Results of a randomized phase I gene therapy clinical trial of nononcolytic fowlpox viruses encoding T cell costimulatory molecules.

Oncolytic viruses have shown promise as gene delivery vehicles in the treatment of cancer; however, their efficacy may be inhibited by the induction of anti-viral antibody titers. Fowlpox virus is a nonreplicating and nononcolytic vector that has been associated with lesser humoral but greater cell-mediated immunity in animal tumor models. To test whether fowlpox virus gene therapy is safe and can elicit immune responses in patients with cancer, we conducted a randomized phase I clinical trial of two recombinant fowlpox viruses encoding human B7.1 or a triad of costimulatory molecules (B7.1, ICAM-1, and LFA-3; TRICOM). Twelve patients (10 with melanoma and 2 with colon adenocarcinoma) enrolled in the trial and were randomized to rF-B7.1 or rF-TRICOM administered in a dose escalation manner (~3.7×10(7) or ~3.7×10(8) plaque-forming units) by intralesional injection every 4 weeks. The therapy was well tolerated, with only four patients experiencing grade 1 fever or injection site pain, and there were no serious adverse events. All patients developed anti-viral antibody titers after vector delivery, and posttreatment anti-carcinoembryonic antigen antibody titers were detected in the two patients with colon cancer. All patients developed CD8(+) T cell responses against fowlpox virus, but few responses against defined tumor-associated antigens were observed. This is the first clinical trial of direct (intratumoral) gene therapy with a nononcolytic fowlpox virus. Treatment was well tolerated in patients with metastatic cancer; all subjects exhibited anti-viral antibody responses, but limited tumor-specific T cell responses were detected. Nononcolytic fowlpox viruses are safe and induce limited T cell responses in patients with cancer. Further development may include prime-boost strategies using oncolytic viruses for initial priming.

Local and distant immunity induced by intralesional vaccination with an oncolytic herpes virus encoding GM-CSF in patients with stage IIIc and IV melanoma.

BACKGROUND: An oncolytic herpes simplex virus engineered to replicate selectively in tumor cells and to express granulocyte-macrophage colony-stimulating factor (GMCSF) was tested as a direct intralesional vaccination in melanoma patients. The work reported herein was performed to better characterize the effect of vaccination on local and distant antitumor immunity. METHODS: Metastatic melanoma patients with accessible lesions were enrolled in a multicenter 50-patient phase II clinical trial of an oncolytic herpesvirus encoding GM-CSF (Oncovex(GM-CSF)). An initial priming dose of 10(6) pfu vaccine was given by intratumoral injection, followed by 10(8) pfu every 2 weeks to 24 total doses. Peripheral blood and tumor tissue were collected for analysis of effector T cells, CD4(+)FoxP3(+) regulatory T cells (Treg), CD8(+)FoxP3(+) suppressor T cells (Ts), and myeloid-derived suppressive cells (MDSC). RESULTS: Phenotypic analysis of T cells derived from tumor samples suggested distinct differences from peripheral blood T cells. There was an increase in melanomaassociated antigen recognized by T cells (MART-1)-specific T cells in tumors undergoing regression after vaccination compared with T cells derived from melanoma patients not treated with vaccine. There was also a significant decrease in Treg and Ts cells in injected lesions compared with noninjected lesions in the same and different melanoma patients. Similarly MDSC were increased in melanoma lesions but underwent a significant decrease only in vaccinated lesions. CONCLUSIONS: Melanoma patients present with elevated levels of Tregs, Ts, and MDSC within established tumors. Direct injection of Oncovex(GM-CSF) induces local and systemic antigen-specific T cell responses and decreases Treg, Ts, and MDSC in patients exhibiting therapeutic responses.

Serum vascular endothelial growth factor and fibronectin predict clinical response to high-dose interleukin-2 therapy.

PURPOSE: High-dose interleukin-2 (IL-2) induces durable therapeutic responses in a small subset of patients with metastatic melanoma and renal cell carcinoma, but simple pretreatment predictors of response have not been identified. PATIENTS AND METHODS: To identify predictive biomarkers of clinical response, sera from patients treated with high-dose IL-2 were collected for analysis using a customized, multiplex antibody-targeted protein array platform that surveyed expression of soluble factors associated with tumor immunobiology. Soluble factors associated with clinical responses were analyzed using a multivariate permutation test, and survival outcomes were determined using Kaplan-Meier and log-rank tests. RESULTS: A training set from 10 patients identified 68 potentially relevant soluble factors that were then tested in an independent validation set of 49 patients. Class comparison revealed a cluster of 11 biomarkers that were associated with therapeutic outcome. Vascular endothelial growth factor (VEGF) and fibronectin were identified as independent predictors of response. In particular, high levels of these proteins were correlated with lack of clinical response and decreased overall survival. CONCLUSION: Serum VEGF and fibronectin are easily measured pretreatment biomarkers that could serve to exclude patients unlikely to respond to IL-2 therapy.

Phase II trial of Modified Vaccinia Ankara (MVA) virus expressing 5T4 and high dose Interleukin-2 (IL-2) in patients with metastatic renal cell carcinoma.

BACKGROUND: Interleukin-2 (IL-2) induces durable objective responses in a small cohort of patients with metastatic renal cell carcinoma (RCC) but the antigen(s) responsible for tumor rejection are not known. 5T4 is a non-secreted membrane glycoprotein expressed on clear cell and papillary RCCs. A modified vaccinia virus Ankara (MVA) encoding 5T4 was tested in combination with high-dose IL-2 to determine the safety, objective response rate and effect on humoral and cell-mediated immunity. METHODS: 25 patients with metastatic RCC who qualified for IL-2 were eligible and received three immunizations every three weeks followed by IL-2 (600,000 IU/kg) after the second and third vaccinations. Blood was collected for analysis of humoral, effector and regulatory T cell responses. RESULTS: There were no serious vaccine-related adverse events. While no objective responses were observed, three patients (12%) were rendered disease-free after nephrectomy or resection of residual metastatic disease. Twelve patients (48%) had stable disease which was associated with improved median overall survival compared to patients with progressive disease (not reached vs. 28 months, p = 0.0261). All patients developed 5T4-specific antibody responses and 13 patients had an increase in 5T4-specific T cell responses. Although the baseline frequency of Tregs was elevated in all patients, those with stable disease showed a trend toward increased effector CD8+ T cells and a decrease in Tregs. CONCLUSION: Vaccination with MVA-5T4 did not improve objective response rates of IL-2 therapy but did result in stable disease associated with an increase in the ratio of 5T4-specific effector to regulatory T cells in selected patients. TRIAL REGISTRATION NUMBER: ISRCTN83977250.

Poxvirus-based vaccine therapy for patients with advanced pancreatic cancer.

PURPOSE: An open-label Phase 1 study of recombinant prime-boost poxviruses targeting CEA and MUC-1 in patients with advanced pancreatic cancer was conducted to determine safety, tolerability and obtain preliminary data on immune response and survival. PATIENTS AND METHODS: Ten patients with advanced pancreatic cancer were treated on a Phase I clinical trial. The vaccination regimen consisted of vaccinia virus expressing tumor antigens carcinoembryonic antigen (CEA) and mucin-1 (MUC-1) with three costimulatory molecules B7.1, ICAM-1 and LFA-3 (TRICOM) (PANVAC-V) and fowlpox virus expressing the same antigens and costimulatory molecules (PANVAC-F). Patients were primed with PANVAC-V followed by three booster vaccinations using PANVAC-F. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used as a local adjuvant after each vaccination and for 3 consecutive days thereafter. Monthly booster vaccinations for up to 12 months were provided for patients without progressive disease. Peripheral blood was collected before, during and after vaccinations for immune analysis. RESULTS: The most common treatment-related adverse events were mild injection-site reactions. Antibody responses against vaccinia virus was observed in all 10 patients and antigen-specific T cell responses were observed in 5 out of 8 evaluable patients (62.5%). Median overall survival was 6.3 months and a significant increase in overall survival was noted in patients who generated anti CEA- and/or MUC-1-specific immune responses compared with those who did not (15.1 vs 3.9 months, respectively; P = .002). CONCLUSION: Poxvirus vaccination is safe, well tolerated, and capable of generating antigen-specific immune responses in patients with advanced pancreatic cancer.

Characterization of CD4+CD25+ regulatory T cells in patients treated with high-dose interleukin-2 for metastatic melanoma or renal cell carcinoma.

PURPOSE: To characterize the number and functional status of CD4+CD25+ regulatory T cells (Tregs) in patients with metastatic melanoma (MM) and renal cell carcinoma (RCC) treated with high-dose bolus interleukin-2 (IL-2). PATIENTS AND METHODS: Patients with MM or RCC treated with high-dose bolus IL-2 (600,000 IU/kg every 8 hours) at a single center provided pre- and post-treatment whole blood specimens. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, separated into cellular subsets, and analyzed by flow cytometry or used for in vitro proliferation assays. RESULTS: Between September 2003 and July 2005 57 patients were enrolled in the study with 48 patients available for analysis (45 MM, 12 RCC). Tregs were defined as CD4+CD25(hi) T cells, and this subset was significantly elevated in the cancer patients compared with normal donors (7.75% v 2.24%). The CD4(+)CD25(hi) T-cell pool in the patients constitutively expressed intracellular FoxP3, CTLA-4, and produced high amounts of IL-10. The Tregs were CCR7+ with 50% representing naïve and 50% central-memory T cells. The cells were functionally suppressive in mixed in vitro proliferation assays. Following IL-2 administration, the number and frequency of Tregs increased in patients with progressive disease but returned to normal levels in patients with objective clinical responses. CONCLUSION: The number of Tregs, defined as CD4+CD25(hi) T cells is increased in patients with MM and RCC. High-dose IL-2 resulted in a significant decrease of Tregs in those patients achieving an objective clinical response to IL-2 therapy.

Local delivery of vaccinia virus expressing multiple costimulatory molecules for the treatment of established tumors.

Successful immunotherapy of established tumors depends on overcoming the suppressive influence of the local tumor microenvironment. The direct injection of vaccinia virus expressing the B7.1 (CD80) costimulatory molecule into melanoma lesions resulted in local and systemic immunity with associated clinical responses. Therefore, we sought to evaluate the effects of a vaccinia virus expressing three costimulatory molecules, B7.1, ICAM-1, and LFA-3 (rV-TRICOM), in patients with metastatic melanoma. A standard dose escalation phase I clinical trial was performed. Thirteen patients were enrolled and 12 were available for follow-up. Local vaccination was feasible, with only low-grade injection site reactions associated with mild fatigue and myalgia reported. There was one occurrence of grade 1 vitiligo. Overall there was a 30.7% objective clinical response, with one patient achieving a complete response for more than 22 months. An inverse association was detected between anti-vaccinia antibody and anti-vaccinia T cell responses. Patients who failed to respond to vaccination but received high-dose interleukin-2 had a trend toward improved survival. Collectively, these results confirm the safety profile and feasibility of direct injection of vaccinia virus expressing multiple costimulatory molecules in patients with established tumors. Further clinical investigation is needed to better define the role of antigen, adjuvant cytokines, costimulation, and cross-presentation in the host immune response to local vaccination with vaccinia viruses expressing immunomodulatory molecules.

Targeting the local tumor microenvironment with vaccinia virus expressing B7.1 for the treatment of melanoma.

Immunotherapy for the treatment of metastatic melanoma remains a major clinical challenge. The melanoma microenvironment may lead to local T cell tolerance in part through downregulation of costimulatory molecules, such as B7.1 (CD80). We report the results from the first clinical trial, to our knowledge, using a recombinant vaccinia virus expressing B7.1 (rV-B7.1) for monthly intralesional vaccination of accessible melanoma lesions. A standard 2-dose-escalation phase I clinical trial was conducted with 12 patients. The approach was well tolerated with only low-grade fever, myalgias, and fatigue reported and 2 patients experiencing vitiligo. An objective partial response was observed in 1 patient and disease stabilization in 2 patients, 1 of whom is alive without disease 59 months following vaccination. All patients demonstrated an increase in postvaccination antibody and T cell responses against vaccinia virus. Systemic immunity was tested in HLA-A*0201 patients who demonstrated an increased frequency of gp100 and T cells specific to melanoma antigen recognized by T cells 1 (MART-1), also known as Melan-A, by ELISPOT assay following local rV-B7.1 vaccination. Local immunity was evaluated by quantitative real-time RT-PCR, which suggested that tumor regression was associated with increased expression of CD8 and IFN-gamma. The local delivery of vaccinia virus expressing B7.1 was well tolerated and represents an innovative strategy for altering the local tumor microenvironment in patients with melanoma.

Initial clinical response predicts outcome and is associated with dose schedule in metastatic melanoma and renal cell carcinoma patients treated with high-dose interleukin 2.

BACKGROUND: High-dose interleukin (IL)-2 is an effective agent for the treatment of metastatic malignant melanoma and renal cell carcinoma. This study evaluated the outcomes of patients receiving two commonly used intravenous IL-2 schedules that have never been directly compared. METHODS: Forty-seven metastatic malignant melanoma and renal cell carcinoma patients were identified from a prospective database who underwent high-dose IL-2 therapy (720,000 or 600,000 IU/kg) during 1999 to 2003. Disease-specific survival (DSS) was calculated by the Kaplan-Meier method with the log-rank test on an intention-to-treat basis. Multivariate Cox regression analysis of prognostic variables associated with outcome was performed. Factors associated with initial response and prevention of disease progression were determined. RESULTS: Objective response (5 partial and 5 mixed) or disease stabilization was noted in 9 (20%) and 10 (22%), respectively, of 46 assessable patients after 1 course of therapy. Four patients (22%) achieved disease-free status after the third course of IL-2 (n = 1) or surgical resection of confined metastatic disease (n = 3). At 19.1 months' median follow-up, factors associated with improved DSS included an initial clinical response to IL-2 therapy (P < .001) and a higher administered dose (P = .04). Patients who received 720,000 IU/kg were more likely to experience an initial major objective response (P = .03) and disease stabilization (P = 0.03) independent of the tumor treated. Objective response early in the course of therapy was the only independent predictor of tumor-related mortality (P = .004). CONCLUSIONS: The initial clinical response to IL-2 therapy is an independent predictor of improved outcome associated with DSS and the 720,000 IU/kg dose. These results support further prospective trials with increased IL-2 dose schedules in a larger cohort of patients.

Management of metastatic malignant melanoma of the bladder.

Recombinant interleukin-2 (IL-2) has demonstrated antitumor activity and durable clinical responses in patients with metastatic melanoma. Careful screening and selection of appropriate patients has improved the safety profile of IL-2 administration. Gross hematuria would ordinarily preclude the safe delivery of IL-2. We report a case of metastatic melanoma to the bladder presenting with hematuria. A complete resection was performed and subsequently allowed the administration of high-dose, bolus IL-2. The combination of resection and IL-2 therapy resulted in a partial response maintained for more than 18 months. Symptomatic bladder melanoma should be aggressively treated to allow for systemic immunotherapy, which can provide durable responses.

Treatment of pediatric ocular melanoma with high-dose interleukin-2 and thalidomide.

Uveal melanoma is the most common primary ocular malignancy, although it is rare in children, and patients presenting with metastatic disease have a median survival of only 2 to 5 months. The tumor is generally unresponsive to systemic chemotherapy, but immunotherapy may be effective in selected patients. This report describes an 8-year-old girl with metastatic uveal melanoma treated with high-dose, bolus interleukin-2 (IL-2) and the antiangiogenic agent thalidomide. She tolerated treatment well and initially responded with stable disease in the liver and pancreas for 23 months. New pulmonary metastases developed and she was re-treated with high-dose IL-2, resulting in regression of her liver lesions and stable pulmonary disease for more than 18 months. These results suggest that IL-2 at high doses, and in combination with thalidomide, may be useful for uveal melanoma with tolerable side effects in children. Further study of this combination in children with immune-responsive tumors is warranted.