Cycles Of IL-2 Research Articles

Tumor-Localized Interleukin-2 and Interleukin-12 Combine with Radiation Therapy to Safely Potentiate Regression of Advanced Malignant Melanoma in Pet Dogs.

Cytokines IL2 and IL12 exhibit potent anticancer activity but suffer a narrow therapeutic window due to off-tumor immune cell activation. Engineering cytokines with the ability to bind and associate with tumor collagen after intratumoral injection potentiated response without toxicity in mice and was previously safe in pet dogs with sarcoma. Here, we sought to test the efficacy of this approach in dogs with advanced melanoma. This study examined 15 client-owned dogs with histologically or cytologically confirmed malignant melanoma that received a single 9-Gy fraction of radiotherapy, followed by six cycles of combined collagen-anchored IL2 and IL12 therapy every 2 weeks. Cytokine dosing followed a 3 + 3 dose escalation design, with the initial cytokine dose chosen from prior evaluation in canine sarcomas. No exclusion criteria for tumor stage or metastatic burden, age, weight, or neuter status were applied for this trial. Median survival regardless of the tumor stage or dose level was 256 days, and 10/13 (76.9%) dogs that completed treatment had CT-measured tumor regression at the treated lesion. In dogs with metastatic disease, 8/13 (61.5%) had partial responses across their combined lesions, which is evidence of locoregional response. Profiling by NanoString of treatment-resistant dogs revealed that B2m loss was predictive of poor response to this therapy. Collectively, these results confirm the ability of locally administered tumor-anchored cytokines to potentiate responses at regional disease sites when combined with radiation. This evidence supports the clinical translation of this approach and highlights the utility of comparative investigation in canine cancers.

Consecutive injections of low-dose interleukin-2 improve symptoms and disease control in patients with chronic spontaneous urticaria

PurposeTo describe the effectiveness and tolerability of low-dose interleukin (IL)-2 in treating patients with chronic spontaneous urticaria (CSU) refractory to H1-antihistamines. MethodsThis retrospective study included CSU patients who received treatment with at least one cycle of IL-2, injected intramuscularly at a dose of 1.0 million international units daily for 7 consecutive days, after failing treatment with H1-antihistamines. Patients were followed up for ≥12 weeks. ResultsOf the 15 patients, 7 (46.7%) and 11 (73.3%) achieved complete response at Week 2 and Week 12, respectively. The mean change of urticaria control test (UCT) and weekly urticaria activity score (UAS7) from baseline was 6.6 (95% CI, 4.2 to 8.9) and − 16.9 (95% CI, −24.0 to −9.8), respectively, at Week 12. Local injection-site reactions were the most common adverse events. No serious adverse events were reported. ConclusionLow-dose IL-2 treatment improves symptoms and disease control for CSU patients refractory to H1-antihistamines.

Immune Monitoring during Therapy Reveals Activitory and Regulatory Immune Responses in High-Risk Neuroblastoma.

Simple SummaryNeuroblastoma is a type of childhood cancer accounting for approximately 15% of childhood cancer deaths. Despite intensive treatment, including immunotherapy, prognosis of high-risk neuroblastoma is poor. Increasing amounts of research show that the fighting capacity of the immune system is very important for the outcome of neuroblastoma patients. Therefore, we investigated the fighting capacity of immune cells in blood at diagnosis and during the different phases of therapy. In this study, we observed both processes that stimulate and processes that decrease fighting capacity of immune cells in neuroblastoma patients during therapy. Despite this, we show that overall fighting capacity of the immune system of neuroblastoma patients is impaired at diagnosis as well as during therapy. In addition, we observed a lot of variation between patients, which might explain differences in therapy efficacy between patients. This study provides insight for improvement of therapy timing as well as new therapy strategies enhancing immune cell fighting capacity.Despite intensive treatment, including consolidation immunotherapy (IT), prognosis of high-risk neuroblastoma (HR-NBL) is poor. Immune status of patients over the course of treatment, and thus immunological features potentially explaining therapy efficacy, are largely unknown. In this study, the dynamics of immune cell subsets and their function were explored in 25 HR-NBL patients at diagnosis, during induction chemotherapy, before high-dose chemotherapy, and during IT. The dynamics of immune cells varied largely between patients. IL-2- and GM-CSF-containing IT cycles resulted in significant expansion of effector cells (NK-cells in IL-2 cycles, neutrophils and monocytes in GM-CSF cycles). Nonetheless, the cytotoxic phenotype of NK-cells was majorly disturbed at the start of IT, and both IL-2 and GM-CSF IT cycles induced preferential expansion of suppressive regulatory T-cells. Interestingly, proliferative capacity of purified patient T-cells was impaired at diagnosis as well as during therapy. This study indicates the presence of both immune-enhancing as well as regulatory responses in HR-NBL patients during (immuno)therapy. Especially the double-edged effects observed in IL-2-containing IT cycles are interesting, as this potentially explains the absence of clinical benefit of IL-2 addition to IT cycles. This suggests that there is a need to combine anti-GD2 with more specific immune-enhancing strategies to improve IT outcome in HR-NBL.

Low-Dose IL-2 for Treating Moderate to Severe Alopecia Areata: A 52-Week Multicenter Prospective Placebo-Controlled Study Assessing its Impact on T Regulatory Cell and NK Cell Populations

Low-Dose IL-2 for Treating Moderate to Severe Alopecia Areata: A 52-Week Multicenter Prospective Placebo-Controlled Study Assessing its Impact on T Regulatory Cell and NK Cell Populations

High-dose interleukin 2 in patients with metastatic renal cell carcinoma with sarcomatoid features.

BackgroundHigh-dose interleukin-2 (HD IL-2) is used in the treatment of metastatic renal cell carcinoma (mRCC) and has an overall response rate (ORR) of 12–20% and a complete response rate (CR) of 8% in unselected populations with predominantly clear cell type renal cell carcinoma. Nearly 10–15% of patients with renal cell carcinoma exhibit sarcomatoid differentiation, a feature which correlates with a median overall survival (OS) of 9 months and overall poor prognosis. We report a single institution experience with 21 patients with mRCC with sarcomatoid features post-nephrectomy who were treated with HD IL-2.MethodsTwenty one patients with mRCC with sarcomatoid features post-nephrectomy who underwent therapy with HD IL-2 were identified at the University of Pittsburgh Medical Center from 2004 to 2016. Baseline patient characteristics, HD IL-2 cycles, time to progression, and subsequent therapies were evaluated. OS and progression-free survival (PFS) in the cohort were calculated using the Kaplan-Meier method. Disease characteristics were evaluated for significance using the Fischer′s exact test and Wilcoxon rank sum test.ResultsPatients were predominantly Caucasian males with a median age of 54 years. A majority, 86% of these patients, had metastatic disease at time of initial presentation, primarily with lung and lymph node involvement. The ORR and CR with HD IL-2 was 10% and 5%, respectively. Initial localized disease presentation is the only variable that was significantly associated with response to HD IL-2 (p = 0.0158). Number of HD IL-2 doses did not correlate with response with a mean of 16.5 and 15.0 total doses in responders and non-responders, respectively (p = 0.53). Median PFS with HD IL-2 was 7.9 months (95% CI, 5.0–21.3). Median OS was 30.5 months (95% CI 13.3–57.66). Within the subset of patients who had progression on IL-2, median OS was 19.4 months (95% CI, 13.3–35.3). In patients who received second-line therapy, median PFS was 7.9 months (95% CI 2.4–10.2).ConclusionIn patients with mRCC with sarcomatoid features, use of HD IL-2 was associated with a modest ORR and a higher survival compared to historical controls (patients with mRCC and sarcomatoid features). Thus, HD IL-2 may have a role in treating selected patients with mRCC with sarcomatoid features.

Negative modulation of suppressive HIV-specific regulatory T cells by IL-2 adjuvanted therapeutic vaccine.

The potential benefit in using IL-2 in immunotherapy for cancer and autoimmunity has been linked to the modulation of immune responses, which partly relies on a direct effect on Tregs populations. Here, we revisited the role of IL-2 in HIV infection and investigated whether its use as an adjuvant with therapeutic vaccination, impacts on HIV-specific responses. Antiretroviral therapy treated-patients were randomized to receive 4 boosts of vaccination (ALVACHIV/Lipo-6T, weeks 0/4/8/12) followed by 3 cycles of IL-2 (weeks 16/24/32) before treatment interruption (TI) at week40. IL-2 administration increased significantly HIV-specific CD4+CD25+CD134+ T-cell responses, which inversely correlated with viral load after TI (r = -0.7, p <0.007) in the vaccine/IL-2 group. IL-2 increased global CD25+CD127lowFoxP3+Tregs (p <0.05) while it decreased HIV- but not CMV- specific CD39+FoxP3+CD25+CD134+Tregs (p <0.05). HIV-specific Tregs were inversely correlated with IFN-γ producing specific-effectors (p = 0.03) and positively correlated with viral load (r = 0.7, p = 0.01), revealing their undesired presence during chronic infection. Global Tregs, but not HIV-specific Tregs, inversely correlated with a decrease in exhausted PD1+CD95+ T-cells (p = 0.001). Altogether, our results underline the negative impact of HIV-specific Tregs on HIV-specific effectors and reveal the beneficial use of IL-2 as an adjuvant as its administration increases global Tregs that impact on T-cell exhaustion and decreases HIV-specific CD39+Tregs by shifting the balance towards effectors.

Abstract 3827: NCI 8628 - A randomized phase II study of Ziv-aflibercept (Z) and high dose Interleukin-2 (IL-2) or IL-2 alone for inoperable stage III or IV melanoma

Abstract Background: IL-2 plays a central role in antitumor immunity. VEGF plays a critical role in angiogenesis and host innate and adaptive immunity. High baseline serum VEGF was associated with non-response to IL-2. Z, a high-affinity soluble decoy VEGF receptor, may deplete VEGF prior to IL-2 to reverse the immunosuppressive impact of VEGF and enhance antitumor T cell response. Methods: NCI8628 was a phase II trial of Z and IL-2 (A) versus IL-2 alone (B) randomized 2:1. Eligible patients (pts): Stage III inoperable or Stage IV melanoma. Up to two prior regimens for metastatic melanoma and stable treated brain metastases were allowed. The primary endpoint was progression-free survival (PFS). Results: A total of 89 pts were enrolled, but 5 who never started study treatment were excluded. Six pts (4 in A and 2 in B) who were treated but withdrew early without a response assessment were considered non-responders in this analysis. Pt and disease characteristics are summarized in Table 1. Median number of IL2 cycles was 3 (A) and 2 (B) and of Z cycles in A was 3 (1 - 31). Median follow up for all alive patients was 19 months. Among 84 treated pts (55 in A and 29 in B), there was significant improvement in PFS in favor of A: median and 95% CI of 6.9 (4.2 - 8.8) months vs 2.1 (1.7 - 4.1), logrank p &amp;lt;0.001. No significant difference in OS: median and 95% CI of 23.1 (14.4 - 35.0) months (A) vs 22.3 (12.6 - NA). Response rate (RECIST) was 22% in A (5CR, 7PR) and 17% in B (5PR). Stable disease or PR or CRwas seen in 69% in A and 48% in B. Adverse events were consistent with the AE profiles of monotherapy with IL2 and Z. Grade 4 events in A included decreased lymphocytes (41 pts) and platelets (6), renal (1), hypophosphatemia (4), hypertension (2) and thromboembolism (1). Table 1Arm A (N=55)Arm B (N=29)Age (median and range)55 (26-73)55 (31-74)GenderFemale2211Male3318Cutaneous primary3616Mucosal54Uveal Unknown6 84 5AJCC StageIII (N3) M1a9 103 5M1b116M1c2515 Conclusions: The combination of Z and IL2 significantly improved PFS over IL2 alone, meeting the study’s primary endpoint. The regimen was relatively safe and manageable. Correlative and mechanistic studies are ongoing. Citation Format: Ahmad A. Tarhini, Paul H. Frankel, Christopher Ruel, Marc S. Ernstoff, Timothy M. Kuzel, Theodore F. Logan, Nikhil I. Khushalani, Hussein A. Tawbi, Kim A. Margolin, Sanjay Awasthi, David F. McDermott, Alice Chen, Primo N. Lara, John M. Kirkwood. NCI 8628 - A randomized phase II study of Ziv-aflibercept (Z) and high dose Interleukin-2 (IL-2) or IL-2 alone for inoperable stage III or IV melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3827. doi:10.1158/1538-7445.AM2017-3827

Long-term (LT) disease-free survival (DFS) of melanoma (MM) and renal cell cancer (RCC) patients following high-dose interleukin-2 (HD IL2).

e21005 Background: HD IL2 treatment (Tx) produces durable complete responses (CRs) and surgical CRs. Patients (pts) achieving partial response (PR) and stable disease (SD) demonstrate improved survival compared with pts who progress. Methods: 7 HD IL2 Tx centers identified pts with survival &gt; 5 years (yrs) after HD IL2. DFS was from end of IL2 to January 2017. Tx courses generally consisted of 2 1-week cycles of HD IL2, 600,000-720,000 U/kg IV every 8 hours. We collected data on pts Tx with HD IL2 alone, or HD IL2 plus local Tx (surgery or radiation (SRS) leading to CR) with survival &gt; 5 yrs after HD IL2 Tx. Results: 72 pts are reported: 29 RCC (male 22, female 7) and 43 mm (male 26, female 17). Median age at Tx of RCC is 54 yrs (range, 47-67 yrs) and of mm is 50 yrs (range, 24-72 yrs). Sites of metastatic disease for 26 mm pts were lymph nodes (LN) -18, lungs-12, bone-6, liver-9, brain-3, other organs-10 and for 22 RCC pts were lung-17, LN-5, adrenal-4, bone-3, other organs-8. Number of IL2 courses received: 1-10 pts, 1.5-4 pts, 2-28 pts, 2.5-5 pts, 3-24 pts, 4-one pt. Among the 29 RCC pts, there are 24 CRs, 3 surgical CRs and 2 PRs with no further Tx. Among 43 mm pts, there are 34 CRs, 2 near CRs, and 7 Surgical/SRS CRs. DFS in these pts after HD IL2 Tx ranges from 5+ yrs to 30+ yrs, median 10.5+ yrs. 10 RCC and 16 mm are alive &gt; 5 - &lt; 10 yrs after IL2, and 19 RCC and 27 mm are alive &gt; 10 yrs after IL2. LT toxicity among these 72 pts includes hypothyroidism-5 pts, arthralgias/arthritis-5 pts, vitiligo-3 pts, and 1 pt each: neuropathy, PVCs, and normal pressure hydrocephalus. Additional pts may be added as records become available. Conclusions: We document LT DFS ( &gt; 5 yrs) after CR or PR from HD IL2 alone. Surgical or SRS conversion of PR to CR can produce durable CRs. LT DFS was observed in pts with visceral and bone metastases, not only those with LN or lung sites. Neither age, sex, nor number of courses of IL2 predicted LT DFS. Chronic toxicity due to IL2 is uncommon in LT survivors.

Phase II trial of sequential subcutaneous interleukin-2 plus interferon alpha followed by sorafenib in renal cell carcinoma (RCC)

Immunotherapy (IL-2 and INF-α) was the treatment of choice for advanced renal cell carcinoma (RCC) until antiangiogenic therapy with tyrosin kinase inhibitors was developed in the early 2000s. This clinical trial explored the efficacy and toxicity of sequential treatment of IL-2 plus INF-α followed by sorafenib. Eligibility criteria included measurable, non-resectable, histologically confirmed predominantly clear cell RCC, no prior systemic treatment, and ECOG PS 0-2. The treatment regimen was a 6-week cycle of subcutaneous IL-2 at 9×10(6) IU on days 1-6 of weeks 1, 2, 4 and 5 plus s.c. INF-α at 6×10(6) IU on days 1, 3 and 5 of weeks 1-6. Responders received 6 additional weeks of this regimen. All patients received oral sorafenib (400mg bid) after immunotherapy until disease progression. The primary endpoint was progression-free survival. Forty-one patients were enrolled, median age 57years. ECOG was 0/1 in 17/20 patients, 35 patients had prior nephrectomy and 18 patients pure clear cell cancer. Median PFS was 7.4months (95% CI 6.5-13.1) and OS was 16.6months (95% CI not reached). In 36 patients evaluable for response, ORR was 44.4% and control rate was 94.4%. Most adverse events (AEs) were Grade 1 or 2 toxicities (84.7%). During immunotherapy the most common AEs were pyrexia (82.9%), asthenia (56.1%) and anorexia (46.3%), whereas during sorafenib were diarrhoea (48.8%) and hand-foot syndrome (46.3%). A sequential regimen of IL-2 and INF-α followed by sorafenib showed effectiveness and manageable toxicity in patients with advanced RCC.

Effect of intermittent interleukin-2 therapy on CD4+ T-cell counts following antiretroviral cessation in patients with HIV

Interleukin (IL)-2 therapy impacts T-cell homeostasis. Whether IL-2 expanded CD4(+) T cells may persist following viral rebound has not been fully investigated. Patients with CD4(+) T cells 500/μl or more and HIV RNA less than 50 copies/ml were randomized to continue antiretroviral therapy (ART) either alone (n = 67) or combined with three IL-2 cycles (n = 81; 6 million units) twice daily for 5 days at weeks 0, 8, and 16 before stopping ART (week 24). Patients were followed up to 168 weeks. At week 24, median CD4(+) T-cell counts were 1198 and 703 cells/μl in the IL-2 and control groups, respectively (P < 0.001). At week 72, 27% (IL-2 group) and 45% (control group; P = 0.03) of patients were in failure (defined as no interruption of ART at week 24, CD4 drop below 350 cells/μl or ART resumption). After week 24, a biphasic decline (before and after week 32) of CD4 was noted -106 and -7 cells/μl per month in controls and -234 and -17 in IL-2 group (all P ≤ 0.0001). At week 96, IL-2-expanded CD4(+)CD25(+) T cells remained higher than in the control group (26 vs. 16%, P = 0.006). In IL-2-treated patients, CD4(+)CD25(+) T cells persisting despite viral replication allow a longer period of ART interruption.

Rescue of HIV-1 long-time archived X4 strains to escape maraviroc

Entry of Human Immunodeficiency Virus type 1 (HIV-1) into target cells is mediated by the CD4 receptor and a coreceptor, CCR5 or CXCR4. Maraviroc interferes with HIV entry by binding the CCR5 coreceptor. Virological failure to maraviroc-containing regimens can occur through the emergence of resistance, or through tropism evolution and broadened coreceptor usage. In the latter case, the physiological relevance of minority strains is a major concern.Here we report a retrospective analysis of coreceptor-usage and evolution based on 454-ultra-deep-sequencing of plasma and Peripheral Blood Mononuclear Cell (PBMC)-derived envelope V3-loops, accounting for coreceptor usage, from a patient who failed a maraviroc-containing regimen through the emergence of X4 strains. The X4 maraviroc-escape variant resulted from recombination between a long time archived proviral sequence from 2003 (5′-portion, including the V3-loop) and the dominant R5 strains circulating in plasma at the time of maraviroc-treatment initiation (3′-portion). Phylogenetic analyses and BEAST modeling highlighted that an early diverse viral quasispecies underwent a severe bottleneck following reinitiation of HAART and repeated IL-2 cycles between 1999 and 2001, leading to the transient outgrowth and archiving of one highly homogeneous X4 population from plasma, and to the expansion in plasma of one PBMC-derived R5 strain. Under maraviroc selective pressure, the early, no longer detectable X4 strains archived in PBMC were partially rescued to provide X4-determinants to the main circulating strain.

Interleukin-2 cycling causes transient increases in high-sensitivity C-reactive protein and D-dimer that are not associated with plasma HIV-RNA levels

To determine the effects of interleukin (IL)-2 treatment on inflammatory and thrombotic biomarkers in chronically HIV-infected adults receiving antiretroviral therapy. Cryopreserved plasma was evaluated retrospectively for C-reactive protein (CRP) and D-dimer at baseline, end of an IL-2 cycle, and long-term follow up from two randomized, controlled trials: 57 IL-2-naive adults receiving either three to six cycles of IL-2 as well as antiretroviral therapy (nucleoside analogues) or antiretroviral therapy alone for 12 months, and 40 IL-2-experienced adults on highly active antiretroviral therapy who either interrupted or continued therapy for 6 months after a baseline IL-2 cycle. High-sensitivity CRP (hsCRP) was measured by immunonephelometry (detection limit 0.175 mg/l) and D-dimer by latex agglutination (detection limit 0.20 mg/l). Median within-group differences and pre and post-IL-2 changes between groups were assessed via nonparametric Wilcoxon signed-rank and Mann-Whitney U-tests. Spearman's rank test was used to assess correlations between changes in hsCRP, D-dimer, and HIV-RNA viral load. Significant increases in hsCRP (study 1: 138.6 mg/l; study 2: 58.9 mg/l) and D-dimer (study 1: 3.1 mg/l; study 2: 0.4 mg/l, all P < 0.0001) occurred by the end of the initial IL-2 cycle, returning to baseline by the end of study. No correlations were seen between changes in hsCRP or D-dimer and HIV-RNA, CD4 T-cell count, or proliferation (Ki67 expression). No thrombotic or cardiovascular serious adverse events occurred during these study periods. IL-2 dosing caused transient increases in plasma hsCRP and D-dimer levels, regardless of HIV-RNA viral load, suggesting the possibility of increased risk for thrombotic events.

Inferiority of IL-2 alone versus IL-2 with HAART in maintaining CD4 T cell counts during HAART interruption: a randomized controlled trial

To evaluate whether interleukin (IL)-2 in patients with chronic HIV infection can maintain CD4 T cell counts during 6 months of HAART interruption. Prospective, randomized, controlled, open-label phase II noninferiority trial comparing IL-2 with HAART interruption or continuous HAART. Forty-one IL-2-experienced (three or more prior cycles) HIV-1-infected adults with CD4 cell count at least 500 cells/microl were randomized in the ratio 2: 1 to interrupted (I = 27) or continuous (C = 14) HAART for 6 months following an initial IL-2 cycle. Subsequent IL-2 cycles were triggered by CD4 T cell counts less than 90% of baseline. Immune, metabolic, and quality of life indices were compared (Mann-Whitney and Fisher's exact tests), defining noninferiority as a percentage difference (C- I) in treatment success (CD4 T cells > or =90% of baseline at 6 months) with a 95% confidence interval (CI) lower limit greater than -20%. Demographic and immune parameters were similar between the groups at baseline. Median CD4 T cell count, HIV viral load, and treatment success differed significantly at 6 months (I: 866 cells/microl, 39,389 copies/ml, 48.1%; C: 1246 cells/microl, <50 copies/ml, 92.3%; P < or = 0.001). Group I was inferior to C (% difference = -44.2%; 95% CI: -64.2%, -11.2%; P = 0.013). Minor statistically significant differences in HgbA1c and energy level occurred at 6 months (I > C). Following HAART interruption, single cases of acute retroviral syndrome, secondary syphilis, non-Hodgkin's lymphoma, and Kaposi's sarcoma recurrence were observed. IL-2 alone was inferior to IL-2 with HAART in maintaining baseline CD4 T cell counts. HAART interruption had a small impact on metabolic parameters and quality of life.

Correlation of effector and regulatory T cell responses with clinical outcome in metastatic renal cell carcinoma patients treated with MVA-5T4 vaccine and high-dose interleukin-2

3004 Background: The attenuated vaccinia virus MVA was engineered to deliver the tumor antigen 5T4 (MVA-5T4; TroVax). 5T4 is a glycoprotein expressed by nearly all RCCs. MVA-5T4 was tested in combination with HD IL-2 to determine the safety of the combination, clinical response rate and humoral and cellular immune responses. Methods: Patients with metastatic RCC who qualified for HD IL-2 were eligible to receive five TroVax vaccinations, which were administered i.m. every three weeks. Each vaccination was followed by a cycle of HD IL-2 (600,000 IU/kg). Blood was collected for analysis of 5T4-specific cellular and humoral responses. The frequency and functional characterization of regulatory T cells (Treg) was determined throughout the treatment time course. Results: 25 patients (mean age 58.4±10.07) were enrolled with a median follow-up of 22.8 months. There were no serious vaccine-related adverse events. The frequency of peripheral Tregs (pre-treatment) was found to be significantly higher than those detected in normal donors (6.44% vs 1.42%, P=0.00018) but cells had comparable suppressive capacity. Despite the presence of elevated levels of Tregs, 5T4-specific cellular and/or humoral responses were detected in all patients who had received ≥1 TroVax immunization (n=22). Two patients who presented with both primary tumors in place and metastases had complete responses of metastatic disease and underwent post-treatment nephrectomy. 10 patients (48%) were stable with a median overall survival that has not yet been reached, while median survival was 22 months for the 10 patients with PD (p=0.04). Patients with stable disease showed a significant increase in 5T4-specific T cell responses by direct ex vivo interferon-gamma ELISPOT assay (p=0.005) and a decrease in Tregs compared to those with PD (p=0.034) and the change in Treg frequency was independently associated with prolonged survival (P=0.006). Conclusions: Local vaccination with MVA-5T4 and HD IL-2 was safe and induced potent 5T4-specific cellular and humoral immune responses. Patients with SD demonstrated a significant increase in survival, reduced Treg frequency and enhanced 5T4-specific cellular responses relative to patients with PD. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Oxford Biomedica Oxford Biomedica

TILT: a randomized controlled trial of interruption of antiretroviral therapy with or without interleukin-2 in HIV-1 infected individuals

We aimed to see if structured treatment interruption (STI) could be supported safely with the use of two cycles of IL-2 (4.5 MIU q12h subcutaneously 5 days) before STI to prolong the time before therapy restarted. Subjects were randomly allocated to either A - continuous ART; B - continue for 9 weeks, then STI; restart with the same ART when the CD4 count falls below 200 cells; or C - two cycles of IL-2, 8 weeks apart, while still on ART; at week 9 stop ART and use a new cycle of IL-2 alone whenever the CD4 count falls < 300 cells. Patients were followed until week 105. 86 mostly white middle aged homosexual men with a baseline median CD4 count of 754 cells/ml (range 240-1400) and a nadir CD4 count of 268 cells/ml (range 62-822) enrolled. By 96 weeks there was a 66% probability of having restarted ART in arm B compared with 34% in arm C (p = 0.002; log rank test). New drugs were used in 60% in arm A, 57% in arm B and 45% in arm C. 4 subjects had a dose modification in the first cycle due to toxicity with 2 interrupting. There were 39 SAEs with 21 in arm C. There were no deaths. The primary aim of the trial was to gain experience in using IL-2. IL-2 delayed restarting drugs and fewer new drugs were used.

Long-term effects of intermittent interleukin-2 therapy in chronic HIV-infected patients (ANRS 048–079 Trials)*

Interleukin (IL)-2 therapy leads to significant CD4 cell increases in HIV-infected patients. Since phase III trials are ongoing, studies supporting the long-term feasibility of this strategy are needed. We studied the long-term outcomes of 131 patients treated with IL-2 in two studies initiated either before (ANRS 048) or following (ANRS 079) the advent of HAART. At the last assessment (median follow-up 3.4 years), these patients experienced a gain of 428 cells/microl and a decrease in plasma HIV RNA to 1.70 log10 copies/ml. In both studies, high CD4 cell counts were maintained with a median of ten 5-day cycles of subcutaneous IL-2. Median time since the last cycle was 2 years. At last assessment, 59% of 048 patients maintained a non-HAART regimen. Detailed analysis at week 170 showed that median CD4 cell counts were 856 (048) and 964 (079) cells/microl. This corresponded to a gain from baseline of 515 (048) and 627 (079) cells/microl. The median viral load decreases from baseline and corresponded to 1.70 (048) and 1.88 (079) log10 copies/ml. Comparisons across the studies showed that CD4 gains and viral load changes were similar whether HAART or non-HAART was used. The frequency of cycling, but not CD4 cell counts, viral loads or antiviral regimen at baseline, was predictive of long-term CD4 gain (P = 0.03). Altogether, these observations support IL-2 as a long-term therapeutic strategy in HIV infection.

CD4+T Cell Responses to Interleukin‐2 Administration in HIV‐Infected Patients Are Directly Related to the Baseline Level of Immune Activation

Intermittent interleukin (IL)-2 administration to human immunodeficiency virus (HIV)-infected patients leads to CD4(+) T cell expansions. The factors potentially affecting these expansions were investigated in the present study. A matched (for baseline CD4(+) T cell count) case-control study was designed. Nonresponders (NRs) were defined as patients with a <or=10% increase in CD4(+) T cell count 2 months after the third IL-2 cycle (week 24), compared with that at baseline (week 0). Control subjects experienced a >or=50% increase in CD4(+) T cell count at week 24. Immunophenotype, Ki67 and forkhead box protein P3 (FoxP3) expression, and T cell receptor excision circle (TREC) measurements in T cells were evaluated at weeks 0 and 24 in both groups. Control subjects and NRs did not differ significantly at baseline in age, viral load, CD4(+) T cell count, nadir CD4(+) T cell count, or CD8(+) T cell count. At week 0, NRs had lower TREC levels per 1x106 T cells and higher levels of T cell proliferation and activation than did control subjects. At week 24, both groups experienced decreases in T cell proliferation and increases in CD25 and FoxP3 expression on CD4(+) T cells, with TREC levels per 1x106 CD4(+) T cells decreasing significantly only in control subjects. Increased immune activation can adversely affect CD4(+) T cell expansions after IL-2 administration. Despite the lack of expansion, other evidence of IL-2-induced biological activity was observed.

CD8 apoptosis may be a predictor of T cell number normalization after immune reconstitution in HIV

BackgroundAs part of the Houston Vanguard study, a subset of 10 patients randomized to receive IL-2 therapy were compared to 4 patients randomized to not receive IL-2, for markers of T cell activation and death during the first three cycles of IL-2. All patients were treated with combination antiretroviral therapy (ART) and were virally suppressed. The purpose of the study was to examine the role of CD8+ T cell death in responses to ART and IL-2 therapy.MethodsLymphocytes were examined at Day 0, 5 and 30 days during three cycles of IL-2 therapy. CD25, CD38, HLA-DR expression and annexin (cell death) were examined on CD4 and CD8 subpopulations. Follow up studies examined CD4 levels and CD4:CD8 reconstitution after 6 years using both univariant and multivariate analyses.ResultsHuman lymphocytes responded to IL-2 therapy by upregulation of CD25 on CD4+ T cells, leading to an increase in CD4 cell counts. CD8+ T cells did not increase CD25 expression, but upregulated activation antigens (CD38 and DR) and had increased death. At baseline, 7 of the 14 patients had high CD8+ T cell apoptosis (mean 17.0% ± 6.0). We did an exploratory analysis of immune status after six years, and found that baseline CD8+ T cell apoptosis was correlated with CD4 cell count gain beginning two years post enrollment. Patients with low levels of CD8+ T cell apoptosis at baseline (mean 2.2% ± 2.1) had significantly higher CD4 cell counts and more normalized CD4:CD8 ratios than patients with high CD8+ T cell apoptosis (mean CD4 cell counts 1,209 ± 164 vs 754 ± 320 cells/mm3; CD4:CD8 ratios 1.55 vs. 0.70, respectively).ConclusionWe postulate that CD8+ T cell apoptosis may reflect inherent activation status, which continues in some patients even though viral replication is suppressed which influences the ability of CD4+ T cells to rebound. Levels of CD8+ T cell apoptosis may therefore be an independent predictor of immune status, which should be shown in a prospective study.

A Novel p27kip1-Smad3 Regulated Pathway Is Involved in Induction of T Cell Tolerance.

Induction and maintenance of peripheral tolerance is essential for homeostasis of the immune system. In vivo studies demonstrate the significance of tolerance induction in preventing autoimmunity, graft rejection and GVHD. Upregulation of the cyclin-dependent kinase inhhibitor, p27, correlates with induction of T cell tolerance in vitro and in vivo. p27 interacts with cdk2, cdc2, grb2, and Rho family GTPases. Extensive studies support an essential role of cdks, particularly cdk2, in cell cycle re-entry. Cdk2 promotes cell cycle progression in part by phosphorylating Rb and related pocket proteins thereby reversing their ability to sequester E2F transcription factors. Recent work indicates that cdk2 phosphorylates Smad2 and Smad3. Smad3 inhibits progression from G1 to S phase, and impaired phosphorylation on the cdk-specific sites renders it more effective in executing this function. In contrast, cdk-mediated phosphorylation of Smad3 reduces Smad3 transcriptional activity and antiproliferative function. In spite the strong correlation between p27 expression level and T cell tolerance, it remains unclear whether p27 has a causative role in induction of tolerance. Here, we examined the role of p27 during induction of tolerance of naïve T cells in vivo, using RAG2 deficient, DO11.10 TCR-transgenic T cells that lack the cyclin-cdk-binding domain of p27 (p27Δ) thereby disrupting only the interactions of p27 with cyclin-cdk complexes. We adoptively transferred CD4+ T cells from RAG2−/−DO11.10 TCR-transgenic mice (DO11.10) or RAG2−/−DO11.10 TCR-transgenic p27Δ mice (DO11.10/p27Δ) into syngeneic wild-type recipients and compared the development of immune responses to immunogenic or tolerizing stimulus in vivo. Following exposure to immunogenic or tolerizing stimulus, DO11.10 and DO11.10/p27Δ CD4+ T cells underwent equal numbers of divisions in vivo, and both cell types exhibited reduced number of divisions in response to tolerizing stimulus. Strikingly, only wild-type DO11.10 TCR-transgenic T cells were tolerized as determined by impaired cyclin E activation, proliferation, and IL-2 production upon antigen-specific rechallenge. Compared to primed wild-type DO11.10 cells, tolerized wild-type DO11.10 cells exhibited impaired cdk2 and cdc2 activity, reduced levels of Smad3 phosphorylation on cdk-specific sites, and increased Smad3-transactivation leading to upregulation of the cdk4/6-specific cdk inhibitor p15. In contrast, after either priming or tolerizing stimulus, DO11.10/p27Δ cells exhibited comparable cdk2 and cdc2 activity, cdk-mediated phosphorylation of Smad3, low-level Smad3 transactivation, and no upregulation of p15. Furthermore, knockdown of Smad3 by expression of Smad3 shRNA in wild-type DO11.10 T cells recapitulated the functional and molecular findings observed in DO11.10/p27Δ cells, preventing induction of tolerance and upregulation of p15, and resulting in production of IL-2 and cell cycle progression. In contrast, expression of Smad3 mutant resistant to cdk-mediated phosphorylation in DO11.10/p27Δ cells recapitulated the molecular and functional effects of tolerance and resulted in inhibition of IL-2 production, upregulation of p15 and blockade of cell cycle progression. These results show that p27 plays a causative role in the induction of tolerance of naïve T cells and Smad3 is a critical component of a pathway downstream of p27 regulating the induction of tolerance in vivo.

Phase II trial of sequential temozolomide (TMZ) and high-dose bolus (HDB) IL-2 in patients with metastatic melanoma

8037 Background: Previous biochemotherapy (BCT) regimens for metastatic melanoma have utilized lower doses of IL-2 and multiple chemotherapeutic agents, adding to toxicity, but not to efficacy. Methods: We designed a 2-stage Simon phase II study testing a unique BCT approach of single agent chemotherapy with TMZ given in an extended schedule (75 mg/m² per day for 3 weeks PO) followed by HDB IL-2 (600,000 U/Kg/dose, maximum 14 doses administered over 5 days). Cycles were repeated every 28 days with a two-week interval between alternate cycles. The first stage accrued 20 patients with promising activity and safety permitting enrollment of additional patients. Results: Thirty-one patients (20 male, 11 female), age 27–74 (median 47) have been enrolled to date. All had AJCC stage IV melanoma (7 M1a, 5 M1b, 19 M1c) and had not previously received therapy for metastatic disease. Twelve had received prior adjuvant interferon. A total of 88 cycles of therapy have been administered (median of 2 cycles per patient; 5 patients continue on therapy). The median number of doses was 9 (range 7–12) during cycle 1, and 6 (range 4–11) during cycle 2. Three patients did not receive any IL-2 due to disease progression, and 6 patients received only one cycle of IL-2. Twenty two patients who received at least 2 cycles are evaluable for response. All 31 patients are evaluable for toxicity. Grade 3 toxicities included hepatic (8), hematologic (4 leukopenia, 2 thrombocytopenia), diarrhea (1). No grade 3–4 cardiovascular or renal toxicities were noted. Overall response rate is 22.7% (2 complete lasting 10.8 and 17+ months, 3 partial lasting 3.7 and 16+ months, 1+ month). Responses were seen in both M1a and M1c disease. Fourteen patients had stable disease after 2 cycles and 10 of these have progressed. As of 12/31/2005, the 4 month PFS rate is 74% [40%, 88%], median TTP is 24 weeks [11, 32] and median OS is 71 weeks (31.4, inf). Conclusions: HDB IL-2 can be safely administered in combination with single agent temozolomide in an extended schedule and appears to have promising efficacy and lower toxicity than previously used BCT regimens. Further follow-up will determine if durability of response exceeds that of single agent HDB IL-2. [Table: see text]