William W. Grosh

Clinical and Immunologic Results of a Randomized Phase II Trial of Vaccination Using Four Melanoma Peptides Either Administered in Granulocyte-Macrophage Colony-Stimulating Factor in Adjuvant or Pulsed on Dendritic Cells

PURPOSE To determine clinical and immunologic responses to a multipeptide melanoma vaccine regimen, a randomized phase II trial was performed. PATIENTS AND METHODS Twenty-six patients with advanced melanoma were randomly assigned to vaccination with a mixture of four gp100 and tyrosinase peptides restricted by HLA-A1, HLA-A2, and HLA-A3, plus a tetanus helper peptide, either in an emulsion with granulocyte-macrophage colony-stimulating factor (GM-CSF) and Montanide ISA-51 adjuvant (Seppic Inc, Fairfield, NJ), or pulsed on monocyte-derived dendritic cells (DCs). Systemic low-dose interleukin-2 (Chiron, Emeryville, CA) was given to both groups. T-lymphocyte responses were assessed, by interferon gamma ELIspot assay (Chiron, Emeryville, CA), in peripheral-blood lymphocytes (PBLs) and in a lymph node draining a vaccine site (sentinel immunized node [SIN]). RESULTS In patients vaccinated with GM-CSF in adjuvant, T-cell responses to melanoma peptides were observed in 42% of PBLs and 80% of SINs, but in patients vaccinated with DCs, they were observed in only 11% and 13%, respectively. The overall immune response was greater in the GM-CSF arm (P <.02). Vitiligo developed in two of 13 patients in the GM-CSF arm but in no patients in the DC arm. Helper T-cell responses to the tetanus peptide were detected in PBLs after vaccination and correlated with T-cell reactivity to the melanoma peptides. Objective clinical responses were observed in two patients in the GM-CSF arm and one patient in the DC arm. Stable disease was observed in two patients in the GM-CSF arm and one patient in the DC arm. CONCLUSION The high frequency of cytotoxic T-lymphocyte responses and the occurrence of clinical tumor regressions support continued investigation of multipeptide vaccines administered with GM-CSF in adjuvant.

Immunologic and Clinical Outcomes of Vaccination With a Multiepitope Melanoma Peptide Vaccine Plus Low-Dose Interleukin-2 Administered Either Concurrently or on a Delayed Schedule

PURPOSE A phase II trial was performed to test whether systemic low-dose interleukin-2 (IL-2) augments T-cell immune responses to a multipeptide melanoma vaccine. Forty patients with resected stage IIB-IV melanoma were randomly assigned to vaccination with four gp100- and tyrosinase-derived peptides restricted by human leukocyte antigen (HLA) -A1, HLA-A2, and HLA-A3, and a tetanus helper peptide plus IL-2 administered daily either beginning day 7 (group 1), or beginning day 28 (group 2). PATIENTS AND METHODS T-cell responses were assessed by an interferon gamma ELIspot assay in peripheral blood lymphocytes (PBL) and in a lymph node draining a vaccination site (sentinel immunized node [SIN]). Patients were followed for disease-free and overall survival. RESULTS T-cell responses to the melanoma peptides were observed in 37% of PBL and 38% of SINs in group 1, and in 53% of PBL and 83% of SINs in group 2. The magnitude of T-cell response was higher in group 2. The tyrosinase peptides DAEKSDICTDEY and YMDGTMSQV were more immunogenic than the gp100 peptides YLEPGPVTA and ALLAVGATK. T-cell responses were detected in the SINs more frequently, and with higher magnitude, than responses in the PBL. Disease-free survival estimates at 2 years were 39% (95% CI, 18% to 61%) for group 1, and 50% (95% CI, 28% to 72%) for group 2 (P = .32). CONCLUSION The results of this study support the safety and immunogenicity of a vaccine composed of four peptides derived from gp100 and tyrosinase. The low-dose IL-2 regimen used for group 1 paradoxically diminishes the magnitude and frequency of cytotoxic T lymphocyte responses to these peptides.

Effect of Granulocyte/Macrophage Colony-Stimulating Factor on Circulating CD8+ and CD4+ T-Cell Responses to a Multipeptide Melanoma Vaccine: Outcome of a Multicenter Randomized Trial

Purpose: Granulocyte/macrophage colony-stimulating factor (GM-CSF) administered locally together with vaccines can augment T-cell responses in animal models. Human experience has been limited to small and uncontrolled trials. Thus, a multicenter randomized phase II trial was done to determine whether local administration of GM-CSF augments immunogenicity of a multipeptide vaccine. It also assessed immunogenicity of administration in one versus two vaccine sites. Experimental Design: One hundred twenty-one eligible patients with resected stage IIB to IV melanoma were vaccinated with 12 MHC class Irestricted melanoma peptides to stimulate CD8+ T cells plus a HLA-DRrestricted tetanus helper peptide to stimulate CD4+ T cells, emulsified in incomplete Freunds adjuvant, with or without 110 g GM-CSF. Among 119 evaluable patients, T-cell responses were assessed by IFN- ELIspot assay and tetramer analysis. Clinical outcomes were recorded. Results: CD8+ T-cell response rates to the 12 MHC class Irestricted melanoma peptides (by day 50) with or without GM-CSF were 34 and 73, respectively (P < 0.001), by direct ELIspot assay. Tetramer analyses corroborated the functional data. CD4+ T-cell responses to tetanus helper peptide were higher without GM-CSF (95 versus 77; P = 0.005). There was no significant difference by number of vaccine sites. Three-year overall and disease-free survival estimates (95 confidence interval) were 76 (67-83) and 52 (43-61), respectively, with too few events to assess differences by study group. Conclusions: High immune response rates for this multipeptide vaccine were achieved, but CD8+ and CD4+ T-cell responses were lower when administered with GM-CSF. These data challenge the value of local GM-CSF as a vaccine adjuvant in humans. (Clin Cancer Res 2009;15(22):703644)

Immunologic and clinical outcomes of a randomized phase II trial of two multipeptide vaccines for melanoma in the adjuvant setting.

Purpose: Human melanoma cells express shared antigens recognized by CD8+ T lymphocytes, the most common of which are melanocytic differentiation proteins and cancer-testis antigens. However, peptide vaccines for melanoma usually target only one or two MHC class I–associated peptide antigens. Because melanomas commonly evade immune recognition by selective antigen loss, optimization of melanoma vaccines may require development of more complex multipeptide vaccines. Experimental Design: In a prospective randomized clinical trial, we have evaluated the safety and immunogenicity of a vaccine containing a mixture of 12 peptides from melanocytic differentiation proteins and cancer-testis antigens, designed for human leukocyte antigen types that represent 80% of the melanoma patient population. This was compared with a four-peptide vaccine with only melanocytic differentiation peptides. Immune responses were assessed in peripheral blood and in vaccine-draining lymph nodes. Results: These data show that (a) the 12-peptide mixture is immunogenic in all treated patients; (b) immunogenicity of individual peptides is maintained despite competition with additional peptides for binding to MHC molecules; (c) a broader and more robust immune response is induced by vaccination with the more complex 12-peptide mixture; and (d) clinical outcome in this peptide vaccine trial correlates with immune responses measured in the peripheral blood lymphocytes. Conclusions: These data support continued investigation of complex multipeptide vaccines for melanoma.

Evaluation of peptide vaccine immunogenicity in draining lymph nodes and peripheral blood of melanoma patients

Many peptide epitopes for cytotoxic T lymphocytes (CTLs) have been identified from melanocytic differentiation proteins. Vaccine trials with these peptides have been limited mostly to those associated with HLA‐A2, and immune responses have been detected inconsistently. Cases of clinical regression have been observed after peptide vaccination in some trials, but melanoma regressions have not correlated well with T‐cell responses measured in peripheral blood lymphocytes (PBLs). We vaccinated stage IV melanoma patients with a mixture of gp100 and tyrosinase peptides restricted by HLA‐A1 (DAEKSDICTDEY), HLA‐A2 (YLEPGPVTA and YMDGTMSQV) and HLA‐A3 (ALLAVGATK) in an emulsion with GM‐CSF and Montanide ISA‐51 adjuvant. CTL responses were assessed in PBLs and in a lymph node draining a vaccine site (sentinel immunized node, SIN). We found CTL responses to vaccinating peptides in the SIN in 5/5 patients (100%). Equivalent assays detected peptide‐reactive CTLs in PBLs of 2 of these 5 patients (40%). CTLs expanded from the SIN lysed melanoma cells naturally expressing tyrosinase or gp100. We demonstrated immunogenicity for peptides restricted by HLA‐A1 and ‐A3 and for 1 HLA‐A2 restricted peptide, YMDGTMSQV. Immune monitoring of clinical trials by evaluation of PBLs alone may under‐estimate immunogenicity; evaluation of SIN provides a new and sensitive approach for defining responses to tumor vaccines and correlating these responses with clinical outcomes. This combination of an immunogenic vaccine strategy with a sensitive analysis of CTL responses demonstrates the potential for inducing and detecting anti‐tumor immune responses in the majority of melanoma patients.

MAGE-A1-, MAGE-A10-, and gp100-Derived Peptides Are Immunogenic When Combined with Granulocyte-Macrophage Colony-Stimulating Factor and Montanide ISA-51 Adjuvant and Administered as Part of a Multipeptide Vaccine for Melanoma

Twelve peptides derived from melanocyte differentiation proteins and cancer-testis Ags were combined and administered in a single mixture to patients with resected stage IIB, III, or IV melanoma. Five of the 12 peptides included in this mixture had not previously been evaluated for their immunogenicity in vivo following vaccination. We report in this study that at least three of these five peptides (MAGE-A196–104, MAGE-A10254–262, and gp100614–622) are immunogenic when administered with GM-CSF in Montanide ISA-51 adjuvant. T cells secreting IFN-γ in response to peptide-pulsed target cells were detected in peripheral blood and in the sentinel immunized node, the node draining a vaccine site, after three weekly injections. The magnitude of response typically reached a maximum after two vaccines, and though sometimes diminished thereafter, those responses typically were still detectable 6 wks after the last vaccines. Most importantly, tumor cell lines expressing the appropriate HLA-A restriction element and MAGE-A1, MAGE-A10, or gp100 proteins were lysed by corresponding CTL. This report supports the continued use of the MAGE-A196–104, MAGE-A10254–262, and gp100614–622 epitopes in peptide-based melanoma vaccines and thus expands the list of immunogenic peptide Ags available for human use. Cancer-testis Ags are expressed in multiple types of cancer; thus the MAGE-A196–104 and MAGE-A10254–262 peptides may be considered for inclusion in vaccines against cancers of other histologic types, in addition to melanoma.

Helper T-Cell Responses and Clinical Activity of a Melanoma Vaccine With Multiple Peptides From MAGE and Melanocytic Differentiation Antigens

PURPOSE A phase I/II trial was performed to evaluate the safety and immunogenicity of a novel melanoma vaccine comprising six melanoma-associated peptides defined as antigenic targets for melanoma-reactive helper T cells. Source proteins for these peptides include MAGE proteins, MART-1/MelanA, gp100, and tyrosinase. PATIENTS AND METHODS Thirty-nine patients with stage IIIB to IV melanoma were vaccinated with this six-peptide mixture weekly at three dose levels, with a preceding phase I dose escalation and subsequent random assignment among the dose levels. Helper T-lymphocyte responses were assessed by in vitro proliferation assay and delayed-type hypersensitivity skin testing. Patients with measurable disease were evaluated for objective clinical response by Response Evaluation Criteria in Solid Tumors. RESULTS Vaccination with the helper peptide vaccine was well tolerated. Proliferation assays revealed induction of T-cell responses to the melanoma helper peptides in 81% of patients. Among 17 patients with measurable disease, objective clinical responses were observed in two patients (12%), with response durations of 1 and 3.9+ years. Durable stable disease was observed in two additional patients for periods of 1.8 and 4.6+ years. CONCLUSION Results of this study support the safety and immunogenicity of a vaccine comprised of six melanoma helper peptides. There is also early evidence of clinical activity.

Randomized Multicenter Trial of the Effects of Melanoma-Associated Helper Peptides and Cyclophosphamide on the Immunogenicity of a Multipeptide Melanoma Vaccine

PURPOSE This multicenter randomized trial was designed to test whether melanoma-associated helper peptides augment CD8(+) T-cell responses to a melanoma vaccine and whether cyclophosphamide (CY) pretreatment augments CD4(+) or CD8(+) T-cell responses to that vaccine. PATIENTS AND METHODS In all, 167 eligible patients with resected stage IIB to IV melanoma were randomly assigned to four vaccination study arms. Patients were vaccinated with 12 class I major histocompatibility complex-restricted melanoma peptides (12MP) to stimulate CD8(+) T cells and were randomly assigned to receive a tetanus helper peptide or a mixture of six melanoma-associated helper peptides (6MHP) to stimulate CD4(+) T cells. Before vaccination, patients were also randomly assigned to receive CY pretreatment or not. T-cell responses were assessed by an ex vivo interferon gamma ELISpot assay. Clinical outcomes and toxicities were recorded. RESULTS Vaccination with 12MP plus tetanus induced CD8(+) T-cell responses in 78% of patients and CD4(+) T-cell responses to tetanus peptide in 93% of patients. Vaccination with 12MP plus 6MHP induced CD8(+) responses in 19% of patients and CD4(+) responses to 6MHP in 48% of patients. CY had no significant effect on T-cell responses. Overall 3-year survival was 79% (95% CI, 71% to 86%), with no significant differences (at this point) by study arm. CONCLUSION Melanoma-associated helper peptides paradoxically decreased CD8(+) T-cell responses to a melanoma vaccine (P < .001), and CY pretreatment had no immunologic or clinical effect. Prior work showed immunologic and clinical activity of 6MHP alone. Possible explanations for negative effects on CD8 responses include modulation of homing receptor expression or induction of antigen-specific regulatory T cells.

Immunity to Melanoma Antigens: From Self‐Tolerance to Immunotherapy

The development of effective immune therapy for cancer is a central goal of immunologists in the 21st century. Our laboratories have been deeply involved in characterization of the immune response to melanoma and translation of laboratory discoveries into clinical trials. We have identified a cohort of peptide antigens presented by Major Histocompatibility Complex (MHC) molecules on melanoma cells and widely recognized by T cells from melanoma patients. These have been incorporated into peptide-based vaccines that induce CD8(+) and CD4(+) T-cell responses in 80-100% of patients. Major objective clinical tumor regressions have been observed in some patients, and overall survival in vaccinated patients exceeds expected stage-specific survival. New clinical trials will determine the value of combination of melanoma helper peptides (MHP) into multipeptide vaccines targeting CD8 cells. New trials will also evaluate new approaches to modulating the host-tumor relationship and will develop new combination therapies. Parallel investigations in murine models are elucidating the immunobiology of the melanoma-host relationship and addressing issues that are not feasible to approach in human trials. Based on the fact that the largest cohort of melanoma antigens are derived from normal proteins concerned with pigment production, we have evaluated the mechanisms of self-tolerance to tyrosinase (Tyr) and have determined how T cells in an environment of self-tolerance are impacted by immunization. Using peptide-pulsed dendritic cells as immunogens, we have also used the mouse model to establish strategies for quantitative and qualitative enhancement of antitumor immunity. This information creates opportunities for a new generation of therapeutic interventions using cancer vaccines.

Molecular Insights on the Peripheral and Intratumoral Effects of Systemic High-Dose rIL-2 (Aldesleukin) Administration for the Treatment of Metastatic Melanoma

Purpose: We have previously shown that within tumors, recombinant interleukin-2 (rIL-2, aldesleukin) consistently activates tumor-associated macrophages and upregulates IFN-stimulated genes while inducing minimal migration, activation, or proliferation of T cells. These effects are independent of tumor response to treatment. Here, we prospectively evaluated transcriptional alterations induced by rIL-2 in peripheral blood mononuclear cells (PBMC) and within melanoma metastases. Experimental Design: We evaluated gene expression changes by serially comparing pre- to posttreatment samples in 13 patients and also compared transcriptional differences among lesions displaying different responsiveness to therapy, focusing on 2 lesions decreasing in size and 2 remaining stable (responding lesions) compared with nonresponding ones. Results: As previously described, the effects of rIL-2 were dramatic within PBMCs, whereas effects within the tumor microenvironment were lesion specific and limited. However, distinct signatures specific to response could be observed in responding lesions pretreatment that were amplified following rIL-2 administration. These signatures match the functional profile observed in other human or experimental models in which immune-mediated tissue-specific destruction (TSD) occurs, underscoring common pathways leading to rejection. Moreover, the signatures observed in pretreatment lesions were qualitatively similar to those associated with TSD, underlining a determinism to immune responsiveness that depends upon the genetic background of the host or the intrinsic genetic makeup of individual tumors. Conclusions: This is the first prospectively collected insight on global transcriptional events occurring during high-dose rIL-2 therapy in melanoma metastases responding to treatment. Clin Cancer Res; 17(23); 7440–50. ©2011 AACR.