IL-2 Toxicity Research Articles

Abstract 2473: A novel anti-PD-1 and proIL-2 bifunctional fusion protein delivers potent anti-tumor activity via PD-1 checkpoint inhibition and tumor selective IL-2R agonism preferentially on antigen specific T cells

Abstract High-dose IL-2 (HD IL-2) induces complete and durable responses in RCC and melanoma but the treatment is associated with severe toxicities. PD-(L)1 blockade represents a recent breakthrough in immunotherapy with solid tumors, but only 20-30% of patients can benefit from the therapy and most responders eventually develop resistance. In more recent clinical studies with small number of subjects, HD IL-2 has shown promising responses in patients who progressed on anti-PD-1 therapy and combination therapy of HD-IL-2 and PD-1 blockade has resulted in high rate of objective response. In an attempt to maximize the clinical benefit of these immunotherapies to a broader patient population with deeper response and in the meantime to minimize the severe toxicity of HD IL-2, we have generated a novel bifunctional PD-1-proIL-2v fusion protein harnessing the mechanisms of PD-1 blockade and PD-1-cis directed tumor conditional IL-2R activation. PD-1-proIL-2v shows dose dependent inhibition of PD-1 immune checkpoint pathway measured by GM-CSF and IFNγ production in an MLR assay. Protease activated PD-1-proIL-2v, but not the intact drug, stimulates the phosphorylation of STAT5 of NK and T cells, the proliferation of CD8 T cells as well as the cytokine production in human whole blood. The potency of pSTAT5 by protease activated product is enhanced by 10-20 folds on PD-1+ T cells than T cells without PD-1. When tested in mouse tumor models resistant to PD-1 blockade, PD1-proIL-2v demonstrated high potency of tumor inhibition at as low as 0.1 mg/kg dose. In contrast, non-targeted proIL-2v at molar equivalent or higher dose showed minimal antitumor effect in either monotherapy or combination with anti-PD-1. Analysis of PD-1-proIL-2v treated mice showed dramatically increased tumor infiltrating CD4+ and CD8+ T cells, including PD-1+Tcf-1+ and PD-1+ IL-18Ra+TIM3− CD8+ T cells, referred to “stem-like” progenitor effector cells and “fresh” effector cells, respectively. Activation of the IL-2 procytokine in PD-1-proIL-2v was observed in mouse tumor tissues but not in the plasma. In monkey GLP-compliant 4-week repeat-dose toxicity study, PD-1-proIL-2v demonstrated excellent safety profiles without overt irAEs that are characteristic of IL-2. In conclusion, our preclinical data demonstrated that PD-1-proIL-2v effectively stimulated the expansion and reinvigoration of antigen specific T cells in the tumor tissue resulting in strong anti-tumor efficacy while it did not cause significant peripheral toxicity. Phase 1 clinical trial of PD-1-proIL-2v is planned to start in early 2024 to evaluate the safety, pharmacokinetics and anti-tumor activity in advanced solid tumors. Citation Format: Zijuan Li, Feifei Zhang, Ze Zhang, Harry Zhou. A novel anti-PD-1 and proIL-2 bifunctional fusion protein delivers potent anti-tumor activity via PD-1 checkpoint inhibition and tumor selective IL-2R agonism preferentially on antigen specific T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2473.

Abstract 561: CAR-targeted IL-2 drives selective CAR-T cell expansion and improves anti-tumor efficacy

Abstract Chimeric antigen receptor T cell (CAR-T) therapies have transformed the treatment of some hematological malignancies and are showing promising preliminary results in solid tumors. Recent studies have shown that in vivo expansion and persistence of CAR-Ts are correlated with improved therapeutic outcomes in patients. Administration of IL-2 enhances CAR-T engraftment, persistence, and functionality in preclinical models. However, the clinical potential of IL-2 stimulation with cell therapies is limited using current molecules due to severe toxicity of high-dose IL-2 and the inadequate selectivity of existing engineered IL-2 variants which expand multiple endogenous cells in addition to CAR-T cells. To address this challenge, we have applied our cis-targeting technology to develop CAR-T selective IL-2 fusion molecules that specifically activate CAR-Ts, while exhibiting minimal activity on CAR-negative cells. Cis-targeted IL-2 fusions are comprised of a fusion between a targeting antibody and an IL-2 mutein having attenuated binding to IL2R⍺ and IL2Rβ. The attenuated cytokine selectively binds and activates IL-2 receptors on CAR-Ts via the avidity provided by the targeting arm. We engineered two cis-targeted CAR-T selective IL-2 fusions, CAR-IL2 or EGFRt-IL2. CAR-IL2 targets the FMC63 CAR directly without blocking CD19 antigen recognition, enabling targeting of approved anti-CD19 CAR-T products, and EGFRt-IL2 targets the EGFRt tag co-expressed with the CAR. Molecules were characterized in vitro using primary human CAR-transduced T cells. The in vivo activity was examined using tumor bearing NSG mice infused with human CAR-Ts. The specificity of the CAR-IL2 or EGFRt-IL2 molecules was demonstrated by their ability to selectively induce pSTAT5 signaling resulting in >1000-fold preferential STAT5 activity in CAR-expressing cells compared to CAR-negative cells. The ability of the fusion proteins to selectively expand CAR-Ts in vivo was shown through a substantial and specific expansion of the infused CAR-Ts. The CAR-T fraction increased from approximately 50% of infused T cells, to over 93% of T cells, demonstrating a nearly 40-fold expansion in vivo. Re-expansion of CAR-Ts was demonstrated after allowing the CAR-Ts to rest in vivo >100 days; re-dosing resulted in significant and selective re-expansion of CAR-T cells. Furthermore, treatment with the CAR-IL2 molecule enhanced tumor regression of CD19+ lymphoma-bearing mice infused with suboptimal doses of CAR-Ts. Together, these data demonstrate that our cis-targeted IL-2 molecules selectively activate CAR-Ts in vitro and enhance CAR-T expansion and anti-tumor efficacy in vivo. Cis-targeted IL-2 fusion molecules directed by anti-CAR or anti-tag antibodies represent a promising approach to confer enhanced CAR-T activity in a specific and temporally controlled manner. Citation Format: Nathan D. Mathewson, Kelly D. Moynihan, Sara Sleiman, Wei Chen, Paul Bessette, Christopher Kimberlin, Eric Wigton, Danielle Pappas, Terrence Park, Ton N. Schumacher, Saar I. Gill, Yik Andy Yeung, Ivana Djuretic. CAR-targeted IL-2 drives selective CAR-T cell expansion and improves anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 561.

Abstract 1448: Combination therapy of anti-sense oligonucleotide targeting TGF-beta2 (TASO) and IL-2 (Proleukin) has anti-cancer effect in solid cancer

Abstract Background: TGF-Β (transforming growth factor-beta) is an essential cytokine for tumor proliferation and metastasis. The expression of TGF-Β correlates with malignancy of various cancers and involves immunosuppression and angiogenesis of a tumor. IL-2 is a major cytokine to proliferate T cells and NK cells which are major players of cancer immunity. However, the toxicity of high dose IL-2 limits its use in cancer therapy. Combination treatment of TGF-Β inhibitor and IL-2 would have an anti-tumor effect by immune cells through diminishing immunosuppression by TGF-Β and enforcement of immune cells by IL-2. Trabedersen is an anti-sense oligonucleotide targeting human TGF-Β mRNA. It is shown that Trabedersen is well tolerable in cancer patients and effective reagent to treat pancreatic cancer, melanoma, and glioblastoma. Proleukin is the only approved IL-2 reagent to treat Renal cell carcinoma and Melanoma. Methods: Trabedersen and Proleukin activated human PBMC (Peripheral Blood Mononuclear Cell) are treated to several solid cancer cell lines, such as Breast cancer, Pancreatic cancer, Melanoma, Lung cancer, and Colon cancer to see the cytotoxicity effect of combination therapy of Trabedersen and Proleukin. NSG mouse (NOD Scid Gamma mouse, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) which are humanized by human PBMC engraftment and the tumor growth of Melanoma and TNBC (Triple Negative Breast Cancer cell) cell line are monitored. Results: The combination treatment of Trabedersen and low dose Proleukin decreased cancer cell viability in vitro experiment in solid cancer cell lines. Melanoma and TNBC tumor growth was delayed in humanized NSG mouse model by Trabedersen and low dose IL-2 combination therapy and tumor growth delay was statistically significant to Trabedersen alone or IL-2 alone group. Tumor infiltrating lymphocyte population was increased in Trabedersen treated group and FoxP3+ regulatory T cell population in blood and tumor microenvironment was decreased by treatment of Trabedersen. Conclusion: TGF-Β inhibitor (Trabedersen) and low dose IL-2 (Proleukin) combination treatment is expected to be an effective regimen in solid cancer treatment than individual treatment by alteration of tumor environment. Modulation of the dose of Proleukin expects to help reduce the toxicity of IL-2 and increase the anti-cancer effect by combination with Trabedersen. Citation Format: Jun-Eui Park, Jihye Koo, Hong-Kyu Lee, Tae Hun Kim. Combination therapy of anti-sense oligonucleotide targeting TGF-beta2 (TASO) and IL-2 (Proleukin) has anti-cancer effect in solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1448.

Abstract 1744: STK-012, an alpha/beta selective IL-2 mutein for the activation of the antigen-activated T cells in solid tumor

Abstract Interleukin-2 (IL-2) is a potent stimulator of T and NK cell proliferation, survival, and cytotoxic function. High dose IL-2 induces complete responses as a single agent in certain cancers. Its use is limited due to toxicities such as severe hypotension and vascular leak syndrome (VLS). A better understanding of the mechanisms of IL-2 efficacy and toxicity and development of more selective therapies are needed to improve the use of IL-2. Next generation IL-2 therapeutics have been engineered to bias activity towards the dimeric form of the IL-2 receptor, which consists of IL-2Rβ (CD122) and IL-2Rγ (CD132), and away from the high affinity trimeric form of the receptor, which also includes IL-2Rα (CD25). This approach is designed to stimulate NK cells and naïve effector T-cells, which express the dimeric, and not the trimeric, form of the receptor. However, this approach largely misses antigen-activated and tumor-specific T-cells, which have high expression of both CD25 and CD122. In addition, this approach expands peripheral NK cells, which have been shown in mice to cause VLS (1-3).To specifically stimulate antigen-activated CD25+ effector T cells in cancer patients and avoid systemic NK and naïve T cell activation, we have developed a PEGylated, CD25/CD122-selective IL-2 mutein (STK-012) and its mouse surrogate (STK-014). Here we present efficacy and safety of STK-014 compared to wild type IL-2 (wtIL-2) and a CD122/CD132 biased IL-2 (βγIL-2). We also present safety of STK-012 in non-human primates (NHP). STK-012/STK-014 selectively induced STAT5 phosphorylation and proliferation in antigen activated T cells but not in NK cells and naïve T cells. In mice, STK-014 showed reduced toxicity compared to wtIL-2 and βγIL-2. In particular, wtIL-2 and βγIL-2 induced VLS while STK-014 did not. Moreover, STK-014 demonstrated complete responses both as single agent and in combination with a PD-1 antibody in syngeneic tumor models. In general, STK-014 demonstrated improved efficacy compared to wtIL-2 and βγIL-2. STK-014 dramatically increased intratumoral T cells and intratumoral cytotoxic activity compared wtIL-2 and βγIL-2 while avoiding T cell activation in the spleen. STK-014 treatment drastically increased the CD8+ T cell to Treg ratio within the tumor when compared to control tumors (25-fold), but also in comparison to wtIL-2 (2.75-fold) and to βγIL-2 (5.2-fold).In NHP, STK-012 was well tolerated at doses supra-efficacious in mice. Weekly dosing of STK-012 led to continuous elevated serum concentrations, demonstrating selectivity for CD25/122+ T cells. STK-012 selectively induced memory T cell expansion, including CD28+ CD95+ CD8 T cells. In summary, STK-012 avoids IL-2 mediated toxicity and may enable the specific expansion of antigen activated memory T cells in cancer patients, leading to durable tumor response. 1 Peace DJ and Cheever MA. JEM, 1989.2 Gatley MK et al. JI, 1988.3 Assier E et al. JI; 2004. Citation Format: Jan Emmerich, Michele Bauer, Marie Semana, Bhargavi Jayaraman, Sandro Vivona, Scott McCauley, Romina Riener, Rene De Waal Malefyt, Paul-Joseph Aspuria, Deepti Rokkam, Patrick Lupardus, Rob Kastelein, Martin Oft. STK-012, an alpha/beta selective IL-2 mutein for the activation of the antigen-activated T cells in solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1744.

Potent immunomodulatory effects of an anti-CEA-IL-2 immunocytokine on tumor therapy and effects of stereotactic radiation

ABSTRACT While anti-CEA antibodies have no direct effect on CEA-positive tumors, they can be used to direct potent anti-tumor effects as an antibody-IL-2 fusion protein (immunocytokine, ICK), and at the same time reduce the toxicity of IL-2 as a single agent. Using a fusion protein of humanized anti-CEA with human IL-2 (M5A-IL-2) in a transgenic murine model expressing human CEA, we show high tumor uptake of the ICK to CEA-positive tumors with additional lymph node targeting. ICK treated CEA-positive tumors exhibit significant tumor eradication. Analysis of tumor-infiltrating lymphocytes shows a high frequency of both CD8+ and CD4+ T cells along with CD11b positive myeloid cells in ICK treated mice. The frequency of tumor-infiltrating FoxP3+ CD4+ T cells (Tregs) is significantly reduced vs anti-CEA antibody-treated controls, indicating that ICK did not preferentially stimulate migration or proliferation of Tregs to the tumor. Combination therapy with anti-PD-1 antibody did not improve tumor reduction over ICK therapy alone. Since stereotactic tumor irradiation (SRT), commonly used in cancer therapy has immunomodulatory effects, we tested combination SRT+ICK therapy in two tumor model systems. Use of fractionated vs single high dose SRT in combination with ICK resulted in greater tumor inhibition and immunity to tumor rechallenge. In particular, tumor microenvironment and myeloid cell composition appear to play a significant role in the response rate to ICK+SRT combination therapy.

Tcell-directed IL-17 production by lung granular γδ T cells is coordinated by a novel IL-2 and IL-1β circuit.

Immune-mediated lung is considered the result of an exacerbated innate injury immune response, although a role for adaptive lymphocytes is emerging. αβ T cells specific for S. aureus enterotoxin A orchestrate a Tγδ17 response during lung injury. However, the mechanism driving IL-17 production is unclear. Here, we show a role for IL-2 triggering IL-17 production by lung granular γδ T cells as IL-17 synthesis and neutrophil recruitment was reduced by IL-2 blocking mAbs in vitro and in vivo. Mass cytometry analysis revealed that lung γδ T cells responded directly to IL-2 as evident from STAT5 phosphorylation and RoRγt expression. IL-2 receptor blocking mAbs and JAK inhibition impaired STAT5 phosphorylation and IL-17 release. Moreover, inhalation of S. aureus enterotoxin A induced IL-2 secretion and caspase-1-dependent IL-1β activation to drive IL-17 production. This T-cell-mediated inflammasome-dependent IL-17 response is maximum when lung Tγδ17 cells were sequentially stimulated first with IL-2 then IL-1β. Interestingly, when IL-2 is given therapeutically to cancer patients it carries a known risk of lung injury that is largely indistinguishable from that seen in sepsis. Hence, this novel mechanism reveals therapeutic targets treating both acute lung injury and high-dose IL-2 toxicity in cancer.

T Cell-Directed IL-17 production by Lung Granular γδ T cells is Coordinated by a Novel IL-2 and IL-1β Circuit

Abstract Immune-mediated lung injury is considered the result of an exacerbated innate immune response, although a role for adaptive lymphocytes is emerging. αβ T cells specific for S. aureus enterotoxin A orchestrates a typical innate-like Tγδ17 response during lung injury. However, the mechanism by which a specific T cell response drives an innate immune response in the lung is unclear. Here, we show a role for IL-2 triggering IL-17 production by lung granular γδ T cells as IL-17 synthesis was reduced by IL-2 blocking mAbs in vitro and in vivo. Granular γδ T cells in the lung constitute a rare population, hence Mass Cytometry (CyTOF) was utilized to characterize this population. CyTOF analysis revealed that lung γδ T cells responded directly to IL-2 as evident from STAT5 phosphorylation and RoRγt expression. IL-2 receptor blocking mAbs and JAK inhibition impaired STAT5 phosphorylation and IL-17 release. Moreover, inhalation of S. aureus enterotoxin A induced IL-2 secretion and caspase-1-dependent IL-1β activation to drive IL-17 production. This T cell-mediated inflammasome dependent IL-17 response is maximum when lung Tγδ17 cells were sequentially stimulated first with IL-2 then IL-1β. Further investigation is still needed to identify the cellular source of IL-1β. Interestingly, when IL-2 is given therapeutically to cancer patients it carries a known risk of lung injury that is largely indistinguishable from that seen in sepsis. Hence, this novel mechanism reveals therapeutic targets treating both acute lung injury and high dose IL-2 toxicity in cancer.

Blockade of CD25 reduces regulatory T cell expansion and some manifestations of IL-2 mediated toxicity but does not improve anti-tumor efficacy in animal models of IL-2 immunotherapy

Abstract High dose IL-2 (HD IL-2) is an approved treatment for melanoma and RCC that can result in long term disease remission, but is associated with severe adverse side effects. Adverse effects may be mediated by non-specific activation of cells expressing the high affinity CD25-containing IL-2 receptor. HD IL-2 also results in the expansion of regulatory T cells (Treg) that may negatively impact tumor immunotherapy. Blocking IL-2 binding to CD25 was explored as a mechanism to reduce expansion of Tregs and adverse effects. We created a high affinity anti-mouse CD25 mAb that effectively blocks the interaction of IL-2 with CD25 and carries the N297A Fc region modification to reduce FcgR binding. Saturation of CD25 ameliorates some but not all biomarkers of IL-2 toxicity. These include behavioral signs and serum ALT elevation. However, lung and spleen weight increase induced by IL-2 was not affected by CD25 blockade. CD25 blockade reduced IL-2 mediated expansion of Tregs. A saturating dose of CD25 antibody prior to 5-6 days of IL-2 therapy resulted in a complete loss of IL-2-mediated tumor efficacy in the CT26 model. CD25 blockade and IL-2 expanded CD8+ and NK cells while reducing blood and TIL Tregs, but did not impact primary tumor growth in the 4T1 or B16F10 tumor models. The loss of anti-tumor activity in the CT26 model correlated with reduced CD8+ CD25+ expansion in the blood and the TIL populations. Expansion of CD25 negative CD8+ and NK cells with concomitant Treg cell suppression in three different syngeneic tumor models did not result in improved anti-tumor efficacy. This result has implications for IL-2 and IL-15 engineering, and suggests that IL-2 mediated toxicity cannot be completely controlled by re-directing IL-2 to the CD122/CD132 receptor.

Regulatory T cells control toxicity in a humanized model of IL-2 therapy

While patient selection and clinical management have reduced high-dose IL-2 (HDIL2) immunotherapy toxicities, the immune mechanisms that underlie HDIL2-induced morbidity remain unclear. Here we show that dose-dependent morbidity and mortality of IL-2 immunotherapy can be modeled in human immune system (HIS) mice. Depletion of human T cell subsets during the HDIL2 treatment reduces toxicity, pointing to the central function of T cells. Preferential expansion of effector T cells secondary to defective suppressive capacity of regulatory T (Treg) cells after HDIL2 therapy further underscores the importance of Treg in the maintenance of immune tolerance. IL-2 toxicity is induced by selective depletion or inhibition of Treg after LDIL2 therapy, and is ameliorated in HDIL2-treated HIS mice receiving the PIM-1 kinase inhibitor, Kaempferol. Modeling IL-2 pathophysiology in HIS mice offers a means to understand the functions of effector and regulatory T cells in immune-mediated toxicities associated with cancer immunotherapy.

IL-33-expanded human Vγ9Vδ2 Tcells have anti-lymphoma effect in a mouse tumor model.

From several years, the anticancer effects of Vγ9 T lymphocytes make these cells good candidates for cancer immunotherapies. However, the proved efficacy of γδΤ cell-based cancer immunotherapies in some clinical trials was minimized due to the inherent toxicity of IL-2, which is essential for the combination therapy with Phosphoantigen (PAg). Recently, we showed that IL-33, a γ chain receptor-independent cytokine, was able to induce the in vitro proliferation of PAg-activated Vγ9 Tcells, which were fully functional expressing IFN-γ and TNF-α and showing in vitro anti-tumor cytotoxicity. We proposed IL-33 as an alternative to IL-2 for Vγ9 Tcell-based cancer immunotherapies, and have therefore evaluated the efficacy of this cytokine in preclinical investigations. This study shows that human Vγ9 Tcells are able to proliferate in a mouse model with the combination of PAg and rhIL-33, and that IL-33-expanded Vγ9 Tcells can prevent tumor growth in a mouse lymphoma model.

The association of tumor infiltrating CD8+ and Foxp3+ cells with overall response rate (ORR) in metastatic renal cell carcinoma (mRCC) patients treated with high-dose aldesleukin (HD IL-2).

4576 Background: In the prospective, biomarker validation HD IL-2 “Select” study, durable remissions and prolonged survival were seen in both proposed “good” and “poor-risk” patients, based on clear-cell histology subclassification and carbonic anhydrase-9 (CA-9) IHC staining. Given the toxicity and limited efficacy of HD IL-2, efforts to improve its therapeutic index are warranted. Since the high-affinity receptor of IL2 is expressed on both T regulatory cells and activated effector T lymphocytes, we explored the association between HD IL-2 response and tumor infiltration of CD8+ T and FoxP3+cells. Methods: Archival tumor tissue was collected for pathologic analysis on 120 mRCC patients enrolled in the HD IL-2 “Select” trial.Density of tumor infiltrating CD8+ T cells and Foxp3+ cells (cell/mm2) were evaluated in the invasive margin (IM) and the tumor center (TC) by IHC and automated image analysis using Aperio algorithms. The association between ORR and immune cell density was assessed using Fisher exact test. Results: Tumor specimens of 89 patients were available for analysis. 24 pts experienced response (R, including 2 CR and 22 PR) and 65 pts did not respond (NR, including 11 SD and 54 PD). Baseline patient characteristics were similar between R and NR groups. A very high density of CD8+ cells in the IM, or a high density of Foxp3+ cells in the TC were significantly associated with ORR. A stronger association with ORR was found for patients having both high density of CD8+ cells in the IM and Foxp3+ cells in the TC. Conclusions: In this prospective, biomarker validation study, response to HD IL-2 was associated with a high density of CD8+ cells in the invasive margin, and a high density of Foxp3+cells in the tumor center. Independent validation is ongoing. To improve the predictive value of Foxp3, characterization of Foxp3 expressing cells (Tregs vs activated effector T cells) in highly infiltrated tumors is underway. [Table: see text]

A retrospective analysis of High-Dose Interleukin-2 (HD IL-2) following Ipilimumab in metastatic melanoma

BackgroundHigh dose interleukin-2 (HD IL-2) can induce durable responses in a subset of patients leading to long-term survival. Immune checkpoint blockade (ICB) has demonstrated similarly durable responses in a larger...

Gefitinib plus interleukin-2 in advanced non-small cell lung cancer patients previously treated with chemotherapy.

The activation of lymphocytes by gefitinib treatment has been described. In this phase II pilot trial, we explored the possible synergism between IL-2 and gefitinib for non-small cell lung cancer (NSCLC) treatment. From September, 2003, to November, 2006, 70 consecutive patients with advanced, progressive NSCLC, previously treated with chemotherapy, received oral gefitinib 250 mg daily. The first 39 patients received gefitinib alone (G group). The other 31 also received subcutaneous IL-2 (GIL-2 group): 1 MIU/m2 (Million International Unit/m2)twice a day on Days 1 and 2, once a day on Days 3, 4, 5 every week for four consecutive weeks with a four-week rest period. Median follow-up was 25.2 months. Grade 3–4 toxicity of gefitinib was represented by skin rash (7%), asthenia/anorexia (6%) and diarrhea (7%); patients treated with IL-2 showed grade 2–3 fever (46%), fatigue (21%) and arthralgia (13%). In the GIL-2 group and G-group, we respectively observed: an overall response rate of 16.1% (6.4% complete response) and 5.1% (only partial response); a disease control rate of 41.9% and 41%; a median time to progression of 3.5 (CI 95% = 3.2–3.8) and 4.1 (CI 95% = 2.6–5.7) months; a median overall survival of 20.1 (CI 95% = 5.1–35.1) and 6.9 (CI 95% = 4.9–8.9) months (p = 0.002); and an actuarial one-year survival rate of 54% and 30%. Skin toxicity (p < 0.001; HR = 0.29; CI 95% = 0.16–0.54) and use of IL-2 (p < 0.001; HR = 0.33; CI 95% = 0.18–0.60) were independently associated with improvement of survival. In this consecutive, non-randomized, series of advanced NSCLC patients, the use of IL-2 increased the efficacy of gefitinib.

846P - High-Dose Interleukin-2 (Hd Il2) Armed with Pathology-Based Selection Criteria: a Real Option in Treatment of Metastatic Renal Cell Carcinoma (Mrcc) After Targeted Therapy

ABSTRACT Aim: HD IL2 as initial therapy in well-selected patients with mRCC can produce durable complete remissions rarely achieved by other therapies, but its efficacy and feasibility after VEGF-targeted therapies is less certain. Response rates (RR) in unselected patients has been reported as low and with excessive toxicity. We undertook a retrospective study of HD IL2 post-targeted therapy in our centre to assess its efficacy and toxicity, plus the impact of our previously reported pathology-based selection criteria. Methods: Patients were stratified by pathology into 'Favorable' and 'Other' groups. ‘Favourable’ was defined as 50% alveolar/solid, or >50% clear cell features. HD IL2 was administered as per standard dosing and schedule. Clinical records were reviewed retrospectively; survivals were analysed by Kaplan-Meir methods. Results: 35 patients were included in this study from July 2007 to December 2013. Median age 58; 77% male. Median follow-up was 1 year 9 months. Only 4 patients had ‘Other’ histology and none responded - while not encouraging, the number is too small to definitely preclude such patients from treatment. ‘Favourable’ group were analysed in more detail. RR are comparable to that reported in first line HD IL2 with 42% ORR and 19% CR. All patients achieving CR remain disease-free to date (duration of up to 50+ months). Clinical correlates for good outcome include low disease burden and tolerance of more doses. Toxicity was generally easily manageable although there was 1 death due to myocarditis and 2 other reversibile cases of myocarditis. Most patients were treated in medical wards but 2 required admission to intensive care for careful monitoring. Conclusions: While toxicity of HD IL2 may be higher than in treatment-naive patients, it seems less problematic than previously reported. Importantly, our pathology selection criterion developed in the first-line setting seems equally effective in mRCC after failure of targeted therapy. Our data shows that HD IL2 is effective and tolerable, and can produce durable remissions (∼19%). It should be considered as a treatment option in mRCC with ‘Favourable’ histology, and preferrably with limited disease, after failure of VEGF-targeted therapy. Disclosure: All authors have declared no conflicts of interest.

A single center experience with high-dose (HD) IL-2 treatment for patients with advanced melanoma and pilot investigation of a novel gene expression signature as a predictor of response

9003 Background: HD IL-2 remains one of two FDA-approved therapies for the treatment of patients (pts) with advanced melanoma. Initial studies conducted 15–20 years ago reported 16% response rate with 8% of pts achieving durable responses. The toxicity of HD IL-2 limits its application to pts treated in specialized centers. We present the clinical outcome of pts treated over a recent 2 year period with HD IL-2 at a single institution and an associated retrospective pilot study evaluating the predictive value of a novel tumor gene expression signature. Methods: Clinical and radiological data were collected and analyzed on 49 consecutive pts treated with HD IL-2 at Beth Israel Deaconess Medical Center from 10/05 - 10/07. Response was evaluated via RECIST. Formalin-fixed paraffin embedded tumor was obtained on consenting pts and classified as either Class 1, defined by melanocyte-specific genes including MITF, or Class 2, represented by immune genes, using the DNA-mediated Annealing, Selection, and Ligation (DASL) technique. Two-sided Fisher's Exact test was used to compare the proportion of responses for pts in the two classes. Results: Clinical response occurred in 16 of the 49 pts (32.6%), with 5 pts (10.2%) having a CR. Two other pts had stable disease &gt; 12 mos; 3 pts who progressed after response had resection to NED. 10 pts remain disease/progression free at a minimum of 16 mos following treatment. 28 pts (including 13 of 16 responders) had sufficient tumor for DASL analysis. Among the 21 categorized as Class 1, 7 (33%) were responders. Of the 7 classified as Class 2, 6 (86%) were responders. This difference in response was statistically significant (p = 0.0286). Conclusions: The overall and CR rates in a contemporary series of pts with metastatic melanoma treated with HD IL-2 are twice that reported in initial studies suggesting some treatment selection on clinical grounds since the 1990s. Pts with tumors expressing an immune signature by DASL appeared more likely to respond. This finding requires prospective validation, but suggests immune-related gene expression might contribute to IL-2 responsiveness. [Table: see text]

IL-2 in the therapy of melanoma and other malignancies

IL-2 as a single agent has been approved for use in both renal cancer and melanoma, and it has achieved widespread use in these malignancies for selected patients with metastatic disease. For these diseases, a role for IL-2-based therapy in the adjuvant setting has not been demonstrated, and current investigations involve the use of less toxic and less complex therapies. The activity of IL-2 has been studied extensively in other malignancies, particularly hematologic diseases such as leukemia and lymphoma. To date, IL-2 used either as a single agent or as a component of regimens containing one or more additional agents for diseases other than melanoma or renal cancer has shown promise, but the rapid emergence of safer agents for these and many other malignancies has tempered enthusiasm for IL-2-based regimens. Many investigations have been directed at enhancing the therapeutic ratio of IL-2, mostly by adding chemical modulators of downstream molecules associated with IL-2 toxicity. Other approaches, including variations on the chemical composition of IL-2 to alter its receptor-binding characteristics or its pharmacokinetic profile have been tried with limited success. The addition of targeting molecules co-administered with IL-2 or covalently bound to produce bispecific IL-2 containing molecules have shown promising activity and remain under investigation.

Phase Ib Trial Assessing Autologous, Tumor-pulsed Dendritic Cells as a Vaccine Administered With or Without IL-2 in Patients With Metastatic Melanoma

Twenty-four subjects with metastatic melanoma were treated on a randomized Phase Ib trial evaluating an autologous tumor lysate-pulsed dendritic cell (DC) vaccine with or without interleukin (IL)-2. The vaccine consisted of autologous DCs obtained from peripheral blood mononuclear cells (PBMCs) cultured in granulocyte macrophage-colony stimulating factor and IL-4 then pulsed with autologous tumor cell lysate and keyhole limpet hemocyanin (KLH). The primary end points of the trial were safety and immune response to vaccine. Subjects were randomized to vaccine administered every other week times 3, vaccine x 3 followed by low-dose IL-2, or vaccine x 3 followed by high-dose IL-2. Immune response was monitored pretreatment and at 2 and 4 weeks after the third vaccine administration. Disease evaluation was performed at 4 weeks after the third vaccination. Therapy was well tolerated with no local vaccine toxicity greater than grade 1 in any arm. IL-2 toxicity was as expected without additional toxicity from the addition of IL-2 to vaccine. Immune response defined as delayed-type hypersensitivity, PBMC interferon-gamma enzyme-linked immunosorbent spot, and PBMC proliferation, to both autologous tumor and KLH were detected in all arms. Interferon-gamma enzyme-linked immunosorbent spot response to KLH (7 of 10 patients) and autologous tumor (4 of 10 patients) were also detected in subjects with available vaccine draining lymph node cells. There were no differences in immune response between treatment arms. No clinical responses were seen. Autologous tumor lysate-pulsed DC vaccine with or without IL-2 was well tolerated and immunogenic but failed to induce clinical response in patients with advanced melanoma.

High-dose bolus interleukin-2 in elderly patients (&gt;60 years old) with metastatic melanoma or renal cell cancer

19559 Background: Although durable complete responses have been reported from using high-dose bolus interleukin-2 (HDB IL-2) in a small number of patients with metastatic melanoma and renal cell cancer (RCC), IL-2 toxicity limits its use especially in the elderly. Methods: the medical records of patients older than 60 years old with melanoma or renal cell carcinoma who received HDB IL-2 at the Moffitt Cancer Center between 2000–2005 were reviewed. The effect of increased age, primary diagnosis, and the HDB IL-2 regimen used on the side effects, number of administered doses, and survival was analyzed. Results: 55 cycles were administered to 35 patients (23 RCC, 12 melanoma, 26 men). Median age was 67 years old (range: 61–77). 17 patients received a traditional regimen (one cycle: 600,000 IU/Kg intravenously every 8 hours for 14 doses repeated in 2 weeks, maximum of 28 doses) and 18 received a clinical trial regimen (one cycle: 600,000 IU/Kg intravenously every 8 hours for 5 doses repeated weekly, maximum of 20 doses). Median number of administered cycles was 1 (range 1–4) and median number of total doses was 24 (range 3–79). Increased age was not related to total number of administered doses. Median percentage of IL-2 administered in a cycle was 75% of planned (range 11%-100%). Reasons to discontinue therapy were: oliguria (35%), hypotension (25%), and arrhythmia (15%). Side effects in all cycles were: hypotension (71%), oliguria (67%), Arrhythmia (18%), Myocardial infarction (7%), pulmonary edema (7%), hypothyroidism (4%), confusion (4%), seizures (2%) and stroke (2%). Pressors were used in 58% of all cycles. 20 patients died within a year from starting treatment and 5 lived more than 2 years (4 had RCC). Conclusions: 1) HDB IL-2 has multiple and life-threatening side effects in the elderly and caution is needed when selecting these patients to such therapy 2) the number of doses administered is comparable to that general population 3) more studies are needed to identify the population that would mostly benefit from HDB IL2. [Table: see text] No significant financial relationships to disclose.

Management of interleukin-2-induced severe bronchoconstriction

To the Editors: The immunostimulatory cytokine, interleukin (IL)-2, is used for the treatment of patients with cancers, such as metastatic renal cell carcinoma and melanoma 1, 2. The toxicity of IL-2 has been well described, including hypotension, a capillary leak syndrome including pulmonary oedema, thrombocytopenia and renal dysfunction 1, 3–5. Interestingly in a rare case, IL-2 administration, especially its inhalation, was reported to cause bronchoconstriction 3, 4, 6. However, little is known about the risk factors and management of IL-2-induced bronchoconstriction. We conducted a study to elucidate the clinical characteristics of patients developing IL-2-induced bronchoconstriction and to examine how to manage this condition. The study subjects included 18 patients with malignant cutaneous haemangioendothelioma (10 males and 8 females). Most of them were elderly with a mean (range) age of 72.3 (52–95) yrs, and all had lesions on the face and head. All patients received IL-2 immunotherapy plus radiotherapy. IL-2 was administered intralesionally in four patients and …

Metabolic Response to Stress in the Neonate Who Has Surgery

After completing this article, readers should be able to: 1. Describe the basic metabolic pathways involved in neonatal nutrition. 2. Describe the effect of stress on nutrition and metabolism in the neonate undergoing surgery. 3. Describe the differences in the metabolic response to stress between an adult and a neonate. Over the past 4 decades, there have been remarkable improvements in our ability to care for critically ill infants. Progress in surgical and critical care of neonates has led to improved survival rates. Our understanding of the nutritional requirements of infants continues to advance. In contrast to years past, practitioners caring for critically ill infants undergoing surgery today have various nutritional options, including specialized parenteral and enteral formulations. Although our understanding of host defense and nutritional support continues to evolve, the interplay between the immune system and metabolism is being investigated. Hence, we are improving the match between the infant’s energy expenditure and nutritional requirements. Critical to further progress is additional understanding of the metabolic response of a neonate to surgical stress. All living organisms require energy to survive. Fuel sources enter into complex metabolic cascades to form the ultimate energy substrate, adenosine triphosphate (ATP), a fuel needed to drive and maintain all physiologic systems. Under stressful conditions, intrinsic substrate mobilizes to synthesize additional energy substrate. In essence, host substrate is mobilized for the promotion of healing after injury. Cytokines, released in response to injury, drive the catabolic response to stress, but in abundance, as with the systemic inflammatory response syndrome (SIRS), cytokines are detrimental to the organism. The nutritional requirements of a neonate are significantly greater than those of an adult because not only are there requirements for substrate intake to fulfill basic metabolic needs, but there are metabolic needs to maintain rapid and continued growth and development. Furthermore, the metabolic response …