RESUMEN
Checkpoint-blockade therapy (CBT) is approved for select colorectal cancer (CRC) patents, but additional immunotherapeutic options are needed. We hypothesized that vaccination with carcinoembryonic antigen (CEA) and Her2/neu (Her2) peptides would be immunogenic and well tolerated by participants with advanced CRC. A pilot clinical trial (NCT00091286) was conducted in HLA-A2+ or -A3+ Stage IIIC-IV CRC patients. Participants were vaccinated weekly with CEA and Her2 peptides plus tetanus peptide and GM-CSF emulsified in Montanide ISA-51 adjuvant for 3 weeks. Adverse events (AEs) were recorded per NIH Common Terminology Criteria for Adverse Events version 3. Immunogenicity was evaluated by interferon-gamma ELISpot assay of in vitro sensitized peripheral blood mononuclear cells and lymphocytes from the sentinel immunized node. Eleven participants were enrolled and treated; one was retrospectively found to be ineligible due to HLA type. All 11 participants were included in AEs and survival analyses, and the 10 eligible participants were evaluated for immunogenicity. All participants reported AEs: 82% were Grade 1-2, most commonly fatigue or injection site reactions. Two participants (18%) experienced treatment-related dose-limiting Grade 3 AEs; both were self-limiting. Immune responses to Her2 or CEA peptides were detected in 70% of participants. Median overall survival (OS) was 16 months; among those enrolled with no evidence of disease (n = 3), median OS was not reached after 10 years of follow-up. These data demonstrate that vaccination with CEA or Her2 peptides is well tolerated and immunogenic. Further study is warranted to assess potential clinical benefits of vaccination in advanced CRC either alone or in combination with CBT.
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Vacunas contra el Cáncer/uso terapéutico , Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/tratamiento farmacológico , Células Dendríticas/inmunología , Fragmentos de Péptidos/uso terapéutico , Receptor ErbB-2/inmunología , Vacunación/métodos , Adulto , Anciano , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Femenino , Estudios de Seguimiento , Proteínas Ligadas a GPI/inmunología , Humanos , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/inmunología , Proyectos Piloto , Pronóstico , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
INTRODUCTION: Methods to induce T cell responses to protein vaccines have not been optimized. The immunostimulant AS15 has been administered with the recombinant MAGE-A3 protein (recMAGE-A3) i.m. but not i.d. or s.c. This study tests hypotheses that the i.d./s.c. route is safe and will increase CD4(+) and CD8(+) T cell responses to MAGE-A3. PATIENTS AND METHODS: Twenty-five patients with resected stage IIB-IV MAGE-A3(+) melanoma were randomized to immunization with recMAGE-A3 combined with AS15 immunostimulant (MAGE-A3 immunotherapeutic) either i.m. (group A, n = 13) or i.d./s.c. (group B, n = 12). Adverse events were recorded. Ab responses to MAGE-A3 were measured by ELISA. T cell responses to overlapping MAGE-A3 peptides were assessed in PBMC and a sentinel immunized node (SIN) after 1 in vitro stimulation with recMAGE-A3, by IFN-γ ELISPOT assay and by flow cytometry for multifunctional (TNF-α/IFN-γ) responses. RESULTS: Both routes of immunization were well tolerated without treatment-related grade 3 adverse events. All patients had durable Ab responses. For all 25 patients, the T cell response rate by ELISPOT assay was 30 % in SIN (7/23) but only 4 % (1/25) in PBMC. By flow cytometry, multifunctional CD8(+) T cell responses were identified in one patient in each group; multifunctional CD4(+) T cell response rates for groups A and B, respectively, were 31 and 64 % in SIN and 31 and 50 % in PBMC. CONCLUSION: The MAGE-A3 immunotherapeutic was well tolerated after i.d./s.c. administration, with trends to higher CD4(+) T cell response rates than with i.m. administration. This study supports further study of AS15 by i.d./s.c. administration.
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Adyuvantes Inmunológicos/uso terapéutico , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Proteínas de Neoplasias/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/uso terapéutico , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/uso terapéutico , Humanos , Inyecciones Intramusculares , Persona de Mediana Edad , Proteínas de Neoplasias/uso terapéutico , Proyectos Piloto , Resultado del TratamientoRESUMEN
INTRODUCTION: Optimal approaches to induce T cell infiltration of tumors are not known. Chemokines CXCL9, CXCL10, and CXCL11 support effector T cell recruitment and may be induced by IFN. This study tests the hypothesis that intratumoral administration of IFNγ will induce CXCL9-11 and will induce T cell recruitment and anti-tumor immune signatures in melanoma metastases. PATIENTS AND METHODS: Nine eligible patients were immunized with a vaccine comprised of 12 class I MHC-restricted melanoma peptides and received IFNγ intratumorally. Effects on the tumor microenvironment were evaluated in sequential tumor biopsies. Adverse events (AEs) were recorded. T cell responses to vaccination were assessed in PBMC by IFNγ ELISPOT assay. Tumor biopsies were evaluated for immune cell infiltration, chemokine protein expression, and gene expression. RESULTS: Vaccination and intratumoral administration of IFNγ were well tolerated. Circulating T cell responses to vaccine were detected in six of nine patients. IFNγ increased production of chemokines CXCL10, CXCL11, and CCL5 in patient tumors. Neither vaccination alone, nor the addition of IFNγ promoted immune cell infiltration or induced anti-tumor immune gene signatures. CONCLUSION: The melanoma vaccine induced circulating T cell responses, but it failed to infiltrate metastases, thus highlighting the need for combination strategies to support T cell infiltration. A single intratumoral injection of IFNγ induced T cell-attracting chemokines; however, it also induced secondary immune regulation that may paradoxically limit immune infiltration and effector functions. Alternate dosing strategies or additional combinatorial treatments may be needed to promote trafficking and retention of tumor-reactive T cells in melanoma metastases.
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Vacunas contra el Cáncer/inmunología , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Factores Inmunológicos/uso terapéutico , Inmunoterapia/métodos , Interferón gamma/uso terapéutico , Melanoma/terapia , Linfocitos T/inmunología , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/inmunología , Movimiento Celular , Células Cultivadas , Ensayo de Immunospot Ligado a Enzimas , Femenino , Estudios de Seguimiento , Humanos , Linfocitos Infiltrantes de Tumor/patología , Masculino , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Fragmentos de Péptidos/inmunología , Análisis de Supervivencia , Vacunas de Subunidad/inmunologíaRESUMEN
INTRODUCTION: Infiltration of cancers by T cells is associated with improved patient survival and response to immune therapies; however, optimal approaches to induce T cell infiltration of tumors are not known. This study was designed to assess whether topical treatment of melanoma metastases with the TLR7 agonist imiquimod plus administration of a multipeptide cancer vaccine will improve immune cell infiltration of melanoma metastases. PATIENTS AND METHODS: Eligible patients were immunized with a vaccine comprised of 12 melanoma peptides and a tetanus toxoid-derived helper peptide, and imiquimod was applied topically to metastatic tumors daily. Adverse events were recorded, and effects on the tumor microenvironment were evaluated from sequential tumor biopsies. T cell responses were assessed by IFNγ ELIspot assay and T cell tetramer staining. Patient tumors were evaluated for immune cell infiltration, cytokine and chemokine production, and gene expression. RESULTS AND CONCLUSIONS: Four eligible patients were enrolled, and administration of imiquimod and vaccination were well tolerated. Circulating T cell responses to the vaccine was detected by ex vivo ELIspot assay in 3 of 4 patients. Treatment of metastases with imiquimod induced immune cell infiltration and favorable gene signatures in the patients with circulating T cell responses. This study supports further study of topical imiquimod combined with vaccines or other immune therapies for the treatment of melanoma.
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Aminoquinolinas/uso terapéutico , Antígenos de Neoplasias/inmunología , Antineoplásicos/uso terapéutico , Vacunas contra el Cáncer/inmunología , Melanoma/terapia , Fragmentos de Péptidos/inmunología , Neoplasias Cutáneas/terapia , Linfocitos T/efectos de los fármacos , Administración Tópica , Anciano , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Terapia Combinada , Citocinas/genética , Citocinas/metabolismo , Ensayo de Immunospot Ligado a Enzimas , Femenino , Humanos , Imiquimod , Linfocitos Infiltrantes de Tumor/patología , Masculino , Melanoma/secundario , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Cutáneas/secundario , Linfocitos T/inmunología , Receptor Toll-Like 7/agonistas , Transcriptoma/inmunología , Vacunas de Subunidad/inmunologíaRESUMEN
Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4(+) T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8(+) T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund's adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4(+) and CD8(+) T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4(+) T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3281-295 (49 %) and tyrosinase386-406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8(+) T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4(+) and CD8(+) T cell responses against melanoma antigens. CD4(+) T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8(+) T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.
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Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Epítopos/inmunología , Antígenos HLA/genética , Antígenos HLA/inmunología , Melanoma/inmunología , Péptidos/inmunología , Alelos , Secuencia de Aminoácidos , Antígenos CD4/biosíntesis , Antígenos CD4/inmunología , Diferenciación Celular , Humanos , Datos de Secuencia Molecular , Neoplasias Cutáneas , Melanoma Cutáneo MalignoRESUMEN
The critical roles of CD4+ T cells have been understudied for cancer vaccines. Here we report long-term clinical outcomes of a randomized multicenter phase II clinical trial (NCT00118274), where patients with high-risk melanoma received a multipeptide vaccine targeting CD8+ T cells (12MP) and were randomized to receive either of two vaccines for CD4+ (helper) T cells: 6MHP (6 melanoma-specific helper peptides), or tet (a nonspecific helper peptide from tetanus toxoid). Cyclophosphamide (Cy) pre-treatment was also assessed. Primary outcomes for T cell responses to 12MP, 6MHP, and tet were previously reported, suggesting immunogenicity of both vaccines but that CD8 T cell responses to 12MP were lower when tet was replaced with 6MHP. Here, in post-hoc analyses, we report durable prolongation of overall survival by adding 6MHP instead of tet. That benefit was experienced only by male patients. A favorable interaction of 6MHP and Cy is also suggested. Multivariable Cox regression analysis of the intent-to-treat population identify vaccine arm (12MP + 6MHP+Cy) and patient sex (male) as the two significant predictors of enhanced survival. These findings support the value of adding cognate T cell help to cancer vaccines and also suggest a need to assess the impact of patient sex on immune therapy outcomes.
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Vacunas contra el Cáncer , Melanoma , Humanos , Masculino , Adyuvantes Inmunológicos , Linfocitos T CD8-positivos , Melanoma/tratamiento farmacológico , Péptidos , FemeninoRESUMEN
We conducted a randomized clinical trial in 45 patients with resected AJCC stage IIB-IV melanoma to characterize cellular and molecular events at sites of immunization with incomplete Freund's adjuvant (IFA) alone, or a melanoma vaccine in IFA. At a primary vaccine site, all patients received a multi-peptide melanoma vaccine in IFA. At a replicate vaccine site, which was biopsied, group 1 received IFA only; group 2 received vaccine in IFA. Lymphocytes isolated from replicate vaccine site microenvironments (VSME) were compared to time-matched peripheral blood mononuclear cells (PBMC) in ELISpot and flow cytometry assays. Compared to PBMC, the VSME had fewer naïve and greater proportions of effector memory CD8(+) T cells (TCD8). The vast majority of TCD8 within the VSME were activated (CD69(+)), with a concentration of antigen-specific (tetramer(pos)) cells in the VSME, particularly in vaccine sites with peptide (group 2). CXCR3(+) lymphocytes were concentrated in the VSME of all patients, suggesting IFA-induced chemokine recruitment. TCD8 expression of retention integrins αEß7 and α1ß1 was elevated in VSME, with the highest levels observed in antigen-specific cells in VSME containing peptide (group 2). TCD8 retained in the VSME of both groups were strikingly dysfunctional, with minimal IFN-γ production in response to peptide stimulation and few tetramer(pos) cells producing IFN-γ. These data suggest that vaccine-induced selective retention and dysfunction of antigen-specific TCD8 within VSME may represent a significant mechanism underlying transient immune responses and low clinical response rates to peptide vaccines administered in IFA.
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Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Adyuvante de Freund/inmunología , Leucocitos Mononucleares/inmunología , Lípidos/inmunología , Melanoma/inmunología , Péptidos/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Femenino , Adyuvante de Freund/administración & dosificación , Humanos , Integrina alfa1beta1/metabolismo , Integrinas/metabolismo , Interferón gamma/metabolismo , Lectinas Tipo C/metabolismo , Lípidos/administración & dosificación , Activación de Linfocitos , Masculino , Melanoma/metabolismo , Persona de Mediana Edad , Receptores CXCR3/metabolismoRESUMEN
BACKGROUND: Performing biopsies for correlative studies in cancer trials raises ethical and regulatory concerns and may impact trial accrual negatively. However, strategies to address these concerns remain largely unexplored. PURPOSE: We sought to assess the perceived risk of mandatory tissue biopsies to be performed for research purposes as part of a clinical trial of a melanoma vaccine by administering a pretrial accrual assessment questionnaire in the population of interest. Furthermore, we explored how such survey data may be used to address potential concerns of regulatory and funding organizations that may not be able to assess the risks of those biopsies. Method A total of 91 melanoma patients, similar to potential participants in a melanoma vaccine pilot study, scored their willingness, on a 9-point Likert scale, to participate in vaccine trials involving no skin biopsy versus a skin biopsy resulting in a 3-, 6-, or 12-cm scar. The vaccine trial was performed with skin biopsies leaving a 6-cm scar. Accrual rate was assessed and that accrual was compared to the accrual of two similar vaccine trials without biopsy requirements. RESULTS: A total of 95% of the participants expressed willingness to enter a vaccine trial (likely to highly likely). This proportion decreased to 74%, 63%, and 59%, respectively, for vaccine trials requiring skin biopsy leaving a 3-, 6-, or 12-cm scar. The trial was designed with an estimated 40% decrease in accrual rate compared to prior studies (2 participants expected/month). The resulting trial with a 6-cm biopsy exceeded that accrual rate estimate and had a similar accrual rate to vaccine trials without a biopsy (4.1 vs 2.7-4.6 participants/month). LIMITATIONS: Potential limitations of this study include the exclusion of some questionnaire responses and the post hoc nature of the analysis. CONCLUSION: Willingness to participate in vaccine trials was decreased by the requirement for skin biopsy, but the size of the biopsy was less of a deterrent than expected. Findings from brief surveys may aid in risk assessment during regulatory review, predict acceptability of tissue collection for correlative studies, and support regulatory approval and meeting accrual goals of the study.
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Vacunas contra el Cáncer/uso terapéutico , Consentimiento Informado , Melanoma/patología , Melanoma/terapia , Aceptación de la Atención de Salud/estadística & datos numéricos , Participación del Paciente , Biopsia , Humanos , Proyectos Piloto , Proyectos de Investigación , Medición de Riesgo , Estadística como Asunto , Encuestas y Cuestionarios , VirginiaRESUMEN
BACKGROUND: A vaccine containing 6 melanoma-associated peptides to stimulate helper T cells (6MHP) is safe, immunogenic, and clinically active. A phase I/II trial was designed to evaluate safety and immunogenicity of 6MHP vaccines plus programmed death 1 (PD-1) blockade. PARTICIPANTS AND METHODS: Participants with advanced melanoma received 6MHP vaccines in an incomplete Freund's adjuvant (6 vaccines over 12 weeks). Pembrolizumab was administered intravenously every 3 weeks. Tumor biopsies at baseline and day 22 were analyzed by multiplex immunohistochemistry. Primary end points were safety (Common Terminology Criteria for Adverse Events V.4.03) and immunogenicity (ex vivo interferon-γ ELISpot assay). Additional end points included changes in the tumor microenvironment (TME) and clinical outcomes. RESULTS: Twenty-two eligible participants were treated: 6 naïve to PD-1 antibody (Ab) and 16 PD-1 Ab-experienced. Median follow-up was 24.4 months. Most common treatment-related adverse events (any grade) included injection site reactions, fatigue, anemia, lymphopenia, fever, elevated aspartate aminotransferase, pruritus, and rash. Treatment-related dose-limiting toxicities were observed in 3 (14%) participants, which did not cross the study safety bound. A high durable T cell response (Rsp) to 6MHP was detected in only one participant, but twofold T cell Rsps to 6MHP were detected in 7/22 (32%; 90% CI (16% to 52%)) by week 13. Objective clinical responses were observed in 23% (1 complete response, 4 partial responses), including 4/6 PD-1 Ab-naïve (67%) and 1/16 PD-1 Ab-experienced (6%). Overall survival (OS) was longer for PD-1 Ab-naïve than Ab-experienced participants (HR 6.3 (90% CI (2.1 to 28.7)). In landmark analyses at 13 weeks, OS was also longer for those with T cell Rsps (HR 6.5 (90% CI (2.1 to 29.2)) and for those with objective clinical responses. TME evaluation revealed increased densities of CD8+ T cells, CD20+ B cells, and Tbet+ cells by day 22. CONCLUSIONS: Treatment with the 6MHP vaccine plus pembrolizumab was safe, increased intratumoral lymphocytes, and induced T cell Rsps associated with prolonged OS. The low T cell Rsp rate in PD-1 Ab-experienced participants corroborates prior murine studies that caution against delaying cancer vaccines until after PD-1 blockade. The promising objective response rate and OS in PD-1 Ab-naïve participants support consideration of a larger study in that setting.
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Vacunas contra el Cáncer , Melanoma , Linfocitos T CD8-positivos , Humanos , Melanoma/tratamiento farmacológico , Receptor de Muerte Celular Programada 1 , Microambiente Tumoral , Vacunas de Subunidad/uso terapéuticoRESUMEN
BACKGROUND: Clinical trials of immunologic therapies provide opportunities to study the cellular and molecular effects of those therapies and may permit identification of biomarkers of response. When the trials are performed at multiple centers, transport and storage of clinical specimens become important variables that may affect lymphocyte viability and function in blood and tissue specimens. The effect of temperature during storage and shipment of peripheral blood on subsequent processing, recovery, and function of lymphocytes is understudied and represents the focus of this study. METHODS: Peripheral blood samples (n = 285) from patients enrolled in 2 clinical trials of a melanoma vaccine were shipped from clinical centers 250 or 1100 miles to a central laboratory at the sponsoring institution. The yield of peripheral blood mononuclear cells (PBMC) collected before and after cryostorage was correlated with temperatures encountered during shipment. Also, to simulate shipping of whole blood, heparinized blood from healthy donors was collected and stored at 15 °C, 22 °C, 30 °C, or 40 °C, for varied intervals before isolation of PBMC. Specimen integrity was assessed by measures of yield, recovery, viability, and function of isolated lymphocytes. Several packaging systems were also evaluated during simulated shipping for the ability to maintain the internal temperature in adverse temperatures over time. RESULTS: Blood specimen containers experienced temperatures during shipment ranging from -1 to 35 °C. Exposure to temperatures above room temperature (22 °C) resulted in greater yields of PBMC. Reduced cell recovery following cryo-preservation as well as decreased viability and immune function were observed in specimens exposed to 15 °C or 40 °C for greater than 8 hours when compared to storage at 22 °C. There was a trend toward improved preservation of blood specimen integrity stored at 30 °C prior to processing for all time points tested. Internal temperatures of blood shipping containers were maintained longer in an acceptable range when warm packs were included. CONCLUSIONS: Blood packages shipped overnight by commercial carrier may encounter extreme seasonal temperatures. Therefore, considerations in the design of shipping containers should include protecting against extreme ambient temperature deviations and maintaining specimen temperature above 22 °C or preferably near 30 °C.
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Laboratorios de Hospital , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Estudios Multicéntricos como Asunto , Manejo de Especímenes , Temperatura , Transportes , Antígenos/farmacología , Recuento de Células Sanguíneas , Conservación de la Sangre , Supervivencia Celular/efectos de los fármacos , Criopreservación , Humanos , Interferón gamma/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Mitógenos/farmacología , Embalaje de Productos , Factores de TiempoRESUMEN
BACKGROUND: Experimental cancer vaccines are traditionally administered by injection in subcutaneous tissue or muscle, commonly with adjuvants that create chronic inflammatory depots. Injection of melanoma-derived peptides induces T cell responses; however, the depots that form following injection may inhibit optimization of the immune response. In skin, epidermal Langerhans cells (LC) are a dominant source of professional antigen presenting cells. We hypothesized that: (1) applying melanoma-derived peptides topically, in proximity to LC, could be immunogenic and safe, with low vaccine-site toxicity and (2) topical toll-like receptor 7 (TLR7) agonist would increase immunogenicity of the peptide vaccine. METHODS: Twelve melanoma peptides plus a tetanus helper peptide were combined with granulocyte macrophage colony stimulating factor (GM-CSF) and were administered topically on days 1, 8, and 15, to 28 patients randomized to one of four adjuvant preparations: (1) incomplete Freund's adjuvant (IFA); (2) IFA plus a TLR7 agonist (imiquimod) administered on days 0, 7, 14; (3) dimethyl sulfoxide (DMSO) or (4) DMSO+ imiquimod administered on day 0, 7, 14. Every 3 weeks thereafter (x 6), the peptides were combined with GM-CSF and were injected into the dermis and subcutis in an emulsion with IFA. Toxicities were recorded and immune responses assayed by ELIspot. RESULTS: CD8+ T cell responses to transdermal vaccination in DMSO occurred in 83% of participants in group 3 and 86% in group 4, and responses to vaccination in IFA were observed in 29% of participants in group 1 and 14% in group 2. Overall, 61% of participants had CD4+ T cell immune responses to the tetanus peptide, with large, durable responses in groups 3 and 4. Five of seven participants in group 4 had a severe rash, one that was dose limiting. Ten-year overall survival was 67% and disease-free survival was 44%. CONCLUSIONS: These data provide proof of principle for immunogenicity in humans of transdermal immunization using peptides in DMSO. Further study is warranted into the pharmacokinetics and immunobiology of TLR agonists as vaccine adjuvants during transcutaneous application. Overall survival is high, supporting further investigation of this immunization approach.
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Adyuvantes Inmunológicos/administración & dosificación , Vacunas contra el Cáncer/administración & dosificación , Imiquimod/administración & dosificación , Inmunogenicidad Vacunal , Antígenos Específicos del Melanoma/administración & dosificación , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Receptor Toll-Like 7/agonistas , Adyuvantes Inmunológicos/efectos adversos , Administración Cutánea , Adolescente , Adulto , Anciano , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/efectos adversos , Vacunas contra el Cáncer/inmunología , Femenino , Adyuvante de Freund/administración & dosificación , Adyuvante de Freund/efectos adversos , Adyuvante de Freund/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Humanos , Imiquimod/efectos adversos , Imiquimod/inmunología , Inyecciones Intradérmicas , Inyecciones Subcutáneas , Lípidos/administración & dosificación , Lípidos/efectos adversos , Lípidos/inmunología , Masculino , Melanoma/inmunología , Melanoma/metabolismo , Antígenos Específicos del Melanoma/efectos adversos , Antígenos Específicos del Melanoma/inmunología , Persona de Mediana Edad , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Factores de Tiempo , Receptor Toll-Like 7/metabolismo , Resultado del Tratamiento , Vacunación , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/efectos adversos , Vacunas de Subunidad/inmunología , Adulto JovenRESUMEN
Elevated immunity to cancer-expressed antigens can be detected in people with no history of cancer and may contribute to cancer prevention. We have previously reported that MHC-restricted phosphopeptides are cancer-expressed antigens and targets of immune recognition. However, the extent to which this immunity reflects prior or ongoing phosphopeptide exposures was not investigated. In this study, we found that preexisting immune memory to cancer-expressed phosphopeptides was evident in most healthy donors, but the breadth among donors was highly variable. Although three phosphopeptides were recognized by most donors, suggesting exposures to common microbial/infectious agents, most of the 205 tested phosphopeptides were not recognized by peripheral blood mononuclear cells (PBMC) from any donor and the remainder were recognized by only 1 to 3 donors. In longitudinal analyses of 2 donors, effector immune response profiles suggested active reexposures to a subset of phosphopeptides. These findings suggest that the immunogens generating most phosphopeptide-specific immune memory are rare infectious agents or incipient cancer cells with distinct phosphoproteome dysregulations, and that repetitive immunogenic exposures occur in individual donors. Phosphopeptide-specific immunity in PBMCs and tumor-infiltrating lymphocytes from ovarian cancer patients was limited, regardless of whether the phosphopeptide was expressed on the tumor. However, 4 of 10 patients responded to 1 to 2 immunodominant phosphopeptides, and 1 showed an elevated effector response to a tumor-expressed phosphopeptide. As the tumors from these patients displayed many phosphopeptides, these data are consistent with lack of prior exposure or impaired ability to respond to some phosphopeptides and suggest that enhancing phosphopeptide-specific T-cell responses could be a useful approach to improve tumor immunotherapy.
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Carcinoma Epitelial de Ovario/inmunología , Genes MHC Clase I/inmunología , Memoria Inmunológica/inmunología , Inmunoterapia/métodos , Fosfopéptidos/metabolismo , Estudios de Casos y Controles , Línea Celular Tumoral , Femenino , Humanos , Donantes de TejidosRESUMEN
BACKGROUND: We performed a clinical trial to evaluate safety and immunogenicity of a novel long peptide vaccine administered in combinations of incomplete Freund's adjuvant (IFA) and agonists for TLR3 (polyICLC) and TLR7/8 (resiquimod). We hypothesized that T cell responses to minimal epitope peptides (MEPs) within the long peptides would be enhanced compared with prior vaccines with MEP themselves and that T cell responses would be enhanced with TLR agonists, compared with IFA alone. METHODS: Participants with resected stage IIB-IV melanoma were vaccinated with seven long melanoma peptides (LPV7) from tyrosinase, gp100, MAGE-A1, MAGE-A10, and NY-ESO-1, each containing a known MEP for CD8+ T cells, plus a tetanus helper peptide (Tet) restricted by Class II MHC. Enrollment was guided by an adaptive design to one of seven adjuvant combinations. Vaccines were administered at weeks 1, 2, 3, 6, 9, 12 at rotating injection sites. T cell and IgG antibody (Ab) responses were measured with IFN-gamma ELIspot assay ex vivo and ELISA, respectively. RESULTS: Fifty eligible participants were assigned to seven study groups, with highest enrollment on arm E (LPV7+Tet+IFA+polyICLC). There was one dose-limiting toxicity (DLT) in Group E (grade 3 injection site reaction, 6% DLT rate). All other treatment-related adverse events were grades 1-2. The CD8+ T cell immune response rate (IRR) to MEPs was 18%, less than in prior studies using MEP vaccines in IFA. The CD8+ T cell IRR trended higher for IFA-containing adjuvants (24%) than adjuvants containing only TLR agonists (6%). Overall T cell IRR to full-length LPV7 was 30%; CD4+ T cell IRR to Tet was 40%, and serum Ab IRR to LPV7 was 84%. These IRRs also trended higher for IFA-containing adjuvants (36% vs 18%, 48% vs 24%, and 97% vs 60%, respectively). CONCLUSIONS: The LPV7 vaccine is safe with each of seven adjuvant strategies and induced T cell responses to CD8 MEPs ex vivo in a subset of patients but did not enhance IRRs compared with prior vaccines using short peptides. Immunogenicity was supported more by IFA than by TLR agonists alone and may be enhanced by polyICLC plus IFA. TRIAL REGISTRATION NUMBER: NCT02126579.
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Melanoma/tratamiento farmacológico , Receptores Toll-Like/uso terapéutico , Vacunas de Subunidad/uso terapéutico , Femenino , Humanos , Masculino , Factores de Riesgo , Vacunas de Subunidad/farmacologíaRESUMEN
BACKGROUND: Peptide vaccines designed to stimulate melanoma-reactive CD4+ T cells can induce T cell and antibody (Ab) responses, associated with enhanced overall survival. We hypothesized that adding toll-like receptor 3 agonist polyICLC to an incomplete Freund's adjuvant (IFA) would be safe and would support strong, durable CD4+ T cell and Ab responses. We also hypothesized that oral low-dose metronomic cyclophosphamide (mCy) would be safe, would reduce circulating regulatory T cells (T-regs) and would further enhance immunogenicity. PARTICIPANTS AND METHODS: An adaptive design based on toxicity and durable CD4+ T cell immune response (dRsp) was used to assign participants with resected stage IIA-IV melanoma to one of four study regimens. The regimens included a vaccine comprising six melanoma peptides restricted by Class II MHC (6MHP) in an emulsion with IFA alone (Arm A), with IFA plus systemic mCy (Arm B), with IFA+ local polyICLC (Arm C), or with IFA+ polyICLC+ mCy (Arm D). Toxicities were recorded (CTCAE V.4.03). T cell responses were measured by interferon γ ELIspot assay ex vivo. Serum Ab responses to 6MHP were measured by ELISA. Circulating T-regs were assessed by flow cytometry. RESULTS: Forty-eight eligible participants were enrolled and treated. Early data on safety and dRsp favored enrollment on arm D. Total enrollment on Arms A-D were 3, 7, 6, and 32, respectively. Treatment-related dose-limiting toxicities (DLTs) were observed in 1/7 (14%) participants on arm B and 2/32 (6%) on arm D. None exceeded the 25% DLT threshold for early closure to enrollment for any arm. Strong durable T cell responses to 6MHP were detected ex vivo in 0%, 29%, 67%, and 47% of participants on arms A-D, respectively. IgG Ab responses were greatest for arms C and D. Circulating T-regs frequencies were not altered by mCy. CONCLUSIONS: 6MHP vaccines administered with IFA, polyICLC, and mCy were well tolerated. The dRsp rate for arm D of 47% (90% CI 32 to 63) exceeded the 18% (90% CI 11 to 26) rate previously observed with 6MHP in IFA alone. Vaccination with IFA+ polyICLC (arm C) also showed promise for enhancing T cell and Ab responses.
Asunto(s)
Carboximetilcelulosa de Sodio/análogos & derivados , Ciclofosfamida/administración & dosificación , Adyuvante de Freund/administración & dosificación , Lípidos/administración & dosificación , Melanoma/tratamiento farmacológico , Poli I-C/administración & dosificación , Polilisina/análogos & derivados , Vacunas de Subunidad/administración & dosificación , Administración Metronómica , Administración Oral , Anticuerpos/sangre , Linfocitos T CD4-Positivos/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/efectos adversos , Vacunas contra el Cáncer/inmunología , Carboximetilcelulosa de Sodio/administración & dosificación , Carboximetilcelulosa de Sodio/efectos adversos , Terapia Combinada , Ciclofosfamida/efectos adversos , Femenino , Adyuvante de Freund/efectos adversos , Humanos , Lípidos/efectos adversos , Masculino , Melanoma/inmunología , Melanoma/patología , Estadificación de Neoplasias , Poli I-C/efectos adversos , Polilisina/administración & dosificación , Polilisina/efectos adversos , Linfocitos T Reguladores/metabolismo , Resultado del Tratamiento , Vacunas de Subunidad/efectos adversos , Vacunas de Subunidad/inmunologíaRESUMEN
BACKGROUND: Phosphorylated peptides presented by MHC molecules represent a new class of neoantigens expressed on cancer cells and recognized by CD8 T-cells. These peptides are promising targets for cancer immunotherapy. Previous work identified an HLA-A*0201-restricted phosphopeptide from insulin receptor substrate 2 (pIRS2) as one such target. The purpose of this study was to characterize a second phosphopeptide, from breast cancer antiestrogen resistance 3 (BCAR3), and to evaluate safety and immunogenicity of a novel immunotherapic vaccine comprising either or both of these phosphorylated peptides. METHODS: Phosphorylated BCAR3 protein was evaluated in melanoma and breast cancer cell lines by Western blot, and recognition by T-cells specific for HLA-A*0201-restricted phosphorylated BCAR3 peptide (pBCAR3126-134) was determined by 51Cr release assay and intracellular cytokine staining. Human tumor explants were also evaluated by mass spectrometry for presentation of pIRS2 and pBCAR3 peptides. For the clinical trial, participants with resected stage IIA-IV melanoma were vaccinated 6 times over 12 weeks with one or both peptides in incomplete Freund's adjuvant and Hiltonol (poly-ICLC). Adverse events (AEs) were coded based on National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) V.4.03, with provision for early study termination if dose-limiting toxicity (DLT) rates exceeded 33%. The enrollment target was 12 participants evaluable for immune response to each peptide. T-cell responses were assessed by interferon-γ ELISpot assay. RESULTS: pBCAR3 peptides were immunogenic in vivo in mice, and in vitro in normal human donors, and T-cells specific for pBCAR3126-134 controlled outgrowth of a tumor xenograft. The pIRS21097-1105 peptide was identified by mass spectrometry from human hepatocellular carcinoma tumors. In the clinical trial, 15 participants were enrolled. All had grade 1 or 2 treatment-related AEs, but there were no grade 3-4 AEs, DLTs or deaths on study. T-cell responses were induced to the pIRS21097-1105 peptide in 5/12 patients (42%, 90% CI 18% to 68%) and to the pBCAR3126-134 peptide in 2/12 patients (17%, 90% CI 3% to 44%). CONCLUSION: This study supports the safety and immunogenicity of vaccines containing the cancer-associated phosphopeptides pBCAR3126-134 and pIRS21097-1105, and the data support continued development of immune therapy targeting phosphopeptides. Future studies will define ways to further enhance the magnitude and durability of phosphopeptide-specific immune responses. TRIAL REGISTRATION NUMBER: NCT01846143.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/efectos adversos , Inmunoterapia/efectos adversos , Melanoma/terapia , Neoplasias Cutáneas/terapia , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos de Neoplasias/genética , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Humanos , Inmunogenicidad Vacunal , Inmunoterapia/métodos , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/inmunología , Masculino , Melanoma/inmunología , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Fosfopéptidos/genética , Fosfopéptidos/inmunología , Proyectos Piloto , Prueba de Estudio Conceptual , Neoplasias Cutáneas/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/efectos adversos , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Cancer vaccines require adjuvants to induce effective immune responses; however, there is no consensus on optimal adjuvants. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC or TLR4 agonist lipopolysaccharide (LPS), combined with CD4 T cell activation, would support strong and durable CD8+ T cell responses, whereas addition of an incomplete Freund's adjuvant (IFA) would reduce magnitude and persistence of immune responses. PATIENTS AND METHODS: Participants with resected stage IIB-IV melanoma received a vaccine comprised of 12 melanoma peptides restricted by Class I MHC (12MP), plus a tetanus helper peptide (Tet). Participants were randomly assigned 2:1 to cohort 1 (LPS dose-escalation) or cohort 2 (polyICLC). Each cohort included 3 subgroups (a-c), receiving 12MP + Tet + TLR agonist without IFA (0), or with IFA in vaccine one (V1), or all six vaccines (V6). Toxicities were recorded (CTCAE v4). T cell responses were measured with IFNγ ELIspot assay ex vivo or after one in vitro stimulation (IVS). RESULTS: Fifty-three eligible patients were enrolled, of which fifty-one were treated. Treatment-related dose-limiting toxicities (DLTs) were observed in 0/33 patients in cohort 1 and in 2/18 patients in cohort 2 (11%). CD8 T cell responses to 12MP were detected ex vivo in cohort 1 (42%) and in cohort 2 (56%) and in 18, 50, and 72% for subgroups V0, V1, and V6, respectively. T cell responses to melanoma peptides were more durable and of highest magnitude for IFA V6. CONCLUSIONS: LPS and polyICLC are safe and effective vaccine adjuvants when combined with IFA. Contrary to the central hypothesis, IFA enhanced T cell responses to peptide vaccines when added to TLR agonists. Future studies will aim to understand mechanisms underlying the favorable effects with IFA. TRIAL REGISTRATION: The clinical trial Mel58 was performed with IRB (#15781) and FDA approval and is registered with Clinicaltrials.gov on April 25, 2012 (NCT01585350). Patients provided written informed consent to participate. Enrollment started on June 24, 2012.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Vacunas contra el Cáncer/administración & dosificación , Carboximetilcelulosa de Sodio/análogos & derivados , Adyuvante de Freund/administración & dosificación , Lípidos/administración & dosificación , Lipopolisacáridos/administración & dosificación , Melanoma/tratamiento farmacológico , Poli I-C/administración & dosificación , Polilisina/análogos & derivados , Receptores Toll-Like/agonistas , Vacunas de Subunidad/administración & dosificación , Adyuvantes Inmunológicos/efectos adversos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/efectos adversos , Carboximetilcelulosa de Sodio/administración & dosificación , Carboximetilcelulosa de Sodio/efectos adversos , Femenino , Adyuvante de Freund/efectos adversos , Humanos , Lípidos/efectos adversos , Lipopolisacáridos/efectos adversos , Masculino , Melanoma/inmunología , Poli I-C/efectos adversos , Polilisina/administración & dosificación , Polilisina/efectos adversos , Vacunas de Subunidad/efectos adversosRESUMEN
BACKGROUND: We hypothesized that lymph nodes draining sites of cutaneous vaccination could be identified by sentinel node biopsy techniques, and that measuring T-cell response with lymphocytes obtained from these lymph nodes would provide a more sensitive measure of immunogenicity than would the same measurement made with peripheral blood lymphocytes (PBL). METHODS: ELISpot analysis was used to determine the magnitude of vaccine-specific T-cell response in the sentinel immunized nodes (SIN), random lymph nodes, and peripheral blood lymphocytes (PBL) obtained from patients enrolled in clinical trials of experimental melanoma vaccines. RESULTS: The SIN biopsy was successful in 97% of cases and morbidity was very low. The T-cell response to vaccination was detected with greater sensitivity in the SIN (57%) than in PBL (39%), and evaluation of T-cell responses in the SIN and the PBL together yielded T-cell responses in 63% of patients. When the T-cell responses from a SIN and a random lymph node were compared in four patients, immune responses were detected to one of the vaccine peptides in three of these four patients. In all of those cases, responses were present in the SIN but absent from the random lymph node. CONCLUSION: Measurements of T-cell responsiveness to cutaneous immunization are more frequently positive in the SIN than they are in the PBL, however evaluation of both the SIN and PBL permit a more sensitive measure of T-cell immunogenicity than use of either single source.
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Vacunas contra el Cáncer/inmunología , Ganglios Linfáticos/inmunología , Linfocitos T Citotóxicos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Ensayos Clínicos como Asunto , Estudios de Factibilidad , Humanos , Melanoma/inmunología , Persona de Mediana Edad , Monitorización Inmunológica , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/inmunología , Biopsia del Ganglio Linfático Centinela , Neoplasias Cutáneas/inmunología , VacunaciónRESUMEN
BACKGROUND: Human cancer vaccines incorporating autologous tumor cells carry a risk of implantation and subsequent metastasis of viable tumor cells into the patient who is being treated. Despite the fact that the melanoma cell preparations used in a recent vaccine trial (Mel37) were gamma-irradiated (200 Gy), approximately 25% of the preparations failed quality control release criteria which required that the irradiated cells incorporate 3H-thymidine at no more than 5% the level seen in the non-irradiated cells. We have, therefore, investigated ultraviolet (UV)-irradiation as a possible adjunct to, or replacement for gamma-irradiation. METHODS: Melanoma cells were gamma- and/or UV-irradiated. 3H-thymidine uptake was used to assess proliferation of the treated and untreated cells. Caspase-3 activity and DNA fragmentation were measured as indicators of apoptosis. Immunohistochemistry and Western blot analysis was used to assess antigen expression. RESULTS: UV-irradiation, either alone or in combination with gamma-irradiation, proved to be extremely effective in controlling the proliferation of melanoma cells. In contrast to gamma-irradiation, UV-irradiation was also capable of inducing significant levels of apoptosis. UV-irradiation, but not gamma-irradiation, was associated with the loss of tyrosinase expression. Neither form of radiation affected the expression of gp100, MART-1/MelanA, or S100. CONCLUSION: These results indicate that UV-irradiation may increase the safety of autologous melanoma vaccines, although it may do so at the expense of altering the antigenic profile of the irradiated tumor cells.
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Vacunas contra el Cáncer , Rayos gamma , Melanoma/terapia , Células Tumorales Cultivadas/efectos de la radiación , Rayos Ultravioleta , Apoptosis/efectos de la radiación , Western Blotting , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , InmunohistoquímicaRESUMEN
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.
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Antígenos de Neoplasias/administración & dosificación , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Inmunidad Celular/inmunología , Melanoma/inmunología , Melanoma/terapia , Autotolerancia/inmunología , Animales , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/uso terapéutico , Humanos , Melanoma/patología , Ensayos Clínicos Controlados Aleatorios como Asunto/métodos , Ensayos Clínicos Controlados Aleatorios como Asunto/tendencias , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/uso terapéuticoRESUMEN
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.