疫调控的协同作用。方法 分离膀胱癌ＴＩＬ,置于含ＩＬ－２和（或）ＩＬ－２的完全培养基因中培养４周,定期计数ＴＩＬ增殖数量。四甲基偶氮唑蓝（ＭＴＴ）比色法检测ＴＩＬ细胞毒性。结果 对比单纯ＩＬ－２的培养条件,ＩＬ－２联合ＩＬ－４后４周时ＴＩＬ扩增数量是前者的１．６５倍（Ｐ〈０．０５）。在交靶比为１０：１时,ＴＩＬ对自体膀胱癌细胞（ＢＴＴ７３９）表现出高水平的杀伤活性（Ｐ〈０．０５）。联合培养的ＴＩＬ抗ＢＴＴ３９或小鼠淋巴瘤瘤株（ＹＡＣ－１）的活性与在单纯ＩＬ－２培养的条件下相比无显著改变（Ｐ均〉０．０５）。结论 ＩＬ－４对ＩＬ－２活化的膀胱癌ＴＩＬ增殖具有较强的正向调节效应,而对ＴＩＬ细胞毒性未见明显影响。     

Preservation of lymphokine-activated killer activity following T cell depletion of human bone marrow.

T cell depletion has decreased the incidence and severity of graft-versus-host disease following transplantation of allogeneic bone marrow. In the treatment of leukemia, decreased GVHD has often been associated with diminished antileukemia or graft-versus-leukemia (GVL) reactivity resulting in higher relapse rates. However, we have not seen a loss of the GVL effect following transplantation of marrow grafts depleted of CD3+ T cells. This suggests that non-T-cell effectors may play a role in preventing leukemic relapse. To study whether natural killer and lymphokine-activated killer (LAK) activity in BM was compromised by T cell depletion, the effect of T-cell-specific monoclonal antibodies against CD3 and CD6 determinants alone, or in combination, on the generation and expansion of NK/LAK cells was examined in vitro and compared to the effect of T depletion on mitogen-driven T cell proliferation. Limiting dilution analysis revealed that T depletion with CD3 and/or CD6 specific antibodies significantly reduced the number of proliferating T lymphocytes but did not significantly affect the frequency of cells able to expand and mediate LAK activity. Bone marrow, depleted of CD3+ or CD6+ T cells, generated levels of LAK activity equivalent to non-T-cell-depleted bone marrow following long-term culture in recombinant interleukin 2. CD3- NKH-1+ cells were the predominant population in rIL-2 expanded marrow cultures prior to transplant and in the peripheral blood of patients who had received a CD3-depleted marrow graft 21-65 days earlier. These studies show that it is possible to selectively reduce GVH-reactive T cells in allogeneic bone marrow while retaining non-T-effector cells with potential to mediate an antileukemia effect in vivo.     

Study on adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) for renal cell carcinoma--amplification of IL-2-elicited TIL proliferation by OKT3-monoclonal antibody

Human autologous peripheral blood lymphocytes (PBL) and lymphocytes infiltrating renal cell carcinoma (TIL) were cultured with medium containing 1000 IU/ml of human interleukin 2 (IL-2). A high cytotoxic activity against fresh autologous as well as cultured allogenic tumor cells was developed. By culturing these lymphocytes with OKT3 monoclonal antibody during the initial 2 days of long-term culture, in terms of T cell activation signal, IL-2-driven lymphocyte proliferation was remarkably accelerated with maintenance of appreciable level of cytotoxic activity. The same culture method also induced an increase in OKT3 and IL-2 receptor positive lymphocyte population in LAK cells and TIL. This method may enable us to gain more autologous TIL in vitro for adoptive immunotherapy of renal cell carcinoma than the usual culture method with IL-2 alone. Five patients with metastatic renal cell carcinoma were treated with adoptive immunotherapy with TIL, LAK and IL-2. One patient with pulmonary metastasis has had a minor response which has lasted for 3 months so far. We have not experienced any serious side effects during the treatment.     

Decreased lymphokine-activated killer cells in kidney transplant recipients. Correlation with a diminished number of CD3-/NKH1+ cells.

The activity of lymphokine-activated killer cells, measured either by a clonal or polyclonal technique, was assessed in 30 kidney transplant recipients (TX), in 13 hemodialyzed patients (HD-CRI), and in 18 normal (N) controls. A highly significant decrease of the LAK activity in TX in comparison with HD-CRI or N (P = 0.0001) was observed. Moreover, the percentage of CD3-/NKH1+ cells was decreased in TX in comparison with N (P = 0.01). LAK activity was strongly correlated (r = 0.72; P = 0.0001) with the percentage of CD3-/NKH1+ cells and not with that of double-positive CD3+/NKH1+ cells. Multivariate analysis showed that the sole independent variable that determined the LAK activity was the percentage of CD3-/NKH1+ cells: the pathological status (TX, HD-CRI, and N) variable was statistically not significant. On the other hand, two T cell-specific functions (IL-2 secretion and specific cytotoxic activity) were, on the whole, preserved in TX. Altogether, these results suggest that TX are LAK deficient predominantly because they have a decreased number of CD3-/NKH1+ cells. The normality of T cell functions suggests that the high rate of malignancies seen in TX is related to this LAK deficiency. Moreover, our study indicates that, in vivo, CD3+ cells do not significantly contribute to the LAK precursors.     

Adoptive Immunotherapy With Tumor-Infiltrating Lymphocytes and Subcutaneous Recombinant Interleukin-2 Plus Interferon Alfa-2a for Melanoma Patients With Nonresectable Distant Disease: A Phase I/II Pilot Trial

Background: On the basis of our previous experience, we designed this study to determine the activity and toxicity of outpatient treatment with autologous tumor-infiltrating lymphocytes (TIL) together with intermediate-dose recombinant interleukin-2 (rIL-2) and low-dose recombinant interferon alfa-2a (rIFN-2a), for patients with metastatic melanoma.Methods: Between April 1992 and October 1994, we processed 38 melanoma samples derived from 36 patients with metastases. Proliferative cultures of expanded lymphocytes (TIL) were infused only once into patients with metastatic melanoma. rIL-2 was administered subcutaneously for 1 month, starting on the day of TIL infusion, at an escalating dose of 6–18 × 10⁶ IU/m²/day for the first week and at the maximum-tolerated dose for the subsequent 3 weeks and then, after a 15-day interval, for 1 week/month for 3 months. rIFN-2a was administered subcutaneously at 3 × 10⁶ IU three times each week until progression.Results: Of 38 melanoma samples, 19 (50%) resulted in proliferative cultures and were infused. The median number of expanded lymphocytes was 18 × 10⁹ (range, 1–43 × 10⁹), and the median period of culture was 52 days (range, 45–60). rIL-2 was administered at doses ranging between 6 and 18 × 10⁶ IU/m²/day. Toxicity was mild or moderate, and no life-threatening side effects were encountered. Two of 19 treated patients experienced complete responses of their metastatic sites (soft tissue), 10 had stable disease, and 7 showed progressive disease. The response rate was 11% (95% confidence interval, 2–35%).Conclusions: Outpatient treatment with TIL plus rIL-2 and rIFN-2a is feasible, although, within the context of the small sample size, the activity of the combination was no different from the reported activity of any of the components used alone.     

Assessment of in vitro lymphokine activated killer (LAK) cell activity against renal cancer cell lines and its suppression by serum factor using crystal violet assay

Summary Lymphokine activated killer (LAK) cell activity against renal cancer cell lines was assessed in vitro using a crystal violet assay. A standard 4-h ⁵¹chromium release assay and a 48-h crystal violet assay showed that both natural killer-susceptible (NC65) and-resistant (ACHN) renal cancer cell lines were sensitive to LAK cells which had been generated by a 3-day incubation of peripheral blood mononuclear cells (PBMC) with recombinant interleukin 2 (rIL-2). Optimal LAK activity was generated by a 5-day culture of PBMC with 1 U rIL-2/ml. LAK activity was enhanced by the presence of IL-2 in the crystal violet assay system, while it was suppressed by fresh autologous serum. The suppressive effect was found in serum from both normal donors and patients with metastatic renal cell carcinoma, suggesting that non-specific suppressive factor(s) affecting LAK cell activity were present in human sera.