Augmentation of natural killer activity, induction of IFN and development tumor immunity during the successful treatment of established murine renal cancer using flavone acetic acid and IL-2

The investigational drug flavone acetic acid (FAA) has been previously shown to systemically augment NK activity in vivo in normal mice within 24 h of i.p. or i.v. administration. The current study investigates the ability of FAA, and/or rIL-2, to augment NK activity and antitumor responses in mice bearing murine renal cancer (Renca). The results demonstrate that FAA potently augments NK activity in the blood, spleen, and liver of Renca-bearing mice and that the administration of rIL-2 in addition to FAA results in a further augmentation of NK activity over that observed with FAA alone. Renca-bearing mice treated with FAA (200 to 250 mg/kg) plus rIL-2 exhibited a significantly increased incidence of long term survivors (59%) over that observed following treatment with FAA (0%) or rIL-2 (5%) alone. Therapeutic synergy between FAA and rIL-2 was observed against primary tumors, minimal residual disease, and experimental-induced pulmonary metastases. Mice cured of Renca by FAA plus rIL-2 treatment were largely resistant to rechallenge with Renca suggesting a role for T lymphocytes. The augmentation of NK activity and the therapeutic effects of FAA coincided with the rapid induction of high titers of serum IFN of the alpha/beta type within 4 h of FAA administration. Subsequent studies demonstrated that the contribution of FAA could be partially replaced by the administration of several doses of human rIFN-alpha A/D Bg1 before the initiation of rIL-2 administration. The observed synergistic antitumor effects of FAA plus rIL-2 coincided with the augmentation of NK activity, induction of IFN-alpha/beta, and induction of long lasting tumor immunity. Overall, these results suggest that this approach may obviate the need for adoptive immunotherapy in association with rIL-2 administration for at least some tumor types.     

Interleukin-21 Enhances Rituximab Activity in a Cynomolgus Monkey Model of B Cell Depletion and in Mouse B Cell Lymphoma Models

Rituximab, a monoclonal antibody targeting CD20 on B cells, is currently used to treat many subtypes of B cell lymphomas. However, treatment is not curative and response rates are variable. Recombinant interleukin-21 (rIL-21) is a cytokine that enhances immune effector function and affects both primary and transformed B cell differentiation. We hypothesized that the combination of rIL-21 plus rituximab would be a more efficacious treatment for B cell malignancies than rituximab alone. We cultured human and cynomolgus monkey NK cells with rIL-21 and found that their activity was increased and proteins associated with antibody dependent cytotoxicity were up-regulated. Studies in cynomolgus monkeys modeled the effects of rIL-21 on rituximab activity against CD20 B cells. In these studies, rIL-21 activated innate immune effectors, increased ADCC and mobilized B cells into peripheral blood. When rIL-21 was combined with rituximab, deeper and more durable B cell depletion was observed. In another series of experiments, IL-21 was shown to have direct antiproliferative activity against a subset of human lymphoma cell lines, and combination of murine IL-21 with rituximab yielded significant survival benefits over either agent alone in xenogeneic mouse tumor models of disseminated lymphoma. Therefore, our results do suggest that the therapeutic efficacy of rituximab may be improved when used in combination with rIL-21.     

Enhancement of anti-tumor activity of recombinant interleukin-2 (rIL-2) by immunocomplexing with a monoclonal antibody against rIL-2

We have investigated biological properties of an immune complex of recombinant interleukin-2 (rIL-2) and a monoclonal antibody against rIL-2 in mice for induction of killer cells and for anti-tumor activity. We have also examined the clearance of subcutaneously-injected immune complex in mice and compared it with that of rIL-2 alone. Plasma rIL-2 levels were sustained longer in mice given the immune complex than in mice given rIL-2 alone at a dose of 10g/mouse, and they were detectable even at 24 hours after the administration of the immune complex, while they fell to undetectable levels by 6 hours after the administration of rIL-2 alone. A more significant portion of rIL-2 was detected in lymph nodes after subcutaneous injection of the immune complex than that of rIL-2 alone. Splenic lymphocytes from mice given the immune complex demonstrated a higher killer cell activity against YAC-1 cells than those from mice given rIL-2 alone. The immune complex also exerted more significant anti-tumor effect in a dose-dependent manner in Meth-A fibrosarcoma-bearing mice than rIL-2 alone. Our results indicate that immunocomplexing of rIL-2 with an antibody against rIL-2 provides a useful tool as the drug delivery system for cancer therapy using rIL-2.Abbreviations rIL-2 recombinant interleukin-2 - NK natural killer - MoAb Monoclonal antibody     

Generation of tumor-specific cytotoxic T lymphocytesin vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8⁺ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8⁺ CTL in vivo.     

Augmentation of murine lymphokine (rIL-2)-activated killer cell activity by indomethacin

The effect of indomethacin on murine lymphokine-activated killer (LAK) cell activity was investigated using a natural killer-resistant, spontaneously developed, weakly immunogenic, and highly tumorigenic syngeneic murine mammary adenocarcinoma, mimicking that of human disease, as the target. When used in combination with human recombinant interleukin-2 (rIL-2), indomethacin was found to augment LAK cell activity, which was generated from culture of the normal mouse splenocytes with rIL-2, as compared to that with rIL-2 alone. This increase in LAK cell activity was shown to be indomethacin dose-dependent, and was demonstrated only when indomethacin was added to the rIL-2-containing medium at the beginning of culture. The enhancement of LAK cell activity by indomethacin was abrogated when the nylon-wool nonadherent "macrophage-poor" splenocytes were incubated with rIL-2 plus indomethacin. These results indicated that the rIL-2-induced LAK cell activity generated from murine splenocytes could be augmented by indomethacin, and the macrophages may be involved as the mediator.     

Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: I. phenotypic and functional analysis of the peripheral blood mononuclear cells

Summary The efficacy of recombinant interleukin-2 (rIL-2) or rIL-2 plus lymphokine-activated killer (LAK) cells in cancer therapy has been demonstrated by several groups both in experimental models in animals and clinical trials in humans, but their effects in vivo have yet to be clarified.Starting February 1988, we have treated 12 patients affected by advanced renal cancer with rIL-2 + LAK cells according to an open, non-randomized, phase II trial. Immediately before each rIL-2 infusion and during the last day of infusion, immunological tests were performed on the patients' peripheral blood mononuclear cells. During rIL-2 infusion we have observed a slight increase of the spontaneous cell proliferation and of natural killer (NK) and LAK activity; phenotypic analysis showed a significant decrease in the CD4⁺ T-lymphocyte subset, both in percentage and in absolute number. Conversely, before each cycle CD4⁺ cells increased when compared to basal values. No significant variations were observed in the CD8⁺ T-lymphocyte subset. Furthermore, a significant increase of the NK cells (CD3^– CD56⁺ CD16⁺) was evident during rIL-2 infusion.     

Lymphokine-activated killer cells in rats. IV. Developmental relationships among large agranular lymphocytes, large granular lymphocytes, and lymphokine-activated killer cells

The developmental relationships among large agranular lymphocytes (LAL) large granular lymphocytes (LGL) and the activation of these cells into lymphokine-activated killer (LAK) cells by rIL-2 was investigated. Highly enriched populations of LAL were isolated from Fischer 344 spleen cells by a combination of nylon-wool filtration (to remove B cells and macrophages), treatment with a pan T cell antibody plus complement (to remove T cells) and incubation in L-leucine methyl ester (to remove LGL). The resultant cells were highly enriched in morphologically identifiable LAL which expressed asialo GM1 and partially expressed the OX8 surface marker. The enriched LAL did not contain detectable NK cytotoxic activity, did not express pan T cell (OX19), Ia, Ig, or laminin surface markers and contained less than 0.2% LGL. Incubation of LAL in a low dose of rIL-2 (100 U/ml) induced the generation of LGL having NK activity within 24 h of culture. Longer culture periods (48 h) resulted in a continued increase in the percentage of LGL and higher levels of NK activity. However, with this low dose of rIL-2, little or no LAK activity (i.e., reactivity against NK-resistant target cells) was generated. With a high dose of rIL-2 (500 U/ml), LAL responded by first generating LGL with NK activity (within 24 h), with subsequent generation of LAK activity by 48 h. Evidence that the development of granular lymphocytes from LAL was responsible first for NK activity and then LAK activity was demonstrated by depletion of the generated granular NK or LAK effector cells by second treatments with L-leucine methyl ester. Concomitant with the induction of LGL with NK or LAK activity, rIL-2 also caused LGL to proliferate and expand four- to five-fold in 48 h. This occurred in the presence of high or low dose rIL-2. These results indicate that LAL are the precursors of LGL/NK cells, that LAL, LGL/NK cells and LAK cells appear to represent sequential developmental or activation stages and that LAL may comprise major source of LAK progenitors in lymphoid populations having few LGL or mature active NK cells.     

Augmentation of antitumor effect by combined administration with interleukin-2 and sizofiran,a single glucan,on murine EL-4 lymphoma

The antitumor effect of the combined administration with recombinant human interleukin-2 (rIL-2) and sizofiran (SPG), a single glucan of Shizophyllum commune Fries, was studied in vivo in C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The effect was evaluated by a) comparing the survival time of the mice, b) analysis of the intraperitoneal cell population in Giemsa-stained specimens, c) surface marker analysis of peritoneal exudative cells with flow cytometry, d) cytotoxic assay of cells against EL-4 and Yac-1 lymphoma, and e) elimination of some cell populations by monoclonal antibodies, to identify the antitumor-effector cells showing cytotoxic activity. The survival of mice given both rIL-2 and SPG was significantly longer than the control mice or those given SPG alone or rIL-2 alone. It was demonstrated that the administration of SPG and/or rIL-2 to the EL-4 lymphoma-bearing mice activated immune-response cells in the peritoneal cavity such as T lymphocytes, NK cells, or macrophages, which might be effective in reducing lymphoma cells. The combination of rIL-2 and SPG administration appears to activate the antitumor- immune response at the tumor site more effectively than when either agent was administered alone.     

Augmentative effect of Nocardia rubra cell-wall skeleton (N-CWS) on lymphokine-activated killer (LAK) cell induction

Summary The present study elucidated that N-CWS augments the cytolytic activity against 3LL tumor cells of LAK cells from N-CWS-immunized mice administered i.p. with rIL-2. This augmentative effect of N-CWS was not seen when the LAK cells were prepared from normal mice. The cytolytic activity was predominantly expressed in the NAPC prepared from the site of injection of rIL-2, and repeated administrations of rIL-2 were required to induce and maintain this potent cytolytic activity in vivo. Serological analysis revealed that the LAK cells were positive for Thy 1.2 and asialo GM1 antigens and that they were not classical CTL or NK cells. The administration of rIL-2 statistically prolonged the MST of mice bearing LAK-sensitive 3LL cells but not the MST of mice bearing LAK-resistant EL-4 leukemia. Furthermore, combination therapy with N-CWS and rIL-2 prolonged the MST of the mice more than the therapy with rIL-2 alone. These results suggest that LAK cells potentiated with N-CWS would be useful for immunotherapy of malignant neoplasms. Abbreviations used: N-CWS, Nocardia rubra cell-wall skeleton; rIL-2, recombinant interleukin 2; LAK, lymphokine-activated killer; RPMI 1640, Roswell Park Memorial Institute 1640; FCS, fetal calf serum; TCM, tumor culture medium; PC, peritoneal cells; NAPC, nonadherent PC; APC adherent PC; MST, mean survival time; NK, natural killer; E:T ratios, effector to target ratios; Poly I:C, polyinosinic-polycytidylic acid; CTL, cytotoxic T lymphocytes; RLNC, regional lymphnode cells     

Therapeutic efficacy of human recombinant interleukin-2 (TGP-3) alone or in combination with cyclophosphamide and immunocompetent cells in allogeneic,semi-syngeneic,and syngeneic murine tumors

Summary The potential for a recombinant human interleukin-2 (rIL-2, TGP-3) alone, in combination with cyclophosphamide, and in combination with cyclophosphamide and normal immunocompetent cells to manifest biological activity in vivo was tested using allogeneic, semi-syngeneic, and syngeneic tumor-host systems in mice. The biological activity of rIL-2 was evaluated by the inhibition of the growth of tumors and the inhibition of metastases in short-term assays and, in long-term assays, the prolongation of the survival time of mice bearing subcutaneously (s.c.) or intradermally transplanted tumors. rIL-2 was injected s. c. daily continuously for up to 40 days or intermittently two to four times into mice bearing established tumors. In the short-term assays, the dose and schedule dependence of activity of rIL-2 alone was significantly manifested against sarcoma 180 in ICR mice (allogeneic) by the regression of the tumor, and was confirmed against Meth-A fibrosarcoma in BALB/c mice (syngeneic) by retarding the growth of the tumor. When assessed using these tumors, it was found that the antitumor activity of rIL-2 was scheduledependent: the growth of tumors was more significantly suppressed when rIL-2 was injected every day for 10 days, starting on the 7th day after tumor transplantation, than when rIL-2 was injected five times every other day or twice every 5th day, even if the total amounts of rIL-2 injected were same. The continuous injection for 10 days was considered to be a standard regimen and the daily effective doses of rIL-2 were 5, 10, and 25 µg/mouse. Using the standard regimen and the effective doses, the activity of rIL-2 alone was also observed against two other syngeneic tumors: Colon carcinoma 26 in BALB/c mice, by retarding the growth of the tumor, and Lewis lung carcinoma in C57BL/6 mice by reducing the formation of lung metastases. When assessed using M5076 reticulum cell sarcoma, in a long-term assay, the activity of rIL-2 alone was not manifested in C57BL/6 mice (syngeneic) even when rIL-2 was injected for a long period (20 days) but it was observed in BDF1 (semi-syngeneic) mice. On the other hand, it was found that rIL-2 was effective in combination with cyclophosphamide in prolonging the survival time of C57BL/6 mice bearing the tumor. After cyclophosphamide (2.0 mg) had been administered orally to mice on the 6th day after tumor transplantation, the tumor regressed temporarily but regrew; however, when rIL-2 at a dose of 10 µg was also injected daily for a long period (40 days), the regrowth was retarded and the survival time of the mice was significantly prolonged. Moreover, when normal immunocompetent cells were transferred at the tumor sites, the regrowth of the tumors was retarded more significantly even at a daily dose of 1 µg or 3 µg rIL-2, and mice were observed to be cured by daily doses over 3 µg. The results obtained in the syngeneic tumor-host systems indicate that the continuous injection of rIL-2 is necessary and important for its activity to be manifest in vivo, and that, when combined with cytotoxic drugs and/or with immunocompetent cells, the potential efficacy of rIL-2 is valuable in cancer therapy.     

Effectivein vivo induction of lymphokine-activated killer (LAK) cells by pretreatment with a streptococcal preparation,OK-432

Effects of a streptococcal preparation, OK-432, on precursors of lymphokine-activated killer (LAK) cells were observedin vivo. Total number of splenocytes and the ratio of asGM ₁ ⁺ cells increased gradually after i.v. administration of OK-432, reaching their peaks at 3 to 4 days. It was found that as GM ₁ ⁺ cells were nonadherent and large in size. There were little differences in the ratios of Thy-1⁺, Lyt-2⁺, and L3T4⁺ cells before and after OK-432 treatment. Mice were injected i.p. with recombinant interleukin 2 (rIL-2) at a dose of 5 × 10⁴ U per mouse 4 days after OK-432 administration and LAK activity in their splenocytes was examined using natural killer (NK) resistant EL-4 target cells. Splenocytes in mice treated with both OK-432 and rIL-2 showed higher LAK activity than those in mice treated with rIL-2 alone.In vivo treatment with anti asGM, antibody prior to rIL-2 injection abolished completely such augmentation of LAK activity in OK-432 treated mice. These results demonstrated that asGM ₁ ⁺ LAK precursor cells induced by OK-432 were effectively differentiated into LAK cells by rIL-2.     

Induction of natural killer effectors from human thymus with recombinant IL-2

The NKH1 Ag is expressed on all cells in human peripheral blood capable of mediating spontaneous non-MHC restricted cytolytic function (i.e., natural killing). The majority of NK cells do not express CD3 Ag and do not express TCR gene products. However, approximately 20 to 25% of NKH1+ cells coexpress CD3 and TCR proteins. Both NKH1+CD3+ and NKH1+CD3- effectors can proliferate in response to IL-2 which also results in enhancement of cytolytic function. In the present studies, we examined thymocytes after incubation with rIL-2 for the presence of NKH1+ cells and for the development of non-MHC restricted cytolytic function. NKH1+ cells and NK activity could not be detected in fresh thymus. After culture with rIL-2 only, NK activity appeared in 3 days, reached a maximum after 7 days, and was effective against a panel of NK-sensitive targets. NK activity was correlated with the expression of NKH1 on the surface of in vitro proliferating thymocytes and immunofluorescent cell sorting demonstrated that almost all cytolytic activity was mediated by NKH1+ cells. As expected given the thymic origin of these cells, the majority of NKH1+ cells in culture expressed CD3. However, all cultures contained NKH1+CD3- effector cells which represent 15 to 40% of the NKH1+ population. As in peripheral blood, both NKH1+CD3- and NKH1+CD3+ exhibited non-MHC-restricted cytotoxicity, but only CD3+ effectors could be inhibited by anti-T3 mAb. These findings demonstrate that rIL-2 alone can induce subpopulations of thymocytes to proliferate, to express the NKH1 marker and become NK active in vitro. Furthermore, they suggest that the thymus which plays a role in the differentiation of NKH1+CD3+ NK effectors may also play a role in the differentiation or maturation of NKH1+CD3- NK effectors.     

Immunogenicity of recombinant human interleukin-2: Biological features and clinical relevance

The immunogenicity of recombinant interleukin-2 (rIL-2, EuroCetus, Amsterdam, Netherlands) was studied in seventy-six patients receiving different subcutaneous immunotherapy regimens. Patients presented with progressive metastatic renal cell carcinoma, malignant melanoma, colorectal cancer, B-cell lymphoma, and Hodgkin's disease. An enzyme immunoassay (EIA) was employed to screen patients for development of non-neutralizing antibodies against rIL-2, antibody specificity was confirmed by a standard Western blot. Neutralizing serum activity against rIL-2 was detected using a standard CTLL mouse proliferation assay. Additionally, serum levels of soluble interleukin-2 receptors and lymphocyte subsets expressing the CD56 natural killer (NK) associated antigen were measured.In a proportion of approximately 35% to 90% of the patients treated, non-neutralizing antibodies against rIL-2 could be detected after all treatment courses were evaluated. Antibodies were of the IgG, IgM, IgA and IgD subtypes. None of the 76 patients exhibited serum neutralizing activity after one treatment course. Five patients exhibited neutralizing anti-rIL-2 serum activity after two or more treatment courses of systemic rIL-2. In three of these patients, antibodies neutralized both recombinant and natural IL-2. Patients developing neutralizing anti-rIL-2 antibodies, exhibited significantly lower serum sIL-2 receptor levels upon the emergence of serum neutralizing activity than patients without antibody. Additionally, NK cell associated CD56 positivity was significantly lower in patients who exhibited neutralizing anti-rIL-2 serum activity than in patients who did not. A significant decrease in levels of soluble IL-2 receptors and CD56 NK cell positivity was observed, when comparing values prior to and after onset of serum neutralizing activity against rIL-2. However, while emergence of neutralizing antibodies to rIL-2 diminished rIL-2 induced biological activation, it did not coincide with abrogation of treatment response.Abbreviations rIL-2 recombinant interleukin-2 - EIA enzyme immuno assay - rIFN-2 recombinant interferon- 2     

Recombinant interleukin-2 treatment in patients with metastatic colorectal cancer: Effect on natural cytotoxicity

Summary Natural cytotoxicity (natural killer, NK, and lymphokine-activated killer, LAK, activity) was documented in 12 patients with metastatic colorectal cancer, both before and after a 5-day course of continuous therapy with intravenous recombinant interleukin-2 (rIL-2). Treatment induced a substantial increase in circulating CD56⁺ lymphocytes (pretreatment: 12.1±6.9%, mean ± SD; posttreatment: 39.2±6.9%. Maximal NK cell activity was induced by treatment with rIL-2 but only suboptimal augmentation of LAK cell cytotoxicity was obtained. This study indicates that although continuous infusion of rIL-2 does have a significant effect on natural cytotoxicity, this is suboptimal and further studies are necessary to define the most efficacious immunity-enhancing regimens of therapy, thereby hopefully improving clinical outcome of rIL-2 treatment.     

Treatment of adenocarcinoma in the peritoneum of mice: Chemoimmunotherapy with IL-2-stimulated cytotoxic lymphocytes as a model for treatment of minimal residual disease

Summary We have used a transplantable murine adenocarcinoma of renal origin (Renca) introduced to the abdomen by i. p. injection of a tumor cell suspension, to study the therapeutic potential of adoptive immunotherapy and/or biological response modifiers (BRMs). This tumor model is therapeutically challenging since the tumor grows progressively resulting in extensive peritoneal carcinomatosis, with hemorrhagic ascites, metastases to abdominal lymph nodes, liver, most serous membranes, spleen, and in some animals, pulmonary metastases. Without therapy, death occurs invariably in 36±3 days. In vitro, the tumor is lysed by lymphocytes obtained from the peritoneal cavity of mice treated with human recombinant interleukin-2 (rIL-2) and by cototoxic lymphocytes stimulated by in vitro culture with human rIL-2. Treatment of i. p. Renca with a single i. p. injection of the chemotherapeutic agent doxorubicin hydrochloride (DOX), or adoptive transfer of in vitro stimulated cytotoxic lymphocytes together with rIL-2 cured 50% and 20% of the tumor-bearing mice, respectively. In contrast, combined therapy with DOX and adoptive transfer of in vitro stimulated cytotoxic lymphocytes and rIL-2 cured the majority (90%) of tumor-bearing mice. These results suggest that administration of immunotherapy with in vitro activated cytotoxic cells together with human rIL-2 substantially enhances the effectiveness of chemotherapy.     

IL-4 regulation of murine lymphokine-activated killer activity in vitro. Effects on the IL-2-induced expansion, cytotoxicity, and phenotype of lymphokine-activated killer effectors

The in vitro incubation of B6 splenocytes with purified, mouse rIL-4 for 4 to 5 days was sufficient to generate lymphokine-activated killer (LAK) activity. In addition, rIL-4 augmented LAK cytotoxic activity when combined with rIL-2, as measured in a 4 h 51Cr-release assay against fresh, syngeneic MCA-sarcoma (MCA-102 and MCA-105) cells. Interestingly, this augmentation was not observed against the cultured YAC-1 target. LAK generation and augmentation of cytotoxicity by rIL-4 was species-specific, because human rIL-4 (up to 20,000 U/ml) failed to elicit these effects in the mouse splenocyte cultures. When 5-day B6 LAK cells (splenocytes incubated in rIL-2 at 1000 U/ml for 5 days) were split and recultured in the combination of rIL-2 plus rIL-4 for 4 additional days at least a twofold greater expansion in cell number resulted compared to similar cells cultured in either rIL-2 or rIL-4 alone. Moreover, LAK cells expanded in rIL-2 plus rIL-4 exhibited substantial increases in in vitro cytolytic activity (on a per cell basis) against MCA-102 and MCA-105 sarcoma cells, but not against YAC-1 targets. FACS analysis or negative selection using Lyt-2 or NK-1.1 mAb plus C revealed no differences in effector phenotype(s) of LAK cells expanded in rIL-2 alone compared to rIL-2 plus rIL-4 to account for the differences observed in both expansion and cytolytic activity by rIL-4. The majority of cells was Thy-1+, Lyt-2+, T3+, and ASGM-1+. However, a marked increase in the granule-associated serine esterase, BLT-E, was found only in LAK cells expanded in the combination of both lymphokines. Collectively, these studies show that rIL-4 has potent regulatory activities on splenic LAK generation, expansion, and cytotoxic function in the mouse.     

Interleukin-2: hope in cases of cisplatin-resistant tumours

 To establish whether or not local low-dose recombinant interleukin-2 (rIL-2) therapy might result in therapeutic benefit for ovarian cancer patients treated with cisplatin, the antitumour effects of rIL-2 and of combined treatment with cisplatin and rIL-2 in a mouse ovarian tumour (MOT) model were studied. In addition, some possible mechanistic aspects underlying the observed antitumour responses were analysed. MOT cells were injected i.p. into syngeneic, immunocompetent, female C3HeB mice. Tumour-bearing mice received i.p. treatment with cisplatin, rIL-2 or both. The MOT tumour appeared to be hardly responsive to treatment with cisplatin only or rIL-2 only. In contrast, combined local treatment with low doses of cisplatin (1 and 5 mg/kg body weight) and rIL-2 (60 000 U/day) resulted in an effective antitumour response in MOT-bearing mice. Complete rejection of the i.p. (local) tumour occurred in up to 60% of the cases. In vitro studies showed that cisplatin and rIL-2 do not have cumulative direct toxic effects on MOT cells. Mice cured after combined treatment with cisplatin and rIL-2 were not able to reject a rechallenge with tumour cells, indicating that these mice had not developed immunity to the tumour. Analysis of tumour-associated leucocytes, however, showed that combined treatment with cisplatin and rIL-2 did result in enhanced non-specific cytolytic activity of peritoneal leucocytes. We have thus demonstrated that, in the MOT model, combined local treatment with low doses of cisplatin and of rIL-2 is far more effective than therapy with cisplatin alone. Non-specific cytotoxicity of leucocytes appears to be involved in antitumour responses induced by combined treatment with cisplatin and rIL-2. These results suggest that, in human ovarian carcinoma, much better results may be obtained with the combined treatment of cisplatin and low (non-toxic) doses of rIL-2 than with cisplatin only. This may also apply to cisplatin-resistant ovarian carcinoma. Received: 6 March 1997 / Accepted: 30 October 1997     

Immunotherapy with intralesional and systemic interleukin-2 of patients with non-small-cell lung cancer

Eight patients affected by non-small-cell lung cancer were treated with intralesional and systemic recombinant IL-2(rIL-2) injection with the aim of activating both tumour-infiltrating lymphocytes and circulating cytotoxic or killer cells. The schedule of treatment was as follows: a daily fine-needle transparietal intralesional rIL-2 injection (1×10⁵ Cetus units) from day 1 to day 5 and systemic rIL-2 infusion (1×10⁵ Cetus units kg^–1 day^–1) from day 6 to day 10. One to four cycles of treatment were received by each patient. Clinical and immunological evaluations were performed (a) before treatment, (b) following the intralesional rIL-2 administration, (c) 1 h after the beginning of rIL-2 infusion and (d) at the end of the systemic rIL-2 infusion. No complete remission was achieved, two patients showed a partial remission, three resulted in stable disease and three patients progressed. Natural killer and lymphokine-activated killer cell activity dramatically decreased 1 h after the beginning of rIL-2 infusion and increased at the end of treatment. A progressive increase of circulating CD8⁺ and HLA class II⁺ T cells as well as of CD8⁺ T cell clones, most of which displayed NK activity, was recorded following rIL-2 infusion. Present data indicate that (a) the local administration of rIL-2 coupled with systemic rIL-2 infusion may be suggested as an alternative approach for the immunotherapy of lung cancer, (b) rIL-2 induces different immunological modifications according to the route and the time of its administration and (c) rIL-2 administration increases the amount of circulating immune cells with potential antitumour activity.     

Protective, restorative, and therapeutic properties of recombinant human IL-1 in rodent models

Human rIL-1 alpha and -1 beta are shown to increase significantly the CFU-culture activity in the spleen as well as at other sites after i.v. or i.p. administration. IL-1 can also significantly increase survival and can "rescue" a number of animals if administered either before or after lethal doses of cyclophosphamide or gamma-irradiation. The protective and reconstitutive activities of the rIL-1 are shown to correlate with increased CFU-culture frequency and total number, as well as increased cellularity in the bone marrow and peripheral blood, suggesting that this is one of their mechanisms of action. The sequence and timing of administration of human rIL-1 is critical for the protection or rescue of animals receiving DNA-damaging agents; maximal activity is achieved when IL-1 is given 20 h before insult or 48 h after alkylating agent administration. Minimal therapeutic activity is observed with IL-1 as a single agent for the treatment of metastatic disease compared with other biologic response modifiers including IFN-gamma.     

Anti-tumor effects of interleukin 2 against genitourinary cancer--basic study and clinical application

In order to establish an optimum mode for systemic administration of recombinant interleukin 2 (rIL-2), the effects of rIL-2 (Biogen, Switzerland) on lymphocyte-mediated cytotoxicity against established renal carcinoma cell line Caki 1. KU-2 and freshly prepared renal carcinoma cells were studied. Augmentation of cell-mediated cytotoxicity by rIL-2 was dose- and time-dependent. The results indicated that the optimal dose of rIL-2 was 100 to 500 units (Jurkat units)/ml, and that cytotoxicity increased significantly even at a low concentration such as 4 units/ml. We thus chose daily administration of multiple repeated dose for inpatients. To prevent withdrawal from the therapy as a result of un-tolerable adverse effects, the daily dose was set at 1 x 10(6) units, and rIL-2 was given to 17 patients with advanced genitourinary cancer. Two-hour intravenous drip infusions containing 5 x 10(5) units of rIL-2 was given daily two times to inpatients and after at least 28 days of this mode of administration, subcutaneous injection at a dose of 1 x 10(6) units was given 6 days a week to outpatients. In 12 patients with renal cell carcinoma, 2 patients showed complete response; 1 patient partial response; 7 patients no change, and 2 patients progressive disease. In patients with carcinoma of the prostate or bladder carcinoma, all patients were no change from criteria of Japan Society for Cancer Therapy, however, marked decrease in serum acid-phosphatase and improvement of performance status in 1 patient with carcinoma of the prostate, and massive necrosis of tumor accompanied by disappearance of severe leg edema in a patient with bladder carcinoma were observed.