Bispecific antibody-directed antitumor activity of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus interleukin 2.

Freshly isolated human CD4+ T cells can not respond to recombinant interleukin 2 (rIL-2) because of their lack of p75 IL-2 receptor expression. However, we succeeded in inducing a marked proliferation of purified CD4+ T cells by activation with rIL-2 plus anti-CD3 monoclonal antibody (mAb) cross-linked to a plastic plate. The proliferated CD4+ T cells produced a significant amount of IL-2 upon stimulation with phorbol ester plus A23187. Interestingly, CD4+ T cells activated with anti-CD3 mAb plus rIL-2 revealed a strong cytotoxic activity against Fc receptor (FcR)-positive tumor cells in the presence of anti-CD3 mAb. Moreover, the CD4+ T cells could lyse FcR-negative glioma cells by targeting with bispecific mAb containing anti-CD3 mAb and anti-glioma mAb. Thus, we demonstrated that rIL-2 and immobilized anti-CD3 mAb allowed the rapid generation of human CD4+ helper/killer T cells, which may be useful for the development of a new adoptive tumor immunotherapy.     

Lymphokine-activated killer cell plus recombinant interleukin-2 therapy of erythroleukemia in mice

The antileukemic effects of lymphokine-activated killer (LAK) cells plus recombinant interleukin-2 (rIL-2) therapy were assessed in mice with Friend virus (FV)-induced erythroleukemia. LAK cells were generated by incubating normal spleen cells for 72 hr in the presence of rIL-2 (1000 units/ml). At the time of injection, the LAK cells were cytotoxic in vitro against FV-infected fibroblasts and NK-sensitive and -resistant tumor targets but not normal controls. To determine in vivo activity, fully leukemic mice (spleen weight greater than 0.75 g) were injected with either PBS or LAK cells (10(8) cells/mouse IV at 14 and 17 days post virus) and rIL-2 (10,000 units/mouse IP every 8 hr on days 14 through 18 post virus). More than 70% of the progressively leukemic mice experienced permanent leukemia regressions (disease-free for greater than 100 days) following LAK cell plus rIL-2 therapy. Regressions were characterized by return of spleen and liver weights to normal and elimination of virus-infected erythroid (CFU-E) and macrophage (CFU-C) progenitor cells from spleen and marrow. Leukemic animals treated with either LAK cells alone or IL-2 alone experienced only transient leukemia regressions. These results demonstrate that LAK cell plus rIL-2 treatment can induce permanent regressions in progressively leukemic mice and provide a responsive and manipulable model system to elucidate the mechanisms involved in this form of immunotherapy.     

Lymphokine-activated killer cells in rats: analysis of progenitor and effector cell phenotype and relationship to natural killer cells

The progenitor and effector cell phenotype of lymphokine-activated killer (LAK) cells generated in F344 rats by recombinant human interleukin 2 (IL-2) (rIL-2) were analyzed. Highly purified populations of peripheral blood large granular lymphocytes (LGL) exhaustively depleted of T-cells were fully capable of generating high levels of LAK activity by 3 to 5 days in culture while purified populations of resting T-cells devoid of LGL could not generate LAK activity. This pure population of LGL expressed surface markers characteristic of rat natural killer (NK) cells [i.e., OX8+, asialomonoganglioside (asialo-GM1+), laminin+, OX19-, R1-3B3-, W3/25-, Ia-, surface immunoglobulin negative (SIg-)]. Further evidence that NK cells were the progenitors of cells with LAK activity was obtained by treatment of spleen or peripheral blood lymphocytes with anti-laminin or anti-asialo-GM1 antibodies plus complement or with the lysosomotropic agent L-leucine methyl ester. These treatments effectively depleted LGL/NK cell activity and the subsequent generation of rIL-2-induced LAK activity. Analysis of the LAK effector phenotype by cell sorting demonstrated that the majority of cells with LAK activity were OX8+, asialo-GM1+, laminin+, OX6+, OX19-, R1-3B3-, W3/25-, and SIg-. Furthermore, treatment of LAK cells with L-leucine methyl ester also significantly reduced their cytolytic activity. Thus, the LAK effector cells were also LGL and expressed surface marker characteristic of activated NK cells and not those of mature T- or B-cells. The proliferative response of rat spleen or blood lymphocytes to rIL-2 appeared to be primarily associated with LGL/NK cells since depletion of NK cells by anti-asialo-GM1 or anti-laminin antibody plus complement or by L-leucine methyl ester significantly (P less than 0.001) reduced the incorporation of [3H]thymidine into DNA. In contrast, depletion of T-cells (by anti-T-cell antibody plus complement) did not significantly affect rIL-2-induced proliferation. Similarly, T-cell-depleted, highly purified populations of LGL gave substantial proliferative responses to rIL-2. These studies clearly indicate that in the rat, the major cell population activated by rIL-2 is the LGL/NK cell and these cells appear to represent the major population of cells in blood or spleen which generate broad antitumor (LAK) cytotoxicity.     

Lymphokine-activated killer cells: lysis of fresh syngeneic natural killer-resistant murine tumor cells by lymphocytes cultured in interleukin 2

Normal splenocytes that are cultured in the lymphokine, interleukin 2 (IL-2), for as short as 2 days develop lytic activity for fresh syngeneic natural killer-resistant tumor cells as well as natural killer-sensitive YAC cells in a 4-hr 51Cr release assay. Lymphokine-activated killer (LAK) cells do not lyse syngeneic fresh lymphocytes but do lyse syngeneic concanavalin A-induced lymphocyte blasts. Lysis is not due to the presence of lectin or xenogeneic serum and appears to be an intrinsic property of lymphocytes activated in IL-2. The activation appears universal in that lymphocytes from all strains of mice activated in this manner exhibited similar patterns of lysis for fresh tumor target cells. To characterize the cells responsible for this lysis, we analyzed the phenotypic expression of surface markers on these cells with depletion techniques using monoclonal antibody and complement. These studies indicate that the precursor of the LAK cell is Thy-1+ and nonadherent to plastic or nylon wool. Lysis of syngeneic tumor was inhibited when LAK cells were treated with an anti-Thy-1.2, or anti-Lyt-2.2 monoclonal antibody and complement but not with anti-Lyt-1.2 monoclonal antibody and complement, indicating that the observed lytic activity was due to a Thy-1+ Lyt-1-2+ cell. Furthermore, LAK cell-mediated lysis could be inhibited by the addition of anti-Lyt-2 or LFA-1 monoclonal antibody to cytotoxicity assays. Cold target inhibition analysis revealed that the syngeneic tumor cells were lysed by recognition of a determinant not present on normal lymphocytes or lymphocyte blasts. This lysis of fresh solid tumor cells by lymphoid cells grown in IL-2 may be of value in the study of tumor-host immunological interactions. The biological significance of tumor lysis by IL-2-activated cells requires further study.     

In vivo antitumor activity of anti-CD3-induced activated killer cells

This study investigates the potential of the alpha CD3-induced killer cells for use in adoptive immunotherapy of tumor growth. The alpha CD3-induced, activated, killer cells (CD3-AK) were generated in DBA/2 (H-2d) splenocytes by preactivation with alpha CD3 and were then cultured in the presence (CD3-AK [alpha CD3+]) or absence (CD3-AK [alpha CD3-]) of alpha CD3. The conventional lymphokine-activated killer (LAK) cells were induced by culturing DBA/2 splenocytes with purified human recombinant interleukin 2. Testing their in vitro cytotoxicity against syngeneic mastocytoma P815, CD3-AK (alpha CD3+) cells gave the highest levels of cytotoxicity and were 20-fold higher than LAK cells and 200-fold higher than CD3-AK (alpha CD3-) cells. However, the cytotoxic activity of LAK or CD3-AK (alpha CD3-) cells was augmented by preincubating them with alpha CD3 for 3 h; then, the difference in cytotoxic activity was reduced from 20- to 4-fold and from 200- to 2-fold, respectively. The in vivo antitumor activity of these killer cells paralleled the in vitro activity. In tests using tumor neutralization experiments, 80-100% of the mice that were challenged with 1 x 10(2) P815 cells remained tumor free after receiving 5 x 10(6) CD3-AK (alpha CD3+) cells. When the challenge dose increased to 1 x 10(3) and to 1 x 10(4) cells, giving CD3-AK (alpha CD3+) cells slowed down the rate of tumor growth but only 20% of the mice remained tumor free. The untreated LAK cells or CD3-AK (alpha CD3-) cells did not induce any protection. After preincubation with alpha CD3 for 3 h, the CD3-AK (alpha CD3-) cells provided protection in 30% of the challenged mice. The phenotype of effectors for mediating the in vitro and in vivo antitumor activities was found to be Thy1+, CD4-, and CD8+ cells. Flow microfluorometry analysis showed that the higher levels of cytotoxic activity obtained with CD3-AK (alpha CD3+) cells could not be simply explained by the increase of CD8+ cells, and the cytotoxic activity of individual CD3-AK (alpha CD3+) cells appeared to be much higher than that of the LAK cells. After tumor growth was established for 1-2 days, giving CD3-AK (alpha CD3+) cells slowed down the rate of tumor growth, and 20% of the mice remained tumor free. These results indicate that CD3-AK cells may be used in the immunotherapy of tumor growth.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lymphokine-activated killer activity induced by in vivo interleukin 2 therapy: predominant role for lymphocytes with increased expression of CD2 and leu19 antigens but negative expression of CD16 antigens

The phenotype and function of lymphocytes from cancer patients treated with repetitive weekly cycles of continuous i.v. infusions of recombinant interleukin 2 (IL-2) were examined. Peripheral blood lymphocytes (PBL) obtained after IL-2 therapy showed an increased percentage of cells bearing the CD16 and leu19 markers which are associated with natural killer cells. These PBL mediated significantly increased levels of IL-2-dependent lymphokine-activated killer (LAK) activity against the Daudi cell line. Depletion of CD16+ cells from PBL obtained after in vivo IL-2 caused only slight inhibition of their LAK activity or their proliferative response to IL-2 in vitro. This indicates that CD16+ cells are involved but play only a minor role in these responses. In contrast, depletion of leu19+ cells, from PBL activated in vivo with IL-2, virtually abrogated their LAK activity and their proliferative response to IL-2. Two-color flow cytometry studies showed that a leu19+/CD16- population was expanded by in vivo IL-2 therapy and was responsible for the majority of LAK activity by in vivo-activated PBL. Moreover, this CD16- population showed an increased density of leu19 and CD2 (E rosette receptor) antigens when compared to the resting PBL obtained prior to IL-2 treatment. These data show that the predominant population mediating in vitro LAK activity, induced by in vivo IL-2 therapy, consists of activated natural killer cells with a high density of leu19 and CD2 antigens but negative for the CD16 antigen.     

Interactive effects of alpha-interferon A/D and interleukin 2 on murine lymphokine-activated killer activity: analysis at the effector and precursor level

Studies from our laboratory and others have demonstrated that alpha-interferon (IFN alpha) can regulate natural killer cells and lymphokine-activated killer (LAK) cell activation. In vitro experiments have shown that IFN alpha has differential effects on both natural killer cells and LAK activity when combined with interleukin 2 (IL2); IFN alpha synergized with IL2 to augment natural killer cells activity while it suppressed the IL2-induced LAK response. Here we demonstrated that IFN alpha A/D can also regulate IL2-induced LAK activity in vivo with enhanced or suppressed activity depending on the IFN alpha A/D dose. The enhanced response was observed with the combination when 80,000 units/day of IFN alpha A/D were used and was detectable in the spleen, lung, and peritoneum. When a high dose of IFN alpha A/D was combined with IL2, a moderate reduction in LAK activity was noted in the spleen and peritoneum. In contrast, a high dose IFN alpha A/D augmented IL2-induced LAK activity in the lung even though it reduced the level of cellular infiltration. We have also evaluated the effect that IL2, IFN alpha A/D, and IL2 plus IFN alpha A/D have on the frequency of LAK precursors in the spleen and lung using limiting dilution analysis. Treatment of normal mice with IL2 alone increased the frequency of LAK precursor (LAKp) in the lung. This increase was associated with an infiltration of Thy-1+, asialo-GM1+, Lyt-2- lymphocytes into the lungs. Moreover, treatment with IL2 plus IFN alpha A/D enhanced the frequency of LAKp over that observed with IL2 alone. Treatment with the combination did not change the phenotype of LAKp in the lung from that seen with IL2. The increase in LAKp frequency induced by the combined treatment may not be a direct effect of IFN alpha A/D on precursor cells since IFN alpha A/D alone did not increase the frequency of LAKp in vivo or in vitro when added to limiting dilution analysis cultures. In contrast to what occurred in the lung, a consistent increase in LAKp was not seen in the spleen after treatment with IL2 or with the combination, although LAK activity was observed. These results demonstrated that in addition to inducing lytic activity from LAK effectors in vivo, IL2 treatment increased the number of precursor cells within the lung. Moreover, IFN alpha A/D in combination with IL2 influenced the level of LAKp in situ.     

Cellular interactions in effector cell generation and tumor regression mediated by anti-CD3/interleukin 2-activated tumor-draining lymph node cells.

Previous studies have demonstrated that progressive growth of the weakly immunogenic MCA 106 murine sarcoma stimulated, in the draining lymph nodes, the production of tumor-sensitized but not fully functional preeffector lymphocytes. These lymphocytes could develop into specific immune effector cells after sequential in vitro activation with anti-CD3 monoclonal antibody and interleukin 2 (IL-2). In this study, we analyzed cellular requirements for in vivo sensitization of preeffector cells, for generation of immune effector cells by the method of anti-CD3/IL-2 activation, and for adoptive immunotherapy mediated by activated cells. By selective depletion of T-cell subsets in vivo, we found that tumor regression after systemic adoptive immunotherapy required the collaboration of activated CD4+ and CD8+ cells. It was further demonstrated that CD8+ immune cells alone could mediate antitumor effects if exogenous IL-2 was provided in vivo. These results suggest that CD8+ cells served as immediate effector cells, whereas CD4+ immune cells provided a helper function via the secretion of IL-2. During in vitro anti-CD3/IL-2 activation, generation of effector cells depended on the collaborative interaction between previously sensitized CD4+ and CD8+ preeffector cells. At the stage of in vitro activation, the addition of IL-2 could not substitute the function of CD4+ cells. We next examined whether the sensitization of preeffector cells in the draining lymph nodes required cellular interactions between CD4+ and CD8+ T-cells. By in vivo depletion of T-cell subsets during tumor growth, we found that CD4+ cells were sensitized independently of CD8+ cells. More interestingly, in vivo sensitization of CD8+ preeffector cells also occurred independently in the absence of a CD4+ helper cell response. The lack of T-cell-T-cell interactions in vivo may explain the failure of effector cell generation during progressive tumor growth. Taken together, these results demonstrate that the anti-CD3/IL-2 activation defines an immune response distinct from many previously described mechanisms of antitumor immune responses.     

Metastatic renal-cell carcinoma patients treated with interleukin 2 or interleukin 2 plus interferon γ: Immunological monitoring

We investigated the biological response of 73 patients with metastatic renal-cell carcinoma (MRCC) treated by repetitive weekly cycles of high-dose interleukin 2 (IL-2) (protocol 1, 40 patients) or IL-2 plus interferon-gamma (IFN-γ) (protocol 2, 33 patients). The objectives of this study were (i) to evaluate the effects of this IL-2 administration schedule on biological response, (ii) to compare the effects of IL-2 alone with those of IL-2 plus IFN-γ, (iii) to search for any correlation between certain biological marker values and the clinical response to treatment. Mean CD56⁺ lymphocyte counts (i. e., NK cells) were significantly higher than those of CD3⁺ cells in the 2 protocols and a subpopulation of CD56^bright cells in protocol I was found to be preferentially expanded in vivo. Cytotoxic activity against K562 and Daudi cell lines as well as TNF-α and sTNF-αR (but not IL-6) significantly increased following treatment. Comparison of the data obtained from patients treated with IL-2 alone vs. IL-2 plus IFN-γ did not show any significant changes except for eosinophilia (higher in protocol I). Therefore, addition of IFN-γ did not affect either lymphocyte distribution or non-MHC-restricted cytotoxicity in vivo. No difference in cell subpopulation or cytotoxicity was detected between responders and non-responders. Pre-treatment sTNF-αR concentration, in contrast to IL-6 and TNF-α, was significantly higher in progressive than in stable and responder groups, suggesting that this parameter may be predictive of the clinical response. © 1994 Wiley-Liss, Inc.     

Lymphokine-activated killer activity of tumor-associated and peripheral blood lymphocytes isolated from patients with ascites ovarian tumors

Peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) were isolated from 36 patients with advanced ovarian adenocarcinoma and peritoneal effusions for study of lymphokine-activated killer activity. PBLs and TALs cultured in vitro for 3-5 days in the presence of interleukin-2 (IL-2, supernatant of the MLA 144 gibbon cell line, or human recombinant IL-2) expressed higher levels of cytotoxicity as compared to cells cultured in medium alone, against natural killer (NK)-susceptible (K562) or NK-resistant targets (Daudi and the human ovarian carcinoma cell line SW626). When ovarian tumor cells, freshly isolated from carcinomatous ascites or surgical specimens, were used as target cells in the cytotoxicity assay, 8 of 14 PBLs and 5 of 7 TAL preparations lysed the autologous tumor after treatment with IL-2, while no spontaneous reactivity was observed in any of the 14 patients tested. Although levels of lysis were usually relatively low, these data demonstrate that PBLs and TALs from ovarian cancer patients (TALs usually exhibiting low NK activity) when stimulated in vitro by IL-2 acquire some cytotoxic potential against the autologous tumor.     

Cytological characteristics of human glioma-infiltrating lymphocytes stimulated with recombinant interleukin 2 and an anti-CD3 antibody.

Tumor-infiltrating lymphocytes (TIL) were generated from 10 glioma specimens by using recombinant interleukin-2 and an anti-CD3 antibody (CD3 + TILs). We obtained more than 1 x 10(9) cells in 5 cases, more than 5 x 10(8) cells in 2 cases, and about 1 x 10(8) cells in 3 cases during three weeks of incubation from small specimens ranging in weight from 0.5 to 2.0 g. In 4 cases, TILs were expanded following stimulation with only rIL-2 (CD3-TILs). The growth rate of CD3-TILs was less than that of CD3 + TILs. Cytotoxicity of CD3 + TILs was lower than that of lymphokine-activated killer (LAK) cells in a standard 4h 51Cr release assay. Cold target inhibition was undertaken in three cases and specific cytotoxicity could be shown in only one case. CD3 + TILs mainly consisted of CD3-positive cells, ranging from 63.2 to 99.9%. The ratio of CD4-positive cells to CD8-positive cells was not constant. The expression of Leu 7 and CD16 was low. The present study did not confirm previous findings that TILs were more tumor-selective and potent than LAK cells. Furthermore, the results on in vitro antitumor activity of those cells were not necessarily consistent with the results on their clinical activity. Further careful work is necessary on the preparation of immunocytes and the subsequent adoptive immunotherapy.     

Influence of schedule of interleukin 2 administration on therapy with interleukin 2 and lymphokine activated killer cells

The purpose of this study was to compare the toxicity, immunomodulatory changes, and antitumor efficacy of interleukin 2 (IL-2) and lymphokine activated killer (LAK) cell therapy with two durations of IL-2 infusion. Patients with progressive melanoma, non-Hodgkin's lymphoma, renal carcinoma, or colon carcinoma received IL-2 at 3 X 10(6) units/m2/day on days 1-5 and 13-17, either by bolus injection every 8 h (q8h) or by continuous i.v. (CIV) administration. Peripheral blood mononuclear cells were harvested by leukapheresis on days 8, 9, and 10, were incubated in vitro for 5 days for generation of LAK cells, and were infused on days 13, 14, and 15. The first 11 patients were treated with IL-2 q8h, and the subsequent 13 patients were treated by CIV infusion. Toxicity consisted primarily of fever, chills, emesis, diarrhea, weight gain, and edema but did not require intensive care unit support and did not differ significantly between treatment groups. IL-2-induced lymphocytosis on day 8 was higher with CIV than with q8h administration with a mean lymphocyte count/microliter of 5610 +/- 700 (SE) versus 3300 +/- 500. Immunomodulatory changes observed on days 8 and 20 were also greater with CIV IL-2 and included an increase in peripheral blood mononuclear cell IL-2 receptor expression as well as a marked rise in the number of Leu-11+ and Leu-19+ peripheral blood mononuclear cells. The total leukapheresis yield per patient and total number of LAK cells infused per patient were higher with CIV than q8h administration, with 49.8 +/- 4.9 X 10(9) versus 39.4 +/- 5.4 X 10(9) and 42.6 +/- 5.0 X 10(9) versus 34.0 +/- 5.4 X 10(9), respectively. The cells infused displayed phenotypic evidence of activation and exhibited marked lytic reactivity to Daudi, Raji, and HT-144 targets. One complete and one minimal response were observed in 2 of 8 patients with metastatic renal cell carcinoma who received CIV IL-2 and LAK cells. The results show that IL-2 is more biologically active by CIV than q8h administration, as demonstrated by greater rebound lymphocytosis, LAK cell yield, and in vivo immunostimulation.     

Exogenous interleukin 2 recruits in vitro lymphokine-activated killer activity by in vivo activated lymphocytes

Cryopreserved and thawed lymphocytes can be used instead of fresh lymphocytes to avoid test-to-test variability in studies of fluctuations of natural killer (NK) and lymphokine-activated killer (LAK) activities as a function of time. We investigated the effects of 1-h versus 18-h resting of lymphocytes on their lytic activities, because the process of cryopreservation and thawing decreases NK and LAK activities. Lymphocytes from renal cell cancer patients receiving adoptive immunotherapy were studied. An 18-h versus 1-h resting period led to a significant increase in NK activity but had no significant effect on LAK activity. The presence of 1200 IU/ml interleukin 2 (IL-2) in the medium 1 h prior to and during the cytotoxicity (CTX) assay increased in vivo and in vitro IL-2-induced LAK activities. This phenomenon has been interpreted as IL-2 dependency of effector lymphocytes (J.A. Hank, P.C. Kohler, G. Weil-Hillman, N. Rosenthal, K. H. Moore, B. Storer, D. Minkoff, J. Bradshaw, R. Bechhofer, and P. M. Sondel. Cancer Res., 48: 1965-1971, 1988). We performed kinetic studies to assess the role of effector lymphocyte recruitment in these experiments. LAK activity was tested in the presence or absence of IL-2 during preincubations and CTX assays varying between 0 and 120 min. These kinetic studies showed that effector lymphocyte recruitment indeed contributed to the increased level of LAK activity when IL-2 was added to the CTX assay. A minimal incubation period of 30 min was required to detect recruitment of lymphocytes. Effector lymphocytes could be recruited for periods varying between 90 and greater than 240 min, depending on the lymphocyte donor. We conclude that: (a) in vitro, IL-2-mediated recruitment of lymphocytes due to presence of IL-2 in the CTX assay may lead to an overestimate of the actual LAK activity; and (b) in vivo, prolonged IL-2 infusion after the administration of activated lymphocytes seems warranted in order to recruit maximal levels of effector lymphocytes with LAK activity.     

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.     

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.     

Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice

The adoptive transfer of lymphokine-activated killer (LAK) cells combined with low dose interleukin 2 (IL-2) mediates the regression of established pulmonary metastases in mice and has efficacy in the treatment of human cancer. Systemic administration of high dose IL-2 alone can mediate tumor regression. Cortisone acetate (CA), 25-75 mg/kg, was administered daily to mice receiving high dose IL-2 for 10 days. CA significantly reduced the toxicity induced by IL-2; 38 of 48 mice receiving CA survived compared to 0 of 30 controls (P less than 0.0001). In addition, CA administration caused a decrease in IL-2-induced 125I-labeled albumin leakage in mouse organs. However, CA abrogated the in vivo antitumor effect of high dose IL-2, and to a lesser extent the therapeutic effect of exogenous LAK cells plus lower dose IL-2. Mice treated with 100,000 units of IL-2 showed 98, 63, and 33% reductions of pulmonary metastases in Hanks' balanced salt solution, 25 mg Ca/kg, and 75 mg Ca/kg groups, respectively; treatment with LAK and 7,500 units of IL-2 resulted in reductions of 94, 77, and 57% in these same groups. CA treatment of animals did not affect LAK generation, although the absolute number of LAK precursors was greatly reduced. These results show that although CA can reduce the toxic effect(s) of IL-2, it can be detrimental to successful immunotherapy using this approach.     

Autocytotoxic activity of lymphokine-activated killer cells: characterization of effector cells and susceptible targets

The specificities and surface markers of murine autocytotoxic cells induced by in vitro culture with interleukin 2 (IL2) were studied. Culturing murine spleen cells with recombinant human IL2 resulted in the generation of cytotoxic cells which killed syngeneic lymphoblasts and syngeneic activated macrophages (M phi). Both lectins and protein antigens were capable of inducing lymphoblasts recognized by lymphokine-activated killer (LAK) cells. B-lymphoblasts as well as T-lymphoblasts were sensitive to lysis by these effector cells. In addition, peritoneal M phi activated in vivo with Bacille Calmette-Guérin (BCB), Corynebacterium parvum (C. parvum), thioglycollate (TG) or lipopolysaccharide (LPS) were shown to be susceptible to lysis by LAK cells. In contrast, neither unstimulated T cells nor resident peritoneal M phi were sensitive to lysis by LAK cells, suggesting that normal cells have to be activated in order to be sensitive to lysis by these effector cells. Surface marker analysis indicated that majority of effector cells which killed syngeneic lymphoblasts and activated M phi were Thy1+, asialo GM1+, L3T4-, Ly2-.     

Interleukin 2 and lymphokine-activated killer cell therapy: analysis of a bolus interleukin 2 and a continuous infusion interleukin 2 regimen

Several groups have described the efficacy of interleukin 2 (IL-2) plus lymphokine-activated killer (LAK) cells in the treatment of cancer patients with significant response rates noted in patients with renal cell cancer and malignant melanoma; however, the optimum regimen remains undefined. The Biological Response Modifiers Program of the National Cancer Institute conducted two consecutive Phase I/II studies evaluating the toxicity and clinical efficacy of different methods of IL-2 and LAK cell therapy. In the first trial, we modified the standard Rosenberg regimen by decreasing the duration of priming in an attempt to reduce the toxicity related to this phase of the therapy and thereby administer more IL-2 doses with the LAK cells. In the second trial, we used a continuous i.v. infusion IL-2 regimen and altered both the leukapheresis procedure and the LAK cell culture techniques based on our in vitro and preclinical studies suggesting that 2-day LAK cells were superior. Thirty cancer patients received i.v. bolus IL-2 at 100,000 units/kg every 8 h for 3 days during priming and for 5 days during LAK cell administration. A second group of 22 cancer patients received IL-2 by continuous i.v. infusion at 3 x 10(6) units/m2 for 5 days during priming and an additional 5 days of IL-2 with the LAK cell phase of the treatment. The timing of the start of the leukapheresis procedures, their duration and number, and the LAK cell culture techniques differed in the two trials. Overall, 52 patients with various cancers were treated. The toxicities associated with each regimen were similar to those seen in other IL-2 plus LAK cell trials. Four patients (one each with melanoma and diffuse large cell lymphoma and two with renal cell cancer) exhibited partial responses lasting 2, 4, 10, and 15+ mo. Serial tumor biopsies from treated patients demonstrated that therapy can produce a marked mononuclear cell infiltrate and an increase in HLA-DR expression on tumor cells. There was no difference in the overall response rate between the two regimens, but toxicity was less with continuous i.v. infusion IL-2. The 5-day continuous i.v. infusion regimen resulted in significantly higher rebound lymphocytosis, cell yield from leukapheresis, and number of LAK cells harvested from culture.     

Prolongation of serum half-life of interleukin 2 and augmentation of lymphokine-activated killer cell activity by pepstatin in mice

We reported previously using a murine model that the kidney is the organ involved in catabolism of exogenous human recombinant interleukin 2 (IL-2) and that cathepsin D, a major renal acid protease, is responsible for the degradation of IL-2. In the present report also using BALB/c mice we have investigated the effect of in vivo pepstatin, an acid protease inhibitor, treatment on serum half-life of IL-2, and generation of lymphokine-activated killer (LAK) cell activity. The in vivo pepstatin treatment by i.p. injection resulted in a significant reduction in the accumulation of 125I-IL-2 by the kidney in a reverse dose-response manner. Pepstatin treatment prolonged the serum half-life of 125I-IL-2, and the increase in serum half-life of 125I-IL-2 was pepstatin dose dependent. A significant reduction in renal cathepsin D activity, as monitored by the degradation of 125I-IL-2, was detected. In vivo pepstatin (0.6 mg/kg) treatment along with IL-2 (300,000 IU/mouse) daily for 3 or 6 days resulted in an augmentation of natural killer activity exhibited by freshly prepared and uncultured splenocytes against YAC-1 cells. An additional culturing of the splenocytes with IL-2 (3,000 IU/ml) in vitro for 1 day significantly enhanced the effect of in vivo pepstatin treatment; i.e., LAK cell activity generated from the splenocytes of animals treated with IL-2 plus pepstatin was greatly augmented in comparison with that treated with IL-2 alone. Phenotypic assessment by cell surface markers (Thy-1.2, Lyt-2, L3T4, and asialo-GM1) on the fresh splenocytes prepared from animals treated in vivo with pepstatin plus IL-2 revealed a decrease in the percentage of cells expressing Thy-1.2 and Lyt-2 and an increase in those carrying asialo-GM1. These results demonstrated that, as a result of in vivo pepstatin treatment, renal cathepsin D activity was greatly inhibited, which in turn reduced the degradation of circulating IL-2, then prolonged serum half-life of IL-2, and subsequently augmented natural killer and LAK cell activity. The in vivo pepstatin and IL-2 treatment decreased the T-cells and increased the natural killer-like LAK precursor cells, possibly also with an increase in its activity, which were further induced by in vitro IL-2 culture to generate an augmented LAK cell activity. This study also suggests the clinical potential of pepstatin in IL-2-related immunotherapy.