Laboratory correlates of adoptive immunotherapy with recombinant interleukin-2 and lymphokine-activated killer cells in humans

Adoptive immunotherapy with interleukin 2 (IL-2) and lymphokine-activated killer (LAK) cells (IL-2/LAK) is a technically demanding cancer therapy dependent upon large scale isolation and culture of lymphocytes. An important question is whether this technology can be accomplished routinely outside of highly specialized centers. In addition, no systematic examination of laboratory correlates of IL-2/LAK therapy in humans has been reported to date. The objectives of this report are to address two issues relevant to IL-2/LAK therapy. (a) Can IL-2/LAK therapy be accomplished outside of previously identified centers of expertise? (b) What are the relevant laboratory/clinical parameter correlations? The six institutions in the National Cancer Institute extramural trial treated 83 evaluable patients with renal cancer, malignant melanoma, or colon cancer with IL-2/LAK by a uniform protocol. Patients received 5 days of IL-2 priming, then daily leukaphereses for 5 days starting 48 h after IL-2 to harvest cells. Mononuclear cells were isolated, then cultured in roller bottles in 1-liter aliquots for 3 to 4 days at a cell density of 1.5 x 10(6) per ml with recombinant IL-2, 1500 units per ml. Cells were harvested and administered to patients with additional IL-2. Administration of IL-2 regularly induced lymphopenia and rebound lymphocytosis. Leukapheresis yields and numbers of LAK cells generated in culture and reinfused into patients correlated directly with peak lymphocyte counts achieved by IL-2 administration. Mean mononuclear cell recovery per 5 days of leukapheresis (+/- SEM) was 14.3 +/- 0.8 x 10(10). Average volume of cells cultured per patient was 95 liters (range, 41 to 235). Mean yield of cells harvested from cultures was 53%. Mean total number of LAK cells infused per patient was 7.6 +/- 0.4 x 10(10) (range, 2 to 15.2 x 10(10]. LAK activity was measured in vitro by lysis of 51Cr-labeled natural killer-resistant Daudi and fresh tumor targets. LAK effector cells regularly lysed these targets in vitro. Neither tumor reduction nor clinical toxicity correlated with dose or with cytolytic activity of LAK cells, or with other laboratory parameters including base-line lymphocyte count and IL-2-induced lymphocytosis. We conclude: (a) large quantities of LAK effector cells with tumoricidal activity can be generated routinely at different centers; (b) neither in vitro LAK activity nor numbers of LAK cells infused were predictive of clinical efficacy or toxicity. There is a need to identify other laboratory or clinical parameters more predictive of IL-2/LAK therapeutic efficacy or toxicity.     

Antitumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: successful immunotherapy of established pulmonary metastases from weakly immunogenic and nonimmunogenic murine tumors of three district histological types

We have recently shown that the systemic administration of lymphokine activated killer cells (LAK cells) plus relatively low doses of recombinant interleukin 2 (RIL-2) or the administration of high doses of RIL-2 alone can reduce the number of established pulmonary metastases from the weakly immunogenic MCA-105 sarcoma in mice. We have now analyzed the therapeutic efficacy of these treatments on both weakly and nonimmunogenic tumors of three distinct histological types in two different mouse strains. In all experiments, LAK cells were administered i.v. on days 3 and 6 and RIL-2 was injected i.p. from days 3 through 8 after tumor induction. The MCA-101 sarcoma was completely nonimmunogenic as defined by its inability to successfully immunize C57BL/6 mice. Nevertheless, administration of LAK cells plus 7,500-10,000 units RIL-2 was highly effective in reducing the number of established 3-day pulmonary metastases from this sarcoma [at 7,500 units RIL-2, mean number of metastases 37 +/- 11 (SE); P less than 0.05; at 100,000 units, 2 +/- 1; P less than 0.05] when compared to Hanks' balanced salt solution treated control animals (116 +/- 9). Likewise, RIL-2 alone at doses of 20,000 units/injection or greater had significant antimetastatic effects (77 +/- 12; P less than 0.05). Established 3-day pulmonary metastases from the MCA-38 adenocarcinoma in C57BL/6 mice and the M-3 melanoma in C3H mice were also susceptible to adoptive immunotherapy with LAK cells plus RIL-2 and with high dose RIL-2 alone. Treatment of mice with LAK cells alone or with low doses of RIL-2 alone (less than or equal to 20,000 units/injection) had little if any antitumor effects. LAK cells were tested for cytolytic activity in vitro against tumor target cells of a variety of histological types; there was no discernible relationship between susceptibility to lysis by LAK cells in vitro and therapeutic efficacy in vivo. These findings have thus demonstrated that the successful immunotherapy of established pulmonary metastases with LAK cells plus RIL-2 or with high dose RIL-2 alone includes: tumors that are immunogenic and nonimmunogenic; tumors of distinct histological types such as sarcoma, adenocarcinoma, and melanoma; and tumors in at least two different mouse strains, C57BL/6 and C3H, and that there is little correlation between the in vitro lysability of tumor cells by LAK effectors and the susceptibility of these same tumors to successful immunotherapy in vivo.     

Adoptive immunotherapy of human cancer using low-dose recombinant interleukin 2 and lymphokine-activated killer cells

The adoptive transfer of recombinant-methionyl human interleukin 2 (rIL-2)-activated autologous peripheral blood mononuclear lymphokine-activated killer (LAK) cells to cancer patients is being evaluated as an alternative to conventional cancer therapy. We have independently developed an alternative regimen to previously reported adoptive immunotherapy protocols using rIL-2 and LAK cells which features the prolonged administration of low-dose rIL-2 (30,000 units/kg) and an automated, entirely enclosed system of peripheral blood cell procurement, culture, harvest, and reinfusion of activated cells. The cell culture system was tested with a murine tumor model in which LAK cells generated in plastic culture bags were reinfused into tumor-bearing mice. Tumor regression was as effective with cells activated in the bags as in conventional culture flasks. Twenty-eight cancer patients were treated for 5 consecutive days with low-dose rIL-2, followed by leukapheresis, infusion of LAK cells, and prolonged IL-2 administration. At least 50% tumor regression was observed in 46% of all patients treated. These data imply that human peripheral blood mononuclear cells retain fully their capacity for rIL-2-induced activation and effector cell function under this alternative approach, and further, that a low-dose rIL-2 regimen with markedly reduced toxicities can be as effective as high-dose rIL-2 regimens if low-dose rIL-2 is given for a prolonged period of time following LAK cell infusion.     

Effect of immunotherapy with allogeneic lymphokine-activated killer cells and recombinant interleukin 2 on established pulmonary and hepatic metastases in mice

The adoptive transfer of lymphokine-activated killer (LAK) cells in conjunction with the systemic administration of recombinant interleukin 2 (RIL-2) results in the regression of established pulmonary and hepatic micrometastases from a variety of immunogenic and nonimmunogenic murine tumors in syngeneic C57BL/6 mice. Recent studies have shown that this therapeutic approach can mediate the regression of cancer in humans as well. Because of the practical difficulties in obtaining syngeneic or autologous LAK cells for the therapy of cancer in humans we have now evaluated the antitumor efficacy of allogeneic LAK cells generated from different strains of mice. The in vitro lysis of fresh tumor targets by LAK cells is not a major histocompatibility complex-restricted phenomenon since LAK cells of BALB/c-H-2d, DBA/2-H-2d, and C3H-H-2k origin all exhibited lytic activity when tested against allogeneic MCA-102-H-2b tumor cells in short term 51Cr release assays. In vivo, the i.v. transfer of allogeneic LAK cells combined with i.p. injections of RIL-2 reduced the number of established pulmonary metastases induced by either MCA-105 or MCA-101 tumors which are syngeneic to C57BL/6 hosts. The extent of reduction of these pulmonary metastases by the allogeneic LAK cells was directly dependent upon the dose of RIL-2 given; increasing doses of systemically administered RIL-2 resulted in increasingly greater reduction in the numbers of established 3-day pulmonary sarcoma metastases. In dose titration experiments, adoptive transfer of at least 2 doses of 10(8) allogeneic LAK cells was necessary to achieve significant antitumor effect in vivo. Allogeneic LAK cells were also successful in mediating significant regression of hepatic micrometastases. Again, the i.v. transfer of allogeneic LAK cells had a smaller therapeutic benefit compared to i.v. transfer of syngeneic LAK cells. When allogeneic LAK cells were injected intraportally, however, they were as effective as syngeneic LAK cells. Allogeneic LAK cells had little, if any, therapeutic effect on established pulmonary and hepatic metastases when administered to recipients previously immunized to the histocompatibility antigens on the donor cells. Taken together, our results indicate that allogeneic LAK cells from several strains of mice are effective in lysing fresh MCA-102 tumor in vitro and that when given i.v. in sufficient numbers, in conjunction with RIL-2, they can mediate significant reduction in the number of established pulmonary and hepatic micrometastases in nonalloimmunized C57BL/6 mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

The requirements for successful immunotherapy of intraperitoneal cancer using interleukin-2 and lymphokine-activated killer cells

In a significant proportion of patients with gastrointestinal and ovarian malignancy the peritoneal cavity is a prominent site at which surgical treatment fails. Adjuvant treatments directed at this site should be investigated in an attempt to improve survival in patients with these cancers. In the study reported here, a model of intraperitoneal tumor in the mouse was established and shows the effectiveness of lymphokine activated killer (LAK) cells and exogenous interleukin-2 (IL-2) in the control of intraperitoneal tumor. A standard regimen was used to treat seven different tumors in three different mouse strains. In all seven cases a significant reduction in the intraperitoneal tumor mass was observed when LAK cells plus IL-2 were used as immunotherapy. A prolonged survival was also demonstrated in mice with intraperitoneal tumor. The relevance of these observations to patients with cancer was demonstrated in that allogeneic and syngeneic LAK cells were equally effective, LAK cells generated from normal and from tumor-bearing donors showed equal reactivity, and this treatment was successful in the immuno-compromised host. Both IL-2 derived from an IL-2-producing subline of the EL-4 thymoma and recombinant IL-2 were equally effective in the control of intraperitoneal tumor. The local-regional effects of the intraperitoneal administration of IL-2 were demonstrated by high levels of LAK cell cytotoxicity in peritoneal exudate cells. Intraperitoneal IL-2 or IL-2 plus LAK cell regimens should be investigated in the treatment of malignancy that spreads by implantation onto peritoneal surfaces.     

Successful immunotherapy of murine experimental hepatic metastases with lymphokine-activated killer cells and recombinant interleukin 2

Lymphokine-activated killer (LAK) cells are generated in vitro by the incubation of normal murine splenocytes in interleukin 2. We have shown previously that the systemic injection of LAK cells in conjunction with recombinant interleukin 2 can reduce the number of established pulmonary metastases in mice. In an attempt to study this approach in the treatment of hepatic metastases, we developed a technique for the induction of hepatic metastases in mice based on the intrasplenic injection of tumor cells and have tested the effects of LAK cells and recombinant interleukin 2 produced in Escherichia coli (RIL-2) therapy on these metastases. Treatment with LAK cells alone in 14 consecutive experiments rarely produced significant reduction in metastases over control (mean percentage reduction, 12%). Therapy with RIL-2 alone produced a dose-dependent reduction in the number of liver metastases. In 20 consecutive experiments when RIL-2 was administered i.p. three times a day at doses varying from 1,000 to 5,000, 10,000 to 15,000, and 25,000 units, a statistically significant (P less than 0.05) reduction in liver metastases was seen in 2 of 12, 2 of 4, and 8 of 12 determinations, respectively (percentage reduction, 0 to 97; mean, 42%). At doses greater than 25,000 units, the reduction in metastases was highly reproducible (percentage reduction, 66 to 95; mean, 83%) and was statistically significant in 14 of 14 determinations. When LAK cells were given i.v. in addition to RIL-2 administration in 16 consecutive experiments, the percentage reduction in liver metastases was markedly increased over that seen with RIL-2 alone (mean percentage reduction, 77% at doses of 5,000 to 25,000 units of RIL-2 and mean reduction, 97% for doses greater than 25,000 units of RIL-2). At doses of 5,000, 10,000, 25,000, and greater than 25,000 units of RIL-2 plus LAK cells, significant reduction of liver metastases (P less than 0.05) was achieved in 3 of 7, 2 of 2, 8 of 8, and 6 of 6 determinations, respectively. When animals were given fresh splenocytes or splenocytes cultured in complete medium without RIL-2 instead of LAK cells, no reduction in liver metastases was seen except for that attributable to the administration of RIL-2 alone. Sublethal total body irradiation of the mice prior to therapy abrogated the therapeutic effects of RIL-2, but the effects of treatment with LAK cells plus RIL-2 were maintained. Thus, treatment with RIL-2 alone or in combination with LAK cells is effective in reducing the number of established hepatic micrometastases in a murine model.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased vascular permeability in organs mediated by the systemic administration of lymphokine-activated killer cells and recombinant interleukin-2 in mice

Significant tumor regressions in mice with established carcinomas, sarcomas, and melanomas and in humans with advanced cancers have been observed following immunotherapy with lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (IL-2). However, dose escalations of LAK cells and IL-2 have been prevented by the development of a vascular leak syndrome (VLS). Although IL-2 alone can produce this VLS, we investigated the role of transferred LAK cells, generated by the incubation of syngeneic splenocytes in IL-2, in mediating this phenomenon. We used a murine model to quantitate the vascular leak by measuring the extravasation of iv injected 125I-bovine serum albumin. A permeability index (PI) was calculated by dividing the mean cpm of tissues from treated mice by those from control animals. The systemic transfer of LAK cells and IL-2 produced a significantly greater extravasation of albumin in the lungs, liver, and kidneys than after Hanks' balanced salt solution, IL-2, or LAK cells alone (in the lungs, for example, PI = 4.7, 1.4, and 1.6 after LAK cells and IL-2, LAK cells alone, and IL-2 alone, respectively). To eliminate the contribution to the leak by host lymphocytes, we irradiated mice before cell transfer. As compared to controls, LAK cells and IL-2 resulted in higher extravasation in the lungs, liver, kidneys, and spleen. However, a similar vascular leak was not observed in the lungs, liver, and kidneys after treatment with IL-2 plus fresh or cultured (without IL-2) splenocytes. Moreover, the combination of IL-2 excipient and LAK cells or IL-2 and irradiated LAK cells did not produce a fluid leak. The development of the VLS by LAK cells was directly related to the dose of concurrently administered IL-2 and the number of injected cells. Depletion of Thy 1.2-positive lymphocytes using antibody and complement prior to generating the LAK cells used in adoptive transfer did not abrogate the VLS in any of the organs tested. Similarly, depletion of L3T4 and Lyt-2 positive cells in vivo using monoclonal antibodies prior to harvesting spleens for generation of LAK cells also had no impact on the VLS. In contrast, in vitro treatment of LAK precursor cells with antibody to asialo-GM-1 plus complement completely eliminated the VLS when the depleted cells were cultured in IL-2 and subsequently transferred.(ABSTRACT TRUNCATED AT 400 WORDS)     

Augmentation of interleukin-2 immunotherapeutic effects by lymphokine-activated killer cells and allogeneic stimulation in murine tumor cells

Interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells were used in intraperitoneal and pulmonary tumor models in C57BL/6 mice. To maintain the immunotherapeutic effects of IL-2 plus LAK treatment but reduce its toxicity, ways were sought to augment IL-2 effects. The investigation showed that the adoptive transfer of LAK cells was a prerequisite for successful therapy of intraperitoneal cancer. When LAK cells were given on consecutive days within one course of immunotherapy, antitumor efficacy was augmented with additional doses of LAK cells. However, with the reduction of 1 complete cycle of IL-2 + LAK cells, no further reduction in intraperitoneal tumor was observed as compared to the reduction after 2 or 4 cycles. LAK cells generated from splenocytes of mice that had received an allogeneic tumor challenge 1 week earlier exerted a highly increased cytotoxicity as compared to normal LAK cells. Furthermore, the potentiation effect of an allogeneic response of the host at the tumor site was demonstrated by decreased numbers of lung implants and improved survival in mice given mixtures of syngeneic and allogeneic tumor cell suspensions. An alloimmune response within the microenvironment of tumor tissue markedly enhanced the antitumor effect of IL-2 against the syngeneic tumor. It was concluded that there is a fundamental need to improve the recruitment of adoptively transferred LAK cells or LAK precursors into tumor tissue. This may be the next step required in the further development of IL-2 and LAK immunotherapy.     

Treatment of hepatocellular carcinoma utilizing lymphokine-activated killer cells and interleukin-2

This paper is a report on adoptive immunotherapy involving consecutive injections of recombinant interleukin-2 and lymphokine-activated killer (LAK) cells in the treatment of hepatocellular carcinoma. Peripheral blood lymphocytes, obtained by leukopheresis, acted as the activated killer cells with a co culture of recombinant interleukin-2 in the culture system. After 4 days, the activated killer cells were returned into the patients' bodies intra-arterially and intravenously. No complete remissions or partial remissions have resulted, although five of the seven patients showed a significant decrease in their serum -fetoprotein levels after treatment. In addition, one case showed a patent portal truncus while another indicated the appearance of central necrosis on the computerized tomograph scan. Although the period of observation was short, there were no recurrences after the combination therapy of tumor resection and LAK adoptive immunotherapy. It might be difficult to treat hepatocellular carcinoma with adoptive immunotherapy alone, but there is some possibility of conducting therapy for hepatocellular carcinoma after removing the majority of the tumor cells by surgical resection and transcatheter arterial embolization therapy. This conclusion indicates, at least theoretically, that adoptive immunotherapy will be suitable in the treatment of hepatocellular carcinoma as one of the combination therapies with the two major forms of treatment mentioned above.     

In vivo induction of lymphokine-activated killer cells by interleukin-2 splenic artery perfusion in advanced malignancy

In an effort to stimulate in vivo LAK cell activity at relatively nontoxic doses, 20 patients with advanced metastatic malignancy (13 renal cell carcinoma, 6 melanoma, 1 lymphoma) were treated with recombinant human interleukin-2 (IL-2) by continuous 5-day splenic artery perfusion using the femoral approach. Two treatment cycles were administered 3 weeks apart; IL-2 doses ranged from 1.5-4 x 10(4) Cetus units/kg/day. Peripheral blood lymphocyte cytotoxicity in a 4-h 51Cr release assay was measured using as tumor cell targets K562 for natural killer (NK) activity, Daudi for LAK, and Daudi plus in vitro IL-2 for inducible LAK (I-LAK). For the 20 patients, an increase in mean peak percent cytotoxicity from pretreatment levels was seen for NK (36% to 53%), LAK (8% to 37%) and I-LAK (20% to 53%) activity, all significant at P = 0.001. On day 43, 16 days after completing the second cycle of treatment, NK activity remained elevated at 47% and I-LAK at 40% (P = 0.008 and 0.01, respectively). Lymphocyte phenotype analysis by flow cytometry demonstrated increases from pretreatment levels in Leu 11+ (13 to 23%), Leu 19+ (10 to 21%), Leu 11+ 19+ (7 to 17%), IL-2r+ (4 to 17%), and HLA-DR+ (12 to 25%) subsets, all significant at P less than or equal to 0.01. Dose effect was studied at 3 dose levels: 1.5, 3, and 4 x 10(4) Cetus units/kg/day. At the higher doses mean peak NK (57%) and I-LAK (57%) activity were greater than at the low dose (42 and 31%, respectively), both significant at P less than 0.05. A trend to positive dose effect was seen in LAK activity (P = 0.08). Splenic artery perfusion with IL-2 can result in significant in vivo peripheral LAK cell generation as well as enhancement of I-LAK and NK activity that persists at least 16 days after the cessation of treatment. Such sustained activity would not be expected with conventional high dose i.v. therapy.     

Phase I trial of intraperitoneal recombinant interleukin-2/lymphokine-activated killer cells in patients with ovarian cancer

Ten patients with ovarian cancer refractory to conventional therapy were treated with intraperitoneal (i.p.) recombinant interleukin-2 (rIL-2) and lymphokine-activated killer cells (LAK). The 28-day protocol consisted of 6 priming i.p. rIL-2 infusions on days 0, 4, 6, 8, 10, and 12. Leukapheresis was performed for mononuclear cell collection on days 15, 16, 17, and 18 and lymphokine-activated killer cells were given i.p. with the rIL-2 on days 19 and 21. Three additional i.p. rIL-2 infusions were given on days 23, 25, and 27. Three dose levels of rIL-2 were tested: 5 X 10(5), 2 X 10(6), and 8 X 10(6) units/m2 body surface area. The dose-limiting toxicity was abdominal pain secondary to ascites accumulation with significant weight gain. Other toxic effects included decreased performance status, fever, nausea and vomiting, diarrhea, and anemia. Peripheral lymphocytosis and eosinophilia were seen at all dose levels. The maximum tolerated dose is 8 X 10(6) units/m2/dose. Peripheral and peritoneal IL-2 levels were measured with a bioassay using an IL-2-dependent cell line. At the highest dose level, serum IL-2 was greater than 10 units/ml for 18 h. After the first infusion, a 2-log dilution of the i.p. IL-2 was measured in the serum. In the postleukapheresis i.p. IL-2-dosing period less IL-2 was detected in the serum than in the earlier i.p. IL-2-priming period. The induction and persistence of LAK activity were studied. Peritoneal LAK activity was detected as early as 4 days after the first i.p. infusion, by day 11 in all evaluable patients, and persisted for the 6-day interval between priming IL-2 and LAK/IL-2 infusion. Peritoneal lytic activity persisted until day 28 in 5 tested patients. These peritoneal cells retained lytic activity 48 h in culture medium without rIL-2 present. Peritoneal LAK activity correlated with the percentage of mononuclear cells and the percentage of CD56-positive mononuclear cells in the peritoneum. The yield of peripheral lymphocytes after the six i.p. priming doses of rIL-2 correlated with the dose level of rIL-2 infused. Peripheral blood LAK activity showed a minimal, however progressive, increase during the treatment protocol. LAK activity could be enhanced if rIL-2 was present during the 4-h assay. These studies indicate that i.p. rIL-2 infusion induced durable regional LAK activity and primes peripheral blood cells for LAK activity if exposed briefly to additional IL-2.     

Local adoptive immunotherapy using lymphokine-activated killer cells and interleukin-2 against malignant pleural mesothelioma: report of two cases.

Malignant pleural mesothelioma is refractory to conventional therapy. We tried local adoptive immunotherapy using lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2) to control malignant pleural effusion due to mesothelioma in two patients: Case 1 was that of a 69-year-old man, and Case 2 that of a 49-year-old woman with complicating chronic idiopathic thrombocytopenic purpura. A systemic survey revealed no sign of metastasis in either case. Intrapleural instillations of 6.7 x 10(9) autologous LAK cells in Case 1, or 11.3 x 10(9) allogeneic LAK cells in Case 2, with daily injections of IL-2 resulted in reductions of the pleural effusions in each case and in a decline in the level of hyaluronic acid in the effusion, in Case 1. The instillations of autologous and allogeneic LAK cells were well tolerated. The results suggest that local adoptive immunotherapy could be useful in the treatment of malignant effusion due to mesothelioma.     

Adoptive immunotherapy using lymphokine-activated killer (LAK) cells and interleukin-2 for recurrent malignant primary brain tumors

Summary Ten patients with recurrent malignant primary brain neoplasms were treated with adoptive immunotherapy using lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2). Nine patients had supratentorial glioma and they received multiple intratumoral instillations of LAK cells through reservoir-catheter system or burrhole. The other patients with disseminated subarachnoid metastases from posterior fossa medulloblastoma received immunotherapy via lumbar subarachnoid route. A partial and transient clinical response was observed in two patients following the therapy, and a cystic transformation of the essentially solid tumor was noted on the CT scans of these two patients. No significant clinical or radiological response to the treatment was observed in the remaining 8 patients. The results of this preliminary study reveal limitations of the regional intratumoral adoptive immunotherapy using currently available techniques and provide sufficient evidence of its effectiveness to warrant further investigations.     

Adoptive immunotherapy for recurrent glioblastoma multiforme using lymphokine activated killer cells and recombinant interleukin-2

Thirteen patients with recurrent glioblastoma were treated with adoptively transferred autologous lymphokine activated killer (LAK) cells and recombinant interleukin-2 (rIL-2). Patients' blood mononuclear cells (MNC) obtained by leukapheresis were cultured at 2.5 million MNC per ml for 3 to 5 days in media containing 1000 U rIL-2/ml. After incubation, the nonadherent MNC from all cultures (0.5-5 X 10(9] were combined and concentrated for infusion in 5 to 10 ml saline containing 10(6) U rIL-2. Nine patients received one injection of LAK cells and rIL-2 into the brain tissue immediately surrounding the tumor cavity during craniotomy for subtotal tumor removal (Group 1). On each of the 3 days after surgery, patients received boosters of 10(6) U rIL-2 delivered into the tumor cavity through a skin flap or via an Ommaya reservoir. Approximately 1 to 2 weeks after this series of injections, these patients were treated with a second cycle of LAK cells and rIL-2 injected into the tumor cavity using the reservoir. Four patients received both adoptive immunotherapy cycles by intracavitary injection (Group 2). In this relatively small patient pool, neither age, sex, Karnofsky score, treatment history, nor anticonvulsant and steroid dosage appeared to influence a patient's ability to make LAK cells. The therapy, itself, was well-tolerated by all patients although they all displayed symptoms of aseptic meningitis and increased intracranial pressure, i.e., headache, fever, malaise on the days of LAK cell and/or rIL-2 infusion. The therapy did not appear to have a significant impact on patient survival (mean, 30 weeks) especially for those patients with a high postsurgical tumor burden. As the therapy is safe, the authors believe its efficacy can best be tested in patients with a newly diagnosed or recurrent glioblastoma which lies in an area where a near-total resection is possible.     

Adoptive immunotherapy of a rat glioma using lymphokine-activated killer cells and interleukin 2

The aim of the present study was to develop an animal model to test the therapeutic potential of purified adherent lymphokine-activated killer (A-LAK) cells against an intracerebrally implanted rat glioma, designated F98. Highly purified A-LAK cells demonstrated greater activity against F98 tumor cells than conventional lymphokine-activated killer cells, as determined by means of 51Cr-release and clonogenic assays. Therapeutic efficacy was evaluated by means of a Winn neutralization assay, in which F98 targets and A-LAK cells or control nonadherent mononuclear cells were incubated for 18 h in vitro and then implanted stereotactically into the right caudate nuclei of Fischer rats. Animals given injections of 4000 F98 cells alone or control nonadherent mononuclear cells had a mean survival time of 22.3 days, compared to 46.1 days (P less than 0.001) for rats treated with A-LAK cells. Increasing the tumor inoculum to 12,500 cells reduced the survival time of A-LAK-treated animals to 27.8 days, compared to 20.8 days for untreated controls. Systemic administration of 50,000 units/kg of interleukin 2 every 12 h for 5 days failed to improve survival. The mean survival time of rats implanted with the F98 tumor ranged from 16 days for 10(5) cells to 29 days for 10(2) cells. Extrapolating from these survival data, treatment with A-LAK cells may have decreased the number of F98 cells to less than 10, but even this small number was still lethal. Supernatants from F98 cells had immunoinhibitory activity that, further, may have modulated the antitumor effects of A-LAK cells. Our results indicate that curative, adoptive immunotherapy of the F98 glioma by means of A-LAK/interleukin 2 is impossible to achieve and provide some explanation for the clinical failures that have been observed in the adoptive immunotherapy of malignant gliomas.     

Induction of lymphokine-activated killer cells in strain 2 guinea pigs with human interleukin-2

Culture of spleen cells from strain 2 guinea pigs with Jurkat interleukin-2 (IL-2) resulted in the induction of lymphokine-activated killer (LAK) cells. Maximum LAK activity was induced using 5000 pmol of IL-2. Incubation of spleen cells with IL-2 for as little as 8 h resulted in detectable LAK activity. LAK cell activity was transient and could be stimulated by adding back IL-2. LAK cell activity was enriched in a 1.085 single-step percoll gradient. Admixture of guinea pig LAK cells with the line 10 hepatoma prior to intradermal injection resulted in inhibition of tumor growth. Systemic passive transfer of LAK cells together with concurrent IL-2 resulted in a significant inhibition of metastatic tumor growth.     

Interaction of recombinant interferons with recombinant interleukin-2: differential effects on natural killer cell activity and interleukin-2-activated killer cells

The ability of recombinant interferons (IFNs) to modulate recombinant interleukin-2 (rIL-2) augmentation of natural killer (NK)-cell activity and to modulate the generation of activated killer (AK) cells was examined. Incubation of murine spleen cells for 18 hr with either human rIL-2 or a human hybrid recombinant IFN alpha, rHuIFN-alpha A/D, which is active on murine cells, resulted in a dose-dependent increase in NK activity; however, recombinant murine IFN gamma, rMuIFN-gamma, had little activity. A more than additive augmentation of cytotoxicity was obtained when spleen cells were incubated with the combination of rIL-2 and rHuIFN-alpha A/D. Incubation of murine spleen cells with rIL-2 for 3 days resulted in a dose-dependent induction of AK cells which were cytotoxic to an NK-resistant tumor target cell. In contrast to the results observed on NK activity, incubation of spleen cells with rHuIFN-alpha A/D and rIL-2 inhibited AK-cell activity. Partially purified murine IFN-alpha had inhibitory activity comparable to that of rHuIFN-alpha A/D. The addition of rHuIFN-alpha A/D at the initiation of culture of spleen cells with rIL-2 (day 0) resulted in maximal inhibition of cytotoxicity; inhibition was reduced or absent if rHuIFN-alpha A/D was added on day 1 or 2 of culture. The proliferation of spleen cells incubated with rIL-2 was also inhibited by rHuIFN-alpha A/D. Addition of rMuIFN-gamma to spleen-cells and rIL-2 increased the cytolytic activity of AK cells and did not inhibit rIL-2-induced proliferation of spleen cells. Similar data were also obtained with human peripheral blood lymphocytes and recombinant cytokines. Incubation of human peripheral blood lymphocytes with rIL-2 and recombinant human IFN-alpha A (rHuIFN-alpha A) or recombinant human IFN-gamma (rHuIFN-gamma) resulted in a more than additive increase in NK activity. Human AK-cell cytotoxicity was inhibited by rHuIFN-alpha A but enhanced by rHuIFN-gamma. Thus recombinant IFNs have differential effects on rIL-2-induced cytotoxic cells, resulting in augmentation or inhibition of activity, which is dependent on both the type of IFN and the cytotoxic activity examined. These results may have important implications for the potential therapeutic use of combinations of these cytokines.     

Local adoptive immunotherapy of human head and neck cancer xenografts in nude mice with lymphokine-activated killer cells and interleukin 2

The efficacy of local adoptive immunotherapy with human lymphokine-activated killer cells and recombinant interleukin 2 (rIL-2) in growth inhibition of established squamous cell carcinoma of the head and neck (SCCHN) was evaluated in a nude mouse model. The model of xenografted SCCHN was established by s.c. injections of in vitro maintained tumor cells (2-10 x 10(6) cells/mouse) into the flank of splenectomized animals pretreated with cyclophosphamide (200 mg/kg). The SCCHN line used was tumorigenic in 95% of the appropriately conditioned nude mice. Inhibition of tumor growth by locally administered effector cells was the end point of the study, since the tumors did not metastasize within 6 weeks of tumor challenge. Either i.p. or local administration of rIL-2 alone (1000 units/day) to the tumor site daily for 2 weeks resulted in a significant inhibition of tumor growth. In the absence of detectable natural killer activity in these mice, a modest dose of rIL-2 had a direct antitumor effect on SCCHN cells in vivo. In addition, complete inhibition of tumor growth was achieved with 3 times weekly injections of 5-10 x 10(6) lymphokine-activated killer cells delivered to the tumor site and 1000 units of rIL-2 administered locally every day for 2 weeks. Our data indicate that local or systemic immunotherapy with rIL-2 alone or local adoptive immunotherapy with an adequate dose of lymphokine-activated killer cells plus rIL-2 may be effective in preventing the growth of established SCCHN tumors in vivo.     

In vivo and in vitro activation of natural killer cells in advanced cancer patients undergoing combined recombinant interleukin-2 and LAK cell therapy

Patients with advanced metastatic cancer were given combined autologous lymphokine activated killer (LAK) cell and recombinant interleukin-2 (rIL-2) therapy on a National Cancer Institute extramural phase II trial. Systemic administration of rIL-2 resulted in pronounced lymphocytopenia. Within two days after completion of in vivo rIL-2 therapy, there was a dramatic increase in absolute numbers of circulating lymphocytes, and cytotoxic activity against tumor cell targets was mediated by peripheral blood lymphocytes, indicating in vivo generation of LAK activity. Patients were leukapheresed and cells cultured for three to four days in rIL-2. rIL-2 cultured cells from all patients demonstrated cytotoxic activity. In order to characterize the effector cell, T cells and natural killer (NK) cells were isolated to greater than 95% purity by flow cytometry. Cytotoxic activity was mediated by rIL-2--activated NK cells, whereas T cells demonstrated no substantial activity. The circulating in vivo cytotoxic effectors detected after in vivo rIL-2 therapy were also shown to be rIL-2--activated NK cells. Results from these studies demonstrate that all patients were capable of generating a cytotoxic response, and that the cytotoxic effector cells were rIL-2--activated NK cells, identified by the phenotype CD3--, Leu 19+.     

In vivo and in vitro effect of adoptive immunotherapy of experimental murine brain tumors using lymphokine-activated killer cells

Adoptive immunotherapy for the experimental murine brain tumor was investigated by using lymphokine-activated killer (LAK) cells both in vitro and in vivo. Supernatants of 48-h culture medium of spleen cells from Wistar rats in the presence of concanavalin A were used as interleukin 2 (IL-2). LAK cells were generated by cocultivation of spleen cells from Fischer rats with IL-2 with the peak reactivity on Day 2 or 3 of culture. Lytic activity was observed against not only syngenic tumor cells but also allogenic and xenogenic tumor cells, while no lytic activity was observed against normal brain cells. The cell depletion test, dye exclusion test, and immunofluorescence method using monoclonal antibodies revealed that LAK cells partially belonged to the population of the activated T-cell group, but the precursor cells did not react with any monoclonal antibodies used. On the basis of these results in vivo study was performed. LAK cells and immune spleen cells were adoptively transferred to the rats i.v. or intratumorally (i.t.) on the seventh day after the inoculation of T9, a gliosarcoma induced by methylcholanthrene from Fischer rats, into the right basal ganglia. Then the survival rate and necrotic foci were compared between the groups treated with those cells and the control. The survival rate of the groups treated with LAK cells was significantly higher than that of the control (administered i.v.; P less than 0.01, administered i.t.; P less than 0.05). But the treatment with immune spleen cells was not effective. The incidence and area of necrotic foci in the tumors treated with LAK cells were greater than those of the others. Microautoradiography was also performed using [3H]thymidine-labeled LAK cells, which were administered i.v. to the models on the 14th day after the inoculation of T9. It was revealed that LAK cells accumulated in the lung shortly after the administration and then in the liver and spleen, especially in the white pulp. IL-2 inhibitor activity of the sera from the tumor-bearing rats was greater than that of normal rats (P less than 0.001), but it was depressed markedly by cyclophosphamide (P less than 0.01). The adoptive transfer of LAK cells may be one of the effective treatments of malignant brain tumor. The nature of IL-2 inhibitors is necessary to be clarified for more effective immunotherapy.