Circulating cytokines in patients with metastatic cancer treated with recombinant interleukin 2 and lymphokine-activated killer cells

Treatment with recombinant interleukin 2 and lymphokine-activated killer cells (rIL-2/LAK) has produced a clinical antitumor effect in preliminary human trials. The cytokines gamma-interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and tumor necrosis factor beta (TNF-beta, lymphotoxin) have potent in vitro antitumor activity and some clinical toxicities similar to interleukin 2 (IL-2)/LAK. This study sought to determine whether these cytokines were detectable in sera of IL-2/LAK-treated patients. Ten patients were treated with a protocol of 5-day i.v. rIL-2 bolus priming (10(5) units/kg, every 8 h), followed by 5 daily phereses with harvested lymphocytes cultured in vitro to generate LAK, and 5 days of rIL-2 bolus with infusion of LAK cells. Five patients were treated with a protocol modified to a 3-day rIL-2 prime and 6-day continuous infusion rIL-2 (3 x 10(6) units/m2/day) with infusion of LAK cells. Serum specimens were obtained prior to and 0.5, 2, 3, and 5 h after IL-2 or LAK cell administrations. IFN-gamma was detected by enzyme-linked immunosorbent assay, TNF-alpha by WEHI 164 bioassay or enzyme-linked immunosorbent assay, and TNF-beta by WEHI 164 cell bioassay. During the prime, few patients manifested in vivo detectable serum cytokines: IFN-gamma, three of ten, 5-day prime (1.03 +/- 0.46 ng/ml), and zero of five, 3-day prime; TNF-alpha, one of ten, 5-day prime, and one of three, 3-day prime; TNF-beta, one of ten, 5-day prime. The supernatants of in vitro LAK generation cultures had detectable levels of cytokines at 24 h which increased progressively until culture harvest at Day 4 (IFN-gamma, 2.56 +/- 0.34 ng/ml; TNF-alpha, 356 +/- 110 pg/ml; TNF-beta, 8.2 +/- 4.4 units/ml). The highest levels of in vivo serum cytokines occurred following LAK cell infusion and were more often elevated in patients receiving rIL-2 by bolus than by continuous infusion: IFN-gamma, four of six bolus, zero of three continuous infusion; TNF-alpha, six of six bolus (maximum 679 pg/ml) versus two of three continuous infusion (maximum, 106 pg/ml). LAK cells in vitro responded with cytokine release on stimulation by tumor cell lines (IFN-gamma, 0.88 +/- 0.06 ng/ml; TNF-alpha, 426 +/- 16 pg/ml; TNF-beta, 0.64 +/- 0.06 units/ml). In summary, this preliminary study has detected circulating cytokines in sera of patients receiving IL-2/LAK therapy. The greatest cytokine elevations followed LAK cell infusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synergy of human recombinant interleukin 1 with interleukin 2 in the generation of lymphokine-activated killer cells

Peripheral blood mononuclear cells cultured in vitro with interleukin 2 (IL-2) become cytolytic towards both autologous and allogeneic tumor cells. We report here that IL-1 synergizes with IL-2 in serum-free conditions to produce increased (1.3-286-fold) lymphokine-activated killer (LAK) activity. The most dramatic synergy is seen with low IL-2 concentrations (10 U/ml, 222 pM) and 50-250 U/ml IL-1 alpha or beta. Kinetics of addition experiments demonstrate a specific requirement for IL-1 at or before addition of IL-2 to the culture. We postulate that one of the mechanisms whereby IL-1 augments LAK activity is by rendering LAK-precursors more responsive to IL-2. Up-regulation of the IL-2 receptor beta chain (Tac) and increased [3H]thymidine incorporation in cultures containing IL-1 and IL-2 support this view. In some instances, IL-1 alone is capable of maintaining/generating a small degree of cytolytic activity. Collectively, our data demonstrate that IL-1 is capable of interacting with low dose IL-2 to significantly augment LAK activity, potentially playing an important role in the early stages of LAK activation and differentiation. Because synergy is observed with dramatically reduced IL-2 concentrations, this system may offer an alternative approach to high dose IL-2 therapy for the treatment of neoplastic disease.     

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.     

Local administration of autologous lymphokine-activated killer cells and recombinant interleukin 2 to patients with malignant brain tumors

Lymphokine-activated killer cells (LAK cells) were induced from lymphocytes from patients with malignant glioma by using interleukin 2 (IL-2), and their killing activity was examined. Their LAK activity against Daudi cells was 66.2 +/- 13.1% and 48.7 +/- 12.7% against self glioma cells, 54.4 +/- 10.1% against K562 cells, 43.1 +/- 7.9% against Raji cells, and 33.5 +/- 16.2% against allogeneic glioma cells. The phenotype of these LAK cells was Leu 1 (++), 2a (+/-), 3a (++), 7 (+), and 11 (++). The phenotype of precursor LAK cells, on the other hand, was Leu 1 (-), 2a (-), 3a (+), 7 (-), and 11 (++). Other activated killer cells, including LAK cells, phytohemagglutinin-activated killer cells, autoactivated killer cells, and their precursor LAK cells, were studied serologically in order to identify their phenotypic characteristics. From these data, the LAK cell populations were considered to be polyclonal. Using these LAK cells plus IL-2, local adoptive immunotherapy was undertaken in 23 patients with recurrent malignant glioma. We injected, that is, autologous LAK cells plus IL-2 directly into the cavities of the brain tumors; 1.2 to 324 x 10(8) LAK cells per ml and 0.8 to 5.4 x 10(3) units of IL-2 were directly injected into the brain tumor by using an Ommaya reservoir. Definite tumor regression, improvement of some clinical symptoms, and continuous remission over 6 mo or more were observed in six, nine, and three patients, respectively. There were no marked side effects, except for slight fever and chill, in eight and three patients, respectively. These results suggested the possibility of induction of a sufficient number of LAK cells from the lymphocytes of the patients with recurrent malignant glioma, indicating that local adoptive immunotherapy by direct injections of LAK cells and IL-2 into the brain tumor will prove to be an effective means of immunotherapy. Additional follow-up of the patients will be required before its therapeutic value can be established.     

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)     

Immunotherapy of murine hepatic metastases with lymphokine-activated killer cells expanded in serum-free media and recombinant interleukin 2

It has been shown that the systemic administration of lymphokine-activated killer (LAK) cells with recombinant interleukin 2 (RIL-2) is effective in reducing the number of established pulmonary and hepatic metastases in murine models. Similarly, this modality of therapy has been proven effective against certain selected human tumors as well. In view of the rising concern with transmission of virally related communicable diseases such as hepatitis and AIDS, we have undertaken the evaluation of a serum-free medium (AIM V) for the generation and expansion of murine LAK cells for use in in vivo tumor immunotherapy against murine hepatic metastases. Day 3 LAK cells generated in AIM V medium demonstrated a greater percentage of viable cells than cells generated in serum containing complete medium (CM) (mean percentage of yield, 59 versus 25%, AIM V medium versus CM, respectively, P less than 0.001, N = 6 consecutive experiments). When day 3 LAK cells were transferred to new medium (CM to CM and AIM V to AIM V), a highly reproducible expansion of these cells was demonstrated which was significantly better for cells expanded in AIM V medium versus cells expanded in CM (mean fold expansion on day 21 of culture; 201 versus 54, AIM V medium versus CM, respectively, P less than 0.005, N = 4 consecutive experiments). When day 3 LAK cells, day 5 expanded LAK cells, and day 13 expanded LAK cells grown in CM or in AIM V medium were given in vivo with RIL-2 to mice harboring hepatic metastases, cells grown in AIM V medium demonstrated an increased antitumor activity compared to cells grown in CM. As an example in experiment 1, the mean number of metastases with day 5 expanded LAK cells grown in CM and given with RIL-2 was 47 while the mean number of metastases with day 5 expanded LAK cells grown in AIM V medium and given with RIL-2 was 5 (P less than 0.002). These experiments demonstrate that AIM V medium can be utilized to generate greater numbers of murine LAK cells with enhanced in vivo antitumor activity compared to cells generated in CM. These findings could be applied to the expansion of cytotoxic cells for human antitumor 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.     

Identification of a novel CD56- lymphokine-activated killer cell precursor in cancer patients receiving recombinant interleukin 2.

Circulating lymphokine-activated killer (LAK) cell activity in cancer patients receiving recombinant interleukin 2 (rIL-2) therapy is confined to cells expressing the CD56- surface marker. However, CD56- cells from these patients but not normal individuals have been reported to exhibit LAK cytotoxicity only following in vitro activation with rIL-2. Studies were performed to document the existence of CD56- LAK precursor cells and to phenotypically characterize this population in patients receiving rIL-2 therapy using fluorescence-activated cell sorter-purified CD56- cell subsets. Initial studies confirmed that CD56- cells exhibit NK activity [20 +/- 7 (SE) LU/10(6) cells] but not LAK activity (0 +/- 0 LU/10(6) cells) when evaluated directly from peripheral blood of patients receiving rIL-2. CD56- cells from patients but not normal individuals developed significant LAK cytolytic activity against NK-resistant COLO 205 targets (16 +/- 3 LU/10(6) cells) when cultured for 3 days with 1500 units/ml rIL-2. The CD56- LAK precursor activity was confined to cells expressing a CD56-CD16+ phenotype and a large granular lymphocyte morphology; little or no NK or LAK precursor activity was detectable in CD56-CD5+ T-cells from patients. Phenotypic characterization of CD16+CD56- cells revealed that this population is uniformly CD11a+,CD18+, and CD38+ and is heterogeneous in its expression of CD11b, CD11c, and CD16/Leu 11c. These results indicate that rIL-2 administration induces enhanced LAK precursor activity in a novel population of CD5-CD16+CD56- cells.     

Induction of lymphokine-activated killer cells by intrapleural instillations of recombinant interleukin-2 in patients with malignant pleurisy due to lung cancer

Intrapleural instillations of recombinant interleukin 2 (RIL-2) were performed in 11 patients with malignant pleurisy due to lung cancer. Kinetic studies on RIL-2 concentration in the pleural effusion and serum revealed relatively long-term maintenance of detectable levels of RIL-2 (over 24 h in the pleural effusion and over 8 h in the serum). Clinically, pleural effusions and cancer cells in the effusions disappeared in 9 of the 11 patients 4 to 10 days after the start of the treatment. Lymphokine-activated killer cells were induced in the effusions of responders who exhibited the disappearance of pleural effusion and cancer cells from the effusion, but not in those of the nonresponders. This induction of lymphokine-activated killer cells may result in the disappearance of cancer cells and pleural effusions. Cytological examination of pleural effusions revealed increases of lymphoblasts, immunoblastic large lymphocytes, and eosinophiles in number and proportion in the responder, although such a phenomenon could not be observed in the nonresponders. Main and frequent side effects of intrapleural instillations of RIL-2 were fever up to 39 degrees C, transient increase of pleural effusion, and eosinophilia. No serious side effect was encountered in our experience.     

Differences between in vivo and in vitro activation of cancer patient lymphocytes by recombinant interleukin 2: possible role for lymphokine-activated killer cell infusion in the in vivo-induced activation

In this study 15 consecutive melanoma patients were treated with two courses of bolus recombinant interleukin 2 (rIL2) and rIL2 plus in vitro-generated lymphokine-activated killers (LAK), respectively. The immunological monitoring performed after 4 days of rIL2 or rIL2 plus LAK, indicate that the in vivo peripheral blood lymphocyte (PBL), activation (spontaneous proliferation, tumor cytotoxicity, number of DR+ PBL, obtained after the second cycle of rIL2 plus LAK is significantly higher than after the first cycle of rIL2 alone. During the 5-day interval between the two courses, PBL activation returns to baseline levels and no evidence for increased sensitivity of PBL to rIL2 is present. To further confirm this, two additional patients were studied, in whom rIL2 was administered by continuous i.v. infusion. In these two patients the in vitro versus in vivo PBL activation could be directly and simultaneously compared by using in vitro the same concentration of rIL2 reached and maintained in the patients' sera. The PBL activation induced in vivo by a cycle of rIL2 alone was significantly less (about 10 times) than that obtained in vitro with a comparable rIL2 concentration. Thus, the infusion of in vitro highly activated PBL could explain the increased in vivo lymphocyte activation of the second cycle of rIL2 plus LAK over the first cycle of rIL2 alone.     

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)     

Monoclonal antibody therapy of murine lymphoma: enhanced efficacy by concurrent administration of interleukin 2 or lymphokine-activated killer cells

Lymphokine-activated killer (LAK) cells have recently been shown to be very efficient effector cells for antibody-dependent cellular cytotoxicity. Thus, we explored, in a murine lymphoma model, administration of LAK-inducing doses of interleukin 2 (IL-2) or adoptive transfer of LAK cells as a means of enhancing therapy with tumor-specific monoclonal antibody (mAb). AKR/Cum (Thy-1.2+) hosts were inoculated on day 1 s.c. with the SL-2 thymoma of AKR/J origin (Thy-1.1+) and developed palpable tumor on day 4. Tumor-specific anti-Thy-1.1 IgG2a mAb, 1A14, was given on days 4 and 8 with 50,000 units/day IL-2 i.p. divided in two doses on days 4-12. Therapy with IL-2 or mAb alone had minimal activity, prolonging control median survival of 22 days to 25 and 29 days, respectively, whereas therapy with IL-2 plus mAb significantly prolonged median survival to 40 days. However, combined therapy did not result in cures and long term survival. The efficacy of combined therapy did not result from alterations in the biodistribution of mAb by concurrent IL-2 infusions, as determined by studies with radiolabeled mAb. The combined effect of in vitro generated LAK (10(8) cells) adoptively transferred i.v. with 1A14 on days 4 and 8 following SL-2 inoculation was also evaluated. This regimen had no detectable toxicity, and treatment of mice with LAK and mAb resulted in 60% long term survival compared with 17% or 0% for mice treated with mAb or LAK alone. Thus, the therapeutic effects of tumor-specific mAb was enhanced by in vivo administration of IL-2 or by adoptively transferred LAK, which may represent means to provide the host with increased antibody-dependent cellular cytotoxicity effector cells. Adoptively transferred LAK has the additional benefit of augmenting mAb therapy of tumor without the toxicity associated with the induction of such cells in vivo with high dose IL-2.     

Functional and phenotypic characteristics of recombinant interleukin-2 or T-cell growth factor-activated splenic lymphoid cells from patients with gastric or hepatocellular carcinoma

Fourteen days' culture of human spleen cells with recombinant interleukin-2 (rIL-2) or T-cell growth factor (TCGF) results in the generation of lymphokine-activated killer (LAK) effector cells that have the unique property of lysing natural killer (NK)-resistant human tumor cells, Daudi, and NK-sensitive K562 cells. LAK cells were generated from patients with advanced cancer or liver cirrhosis. The splenic LAK-effector cell types were analyzed by two-color flow cytometry. The rIL-2-induced LAK cells showed an increased proportion of CD8+CD11- and CD57+CD16- and a decreased proportion of CD4+Leu-8- cells. In contrast, TCGF-induced LAK cells revealed a significantly increased proportion of CD8+CD11- and CD4+Leu-8- cells and a decreased proportion of CD57+CD16- cells. Thus, splenic LAK cells with different surface phenotypes were induced by the cultivation with rIL-2 or TCGF. Furthermore, TCGF-induced LAK cell activities in patients with cancer were found to be lower than the rIL-2-induced LAK cell activities. It was noted that the TCGF-activated splenic lymphoid cells did not inhibit the effector process of tumor cell lysis by LAK cells that had been activated by rIL-2. Other mechanisms of lower LAK cell activities of TCGF-activated splenic lymphoid cells from patients with cancer were discussed. The findings suggest that spleens of examined patients with gastric or hepatocellular carcinoma do not seem to be responsible for suppression of cell-mediated antitumor immunity.     

Combination treatment with irritant and recombinant interleukin 2 in the peritoneal cavity for evoking effective anti-tumor activity: generation of lymphokine-activated killer cells and tumor-specific killer cells

Meth A sarcoma, growing in the subcutaneous tissue of syngeneic BALB/c mice, regressed completely after an intraperitoneal (ip) injection of proteose peptone (PP) (on day 6) followed by 2 ip administrations (on days 7 and 8) of human recombinant interleukin-2 (IL-2, 25 micrograms/day), whereas one such treatment alone had little effect on the tumor growth. While this combination treatment was effective in anti-asialo GM1 antibody-treated mice, no such effect was noted in T cell-depleted ATXFL (thymectomized, irradiated and fetal liver cell-reconstituted) mice. These results show that T cells are mainly responsible for this antitumor effect. Treatment with a combination of PP and IL-2, but not with either PP or IL-2 alone, resulted in a marked increase in the T cell population in the peritoneal cavity after the treatment. At an early stage after the combination treatment, both peritoneal exudate cells (PEC) and spleen cells exhibited killing activity with a promiscuous specificity. However, at a later stage, 7 days after the treatment, Meth A-specific killer activity was observed in both PEC and the spleen. Meth A rechallenge was rejected by the mice in which the tumor had regressed, but the antigenically different Meth 1 was accepted by them. A similar result was obtained in Winn's neutralization test. These results suggest that this combination treatment, which is effective in the generation of lymphokine-activated killer cells in the peritoneal cavity, finally resulted in the induction of tumor-specific killer cells in the periphery. These results clearly show the anti-tumor efficacy of combination treatment with PP and rIL-2.     

Synergistic effect of Nocardia rubra cell wall skeleton and recombinant interleukin 2 for in vivo induction of lymphokine-activated killer cells

C57BL/6 mice inoculated i.p. with 3LL tumor cells were treated by local combination therapy with Nocardia rubra cell wall skeleton (N-CWS) and recombinant interleukin 2 (rIL-2). The combination treatment significantly prolonged their survival and augmented lymphokine-activated killer (LAK) activity of peritoneal cavity lymphocytes (PCL), compared with treatments with rIL-2 alone or N-CWS alone. After in vitro culture of peritoneal exudate mononuclear cells with rIL-2, the nonadherent population derived from N-CWS-injected tumor-bearing mice showed a significantly higher LAK activity than did that population derived from saline solution-injected mice. When N-CWS-induced PCL were cocultured with either N-CWS-induced macrophages or control macrophages in the presence of rIL-2, their LAK activity was higher than that of control PCL. Therefore, it was suggested that N-CWS-induced PCL themselves have a more potent ability as precursors of LAK cells. Phenotypic analysis on PCL populations revealed that N-CWS-induced PCL contained increased proportions of CD3+CD4-CD8- cells and asialo GM-1+ cells compared with control PCL. These results suggest that N-CWS selectively accumulates potent LAK precursors, namely, CD3+CD4-CD8- T-cells and asialo GM-1+ natural killer cells, at the injection site and that LAK cells are efficiently induced by subsequent administration of rIL-2.     

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.     

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 interleukin 2-induced activation of lymphokine-activated killer cells and tumor cytotoxic T-cells in cervical lymph nodes of patients with head and neck tumors

To study whether regional injection of recombinant interleukin 2 (rIL-2) can induce an in vivo lymphocyte activation in cervical lymph nodes (LNs) of patients with head and neck carcinoma, 12 patients, candidates for prophylactic dissection, were treated for 7-10 days prior to surgery with rIL-2, 10(5) units/day, injected in the perimastoid region. A marked induction of cytotoxic activity against allogeneic (K562 and Daudi lines) and autologous target cells (fresh spindle cell carcinomas of the tongue) was observed in lymphocytes obtained from jugular, spinal, and, to a lesser extent, submandibular LNs of all treated patients. An increase of cytotoxicity was also present in LNs contralateral to the rIL-2 injection side. On the other hand, only a borderline increase in spontaneous proliferation was detected. Moreover, in the two cases tested, a marked and apparently autologous tumor (Auto-Tu)-specific lysis was found in CD5+ lymphocytes obtained from LNs, whereas lymphokine-activated killer activity was mainly exerted by CD16+ natural killer cells. T-lymphocytes, when cultured with irradiated Auto-Tu cells and low doses of rIL-2, showed an increased Auto-Tu lysis, while cytotoxicity against allogeneic tumor cells (including K562) was not observed. These data indicate that regional injection of rIL-2 can activate lymphokine-activated killer cells from LN lymphocytes but also induce and/or expand a T-cell population expressing a restricted Auto-Tu cytotoxicity.     

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.     

Immunotherapy of cancer with lymphokines and lymphokine-activated killer cells

Our expanding knowledge of the immune system has provided a basis of rationality for immunotherapy. Some non-specific immunotherapy has achieved the status of standard treatment: interferon in hairy cell leukemia and chronic myelogenous leukemia, BCG in bladder cancer, and levamisole in colon cancer adjuvant therapy. Tumor infiltrating lymphocytes, moreover, offer a level of specificity heretofore unknown. Combined with the newly available synthetic cytokines that regulate the normal immune system there is the potential for a major breakthrough in biotherapeutics. Problems remain. We have yet to identify tumor antigens with the precision necessary for effective immunotherapy. Indeed, we have no assurance that tumors will regularly synthesize new antigens. In the broad spectrum of immune deficiency syndromes, we have yet to see an increase in the common epithelial tumors that account for the great bulk of human cancer. This suggests that we still have a great deal more to learn.