Nancy A. Crosby

Three Phase II Cytokine Working Group Trials of gp100 (210M) Peptide Plus High-Dose Interleukin-2 in Patients With HLA-A2–Positive Advanced Melanoma

PURPOSE High-dose interleukin-2 (IL-2) induces responses in 15% to 20% of patients with advanced melanoma; 5% to 8% are durable complete responses (CRs). The HLA-A2-restricted, modified gp100 peptide (210M) induces T-cell immunity in vivo and has little antitumor activity but, combined with high-dose IL-2, reportedly has a 42% (13 of 31 patients) response rate (RR). We evaluated 210M with one of three different IL-2 schedules to determine whether a basis exists for a phase III trial. PATIENTS AND METHODS In three separate phase II trials, patients with melanoma received 210M subcutaneously during weeks 1, 4, 7, and 10 and standard high-dose IL-2 during weeks 1 and 3 (trial 1), weeks 7 and 9 (trial 2), or weeks 1, 4, 7, and 10 (trial 3). Immune assays were performed on peripheral-blood mononuclear cells collected before and after treatment. RESULTS From 1998 to 2003, 131 patients with HLA-A2-positive were enrolled. With 60-month median follow-up time, the overall RR for 121 assessable patients was 16.5% (95% CI, 10% to 26%); the RRs were 23.8% in trial 1 (42 patients), 12.5% in trial 2 (40 patients), and 12.8% in trial 3 (39 patients). There were 11 CRs (9%) and nine partial responses (7%), with 11 patients (9%) progression free at >or= 30 months. Immune studies including assays of CD3-zeta expression and numbers of CD4(+)/CD25(+)/FoxP3(+) regulatory T cells, CD15(+)/CD11b(+)/CD14(-) immature myeloid-derived cells, and CD8(+)gp100 tetramer-positive cells in the blood did not correlate with clinical benefit. CONCLUSION The results again demonstrate efficacy of high-dose IL-2 in advanced melanoma but did not demonstrate the promising clinical activity reported with vaccine and high-dose IL-2 in any of three phase II trials.

Clinical and immunologic effects of intranodal autologous tumor lysate-dendritic cell vaccine with aldesleukin (interleukin 2) and IFN-α2a therapy in metastatic renal cell carcinoma patients

Purpose: To evaluate the clinical and immunologic outcomes of DC (dendritic cell) vaccine with interleukin (IL)-2 and IFN-α 2a in metastatic renal cell carcinoma patients. Experimental Design: Eighteen consented and eligible patients were treated. Peripheral blood monocytes were cultured ex vivo into mature DCs and loaded with autologous tumor lysate. Treatment consisted of five cycles of intranodal vaccination of DCs (1 × 107 cells/1 mL Lactated Ringers solution), 5-day continuous i.v. infusion of IL-2 (18MiU/m2), and three s.c. injections of IFN-α 2a (6MiU) every other day. Response Evaluation Criteria in Solid Tumors criteria were used for disease assessment. Correlative immunologic end points included peripheral blood lymphocyte cell phenotype and function as well as peripheral blood anti–renal cell carcinoma antibody and cytokine levels. Results: All patients received between two and five treatment cycles. Toxicities consisted of known and expected cytokine side effects. Overall objective clinical response rate was 50% with three complete responses. Median time to progression for all patients was 8 months, and median survival has not been reached (median follow up of 37+ months). Treatment-related changes in correlative immunologic end points were noted and the level of circulating CD4+ T regulatory cells had a strong association with outcome. Pre–IP-10 serum levels approached significance for predicting outcome. Conclusions: The clinical and immunologic responses observed in this trial suggest an interaction between DC vaccination and cytokine therapy. Our data support the hypothesis that modulation of inflammatory, regulatory, and angiogenic pathways are necessary to optimize therapeutic benefit in renal cell carcinoma patients. Further exploration of this approach is warranted.

Developing a rational tumor vaccine therapy for renal cell carcinoma: immune yin and yang.

In patients with progressive malignancy, the natural balance between proinflammatory (Yang) and inhibitory (regulatory or Yin) immune pathways is disrupted and favors cancer-specific immune suppression. Therapy with interleukin 2 (IL-2) can mobilize immune effector cells that recognize and destroy cancer. High-dose IL-2 is the only therapy that has consistently induced complete durable remissions in patients with metastatic renal cell carcinoma (RCC) but only in a few of them. The lack of benefit in most metastatic RCC patients is likely due to the ineffective manipulation of other immune circuits critical in regulating tumor cytotoxic pathways. The limited clinical activity of IL-2, RCC vaccines, and other immune therapies to date leads us to postulate that effective clinical treatment strategies will need to simultaneously enhance proinflammatory pathways and disrupt regulatory pathways. We present preliminary studies in RCC patients to highlight the complexity of the regulatory pathways and our approach to shifting the balance of proinflammatory and regulatory immune pathways using dendritic cell–tumor lysate vaccine followed by cytokine therapy.

A phase II study of bevacizumab and high-dose interleukin-2 in patients with metastatic renal cell carcinoma: a Cytokine Working Group (CWG) study.

Overexpression of vascular endothelial growth factor in renal cell carcinoma (RCC) leads to angiogenesis, tumor progression, and inhibition of immune function. We conducted the first phase II study to estimate the efficacy and safety of bevacizumab with high-dose interleukin-2 (IL-2) therapy in patients with metastatic RCC. Eligible patients had predominantly clear cell metastatic RCC, measurable disease, a Karnofsky Performance Status of ≥80%, and adequate end-organ function. IL-2 (600,000 IU/kg) was infused intravenously every 8 hours (maximum 28 doses) during two 5-day cycles on days 1 and 15 of each 84-day course. Bevacizumab (10 mg/kg) was infused intravenously every 2 weeks beginning 2 weeks before initiating IL-2. Fifty of 51 eligible patients from 8 centers were enrolled. Median progression-free survival (PFS) was 11.2 months (90% confidence interval, 5.7–17.7), and 2-year PFS was 18% (90% confidence interval, 8%–27%). Responses included 4 complete (8%) and 11 partial (22%) responses. Toxicities did not exceed those expected from each agent alone. Combining IL-2 plus bevacizumab is feasible, with a response rate and PFS at least as high as reported previously for the single agents. The regimen did not appear to enhance the rate of durable major responses over that of IL-2 alone.

Regulatory T-Cells and Associated Pathways in Metastatic Renal Cell Carcinoma (mRCC) Patients Undergoing DC-Vaccination and Cytokine-Therapy

Purpose To evaluate CD4+CD25+FOXP3+ T regulatory cells (TREG) and associated immune-regulatory pathways in peripheral blood lymphocytes (PBL) of metastatic renal cell carcinoma (mRCC) patients and healthy volunteers. We subsequently investigated the effects of immunotherapy on circulating TREG combining an extensive phenotype examination, DNA methylation analysis and global transcriptome analysis. Design Eighteen patients with mRCC and twelve volunteers (controls) were available for analysis. TREG phenotype was examined using flow cytometry (FCM). TREG were also quantified by analyzing the epigenetic status of the FOXP3 locus using methylation specific PCR. As a third approach, RNA of the PBL was hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays and the gene signatures were explored using pathway analysis. Results We observed higher numbers of TREG in pre-treatment PBL of mRCC patients compared to controls. A significant increase in TREG was detected in all mRCC patients after the two cycles of immunotherapy. The expansion of TREG was significantly higher in non-responders than in responding patients. Methylation specific PCR confirmed the FCM data and circumvented the variability and subjectivity of the FCM method. Gene Set Enrichment Analysis (GSEA) of the microarray data showed significant enrichment of FOXP3 target genes, CTLA-4 and TGF-ß associated pathways in the patient cohort. Conclusion Immune monitoring of the peripheral blood and tumor tissue is important for a wide range of diseases and treatment strategies. Adoption of methodology for quantifying TREG with the least variability and subjectivity will enhance the ability to compare and interpret findings across studies.

Cytokine Working Group Study of Lymphodepleting Chemotherapy, Interleukin-2, and Granulocyte-Macrophage Colony-Stimulating Factor in Patients With Metastatic Melanoma: Clinical Outcomes and Peripheral-Blood Cell Recovery

PURPOSE Recovery of lymphocyte populations after lymphocyte depletion is implicated in therapeutic immune pathways in animal models and in patients with cancer. We sought to evaluate the effects of chemotherapy-induced lymphodepletion followed by granulocyte-macrophage colony-stimulating factor (GM-CSF) and high-dose interleukin-2 (IL-2) therapy on clinical response and the recovery of lymphocyte subcompartments in patients with metastatic melanoma. PATIENTS AND METHODS This was a two-stage phase II trial design. Patients with measurable metastatic melanoma were treated with intravenous cyclophosphamide (60 mg/kg, days 1 and 2) and fludarabine (25 mg/m(2), day 3 through 7) followed by two 5-day courses of intravenous high-dose bolus IL-2 (600,000 U/kg; days 8 through 12 and 21 through 25). GM-CSF (250 microg/m(2)/d beginning day 8) was given until granulocyte recovery. Lymphocyte recovery profiles were determined by flow cytometric phenotyping at regular intervals, and clinical outcome was assessed by Response Evaluation Criteria in Solid Tumors (RECIST). RESULTS The trial was stopped at the end of stage 1 with four of 18 objective responses noted. Twelve patients had detailed lymphocyte subcompartments evaluated. After lymphodepletion, we observed an induction of regulatory cells (CD4+ T regulatory cells; CD8+ T suppressor cells) and of T memory cells (CD8+ T central memory cells; T effector memory RA+ cells). Expansion of circulating melanoma-specific CD8(+) cells was observed in one of four HLA-A2-positive patients. CONCLUSION Chemotherapy-induced lymphodepletion modulates the homeostatic repopulation of the lymphocyte compartment and influences recovering lymphocyte subpopulations. Clinical activity seems similar to standard high-dose aldesleukin alone.

A phase I, dose-escalation study of cyclical weekly oral temozolomide and weekly PEG-interferon alpha-2b in patients with refractory or advanced solid tumours

Abstract Background: Temozolomide (TMZ) is an oral alkylating agent used in the treatment of central nervous system neoplasms and metastatic melanoma. Preclinical and clinical data suggested that combining TMZ with interferon alpha-2b (IFN-alpha-2b) may result in increased anti-tumour efficacy. Methods: This was a phase I, dose-escalation study to define the maximum tolerated dose (MTD) and dose-limiting toxicities (DLT) of cyclical oral TMZ (days 1–7 and 15–21) in combination with pegylated IFN-alpha-2b (PEG-IFN-alpha-2b) in patients with advanced solid tumours. Results: We treated 19 patients (10 female and nine male), median age 58 years (range: 41–79 years). Ten patients tolerated TMZ at 100 mg/m2 on days 1–7 and 15–21 plus PEG-IFN-alpha-2b at 1·5 mcg/kg/week on 28-day cycles which was the MTD of the combination. The pharmacokinetic parameters of PEG-IFN-alpha-2b were not altered by TMZ, at the MTD. Conclusion: The MTD of cyclical oral TMZ was 100 mg/m2 on days 1-7 and 15-21 when combined with weekly subcutaneous PEG-IFNα-2b at 1.5 mcg/kg/week on 28 days cycles. The PK of PEG-IFN-alpha-2b appeared consistent with those when it is used as monotherapy.

Cross-presentation of Tumor Antigens Is Increased by UVC Light Tumor Treatment Response

In Response: We appreciate the thoughtful comments from Drs. Matera and Garetto. We, too, have wrestled with the source and preparation of antigens and human dendritic cells (DC) for use in clinical trials. Although defined renal cell cancer antigens such as G250 are being used for therapeutic immunizations, we agree with Matera and Garetto that, for renal cancers, whole tumor as a source of antigens remains a gold standard (1). As there is a paucity of data for established immunogenic peptides for renal cancer, the use of autologous renal tumor as a source of antigen presents unique challenges for effectively defining the tumor-associated peptide-MHC complex. In addition, the affinities of ligands for MHC molecules have an impact on the generation of protective immunity (2). It has recently been reported that the low-affinity epitope from tryosinase-related protein 1 versus the high-affinity epitope provides protection to B16 tumors. As described by Matera and Garetto, the antigen preparation method from whole tumor cells (apoptosis, necrosis, autophagy) for DC loading can affect DC function. Before our study, we determined if our method of DC and antigen preparation would induce immunity. We tested various approaches, and found the method used in our study equipotent at generating protective antitumor immunity (3). Nevertheless, the best method for the preparation of antigen and therapeutic DCs is evolving and remains a dynamic area of research. Although we did find a 3-fold treatment-related increase in the precursor frequency of renal cell carcinoma–specific CD8 CTLs, the frequency was low overall and, as noted, we did not find a correlation to clinical outcome. We were not able to assess the antigen specificity of these CTLs, and thus were not able to identify an immunogenic peptide target that would facilitate the type of DC studies that Matera and Garetto suggest. In addition, different DC subpopulations are responsible for skewing the immune system for cellular (Th1) or humoral response (Th2), and subpopulations of DCs may also act as inhibitors of immune response. To assess whether different DC or antigen preparations, or for that matter, different routes and schedules of vaccination, would improve outcome in a clinical setting would require large randomized studies, which have their own challenges. We agree with Matera and Garetto that the field of cancer vaccine trials would benefit tremendously from a consensus reporting procedure for defining DC and antigen source and preparation. Marc S. Ernstoff Jan Fisher John D. Seigne Zbigniew M. Szczepiorkowski Nancy A. Crosby Alan R. Schned Robert D. Harris Richard J. Barth, Jr. John A. Heaney Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire

Induction of T cell precursors in advanced melanoma treated with autologous tumor lysate loaded dendritic cells

2623 Background: Immunotherapy in melanoma patients (pts) is limited by tumor-induced effector cell inhibition. Dendritic cells (DC) are powerful initiators of tumor-specific immune responses. We proposed a pilot study that hypothesized that DC matured ex vivofrom the peripheral blood (PB) and pulsed with autologous tumor lysate (TuLy) would stimulate tumor-specific immune activation. METHODS 6 stage III/IV melanoma pts were enrolled and CD14+ precursors were obtained from PB by apheresis and elutriation prior to each treatment. DC were cultured ex vivo for 9 days with IL-4 and GM-CSF, pulsed with autologous TuLy and matured with TNFa. Pts received 3 treatments administered intravenously (IV) over 5-10 minutes at 4 week intervals. Efficiency of DC generation was determined by measuring total DC per apheresis and phenotype. NCI Common Toxicity Criteria v2.0 was used. Tumor-specific immune induction was evaluated using the Dye Dilution Proliferation Assay which measures T cell precursor frequencies (Tp) in response to stimuli; in this case pulsed or unpulsed mature DC. RESULTS 3 pts each have been treated with 1x106and 5x106DC respectively. Adequate yields of DC were achieved for all treatments. Toxicity was minimal with transient grade 2 ataxia. Mature DC highly expressed MHC class I and II, costimulatory markers (CD80, CD86, CD40) and the maturity marker CD83. We observed a 6-fold increase in CD8+ Tp and 1.5-fold increase in CD4+ Tp after 2 treatments (n=2) as shown in the table. Elevated Tp persisted 30 days following completion of treatment. CONCLUSIONS Ex vivogeneration and IV administration of mature DC is feasible and safe. Preliminary data support induction of tumor-specific T cells evidenced by increased Tp. [Figure: see text] No significant financial relationships to disclose.