Cytokine-based Immunotherapy Research Articles

Dividing and conquering: controlling advanced melanoma by targeting oncogene-defined subsets

For decades, therapy for advanced melanoma has lagged behind most of the cancer field owing to its intrinsic resistance to conventional cytotoxic chemotherapy and limited impact of cytokine-based immunotherapy. The opportunity to develop molecularly targeted therapy emerged with the discovery of activating mutations in BRAF, a component of the long studied MAP kinase pathway. These mutations are found in approximately 50 % of patients with regionally advanced or metastatic melanoma and appear to be one of the initiating steps in the development of primary melanoma. Additional oncogenic events, particularly those that affect tumor suppressor genes, are essential for development of invasive and metastatic melanoma. Nonetheless, mutated BRAF retains its central contribution to melanoma pathophysiology even in advanced stage disease as manifested by the remarkable antitumor effects and alteration the natural history of metastatic melanoma of selective BRAF inhibitors. After initial response, resistance commonly emerges within a few months' time and the field has focused on delineating molecular mechanisms of resistance toward the goal of improving upon the early therapeutic effects of single agent BRAF inhibition. Combination regimens are currently undergoing clinical investigation. NRAS and CKIT mutant melanoma represent the next oncogene defined melanoma subsets for which initial targeted therapy approaches are being explored, with early evidence suggesting progress with MEK and CKIT inhibitors, respectively. A considerable subset of patients have melanomas that are not defined by the presence of BRAF, NRAS, or CKIT mutations and, thus, the elucidation of the entire melanoma genome is being pursued with the hope of identifying additional therapeutic targets.

Delineation of antigen-specific and antigen-nonspecific CD8+ memory T-cell responses after cytokine-based cancer immunotherapy

AbstractMemory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8+ T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8+ T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8+ T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8+ CD25− cells within the tumor site. These findings demonstrate that memory CD8+ T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.

Cytokine-based cancer immunotherapies induce differential effects on CD4+ and CD8+ T cell phenotype and function (156.9)

Abstract We have recently observed that cytokine-based immunotherapy (IT) regimens induced an Ag-nonspecific expansion of the memory T cell pool which was responsible for anti-tumor efficacy. We therefore sought to determine the effects of systemic IT on subsequent Ag-specific responses. During IT, there was a significant expansion of the memory CD8 T cell population with the cells in active cell cycle. Splenocytes from treated mice proliferated less in response to TCR engagement, yet they proliferated to a greater extent when stimulated with IL2 alone. Flow cytometric analysis of stimulated splenocytes revealed that CD8 T cells from IT treated mice appropriately upregulated activation markers (ie, CD25, CD44, NKG2D) and expanded normally. Conversely, CD4 T cells from the same mice exhibited markedly less proliferation, activation marker upregulation, and IFNγ production in response to the same stimuli. In a vaccine model where mice were immunized against allogeneic splenocytes while undergoing IT, CD8 T cells were capable of responding to antigen earlier and more robustly upon secondary in vitro restimulation than immunized, untreated mice. Together, these data show that immune stimulatory therapies allow CD8 T cells to function as Ag-specific or nonspecific effectors while functionally exhausting CD4 T cells. We also show that neither CD4 exhaustion after IT, nor the nonspecific expansion of memory CD8 T cells impairs the ability of CD8 T cells to generate new Ag-specific responses.

Next generation therapies change the landscape in melanoma

Melanoma is among the leading causes of years of life lost due to cancer. Current chemotherapy and cytokine-based immunotherapy approaches benefit only a small percentage of patients with advanced disease. However, the recent discovery of mutations in the gene encoding the serine-threonine kinase B-RAF (BRAF) raises the possibility that oncogene-targeted therapy may provide a new point of vulnerability. In parallel, a deeper understanding of the molecular mechanisms underlying antitumor T-cell activation and tolerance has provided a basis for developing therapies targeted against these processes. Results from an early phase trial with a BRAF inhibitor and a phase III trial with a novel agent that activates T cells have radically altered the prospects for improving outcomes for patients with this historically treatment-refractory disease.

Multicenter Phase II Trial of S-1 in Patients With Cytokine-Refractory Metastatic Renal Cell Carcinoma

This phase II multicenter trial was conducted to evaluate the activity and safety of S-1 in Japanese patients with metastatic renal cell carcinoma (mRCC). We also examined the relation between response and mRNA expression levels of enzymes involved in the metabolism of fluorouracil (FU). Patients with mRCC who had received nephrectomy in whom cytokine-based immunotherapy was ineffective or contraindicated were studied. S-1 was administered orally at 80-, 100-, or 120-mg daily, assigned according to body surface area, on days 1 to 28 of a 42-day cycle. The primary end point was the objective response rate. The mRNA expression levels of FU-related enzymes were measured by reverse-transcriptase polymerase chain reaction in formalin-fixed, paraffin-embedded specimens of tumors obtained at nephrectomy. A total of 45 eligible patients were enrolled. Eleven (24.4%) of 45 patients had partial responses to S-1, and 28 (62.2%) had stable disease. Median progression-free survival was 9.2 months. The severity of most adverse events was mild to moderate. The most common grade 3/4 drug-related adverse events were neutropenia (8.9%) and anorexia (8.9%). The expression level of thymidylate synthase (TS) mRNA was significantly lower in patients who responded to treatment (t-test, P = .048), and progression-free survival was significantly longer in patients whose TS mRNA expression levels were below the median value, as compared with those with higher levels (log-rank test, P = .006). S-1 is active against cytokine-refractory mRCC. Quantification of TS mRNA levels in tumors before treatment may facilitate prediction of the response of mRCC to S-1.

An early phase II trial of S-1 in Japanese patients with cytokine-refractory metastatic renal cell carcinoma

S-1, an oral anticancer agent, contains tegafur (FT), 5-chloro-2,4-dihydroxypyridine (CDHP), and potassium oxonate (Oxo) at a molar ratio of FT:CDHP:Oxo = 1:0.4:1. The aim of this trial was to investigate the efficacy and safety of S-1 in Japanese patients with cytokine-refractory metastatic renal cell carcinoma (RCC). We conducted a non-randomized, open-label trial in Japanese patients with metastatic RCC who had received nephrectomy and had failed cytokine-based immunotherapy. The primary endpoint was response rate. S-1 40-60 mg based on the body surface area was administered twice daily (80-120 mg/day) for 4 consecutive weeks, followed by a 2-week rest period; cycles were repeated every 6 weeks. Patients continued treatment until disease progression, unacceptable toxicity, or withdrawal of consent. A total of 20 eligible patients were enrolled. Among these, 3 patients had partial response, yielding objective response rate of 15%; 13 patients had no change; 4 patients had progressive disease. The median time-to-progression and median overall survival were 12.0 and 25.7 months, respectively. The initial adverse event was generally mild to moderate in severity. The most common grade 3/4 drug-related hematological and non-hematological adverse events were neutropenia (20.0%) and anorexia (20.0%), respectively. S-1 is active and well tolerated for the treatment of cytokine-refractory metastatic RCC.

Les lymphocytes CD4 cibles et acteurs dans la pathogenèse de l’infection à VIH — Conséquences thérapeutiques

CD4 T lymphocytes play a central role in the pathogenesis of human immunodeficiency virus (HIV) infection. In viremic patients, these cells are direct targets for the virus: HIV multiplies intensely in this subpopulation during the acute phase of the disease and continues to propagate in these same cells during the clinical latency phase. Uninfected CD4 T lymphocytes are also indirectly affected during this second phase: they are activated, become anergic, then disappear by apoptosis, ultimately contributing to CD4 lymphopenia. Our studies have shown that dysfunction of the interleukin-2 system and its receptors largely explain the early immune deficiency seen in viremic patients. Later, blockade of the homeostatic interleukin-7/CD4 loop contributes to rendering this CD4 lymphopenia irreversible. Our studies suggest that the inflammatory reaction which accompanies this infection gives rise to a state of abnormal activation, with receptor desensitization to the main cytokines regulating CD4 lymphocyte functions and numbers. In the few rare patients who manage to control HIV in the absence of any treatment, the CD4 T cell compartment has a very characteristic configuration. We have shown that an extensive subpopulation of "central memory" CD4 T lymphocytes expresses large quantities of the CCR7 homing receptor and that this helps drive these lymphocytes to infected lymph nodes. Also, these "central memory" CD4 T lymphocytes produce large quantities of IL-2, that they use in an autocrine manner, stimulating their self-renewal and ensuring their long-term survival. Our studies have opened the door to cytokine-based immunotherapy and to the development of new drug- and vaccine-based strategies.

The clinical application of COX-2 inhibitors may strengthen the sensitivity of renal cell carcinoma to immunotherapy

Renal cell carcinoma (RCC) remains a difficult management problem, because of the high metastatic rate and insensitivity of metastatic RCC to the traditional cytokine-based immunotherapy such as interleukin-2 and interferon-alpha. Regulatory T cells had a crucial role in impeding immune surveillance against cancer and hampering the development of effective anti-tumor immunity. At the same time cyclooxygenase-2 derived prostaglandin E2 played an important role in the transformation of regulatory T cells and could strengthen the inhibitory function of regulatory T cells. Previous experiment characterized that cyclooxygenase-2 was highly expressed in RCC. Herein, we draw the hypothesis that cyclooxygenase-2 inhibition may dampen the transformation of regulatory T cells and in turn contribute to eradicating RCC by the immune system. The clinical application of cyclooxygenase-2 inhibitors may strengthen the sensitivity of RCC to cytokine-based immunotherapy.

Immunotherapy for Renal Cell Carcinoma

Since spontaneous regression of metastatic renal cell carcinoma (mRCC) has been reported, immunotherapy for mRCC has been the therapeutic option. The goal of modulating an immune response to the tumor cell with passive and/or active immunotherapy can be achieved through the increasing technological sophistication and the understanding of the immune system. Currently, among the several available cytokines to treat mRCC, high-dose interleukin-2 (IL-2) administration is the only way to obtain complete remission. However, due to the lack of prominent benefit and toxicity of high dose IL-2 therapy, cytokine-based immunotherapy for the treatment of mRCC is threatened by the intriguing molecularly targeted agents, which are still under the trials. Different types of cellular (autologous tumor cell, gene modified tumor cell, dendritic cell) and non-cellular therapeutic vaccines of mRCC have been applied in the clinical setting, and the success of clinical effectiveness in selected population has been reported. Future treatment approaches for mRCC or locally advanced RCC would be directed with combined therapy with immunotherapy and targeted agent. Additionally, molecularly targeted agents and vaccines modulating tumor immunology cascade will be another immunotherapeutic approach for RCC.

Depot Formulation of Interleukin-2 as an Immunotheraphy for B-Cell Lymphoma.

Relapses occurring in most lymphoma patients after treatment highlight the need for effective immunotheraphy approaches. Induction of tumor-specific adaptive immunity in cancer patients is an ideal approach because its selectivity and memory for the same tumors eventually prevent the relapse after conventional therapies. Vaccination with cytokines and tumor cells represents an attractive therapeutic approach for patients with B-cell lymphoma. Interleukin-2 (IL-2) has a wide range of immunologic effects, including the activation of cytotoxic T cells and natural killer cells (NK). The therapeutic use of IL-2 has generated substantial interest based on its ability to induce the regression of metastatic renal cell carcinomas and malignant melanomas tumors in humans. However, toxicity associated with systemic administration of large doses of cytokine is a major drawback for clinical application. In this work, we investigated whether in vivo vaccination with a cytokine-based immunotherapy using IL-2 adsorbed in alum, as a depot system, with lysed lymphoma cells could stimulate lymphoma-specific immunity and improve survival. We developed a reproducible syngenic model of B-cell lymphoma in Balb/c mouse based upon the A20 cell line. In this model animals died between day 35 and 40 after tumor cells inoculation. Mice were first injected subcutaneously with 1 x 106 A20 cell in right flank. Four groups of mice received at days 3 and 7 after tumor cell inoculation by subcutaneous injections one of the following: lysed A20 cells with IL-2 in alumn (A20-IL-2); Lysed A20 cells in alumn (A20); IL-2 in alumn (IL-2); and phosphate-buffered saline (PBS) as control. Mice were followed for survival and immune response was evaluated. At day 22, 17% of A20-IL-2 group had systemic disseminated disease in comparison with 33% A20 group, 83% for IL-2 group and 100% of PBS group (p=0.009). Mice vaccinated with A20-IL-2 had longer survival compared with mice vaccinated with A20 or PBS (p=0.0019). Prolonged survival was related with a marked increase in the number of intratumoral CD4+ T cells (p=0.0001), CD8+ T cells (p=0.001) and NK cells (p=0.001). Aditionally, at day 22 groups of mice vaccinated with A20 showed a significantly lower percentages of intratumoral CD4+ CD25+ CD127- Treg cells in the tumor as compared with PBS group (p=0.0001). At day 39 the percentage of intratumoral Treg cells increased in the groups vaccinated with IL-2 (A20-IL-2 and IL-2 groups). Intratumoral CD4+, CD8+, NK and Treg cells were evaluated by flow cytometry. Interestingly, intracellular cytokine analysis showed a greater number of intratumoral INF-γ producing CD4+ T cells in mice vaccinated with A20-IL-2. Proliferation assays by flow cytometry (using carboxy-fluorescein diacetate, succinimidyl ester and propidium iodide) showed enhanced proliferation upon stimulation with irradiated A20 cells in splenocytes from A20-IL-2 vaccinated mice (p=0.004). In conclusion, the results of this study indicate that vaccination with A20 antigens combined with a depot formulation of IL-2 elicits strong anti-tumor specific immunity and extended survival. This approach may be an interesting strategy to promote systemic immunity against B-cell lymphoma with therapeutic value.

Assessment of cancer immunotherapy outcome in terms of the immune response time features

A cytokine-based periodic immunotherapy treatment is included in a model of tumour growth with a delay. The effects of dose schedule are studied in the case of a weak immune system and a growing tumour. We find the existence of 'metastable' states (that may last for tens of years) induced by the treatment and also potentially adverse effects of the dosage frequency on the stabilization of the tumour. These two effects depend on the delay between the tumour growth and the immune system response, the cytokine dose burden, and other parameters considered in the model.

Cytokine-Based Immunotherapy for Advanced Kidney Cancer: Past Results and Future Perspectives in the Era of Molecularly Targeted Agents

Cytokine-Based Immunotherapy for Advanced Kidney Cancer: Past Results and Future Perspectives in the Era of Molecularly Targeted Agents

The Role of Cytokine Therapy in Metastatic Renal Cell Cancer

Objectives Treatment of metastatic renal cell cancer (mRCC) is rapidly evolving since evidence concerning efficacy of novel molecular targeted drugs recently became available. This paper provides a critical overview of the current and future role of cytokine-based immunotherapy in mRCC in light of these new developments. Methods Published data from phase 3 studies and meta-analyses were analyzed by means of a systematic literature search in PubMed and Medline databases. Results Randomized trials using interferon-α (IFN-α), interleukin-2 (IL-2), and combinations have produced overall response rates of 4–31%. For IFN-α, a modest but significant survival advantage of 2.5 mo over hormonal therapy has been demonstrated. No significant survival advantage was shown for IL-2. High-dose IL-2 may induce complete and durable regression of mRCC in up to 5% of patients, but is characterized by high toxicity. Placebo-controlled prospective studies with these cytokines have not been published. Results from cytokine combination trials are difficult to compare because of differences in administration, dosing, and schedules. No internationally recognized standard immunotherapy treatment protocol could be defined for mRCC. Tyrosine kinase inhibitors achieve objective response or disease stabilization in a higher proportion of patients, but neither cure nor significant survival advantage has been shown for these agents. Combined and sequential treatments with cytokines and targeted agents are being investigated. Conclusions For selected patients, first-line cytokine therapy may still be an option in the repertoire of therapies for mRCC. A better knowledge of molecular markers could allow rational use of different therapies in individual patients in the future.

The Role of Residual Tumor Resection in Patients with Metastatic Renal Cell Carcinoma and Partial Remission following Immunochemotherapy

Objectives Cytokine-based immunotherapy (IMT) is a first-line treatment for metastatic renal cell carcinoma (mRCC). Radical nephrectomy and interferon monotherapy were shown to prolong survival of patients with mRCC compared with interferon treatment alone. We prospectively compared the outcome of patients with mRCC who achieved partial remission (PR) under IMT with patients achieving PR under IMT, followed by complete resection of tumor residues. Methods From January 1997 to June 2003, 34 patients with mRCC were treated with IMT and achieved PR. Of these patients, 18 continued IMT until disease progression (group A). In 16 patients with PR, residual tumor tissue was surgically resected (group B). Surgical complications in group B and survival time were evaluated. Results All patients from group B achieved a surgical complete remission (sCR), which is ongoing in 6 of 16 patients. Tumor-related death has occurred in all patients from group A and in 9 of 16 patients from group B. Surgery post-IMT was not associated with intraoperative complications, wound infections, or prolonged postoperative stay. Median overall survival was 50 mo (range: 18–104) versus 58 mo (range: 9–104) for patients in groups A and B, respectively. Conclusions Median survival benefit after IMT and surgery compared with IMT alone was 8 mo for patients who achieved PR. The resection of tumor residues after PR under IMT seems to extend the long-term survival of patients with mRCC and should be attempted when surgically feasible.

Treatment-Enhanced CD4+Foxp3+ Glucocorticoid-Induced TNF Receptor Family RelatedHigh Regulatory Tumor-Infiltrating T Cells Limit the Effectiveness of Cytokine-Based Immunotherapy

Regulatory T cells can suppress activated CD4+ and CD8+ T effector cells and may serve as an impediment to spontaneous or therapeutic type 1 antitumor immunity. In a previous study, we observed minimal therapeutic impact, but significantly enhanced T cell cross-priming and lesional infiltration of tumor-reactive CD8+ T cells into established CMS4 sarcomas after combined treatment of BALB/c mice with rFLt3 ligand (rFL) and recombinant GM-CSF (rGM-CSF). In this study, we show that this cytokine regimen also results in the profound enhancement of CD4+ tumor-infiltrating lymphocytes (TIL) expressing FoxP3, IL-10, and TGF-beta mRNA, with 50 or 90% of CD4+ TIL coexpressing the CD25 and glucocorticoid-induced TNFR family related molecules, respectively. Intracellular staining for Foxp3 protein revealed that combined treatment with rFL plus rGM-CSF results in a significant increase in CD4+Foxp3+ T cells in the spleen of both control and tumor-bearing mice, and that nearly half of CD4+ TIL expressed this marker. In addition, CD4+ TIL cells were of an activated/memory (ICOS(high)CD62L(low)CD45RB(low)) phenotype and were capable of suppressing allospecific T cell proliferation and IFN-gamma production from (in vivo cross-primed) anti-CMS4 CD8+ T cells in vitro, via a mechanism at least partially dependent on IL-10 and TGF-beta. Importantly, in vivo depletion of CD4+ T cells resulted in the ability of previously ineffective, rFL plus rGM-CSF therapy-induced CD8+ T cells to now mediate tumor regression.

Cytokine-Based Immunotherapy for Advanced Kidney Cancer: Past Results and Future Perspectives in the Era of Molecularly Targeted Agents

Until recently, immunotherapy has been the only therapeutic option available for patients with advanced kidney cancer, even though different choices were often made on the two sides of the Atlantic Ocean. The absence of alternatives made different immunotherapeutic approaches common practice, even with few adequate randomized studies that addressed key questions, such as the best treatment and schedule, and so on. The recent registration of the first two, molecularly targeted, agents Sorafenib and Sunitinib could (and will) render many therapeutic approaches, e.g., single-agent Interferon, obsolete. In this review, we shall cover the past achievements obtained so far with cytokine-based immunotherapy and discuss the present role of immunotherapy in the era of molecularly targeted agents. In particular, specific indications for immunotherapy are emerging (e.g., the use of Interleukin-2 in patients with high CAIX expression), while new trials are ongoing to test immunotherapy in combination with molecularly targeted agents, such as Sorafenib, Sunitinib, or Bevacizumab.

Cytoreductive nephrectomy in metastatic renal cell carcinoma

Metastatic renal cell carcinoma is associated with a poor prognosis and a median survival time of only 6–12 months. However, the emergence of immunotherapies has rekindled interest in cytoreductive nephrectomy as a therapeutic option. Phase III randomized trials have demonstrated that cytoreductive nephrectomy significantly improves overall survival in selected patients with metastatic renal cell carcinoma treated with interferon immunotherapy. While cytokine-based immunotherapy may be considered the standard systemic therapy, clinical studies are ongoing to develop molecular biomarkers and new therapies with improved efficacy and tolerability. With further advances in our understanding of the pathogenesis, behavior and molecular biology of renal cell carcinoma, cytoreductive nephrectomy, in combination with molecular targeted therapies, may become the new standard of care for patients with metastatic renal cell carcinoma.

Interleukin-23 and Interleukin-27 Exert Quite Different Antitumor and Vaccine Effects on Poorly Immunogenic Melanoma

Recent studies revealed that two novel interleukin (IL)-12-related cytokines, IL-23 and IL-27, have potent antitumor activities. However, the antitumor effects were mainly evaluated in relatively highly immunogenic tumors and have not been fully evaluated against nonimmunogenic or poorly immunogenic tumors. In this study, we investigated the antitumor efficacies of IL-23 and IL-27 on poorly immunogenic B16F10 melanoma and found that the antitumor responses mediated by IL-23 and IL-27 were clearly different. In syngeneic mice, mouse single-chain (sc) IL-23-transfected B16F10 (B16/IL-23) tumors exhibited almost the same growth curve as B16F10 parental tumor about until day 20 after tumor injection and then showed growth inhibition or even regression. In contrast, scIL-27-transfected B16F10 (B16/IL-27) tumors exhibited significant retardation of tumor growth from the early stage. In vivo depletion assay revealed that the antitumor effect of B16/IL-23 was mainly mediated by CD8+ T cells and IFN-gamma whereas that of B16/IL-27 mainly involved natural killer cells and was independent of IFN-gamma. We also found that antitumor effects of B16/IL-23 and B16/IL-27 were synergistically enhanced by treatment with IL-18 and IL-12, respectively. Furthermore, B16/IL-23-vaccinated mice developed protective immunity against parental B16F10 tumors but B16/IL-27-vaccinated mice did not. When combined with prior in vivo depletion of CD25+ T cells, 80% of B16/IL-23-vaccinated mice completely rejected subsequent tumor challenge. Finally, we showed that the systemic administration of neither IL-23 nor IL-27 induced such intense toxicity as IL-12. Our data support that IL-23 and IL-27 might play a role in future cytokine-based immunotherapy against poorly immunogenic tumors.

Systemische Therapie des metastasierten Nierenzellkarzinoms

Cytokine-based immunotherapy was the only viable option in metastatic, nonresectable renal cell carcinoma (RCC) for many years. Systemic immunotherapy has become increasingly established as a standard therapy during the last 15 years. In this context, interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) turned out to be the most effective single agents in RCC. Subsequently, the approved subcutaneous application of these compounds was the preferred administration route in Germany. Response rates with cytokine combination therapy were almost similar to those of more aggressive concepts using additional chemotherapeutic agents.Currently, new compounds targeting specific signaling pathways are readily available and have passed clinical testing. Such small molecules like tyrosine kinase inhibitors, monoclonal antibodies, or the mTOR inhibitor CCI-779 may dramatically change the established concepts of systemic RCC treatment. This paper gives an overview of established, current, and evolving concepts of systemic therapy in RCC.

Cytokine-Based Therapy and Biochemotherapy for Advanced Melanoma

A variety of immunotherapeutic approaches have shown activity in patients with metastatic melanoma with the best results being observed with interleukin 2 (IL-2). Follow-up data through 2004 confirm the durability of responses produced by the Food and Drug Administration-approved high-dose IL-2 regimen in this patient population. Efforts to develop more tolerable and/or effective IL-2-based treatment regimens by either prolonged administration of lower doses or the combination of IL-2 with other cytokines, monoclonal antibodies, or vaccines have yet to produce results superior to those seen with high-dose IL-2 alone. Recent investigations have suggested that, in some patients, IL-2 may expand regulatory T-cell populations leading to immune tolerance rather than antitumor immunity. Efforts to shift this balance in favor of immune rejection by reducing the confounding effects of regulatory T cells on IL-2 therapy or the use of novel and potentially more purely immunostimulatory cytokines are ongoing. Despite promising phase 2 data, phase 3 studies have failed to show meaningful clinical benefit for the combination of cytokines with cytotoxic chemotherapy, so-called "biochemotherapy." Nonetheless, recent investigations with biochemotherapy followed by maintenance immunotherapy suggest that biochemotherapy may still have a role as a "bridge to immunotherapy" in some patients with rapidly progressive disease. Given the low number of patients achieving durable benefit with cytokine-based immunotherapy, considerable recent effort has focused on identifying predictors of therapeutic response. Investigations suggest that immune responsiveness may be predetermined by a tumor microenvironment conducive to immune recognition and the host propensity to develop autoimmunity. Efforts to understand and further define pretreatment predictors of response through the use of gene expression and proteomic techniques are ongoing and raise the potential for eventually limiting cytokine-based immunotherapy to those most likely to benefit.