Survival after intratumoral interleukin-2 treatment of 72 melanoma patients and response upon the first chemotherapy during follow-up

Systemic high-dose interleukin-2 (IL-2) treatment achieves long-term survival in a subset of advanced patients with melanoma. As we reported previously, intratumoral IL-2 induced complete local responses in more than 60% of melanoma patients. This study aimed to analyze the long-term outcome of 72 patients treated in two prior trials. Melanoma patients (49 stage III, 23 stage IV) with injectable metastases received intratumoral IL-2 injections thrice weekly at individually escalated doses (median duration, 6.5 weeks; median total IL-2 dose, 72 MIU; median number of injected metastases, 10). The observed 2-year overall survival rates were 95.5% for stage III patients with cutaneous metastases only (stage IIIB), 72% for those with combined cutaneous and lymph node involvement (stage IIIC), 66.7% for stage IV patients with disease limited to distant soft-tissue metastases (stage IV M1a), and 9.1% for those with visceral metastases (stage IV M1b and stage IV M1c). Thirty patients who reported recurrence of unresectable distant metastases subsequently received chemotherapy in the further course of disease and showed an overall response rate of 36.7% (16.7% complete responses, 20% partial responses). A high total dose of IL-2 and a dacarbazine/temozolomide-based chemotherapy regimen were variables correlated with a clinical response. In conclusion, patients with cutaneous metastasis without lymph node involvement in stage III and with soft-tissue metastasis without visceral involvement in stage IV showed unexpected favorable survival rates after intratumoral treatment with IL-2. Furthermore, the intratumoral IL-2 treatment seemed to be associated with increased complete and partial responses in subsequent chemotherapies.     

Sequential immunotherapy using interleukin-1 followed by interleukin-2 of ascitic MOPC104E-bearing mice

This study shows that intraperitoneal injection of interleukin-1 (IL-1), followed by interleukin-2 (IL-2), can effectively eradicate murine ascitic tumor cells. This antitumor effect of IL-1 and IL-2 was abolished when administration of IL-2 preceded that of IL-1. Solid tumors inoculated subcutaneously (s.c.) into the back of mice were also sensitive to this combined i.p. therapy, indicating a systemically-operating antitumor mechanism. Splenocytes from tumor-bearing mice treated with IL-1 followed by IL-2 showed a strong tumor-neutralizing activity. The population responsible proved to be Lyt2.2 (CD8)-positive cells.Abbreviations IL interleukin - LAK lymphokine activated killer - LU lytic unit - MST median survival time - SE sonicated tumor extract     

Serum levels of interleukin-2 in cancer patients: preliminary considerations

In order to investigate the production of interleukin-2 (IL-2) in human neoplasms, we determined IL-2 and soluble IL-2 receptors (sIL-2R) in serum from 18 patients with lymphoma and 28 patients with solid tumors, with (15 cases) or without (13 cases) metastases. As controls, 58 healthy subjects were evaluated. Low levels of IL-2 were not observed in patients with lymphoma or limited solid tumor but abnormally low concentrations of IL-2 were seen in 4/15 metastatic solid tumors, associated with abnormally high values of sIL-2R. This preliminary study confirms in vivo the reduced IL-2 production in metastatic solid neoplasms, previously documented in vitro.     

Phase I/II study of sequential chemoimmunotherapy (SCIT) for metastatic melanoma: outpatient treatment with dacarbazine,granulocyte-macrophage colony-stimulating factor,low-dose interleukin-2, and interferon-alpha

A regimen of sequential chemoimmunotherapy (SCIT) was studied in a phase I/II study to analyze toxicity, anti-tumor and immunomodulatory effects in patients with previously untreated metastatic cutaneous melanoma. Treatment consisted of dacarbazine (DTIC) 800 mg/m2 administered intravenously (i.v.) on day 1, followed by subcutaneous (s.c.) molgramostim (GM-CSF), 10 times 2.5 micro g/kg on days 2 to 12, s.c. low-dose interleukin-2 (IL-2), 10 times 1.8 MU on days 8 to 18, and s.c. interferon-alpha-2b (IFN-alpha), 5 times 6 MU on days 15 to 20. Dosages were not escalated. Therapy was given in the form of outpatient treatment. Changes in T-lymphocyte phenotype and in soluble mediators were monitored during treatment. A total of 32 patients with stage IV melanoma were enrolled in the study. Treatment was well tolerated, without serious toxicity. In all cases, it could be given as outpatient treatment. Ten subjects out of the 31 patients evaluated showed an objective response, with 4 complete responses (CR) and 6 partial responses (PR); the response rate amounted to 32% (95% CI: 16-49%). Median survival of all patients was 8 months, with those patients who responded to treatment living longer than the non-responding group. Survival rate at 1 year was 22%. Monitoring of the effects of treatment revealed increased numbers of activated T-lymphocytes, both in the CD4 and in the CD8 subsets. The levels of soluble mediators such as sIL-2R and sCD8 were also increased. Changes were noted as early as the GM-CSF treatment period, and were observed to a further extent during IL-2 treatment. In the present study, it was found that this sequential chemoimmunotherapy regimen consisting of 4 agents (DTIC, GM-CSF, IL-2, IFN-alpha) has acceptable toxicity, can be administered on an outpatient basis, results in increased numbers of activated T-lymphocytes, and induces activity against metastatic melanoma that warrants further investigation.     

Cytomodulation of interleukin-2 effect by l-2-oxothiazolidine-4-carboxylate on human malignant melanoma

Glutathione (GSH), the most prevalent intracellular non-protein thiol, plays an important role in the interleukin-2 (IL-2)-induced proliferative activity of normal and tumour cells expressing IL-2 receptor (IL-2R). In the present study, we investigate the effect of IL-2 on proliferation of the human melanoma A375 cell line, and the possible selective cytomodulation effect of this cytokine by l-2-oxothiazolidine-4-carboxylate (OTZ) on these melanoma cells and on human peripheral blood mononuclear cells (PBMCs). We found that recombinant IL-2 (rIL-2) significantly increased the proliferation rate of A375 melanoma cells, which was associated with an increase in GSH levels, the enhancement of IL-2Rα expression and the endogenous production of IL-2 in these tumour cells. In contrast, OTZ decreased GSH content and the proliferation rate of A375 cells, and abrogated the growth-promoting effects of rIL-2. Thus, compared to cells treated with rIL-2, pre-treatment with OTZ reduced IL-2Rα expression, and also decreased the consumption of rIL-2 and the endogenous secretion of IL-2 by these tumour cells. With regard to PBMCs, the combination of OTZ plus rIL-2 resulted in a more rapid and greater increase of IL-2Rα expression than rIL-2 alone, with the proliferation rate being similar in the first 24 h, but with a lower PBMC′ count found thereafter compared to rIL-2 treatment alone. These results suggest that OTZ plays a crucial role in obtaining a selective cytomodulation of rIL-2, enabling it to exert its growth-promoting effect on normal cells, but not on melanoma cells, thereby possibly improving biochemotherapy with rIL-2.     

RNA editing signatures identify melanoma patients who respond to Pembrolizumab or Nivolumab treatment

Immunotherapy has improved the prognosis for many melanoma patients; however, our capacity to predict patient responses and to understand the biological differences between patients who will or will not respond is limited. Gene expression profiling of tumors from patients who respond to immunotherapy has focused on deriving primarily immune-related signatures; however, these have shown limited predictive power. Recent studies have highlighted the role of RNA editing in modulating resistance to immunotherapy. To evaluate the utility of RNA editing activity as a discriminative tool in predicting immunotherapy response, we conducted a retrospective analysis of RNA-sequencing data from melanoma patients treated with Pembrolizumab or Nivolumab. Here, we developed RNA editing signatures that can identify patients who will respond to immunotherapy with very high accuracy and confidence. Our analysis demonstrates that RNA editing is a strong discriminative tool for examining sensitivity of melanoma patients to immunotherapy.     

Active immunization of metastatic melanoma patients with interleukin-2-transduced allogeneic melanoma cells: evaluation of efficacy and tolerability

 From January 1994 to July 1996 we immunized metastatic melanoma patients with HLA-A2-compatible, interleukin-2 (IL-2)-secreting, immunogenic melanoma lines in an attempt to induce a systemic reaction that might also affect distant melanoma lesions. Twelve patients (6 male and 6 female) aged from 28 to 72 years, affected with visceral and/or subcutaneous (s.c.) melanoma metastases, were treated. Two different HLA-A2⁺ melanoma lines were transduced with the human IL-2 gene (14932/IL-2 and 1B6/IL-2) and used as vaccine. Two groups of 4 patients each were injected s.c. with 5×10⁷ and 15×10⁷ irradiated 14932/IL-2 melanoma cells respectively, whereas a third group received 5×10⁷ cells of the second line (1B6/IL-2). All patients received the vaccine on days 1, 13, 26; if no progression was evident, further immunizations were administered at monthly intervals. All patients were assessable for clinical response after at least three injections of the vaccine. In 4 cases a stabilization of disease lasting from 2 to 6 months was observed; in 2 of them a mixed type of response to treatment was noted with simultaneous evidence of regressing and non-responding lesions in the same patients. No signs of clinical response were found in the remaining patients. Nine patients died of disease between 3 and 24 months after the onset of therapy, whereas 3 were alive 3 months after the end of therapy. The local and systemic side-effects of treatment were mild. These results indicate that vaccination with cells bearing the appropriate antigens and releasing IL-2 locally can produce weak clinical responses, but also indicate that better results may be achieved through modifications of the vaccine, the schedule of immunization and/or a more appropriate selection of patients. Received: 20 December 1996 / Accepted: 27 February 1997     

Blood concentrations of interleukin-15 in cancer patients and their variations during interleukin-2 immunotherapy: preliminary considerations

In addition to the better known cytokines IL-2 and IL-12, IL-15, which is mainly produced by macrophages, is a new antitumor cytokine with a mechanism of action similar to that of IL-2. At present, however, there are no data about IL-15 secretion in cancer patients. This study was carried out to evaluate IL-15 blood concentrations in patients with early or advanced cancer and their possible variations in response to IL-2 cancer immunotherapy. The study included 40 patients with solid tumors, 24 of whom had metastatic disease. In addition, IL-15 secretion was evaluated during subcutaneous low-dose IL-2 therapy (6 million IU/day for 6 days/week for 4 weeks) in 14 metastatic renal cell cancer patients by collecting blood samples at weekly intervals. The control group consisted of 40 age-matched healthy subjects. Serum levels of IL-15 were measured by an enzyme immunoassay. No significant difference in mean serum levels of IL-15 was observed between cancer patients and controls. Moreover, the mean serum levels of IL-15 found in metastatic cancer patients were not significantly different from those found in patients with limited disease. Finally, no significant changes in mean levels of IL-15 occurred during IL-2 cancer immunotherapy. This preliminary study would suggest that IL-15 secretion is substantially within the normal range in cancer patients, both in early and advanced disease, and no variation seems to occur in response to IL-2 administration.     

Crystallization and preliminary X-ray studies of human recombinant interleukin-2

Two different forms of crystals (potentially) suitable for x-ray structure analysis were obtained for recombinant human interleukin-2 (IL-2) using ammonium sulfate as a precipitant in the pH range of 6.3-7.3 (in the case of hexagonal bipyramidal crystals) and 4.5-5.5 (in the case of plate crystals). The hexagonal bipyramidal crystal belongs to a hexagonal space group P6(2)22 or P6(4)22 with a = b = 105.8 A and c = 122.2 A. The crystal diffracts up to 3.4 A resolution and contains 2 or 3 IL-2 molecules in an asymmetric unit. The plate crystal belongs to an orthorhombic space group P2(1)2(1)2 with a = 47.9 A, b = 79.6 A, and c = 31.9 A. The crystal diffracts up to 2.5 A resolution and contains only 1 IL-2 molecule in an asymmetric unit. These facts reconfirmed crystallographically the high homogeneity of the present preparation of human recombinant IL-2.     

Phase I trial of combined treatment with ch14.18 and R24 monoclonal antibodies and interleukin-2 for patients with melanoma or sarcoma

Purpose: We conducted a phase I trial of interleukin 2 (IL-2) in combination with chimeric 14.18 (ch14.18) and murine R24 antibodies to determine the maximal tolerated dose (MTD), immunological effects, and toxicity of this treatment combination. Experimental Design: Twenty-seven patients with either melanoma (23 patients) or sarcoma (4 patients) were enrolled to receive a combination therapy with ch14.18 and R24 antibodies together with continuous infusion of Roche IL-2 (1.5×10⁶ U/m²/day, 26 patients) or Chiron IL-2 (4.5×10⁶ U/m²/day, 1 patient) given 4 days/week for 3 weeks. The antibodies ch14.18 (2–7.5 mg/m²/day) and R24 (1–10 mg/m²/day) were scheduled to be administered for 5 days during the second week of IL-2 therapy. Results: When given in combination in this study, the MTD for ch14.18 was 5 mg/m²/day and the MTD for R24 was 5 mg/m²/day. Dose-limiting toxicities were severe allergic reactions to both ch14.18 and R24 as well as pain related to ch14.18. This ch14.18 MTD was lower than the 7.5 mg/m²/day MTD previously determined for ch14.18 given alone with the same dose and schedule of IL-2. Immunological effects included the induction of lymphokine-activated killer (LAK) activity and antibody-dependent cell-mediated cytoxicity (ADCC). Anti-idiotype response to ch14.18 was seen in six patients, including two melanoma patients who had a partial response to treatment. In addition to two partial responses, four patients had a stable disease and one patient remained without any evidence of disease. Conclusions: Immunotherapy with IL-2 in combination with ch14.18 and R24 antibodies augments LAK function and ADCC measured in vitro in all patients. While there exist theoretical advantages of combining these two antibodies, the MTD of ch14.18 and of R24 were lower than the MTD of each antibody in prior studies evaluating single antibody therapy with IL-2. As such, the combination of these two antibodies together with IL-2 therapy appeared to influence the MTD and toxicity of each of the administered antibodies. This work is supported by NIH grants M01-RR03186, R01-CA32685, and P30-CA14520     

Augmentation of interleukin-2-induced activation of human melanoma tumor-infiltrating lymphocytes by heteroconjugate antibody

Summary Heteroconjugate (HC) antibody (anti-CD3 mAb × anti-p97 melanoma mAb) or monomeric anti-CD3 mAb by itself did not induce proliferation of uncultured melanoma tumor-infiltrating lymphocytes (TILs). They also failed to induce IL-2 production in uncultured TILs, although anti-CD3 mAb, but not HC antibody, stimulated IL-2 production in peripheral blood mononuclear cells (PBMCs). Sequential treatment of uncultured TILs from p97-antigen-positive (p97⁺) melanomas with HC antibody, followed by washing and incubation with interleukin-2 (IL-2), induced significantly higher proliferation than incubation with IL-2 alone. HC antibody pretreatment led to significantly greater results than with anti-CD3 mAb at a 1 ng/ml level in IL-2-induced proliferation of TILs from p97⁺ melanomas, similar to those with anti-CD3 mAb at a level of 100 ng/ml. HC antibody (1 ng/ml) pretretment did not enhance IL-2-induced proliferation of either TILs from p97^– melanomas or PBMCs, while anti-CD3 mAb enhanced the proliferation of TILs from some p97^– melanomas and PBMCs. Regardless of the pretreatment of uncultured TILs with HC antibody or anti-CD3 mAb, IL-2-activated TILs were cytotoxic primarily only to autologous tumor cells, and their phenotypes remained the same. Thus, HC antibody can augment IL-2-induced activation of TILs only from p97⁺ melanomas, without altering their pattern of cytotoxicity or phenotype. The findings were consistent with observations at the clonal level. In contrast to anti-CD3 mAb, HC pretreatment of uncultured TILs from only p97⁺ melanoma prior to limiting-dilution analysis increased the number of proliferating TIL clones, including autologous tumor-specific cytotoxic T lymphocyte clones. These results suggest that use of HC antibody in vivo would be more advantageous than anti-CD3 mAb, with regard to augmentation of IL-2-induced TIL activation.This work was supported in part by grants CA47 891, CA09 599, and RR5511-27 from the National Institutes of Health     

Treatment with tumour infiltrating lymphocytes and interleukin-2 in patients with metastatic melanoma: A pilot study

Tumour infiltrating lymphocytes (TIL) were isolated and expanded from biopsy samples of 4 patients with metastatic melanoma. The patients were treated with autologous expanded TIL and continuous or bolus infusion of Interleukin 2 (IL-2) at a dose of 18 × 10⁶ International Units/m²/day for 5 days starting 36–48 hours after administration of cyclophosphamide at a dose of 1 g/m². The number of TIL infused ranged from 10¹⁰ to 5,56 × 10¹⁰ cells. Two patients had stable disease (SD) lasting for 2 1/2 and 4 months respectively and they died 24 and 13 months after therapy. One patient died during therapy due to a pseudomonas septicaemia and another patient developed progressive disease (PD). He died 3 months after the start of therapy. The side effects were substantial but most of them were reversible upon cessation of the treatment.The majority of the expanded TIL of all patients were of the CD8+ phenotype. Cutaneous metastases from two patients, removed after treatment with IL-2 and TIL, showed moderate lymphocytic infiltration also mainly of CD8+ T cells.The treatment with IL-2 and TIL is feasible, but further investigations should continue in an attempt to improve the efficacy of the therapy, to reduce toxicity and to diminish the costs and labour of the culture methods.     

Prevention of superantigen-induced tolerance in vivo by interleukin-2 treatment

 Injection of the superantigen staphylococcal enterotoxin A (SEA) activates both CD4⁺ and CD8⁺ T cells expressing certain families of T cell receptor (TCR) variable-region β (V_β) chain. T cells respond with profound cytokine production and induction of cytotoxicity. Repeated injections, however, cause deletion and anergy of both CD4⁺ and CD8⁺ T cells, resulting in reduced frequency of SEA-responsive cells TCR-V_β11⁺ as well as reduced cytokine levels in serum upon challenge with SEA. Exogenous interleukin-2 (IL-2) in vivo rescued SEA-responsive CD4⁺ and CD8⁺ cells from SEA-induced deletion and/or increase expansion of SEA-primed cells as well as preventing down-regulation of endogenous IL-2 production in vivo. Combined treatment with SEA and IL-2 also superinduced production of important cytokines for the cytotoxic function of T cells, tumour necrosis factor α, interferon γ and IL-6, on a cellular level. These studies show that continuous stimulation with IL-2 in vivo could be useful for superantigen-based immunotherapy by induction of excessive T cell activation and by prevention of the development of T cell deletion and anergy. Received: 29 August 1996 / Accepted: 16 January 1997     

Functional and phenotypic modifications induced by IL-4, as single agent or in combination with IL-2, on PBMC preactivated in vivo by alpha-interferon + interleukin-2 therapy

The effect of human IL-4, used as a single agent or in combination with low or high dose IL-2, upon LAK-cell proliferation and activation has been tested on PBMC from patients treated with alpha 2-IFN and IL-2. Four days in vitro culture with IL-4 did not induce any LAK-cell activation; IL-4 induced the proliferation of CD3+ CD4+ T-cells, but decreased the percentage of NK cells in culture samples. When combined with high dose IL-2, IL-4 improved the recovery of MN cell without modification of T-cell subsets; however, IL-4 had no major effect on IL-2-induced NK or LAK cell activity. The combination of IL-4 and low dose IL-2 still significantly improved the total MN cell recovery but did not modify the distribution of T and NK lymphocytes; IL-4 inhibited low dose IL-2-induced NK and LAK cell activity, and increased the BL-esterase activity induced by high or low dose IL-2. The combination of IL-4 and IL-2 did not induce any large variation in the percentage of IL-2R (p55) expressing cells. In all tested conditions, IL-2R (p55) was mainly expressed on CD4+ T cells; less than 2% of the cells coexpressed the NK cell marker CD56 and IL-2R (p55). The effect of IL-4 upon IL-2-induced LAK cell expansion is thus very different on PBMC pre-activated in vivo by alpha IFN + IL-2 therapy than on PBMC pre-treated in vitro with IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased expression of perforin and granzyme B genes in patients with metastatic melanoma treated with recombinant interleukin-2

The frequency of peripheral blood cells expressing the perforin gene or the granzyme B gene was evaluated by in situ hybridization in nine patients suffering from metastatic melanoma and treated with recombinant interleukin-2 (rIL-2). A spontaneous expression of both genes was detected in five to seven patients, rIL-2 administration increased the frequency of positive cells in all patients (P<0.03 for each gene), the highest frequency being reached in the patients who already expressed these genes prior to rIL-2 treatment (P<0.02). Expressions of the granzyme B gene and of the perforin gene were strongly correlated before IL-2 treatment and they were similarly affected by rIL-2 administration. In contrast, their modification under treatment did not correlate with that of CD56⁺ cell counts, of natural killer activity and of sCD8 release. This indicates that perforin and granzyme B gene expressions are markers of cytotoxic cell activation independent of those previously described, and that they should be further evaluated in patients with malignancies to delineate their potential value in predicting clinical outcome.     

Antitumor and antimetastatic effects of dacarbazine combined with cyclophosphamide and interleukin-2 in Lewis lung carcinoma (3LL)

The antitumor and antimetastatic activity of dacarbazine (DTIC) alone or in combination with cyclophosphamide (CY) was tested in C57BL/6 mice bearing Lewis lung carcinoma (3LL). Treatment with both agents significantly reduced tumor growth and the number of metastases. These effects were associated with marked changes of the biochemical and immunological properties of drug-treated 3LL cells, i.e. (a) reduction of 6 integrin expression, (b) increased susceptibility to natural immunity in vivo, as measured in terms of rapid clearance from mouse lungs of prelabeled 3LL cells injected i.v. and (c) increased immunogenicity, as assessed by T-cell-mediated immune responses (i.e. graft rejection by intact syngeneic mice, and frequency of specific CTL precursors recognizing DTIC/CY-treated cancer cells). The immunotherapeutic advantage afforded by increased immunosensitivity and immunogenicity of 3LL cells exposed to DTIC+CY appears to be markedly reduced in vivo by the profound immunodepressive effects of these drugs. Within this context, addition of interleukin-2 was found to increase the antitumor and antimetastatic activity of this chemotherapeutic regimen. The present study shows, for the first time in a solid tumor model, that a biological response modifier increases the antitumor efficacy of drugs that are able to affect the immunological properties of cancer cells.